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Decellularized ECMs have been used as biological scaffolds for tissue repair due to their tissue-specific biochemical and mechanical composition, poorly simulated by other materials. It is used as patches and powders, and it could be further processed via enzymatic digestion under acidic conditions using pepsin. However, part of the bioactivity is lost during the digestion process due to protein denaturation. Here, stepwise digestion was developed to prepare a competent biomaterial for osteogenesis from three different ECM sources. In addition, three different proteases were compared to evaluate the most effective digestion protocol for specific cellular processes. GAGs and peptide quantification showed that the stepwise method yielded a higher concentration of bioactive residues. Circular dichroism analysis also showed that the stepwise approach preserved the secondary structures better. The protein profiles of the digested ECMs were analyzed, and it was found to be highly diverse and tissue-specific. The digestion of ECM from pericardium produced peptides originated from 94 different proteins, followed by 48 proteins in ECM from tendon and 35 proteins in ECM from bone. In addition, digested products from pericardium ECM yielded increased proliferation and differentiation of bone marrow mesenchymal stem cells to mature osteoblasts.

This study aimed to quantitatively and qualitatively determine the proteomic profile of apical periodontitis (AP) in type 2 diabetes mellitus (T2DM) patients in comparison with systemically noncompromised patients and to correlate the protein expression of both groups with their biological functions.The sample consisted of 18 patients with asymptomatic AP divided into two groups according to the presence of T2DM: diabetic group-patients with T2DM (n = 9) and control group-systemically healthy patients (n = 9). After sample collection, the root canal samples were prepared for proteomic analysis using reverse-phase liquid chromatography-mass spectrometry. Label-free quantitative proteomic analysis was performed by Protein Lynx Global Service software. Differences in protein expression between groups were calculated using t-test (p .05). Biological functions were analysed using the Homo sapiens UniProt database.A total of 727 human proteins were identified in all samples. Among them, 124 proteins common to both groups were quantified, out of which 65 proteins from the diabetic group showed significant differences compared with the control: 43 upregulated (p .05) and 22 downregulated (p .05) proteins. No significant differences in protein expression were seen for the remaining 59 proteins (p .05). Most proteins with differences in expression were related to immune/inflammatory response. Neutrophil gelatinase-associated lipocalin, Plastin-2, Lactotransferrin and 13 isoforms of immunoglobulins were upregulated. In contrast, Protein S100-A8, Protein S100-A9, Histone H2B, Neutrophil defensin 1, Neutrophil defensin 3 and Prolactin-inducible protein were downregulated.Quantitative differences were demonstrated in the expression of proteins common to diabetic and control groups, mainly related to immune response, oxidative stress, apoptosis and proteolysis. These findings revealed biological pathways that provide the basis to support clinical findings on the relationship between AP and T2DM.

Metalloproteomics is an innovative methodology for identifying of protein-associated mercury. Thus, we analyzed the muscle proteome of Arapaima gigas (pirarucu), collected in the Madeira River of the Brazilian Amazon, to identify protein-associated mercury, with the aim of identifying possible mercury biomarkers in fish muscle tissue. After obtaining the protein pellet, we conducted two-dimensional electrophoresis (2D PAGE) to fractionate the muscle proteome. Total mercury in muscle tissue and protein pellets and mapping of mercury content in protein spots of the 2D PAGE gels was determined using graphite furnace atomic absorption spectrometry (GFAAS). The protein-associated mercury identification was performed using liquid chromatography coupled with sequence mass spectrometry (LC‒MS/MS). Total mercury determinations by GFAAS indicated concentrations on the order of 153 ± 1.90 mg kg

The HDL proteome has been widely recognized as an important mediator of HDL function. While a variety of HDL isolation methods exist, their impact on the HDL proteome and its associated function remain largely unknown. Here, we compared three of the most common methods for HDL isolation, namely immunoaffinity (IA), density gradient ultracentrifugation (UC), and dextran-sulfate precipitation (DS), in terms of their effects on the HDL proteome and associated functionalities. We used state-of-the-art mass spectrometry to identify 171 proteins across all three isolation methods. IA-HDL contained higher levels of paraoxonase 1, apoB, clusterin, vitronectin, and fibronectin, while UC-HDL had higher levels of apoA2, apoC3, and α-1-antytrypsin. DS-HDL was enriched with apoA4 and complement proteins, while the apoA2 content was very low. Importantly, size-exclusion chromatography analysis showed that IA-HDL isolates contained subspecies in the size range above 12 nm, which were entirely absent in UC-HDL and DS-HDL isolates. Analysis of these subspecies indicated that they primarily consisted of apoA1, IGκC, apoC1, and clusterin. Functional analysis revealed that paraoxonase 1 activity was almost completely lost in IA-HDL, despite high paraoxonase content. We observed that the elution conditions, using 3M thiocyanate, during IA resulted in an almost complete loss of paraoxonase 1 activity. Notably, the cholesterol efflux capacity of UC-HDL and DS-HDL was significantly higher compared to IA-HDL. Together, our data clearly demonstrate that the isolation procedure has a substantial impact on the composition, subclass distribution, and functionality of HDL. In summary, our data show that the isolation procedure has a significant impact on the composition, subclass distribution and functionality of HDL. Our data can be helpful in the comparison, replication and analysis of proteomic datasets of HDL.

The neocortex of P301S mice, used as a model of fronto-temporal lobar degeneration linked to tau mutation (FTLD-tau), and wild-type mice, both aged 9 months, were analyzed with conventional label-free phosphoproteomics and SWATH-MS (sequential window acquisition of all theoretical fragment ion spectra mass spectrometry) to assess the (phospho)proteomes. The total number of identified dysregulated phosphoproteins was 328 corresponding to 524 phosphorylation sites. The majority of dysregulated phosphoproteins, most of them hyperphosphorylated, were proteins of the membranes, synapses, membrane trafficking, membrane vesicles linked to endo- and exocytosis, cytoplasmic vesicles, and cytoskeleton. Another group was composed of kinases. In contrast, proteins linked to DNA, RNA metabolism, RNA splicing, and protein synthesis were hypophosphorylated. Other pathways modulating energy metabolism, cell signaling, Golgi apparatus, carbohydrates, and lipids are also targets of dysregulated protein phosphorylation in P301S mice. The present results, together with accompanying immunohistochemical and Western-blotting studies, show widespread abnormal phosphorylation of proteins, in addition to protein tau, in P301S mice. These observations point to dysregulated protein phosphorylation as a relevant contributory pathogenic component of tauopathies.

Proteomic profile; Organ dysfunction; Septic shock Perfil proteòmic; Disfunció orgànica; Xoc sèptic Perfil proteómico; Disfunción orgánica; Choque séptico Introduction The search for new biomarkers that allow an early diagnosis in sepsis and predict its evolution has become a necessity in medicine. The objective of this study is to identify, through omics techniques, potential protein biomarkers that are expressed in patients with sepsis and their relationship with organ dysfunction and mortality. Methods Prospective, observational and single-center study that included adult patients (≥ 18 years) who were admitted to a tertiary hospital and who met the criteria for sepsis. A mass spectrometry-based approach was used to analyze the plasma proteins in the enrolled subjects. Subsequently, using recursive feature elimination classification and cross-validation with a vector classifier, an association of these proteins with mortality and organ dysfunction was established. The protein-protein interaction network was analyzed with String software. Results 141 patients were enrolled in this study. Mass spectrometry identified 177 proteins. Of all of them, and by recursive feature elimination, nine proteins (GPX3, APOB, ORM1, SERPINF1, LYZ, C8A, CD14, APOC3 and C1QC) were associated with organ dysfunction (SOFA > 6) with an accuracy of 0.82 ± 0.06, precision of 0.85 ± 0.093, sensitivity 0.81 ± 0.10, specificity 0.84 ± 0.10 and AUC 0.82 ± 0.06. Twenty-two proteins (CLU, LUM, APOL1, SAA1, CLEBC3B, C8A, ITIH4, KNG1, AGT, C7, SAA2, APOH, HRG, AFM, APOE, APOC1, C1S, SERPINC1, IGFALS, KLKB1, CFB and BTD) were associated with mortality with an accuracy of 0.86 ± 0.05, a precision of 0.91 ± 0.05, a sensitivity of 0.91 ± 0.05, a specificity of 0.72 ± 0.17, and an area under the curve (AUC) of 0.81 ± 0.08 with a confidence interval of 95%. Conclusion In sepsis there are proteomic patterns associated with organ dysfunction and mortality. To Toni del Pino, Rosa Ras and Pol Herrero from the Proteomics and Metabolomics Area of the Center for Omic Sciences (COS), a Joint between Rovira I Virgili University and Eurecat (Reus, Spain), for their contribution to the proteomics analysis. Samples and data from patients included in this study were provided by the Vall d’Hebron University Hospital Biobank (PT20/00107), integrated in the Spanish National Biobanks Network, and they were processed following standard operating procedures with the appropriate approval of the Ethical and Scientific Committees. The authors kindly appreciate the generous donation of samples and clinical data of the donors of the Sepsis Bank of HUVH Biobank.

Glutamatergic neurotransmission dysfunction and the early involvement of the hippocampus have been proposed to be important aspects of the pathophysiology of schizophrenia. Here, we performed proteomic analysis of hippocampus postmortem samples from schizophrenia patients as well as neural cells-neurons and oligodendrocytes-treated with MK-801, an NMDA receptor antagonist. There were similarities in processes such as oxidative stress and apoptotic process when comparing hippocampus samples with MK-801-treated neurons, and in proteins synthesis when comparing hippocampus samples with MK-801-treated oligodendrocytes. This reveals that studying the effects of glutamatergic dysfunction in different neural cells can contribute to a better understanding of what it is observed in schizophrenia patients' postmortem brains.

Testosterone is essential to maintain qualitative spermatogenesis. Nonetheless, no studies have been yet performed in humans to analyze the testosterone-mediated expression of sperm proteins and their importance in reproduction. Thus, this study aimed to identify sperm protein alterations in male hypogonadism using proteomic profiling. We have performed a comparative proteomic analysis comparing sperm from fertile controls (a pool of 5 normogonadic normozoospermic fertile men) versus sperm from patients with secondary hypogonadism (a pool of 5 oligozoospermic hypogonadic patients due to isolated LH deficiency). Sperm protein composition was analyzed, after peptide labelling with Isobaric Tags, via liquid chromatography followed by tandem mass spectrometry (LC-MS/MS) on an LTQ Velos-Orbitrap mass spectrometer. LC-MS/MS data were analyzed using Proteome Discoverer. Criteria used to accept protein identification included a false discovery rate (FDR) of 1% and at least 1 peptide match per protein. Up to 986 proteins were identified and, of those, 43 proteins were differentially expressed: 32 proteins were under-expressed and 11 were over-expressed in the pool of hypogonadic patients compared to the controls. Bioinformatic analyses were performed using UniProt Knowledgebase, and the Gene Ontology Consortium database based on PANTHER. Notably, 13 of these 43 differentially expressed proteins have been previously reported to be related to sperm function and spermatogenesis. Western blot analyses for A-Kinase Anchoring Protein 3 (AKAP3) and the Prolactin Inducible Protein (PIP) were used to confirm the proteomics data. In summary, a high-resolution mass spectrometry-based proteomic approach was used for the first time to describe alterations of the sperm proteome in secondary male hypogonadism. Some of the differential sperm proteins described in this study, which include Prosaposin, SMOC-1, SERPINA5, SPANXB1, GSG1, ELSPBP1, fibronectin, 5-oxoprolinase, AKAP3, AKAP4, HYDIN, ROPN1B, ß-Microseminoprotein and Protein S100-A8, could represent new targets for the design of infertility treatments due to androgen deficiency.

Financial support was provided by the Instituto de Salud Carlos III de Madrid (COV20/00110), co-funded by the European Development Regional Fund (A Way to Achieve Europe programme) and Centro de Investigación Biomedica En Red Enfermedades Respiratorias(CIBERES). CIBERES is an initiative of the Instituto de Salud Carlos III. Suported by: Programa de donaciones “estar preparados” UNESPA (Madrid, Spain); and Fundación Francisco Soria Melguizo (Madrid, Spain).Finançat per La Fundació La Marató de TV3, projecte amb codi 202108-30/-31. COVIDPONENT is funded by Institut Català de la Salut and Gestió de Serveis Sanitaris. APT was funded by the Sara Borrell Research Grant CD018/0123 funded by the Instituto de Salud Carlos III and co-financed by the European Development Regional Fund (A Way to Achieve Europe programme). MCGH is the recipient of a predoctoral fellowship from the “University of Lleida”.DdGC (Miguel Servet 2020: CP20/00041) and MM (PFIS:FI21/00187) have received financial support from the Instituto de Salud Carlos III, cofunded by the European Social Fund (ESF) / “Investing in your future”

Similar to dementia, the risk for developing type 2 diabetes mellitus (T2DM) increases with age, and T2DM also increases the risk for dementia, particularly Alzheimer's disease (AD). Although T2DM is primarily a peripheral disorder and AD is a central nervous system disease, both share some common features as they are chronic and complex diseases, and both show involvement of oxidative stress and inflammation in their progression. These characteristics suggest that T2DM may be associated with AD, which gave rise to a new term, type 3 diabetes (T3DM). In this study, we searched for matching peripheral proteomic biomarkers of AD and T2DM based in a systematic review of the available literature. We identified 17 common biomarkers that were differentially expressed in both patients with AD or T2DM when compared with healthy controls. These biomarkers could provide a useful workflow for screening T2DM patients at risk to develop AD. Semelhante à demência, o risco de desenvolver diabetes mellitus tipo 2 (DM2) aumenta com a idade, e o DM2 também aumenta o risco de demência, particularmente a doença de Alzheimer (DA). Embora o DM2 seja primariamente um distúrbio periférico e a DA seja uma doença do sistema nervoso central, ambas compartilham algumas características comuns, pois são doenças crônicas e complexas, e ambas apresentam envolvimento de estresse oxidativo e inflamação em sua progressão. Essas características sugerem que o DM2 pode estar associado à DA, o que deu origem a um novo termo, diabetes tipo 3 (DM3). Neste estudo, procuramos combinar biomarcadores proteômicos periféricos de DA e DM2 com base em uma revisão sistemática da literatura disponível. Identificamos 17 biomarcadores comuns que foram expressos diferencialmente em ambos os pacientes com DA ou DM2 quando comparados com controles saudáveis. Esses biomarcadores podem fornecer um fluxo de trabalho útil para triagem de pacientes com DM2 em risco de desenvolver DA.

Introduction: With the evolution of diagnostic techniques in traumatic brain injury (TBI), the study of neurological injury has made progress based on the concepts of primary and secondary injury, leading to the era of proteomics to understand the complex molecular events involved in the process. Objectives: This narrative review is intended to discuss the state of the art of the most frequently used biomarkers in TBI, their clinical utility, and the implications for therapeutic decision-making protocols. Materials and methods: In order to fulfill the objective of this paper, a literature review was conducted of the most important databases. Results: Several biomarkers have been studied as prognostic factors in patients with TBI. Learning about their sensitivity and specificity in neurological injury, and its post-trauma evolution over time, has been the goal of various papers in the past few years. Conclusion: Breakthroughs in the study of protein degradation make it necessary to broaden the spectrum and knowledge of new diagnostic methods in TBI. Further studies are needed to define the role of biomarkers and to promote protocols integrating specific values. Resumen Introducción: Con la evolución de las técnicas diagnósticas en el trauma craneoencefálico, el estudio de la lesión neurológica ha progresado sobre los conceptos de lesión primaria y secundaria, para entrar así en la era de la proteómica y, con ella, entender los complejos eventos moleculares existentes en su proceso. Objetivos: En esta revisión narrativa se pretende presentar el estado actual de los biomarcadores que más se usan en lesión cerebral traumática, su utilidad clínica y las implicaciones en protocolos de decisión terapéutica. Materiales y métodos: Para dar respuesta al objetivo de este trabajo, se realizó una revisión de la literatura en las principales bases de datos. Resultados: Se han estudiado varios biomarcadores como factor pronóstico en pacientes con trauma craneoencefálico. Conocer su sensibilidad y especificidad para la lesión neurológica, así como su evolución en el tiempo tras el traumatismo, ha sido el objetivo de diversos trabajos en los últimos años. Conclusión: El avance en el estudio de los productos de degradación de las proteínas hace necesario ampliar el espectro y el conocimiento en el campo de los nuevos métodos diagnósticos en el trauma craneoencefálico. Se requieren más estudios para definir la función de los biomarcadores y proponer protocolos que integren valores específicos.

Una review sulle scienze OMICHE nella gestione del paziente con sindrome dell’apnea ostruttiva del sonno.La sindrome da apnea ostruttiva nel sonno (OSA) è una condizione caratterizzata dalla presenza di completo o parziale collasso delle vie aeree superiori durante il sonno, con conseguente frammentazione del sonno associata a rapidi episodi di ipossia intermittente (IH) e attivazione del sistema nervoso simpatico e dello stress ossidativo. L’OSA è associata ad un ampio spettro di patologie cardiovascolari, metaboliche, neurocognitive e comorbidità che appaiono particolarmente evidenti nei pazienti obesi, interessando entrambi i sessi in modo diverso e variando la gravità a seconda del sesso e dell’età. Negli ultimi anni, gli studi sull’OSA sono aumentati considerevolmente, ma nella pratica clinica, si tratta ancora di una malattia altamente sottodiagnosticata. Ad oggi, il gold standard per la diagnosi di OSA è la polisonnografia notturna (PSG). Tuttavia, poiché non è adatto ad un gran numero di pazienti, anche l’Home Sleep Test (HST) è un metodo diagnostico accettato. Attualmente, l’obiettivo principale della ricerca è quello di identificare metodi non invasivi per ottenere un sistema di screening altamente predittivo e non invasivo per questa categoria di soggetti. I lavori più recenti indicano che la ricerca in questo campo ha compiuto progressi significativi nell’identificazione di possibili biomarcatori in OSA, utilizzando approcci OMICI, in particolare nel campo della proteomica e della metabolomica. In questa review, analizziamo una lista di questi biomarcatori presenti in letteratura.Obstructive sleep apnaea (OSA) syndrome is a condition characterised by the presence of complete or partial collapse of the upper airways during sleep, resulting in fragmentation of sleep associated with rapid episodes of intermittent hypoxia (IH), activation of the sympathetic nervous system and oxidative stress. OSA is associated with a broad spectrum of cardiovascular, metabolic and neurocognitive comorbidities that appear to be particularly evident in obese patients, while affecting both sexes in a different manner and varying in severity according to gender and age. In recent years, studies on OSA have increased considerably, but in clinical practice, it is still a highly underdiagnosed disease. To date, the gold standard for the diagnosis of OSA is nocturnal polysomnography (PSG). However, since it is not well suited for a large number of patients, the Home Sleep Test (HST) is also an accepted diagnostic method. Currently, the major aim of research is to identify non-invasive methods to achieve a highly predictive, non-invasive screening system for these subjects. The most recent reports indicate that research in this field has made significant progress in identifying possible biomarkers in OSA, using -OMIC approaches, particularly in the fields of proteomics and metabolomics. In this review, we analyse these OMIC biomarkers found in the literature.

Objectives To evaluate the oral condition of alcohol and tobacco dependents and identify salivary protein candidates for biomarkers of oral disorders. Subjects and methods Thirty-three male volunteers were evaluated for alcohol abuse rehabilitation; nine were selected for proteomic analysis. Intraoral examination was performed, and non-stimulated saliva was collected. Salivary proteins were extracted and processed for analysis. A list of proteins identified in saliva was generated from the database and manually revised, obtaining the total number of candidate biomarkers for oral disorders. Results The mean age (n = 33) was 42.94 ± 8.61 years. Fourteen (42.4%) subjects presented with 23 oral mucosa changes, and 31 (94%) had dental plaque. A total of 282 proteins were found in saliva (n = 9), of which 26 were identified as candidates for biomarkers of oral disorders. After manual review, 21 proteins were selected. The highest number of candidates for biomarkers was associated with carcinoma of head and neck (n = 10), nasopharyngeal carcinoma (n = 6), and periodontal disease (n = 6). Conclusion Alcohol and tobacco dependents showed gingival inflammation, and less than half of them showed oral mucosa changes. Twenty-one protein candidates for biomarkers of oral disorders were identified in saliva. The two major oral disorders in number of candidates for biomarkers were head and neck cancer and periodontal disease.

We report a quantitative proteomics data analysis pipeline which, coupled to protein-directed dynamic combinatorial chemistry (DDC) experiments, enables the rapid discovery and direct characterization of protein-protein interaction (PPI) modulators. A low-affinity PD-1 binder was incubated with a library of >100 D-peptides under thiol-exchange favoring conditions, in the presence of the target protein PD-1, and we determined the S-linked dimeric species that resulted amplified in the protein samples versus the controls. We chemically synthesized the target dimer candidates and validated them by thermophoresis binding and protein-protein interaction assays. The results provide a proof-of-concept for using this strategy in the high-throughput search of improved drug-like peptide binders that block therapeutically relevant protein-protein interactions.

El objetivo de la presente Tesis doctoral ha sido analizar e interpretar la información relativa a la ecofisiología de varias poblaciones españolas de mejillón cebra (Dreissena polymorpha) en diferentes puntos del río Ebro y en el embalse de Sitjar del rio Mijares recogidas en el año 2009 dentro del proyecto:” Estudio de la estructura genética y diferenciación ecológica de las poblaciones españolas del mejillón cebra (Dreissena polymorpha) frente a factores ambientales de origen natural y antropogénico. Bases científicas para su control ambiental sostenible (CEBRAPOP)”. Se han comparado los parámetros fisiológicos, bioquímicos y de expresión proteica de cada una de las poblaciones, así como los niveles de metales y metalotioneina. En este estudio se seleccionaron siete poblaciones de mejillón cebra localizadas en diferentes puntos del río Ebro y una en el rio Mijares, caracterizados por altos niveles de contaminación por metales de origen industrial. Se recogieron ejemplares de mejillón cebra en varias localizaciones del río Ebro próximas a la planta química, cuatro de ellas corriente arriba (Caspe puente y Caspe Lago, Fayón y Mequinenza) y tres situadas corriente abajo (Embalse de Flix, fábrica de Flix y Riba-roja). Por último, se incluyó el embalse de Sitjar en el río Mijares ya que en los informes de la Cuenca Hidrográfica del Júcar aparecía como zona afectada por la expansión de esta especie. Los animales fueron diseccionados en el punto de recogida, separando la concha del tejido blando (y en algunos casos almacenando la glándula digestiva y las branquias independientemente) para con posterioridad estudiar la acumulación de metales, así como para determinar los parámetros fisiológicos seleccionados y realizar el estudio proteómico. Respecto a la acumulación de metales se encontró que las poblaciones situadas corriente arriba de Flix (Caspe, Mequinenza y Fayón) presentaban niveles de metales tóxicos bajos en la glándula digestiva, que en el caso del cadmio (Cd) estaban entre 0,12 y 0,30 µg.g- 1 y en el caso del mercurio (Hg) entre 0,87 y 2,06 µg.g- 1 . Flix y las poblaciones situadas corriente abajo (Ribaroja y embalse de Flix) y próximas a la planta cloro-alcalina tenían elevados niveles de Cd de 0,98 µg.g-1 y de Hg (14,91 µg.g- 1 ). Además, los parámetros indicativos de su estado osmorregulatorio (índice de contenido hídrico, DWI por sus siglas en inglés) eran inferiores a los de las poblaciones situadas corriente arriba. La población de Sitjar es la que presentaba un contenido mayor de plomo respecto de todas las poblaciones estudiadas, con un valor medio de 0,24 µg.g-1 en glándula digestiva. Se encontraron niveles de cobre (Cu) y Zinc (Zn) en los dos tejidos bastante uniformes entre todas las poblaciones de estudio en otoño siendo coherente con 8 la existencia de mecanismos de regulación para los metales esenciales. Sin embargo, el contenido de Cu en la glándula digestiva en primavera y el de Zn en ambos tejidos en otoño fue variable entre poblaciones. Al igual que ocurre con el contenido en metales, el contenido de metalotioneína (MT) fue mayor en la glándula digestiva que en las branquias en todas las poblaciones. El contenido de MT en las branquias se correlacionó de forma negativa con el contenido de Cd y Hg. En la glándula digestiva los valores de concentración de MT presentaron una correlación positiva con el contenido de Cd y Hg, de forma que las poblaciones situadas corriente abajo de Flix (con mayor cantidad de Hg y Cd) presentaron mayores valores de MT, posiblemente como consecuencia de la activación de mecanismos de defensa celular ante el estrés químico. En la población próxima a la Fábrica de Flix se alcanzaron valores de MT en glándula digestiva de 2439 µg.g -1 mientras que en las localidades corriente arriba estos valores fueron cuatro veces inferiores. La actividad Na+/K+-ATPasa en las branquias fue mayor y más variable entre poblaciones en primavera que en otoño. El estudio proteómico realizado en las branquias de los mejillones mediante metodología DIGE permitió definir patrones de expresión proteica característico de cada población de estudiada y relacionarlo con factores ambientales incluyendo la contaminación por metales. Se detectaron 3626 proteínas, encontrándose diferencias significativas en su abundancia entre las diferentes poblaciones en 161 de ellas. A través del método de jerarquía de grupos se comprobó que las poblaciones de Fayón (río Ebro), cuyos tejidos presentan niveles elevados de metales esenciales Cu, Zn y de potasio (K), y la población de Sitjar (río Mijares), con altos valores de Pb en las branquias, presentaron un patrón de expresión similar entre ambas y bastante diferente al resto de las poblaciones. Por otra parte, las poblaciones de Caspe y Mequinenza, corriente arriba de Flix, donde la contaminación por Cd, Pb y Hg fue menor que en el resto de las poblaciones, mostraron un patrón de expresión de proteínas similar entre ellas. La población de Caspe Lago fue algo diferente de las otras dos. Los perfiles proteicos de las poblaciones de Flix Fábrica, Embalse de Flix y Riba-roja (todas ellas en el río Ebro), con valores más altos de metales tóxicos en sus tejidos que en el resto de las poblaciones, no permitieron una buena separación entre las poblaciones de las diferentes localidades al existir un solapamiento que dificulta diferenciarlas entre sí al no haber un predominio de proteínas con mayor o menor abundancia. De las 35 proteínas seleccionadas por su expresión diferencial entre poblaciones se encontró que las funciones biológicas más relevantes fueron: procesos celulares (25%), respuesta a estímulos y metabolismo (14%), y por último regulación y el transporte (8%). Las 9 funciones moleculares más significativas fueron: actividad catalítica (37%), unión (24%) y actividad de un transportador (3%). Los componentes celulares principales fueron: citoplasma (22%), regiones anatómicas celulares (22%), membrana (14%) y orgánulo (8%). Estas 35 proteínas pueden agruparse en 4 clases: proteínas estructurales (ej. actina, espectrina, y tubulina), de respuesta al estrés térmico (ej.: HSP90, HSP83), de transporte iónico (ej. V-tipo protón ATPasa) y de metabolismo (ej. proteosoma, tiolasa). En general, este estudio ha permitido diferenciar las poblaciones de mejillón cebra en función de los valores de los contaminantes presentes en los tejidos específicos de los mejillones cebra (glándula digestiva y branquias) así como de los niveles de MT y los cambios apreciados en el patrón de expresión proteica de cada localidad. También se ha podido relacionar el estado osmorregulatorio de estos organismos en función de las características de su hábitat natural, como la disponibilidad de nutrientes (fósforo), de metales esenciales (Cu y Zn) y de K. Aquellas poblaciones situadas en el curso alto del río Ebro con menor afectación por contaminantes y mayor cantidad de nutrientes son las que presentan individuos con mejor estado fisiológico. Sin embargo, las localidades próximas geográficamente al municipio de Flix (corriente abajo), ofrecen un hábitat más hostil, debiendo los mejillones cebra adaptarse celular y bioquímicamente a la toxicidad de los metales pesados mediante defensas como la MT. Por otra parte, la población del embalse de Sitjar es un ejemplo de la adaptación de esta especie a factores abióticos desfavorables como es la presencia de plomo. The aim of this doctoral thesis has been to analyze and interpret the information related to the ecophysiology of several Spanish populations of zebra mussel (Dreissena polymorpha) in different locations of the Ebro River and in the Sitjar reservoir of the Mijares River collected in 2009 within the project: "Study of the genetic structure and ecological differentiation of Spanish populations of the zebra mussel (Dreissena polymorpha) against environmental factors of natural and anthropogenic origin. Scientific bases for its sustainable environmental control (CEBRAPOP)". The physiological, biochemical and protein expression parameters, as well as metal and metallothionein levels of each of the selected populations were compared. In this study, seven populations of zebra mussels located in different points of the Ebro River and one in the Mijares river, characterized by high levels of contamination by metals of industrial origin, were selected. Zebra mussel specimens were collected in several locations of the Ebro River near the chemical plant, four of them upstream (Caspe bridge and Caspe Lago, Fayón and Mequinenza) and three downstream (Flix reservoir, Flix factory and Riba-roja). Finally, the Sitjar reservoir on the Mijares River was included since it appeared in the reports of the Júcar River Basin as an area affected by the expansion of this species. The animals were dissected at the collection point, separating the shell from the soft tissue (and in some cases storing the digestive gland and gills independently) to subsequently study the accumulation of metals, as well as to determine the selected physiological parameters and carry out the proteomic study. Regarding the accumulation of metals, it was found that the populations located upstream of Flix (Caspe, Mequinenza and Fayón) presented low levels of toxic metals in the digestive gland, which in the case of cadmium (Cd) were between 0.12 and 0.30 µg.g-1 and in the case of mercury (Hg) between 0.87 and 2.06 µg.g-1. Flix and the populations located downstream (Riba-roja and Flix reservoir) and close to the chlor-alkali plant had elevated levels of Cd (0.98 µg.g-1) and Hg (14.91 µg.g-1). In addition, parameters indicative of their osmoregulatory status (water content index, DWI) were lower than those of upstream populations. The Sitjar population had the highest lead content of all the populations studied, with a mean value of 0.24 µg.g-1in the digestive gland. Copper (Cu) and Zinc (Zn) levels in the two tissues were found to be fairly uniform among all study populations in autumn, consistent with the existence of regulatory mechanisms for essential metals. However, the content of Cu in the digestive gland in spring and that of Zn in both tissues in autumn was variable among populations. 11 As with metal content, metallothionein (MT) content was higher in the digestive gland than in the gills in all populations. MT content in the gills was negatively correlated with Cd and Hg content. In the digestive gland, MT concentration values presented a positive correlation with Cd and Hg content, so that populations downstream of Flix (with higher Hg and Cd) presented higher MT values, possibly as a consequence of the activation of cellular defense mechanisms against chemical stress. In the population near the Flix factory, MT values in the digestive gland reached 2439 µg.g-1 , while in the upstream localities these values were four times lower. Na+/K+-ATPase activity in gills was higher and more variable among populations in spring than in autumn. The proteomic study carried out on mussel gills using DIGE methodology allowed us to define protein expression patterns characteristic of each population studied and to relate them to environmental factors including metal contamination. A total of 3626 proteins were detected, finding significant differences in their abundance among the different populations in 161 of them. Using the hierarchy method, it was found that the populations of Fayón (Ebro River), whose tissues present high levels of essential metals Cu, Zn and potassium (K), and the population of Sitjar (Mijares River), with high values of Pb in the gills, presented a similar expression pattern between them and quite different from the rest of the populations. On the other hand, the Caspe and Mequinenza populations, upstream of Flix, where Cd, Pb and Hg contamination was lower than in the rest of the populations, showed a similar protein expression pattern between them. The Caspe Lago population was somewhat different from the other two. The protein profiles of the populations of Flix Fábrica, Embalse de Flix and Riba-roja (all of them in the Ebro river), with higher values of toxic metals in their tissues than in the rest of the populations, did not allow a good separation between the populations of the different localities as there was an overlap that made it difficult to differentiate them from each other as there was no predominance of proteins with higher or lower abundance. Among the 35 proteins selected for their differential expression between populations, it was found that the most relevant biological functions were: cellular processes (25%), response to stimuli and metabolism (14%), and finally regulation and transport (8%). The most significant molecular functions were: catalytic activity (37%), binding (24%) and transporter activity (3%). The main cellular components were: cytoplasm (22%), cellular anatomical regions (22%), membrane (14%) and organelle (8%). These 35 proteins can be grouped into 4 classes: structural proteins (e.g. actin, spectrin, and tubulin), heat stress response proteins (e.g. HSP90, HSP83), ion transport proteins (e.g. V-type proton ATPase) and metabolism proteins (e.g. proteosome, thiolase). 12 In general, this study has allowed us to differentiate the zebra mussel populations according to the values of contaminants present in the specific tissues selected (digestive gland and gills), as well as the levels of MT and the changes seen in the pattern of protein expression in each locality. It has also been possible to relate the osmoregulatory status of these organisms to the characteristics of their natural habitat, such as the availability of nutrients (phosphorus), essential metals (Cu and Zn) and K. Those populations located in the upper reaches of the Ebro River with a lower affectation by pollutants and a higher amount of nutrients are those with individuals with a better physiological status. However, the localities geographically close to the municipality of Flix (downstream), offer a more hostile habitat, and the zebra mussels must adapt cellularly and biochemically to the toxicity of heavy metals by means of defences such as MT. On the other hand, the population of the Sitjar reservoir is an example of the adaptation of this species to unfavourable abiotic factors such as the presence of lead.

La sepsis es una de las patologías más frecuentes y la primera causa de muerte en las Unidades de Cuidados Intensivos, siendo el shock séptico su forma más grave de presentación. En la actualidad la sepsis constituye un problema de salud importante por su elevada mortalidad empeorando el pronóstico cuando asocia una lesión renal aguda. El diagnóstico precoz y la aplicación inmediata de un tratamiento adecuado han demostrado ser esenciales para frenar el fallo multiorgánico y reducir la mortalidad. Aunque se ha avanzado en el conocimiento de la fisiopatología de la sepsis, la identificación de biomarcadores para guiar terapias farmacológicas eficaces sigue siendo un desafío, debido fundamentalmente a que se trata de un síndrome clínicamente muy heterogéneo en el que intervienen múltiples mediadores moleculares. La sepsis con lesión renal aguda asociada conforma un grupo de pacientes relativamente homogéneo en cuanto al pronóstico. La presencia de sepsis y lesión renal aguda se asocia a mayores costos, estancias hospitalarias más prolongadas y peores resultados clínicos. Este grupo necesita habitualmente terapias específicas de soporte vital que incluyen técnicas continuas de depuración extrarrenal (TCDE) no exentas de riesgos para el paciente, siendo un aspecto importante la elección del momento óptimo para el comienzo de las mismas. Los biomarcadores actuales de uso común (creatinina sérica y diuresis) no son sensibles ni específicos para indicar la necesidad de iniciar las mismas, siendo necesario descubrir nuevos biomarcadores que mejoren a los actuales y en este sentido el papel de la proteómica parece prometedor. La hipótesis del presente estudio es que el conocimiento detallado cuantitativo y cualitativo de las proteínas plasmáticas y de las proteínas eliminadas en el ultrafiltrado durante la terapia continua de depuración extrarenal, obtenidas simultáneamente y analizadas mediante técnicas de proteómica no dirigida basadas en SWATH (Sequential window acquisition of all theorical fragment ion spectral) en pacientes con sepsis o shock séptico con lesión renal aguda asociada permitirá identificar paneles de proteínas que potencialmente servirán como biomarcadores pronósticos que ayudarán a decidir el momento idóneo de inicio de estas terapias, además de permitir profundizar en el conocimiento de los procesos biológicos en los que participan.

[spa] El papel que juega el metabolismo lipídico en la progresión del cáncer está siendo revisado. Estudios recientes de nuestro laboratorio demuestran que el tratamiento con ácido palmítico (AP) de células en cultivo o una dieta rica en AP en ratones incrementan la capacidad metastática de las células cancerosas. Sin embargo, los mismos experimentos realizados con ácido oleico (AO) disminuyen la capacidad metastática, lo cual sugiere que cada ácido graso (AG) de nuestra dieta tiene un efecto diferente en las células tumorales. El efecto pro-metastático del AP es mediado por una memoria epigenética, pero su mecanismo no se ha comprendido completamente todavía. Para caracterizar el mecanismo por el cual el AP induce un estado pro-metastático en las células, decidimos estudiar mediante análisis proteicos los cambios en la señalización celular provocados por el AP y el AO. Observamos que el proteoma de las líneas celulares de carcinoma oral de células escamosas (COCE) cambia en direcciones opuestas en respuesta a los tratamientos con AP o AO en cultivo. Por el contrario, el análisis del proteoma de células tumorales creciendo ortotópicamente en animales alimentados con dietas enriquecidas con aceite de palma (rico en AP) o aceite de oliva (rico en AO) mostró que ambas dietas inducen cambios similares, aunque la magnitud de dichas alteraciones es mayor en la dieta rica en AP. Esta diferencia podría ser causada por el hecho de que los aceites de la dieta (palmítico y oleico) están compuestos por diversos AG, no son AG puros. A pesar de ello, observamos ciertas diferencias en los cambios inducidos por cada dieta. Una de las principales categorías específicamente incrementadas con la dieta rica en AP fue la S-palmitoilación: las acil-proteina transferasas DHHC son aumentadas por esta dieta y su expresión génica incrementa con el tratamiento con AP en cultivo y en las metástasis en nódulos linfáticos en comparación con los tumores primarios. El estudio del palmitoiloma de células COCE pro-metastáticas, indicó que está enriquecido en proteínas involucradas en funciones de la membrana celular como movilidad o adhesión celular. Pudimos observar que las células tratadas con AP muestran un aumento en el nivel de palmitoilación de su proteoma comparadas con las tratadas con AO o las células control. Validamos y caracterizamos la palmitoilación de una proteína con especial relevancia en la biología celular y posible implicación en el incremento de la capacidad metastática inducido por el tratamiento con AP.

This article belongs to the Special Issue Potential Anti-SARS-CoV-2 Molecular Strategies., In the last two years, the coronavirus disease 19 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been a scientific and social challenge worldwide. Vaccines have been the most effective intervention for reducing virus transmission and disease severity. However, genetic virus variants are still circulating among vaccinated individuals with different disease symptomatology. Understanding the protective- or disease-associated mechanisms in vaccinated individuals is relevant to advances in vaccine development and implementation. To address this objective, serum-protein profiles were characterized by quantitative proteomics and data-analysis algorithms in four cohorts of uninfected and SARS-CoV-2-infected vaccinated individuals with asymptomatic, non-severe, and severe disease symptomatology. The results show that immunoglobulins were the most overrepresented proteins in infected cohorts when compared to PCR-negative individuals. The immunoglobulin profile varied between different infected cohorts and correlated with protective- or disease-associated capacity. Overrepresented immunoglobulins in PCR-positive individuals correlated with protective response against SARS-CoV-2, other viruses, and thrombosis in asymptomatic cases. In non-severe cases, correlates of protection against SARS-CoV-2 and HBV together with risk of myasthenia gravis and allergy and autoantibodies were observed. Patients with severe symptoms presented risk for allergy, chronic idiopathic thrombocytopenic purpura, and autoantibodies. The analysis of underrepresented immunoglobulins in PCR-positive compared to PCR-negative individuals identified vaccine-induced protective epitopes in various coronavirus proteins, including the spike receptor-binding domain RBD. Non-immunoglobulin proteins were associated with COVID-19 symptoms and biological processes. These results evidence host-associated differences in response to vaccination and the possibility of improving vaccine efficacy against SARS-CoV-2., This research was funded by the Ministerio de Ciencia e Innovación/Agencia Estatal de Investigación MCIN/AEI/10.13039/501100011033, Spain, and EU-FEDER (Grant BIOGAL PID2020-116761GB-I00); and the Junta de Comunidades de vncha (JCCM), Spain, and EU-FEDER (grant MYCOTRAINING SBPLY/19/180501/000174).

Abnormal uterine bleeding (AUB) is the main symptom of endometrial cancer (EC), but it is highly nonspecific. This represents a huge burden for women's health, since all women presenting with bleeding will undergo sequential invasive tests, avoidable in 90-95% of those women who do not have EC. This study aimed to evaluate the potential of cervical samples collected with five different devices as a source of EC diagnostic protein biomarkers. Samples collected with a Rovers Cervex Brush ® and the HC2 DNA collection device, Digene, were the most suitable for EC proteomic studies. A clinical retrospective study assessing the expression of 52 EC-related proteins in 41 patients (22 EC; 19 non-EC), by targeted proteomics, identified SERPINH1, VIM, TAGLN, PPIA, CSE1L, and CTNNB1 as potential EC protein biomarkers in cervical fluids (AUC > 0.8). This study opens an avenue for developing non-invasive protein-based EC diagnostic tests, which will improve the standard of care for gynecological patients. Background: Abnormal uterine bleeding is the main symptom of endometrial cancer (EC), but it is highly nonspecific. This represents a huge burden for women's health since all women presenting with bleeding will undergo sequential invasive tests, which are avoidable for 90-95% of those women who do not have EC. Methods: This study aimed to evaluate the potential of cervical samples collected with five different devices as a source of protein biomarkers to diagnose EC. We evaluated the protein quantity and the proteome composition of five cervical sampling methods. Results: Samples collected with a Rovers Cervex Brush ® and the HC2 DNA collection device, Digene, were the most suitable samples for EC proteomic studies. Most proteins found in uterine fluids were also detected in both cervical samples. We then conducted a clinical retrospective study to assess the expression of 52 EC-related proteins in 41 patients (22 EC; 19 non-EC), using targeted proteomics. We identified SERPINH1, VIM, TAGLN, PPIA, CSE1L, and CTNNB1 as potential protein biomarkers to discriminate between EC and symptomatic non-EC women with abnormal uterine bleeding in cervical fluids (AUC > 0.8). Conclusions: This study opens an avenue for developing non-invasive protein-based EC diagnostic tests, which will improve the standard of care for gynecological patients.

The qualitative and quantitative evaluation of proteome changes that condition cancer development can be achieved with liquid chromatography–mass spectrometry (LC-MS). LC-MSbased proteomics strategies are carried out according to predesigned workflows that comprise several steps such as sample selection, sample processing including labeling, MS acquisition methods, statistical treatment, and bioinformatics to understand the biological meaning of the findings and set predictive classifiers. As the choice of best options might not be straightforward, we herein review and assess past and current proteomics approaches for the discovery of new cancer biomarkers. Moreover, we review major bioinformatics tools for interpreting and visualizing proteomics results and suggest the most popular machine learning techniques for the selection of predictive biomarkers. Finally, we consider the approximation of proteomics strategies for clinical diagnosis and prognosis by discussing current barriers and proposals to circumvent them., Research Council of Norway INFRASTRUKTUR-program (project number: 295910)

G-protein-coupled receptors (GPCRs) are cell membrane proteins of relevance as therapeutic targets, and are associated to the development of treatments for illnesses such as diabetes, Alzheimer’s, or even cancer. Therefore, comprehending the underlying mechanisms of the receptor functional properties is of particular interest in pharmacoproteomics and in disease therapy at large. Their interaction with ligands elicits multiple molecular rearrangements all along their structure, inducing activation pathways that distinctly influence the cell response. In this work, we studied GPCR signaling pathways from molecular dynamics simulations as they provide rich information about the dynamic nature of the receptors. We focused on studying the molecular properties of the receptors using deep-learning-based methods. In particular, we designed and trained a one-dimensional convolution neural network and illustrated its use in a classification of conformational states: active, intermediate, or inactive, of the ß2 -adrenergic receptor when bound to the full agonist BI-167107. Through a novel explainability-oriented investigation of the prediction results, we were able to identify and assess the contribution of individual motifs (residues) influencing a particular activation pathway. Consequently, we contribute a methodology that assists in the elucidation of the underlying mechanisms of receptor activation–deactivation. This research was funded by Spanish PID2019-104551RB-I00 research project.

Ceramide; Sphingomyelinase Ceramida; Esfingomielinasa Ceramida; Esfingomielinasa Electronegative low-density lipoprotein (LDL(−)) is a minor modified fraction of human plasma LDL with several atherogenic properties. Among them is increased bioactive lipid mediator content, such as lysophosphatidylcholine (LPC), non-esterified fatty acids (NEFA), ceramide (Cer), and sphingosine (Sph), which are related to the presence of some phospholipolytic activities, including platelet-activating factor acetylhydrolase (PAF-AH), phospholipase C (PLC), and sphingomyelinase (SMase), in LDL(−). However, these enzymes’ activities do not explain the increased Sph content, which typically derives from Cer degradation. In the present study, we analyzed the putative presence of ceramidase (CDase) activity, which could explain the increased Sph content. Thin layer chromatography (TLC) and lipidomic analysis showed that Cer, Sph, and NEFA spontaneously increased in LDL(−) incubated alone at 37 °C, in contrast with native LDL(+). An inhibitor of neutral CDase prevented the formation of Sph and, in turn, increased Cer content in LDL(−). In addition, LDL(−) efficiently degraded fluorescently labeled Cer (NBD-Cer) to form Sph and NEFA. These observations defend the existence of the CDase-like activity’s association with LDL(−). However, neither the proteomic analysis nor the Western blot detected the presence of an enzyme with known CDase activity. Further studies are thus warranted to define the origin of the CDase-like activity detected in LDL(−). This research was funded by grants PI13/00364, PI16/00471, FIS PI019/00421, and PI20/00334 from the Instituto de Salud Carlos III, Spanish Ministry of Health (co-financed by the European Regional Development Fund). N.P. is the recipient of FI20/00252 from Instituto de Salud Carlos III. This research was supported by CIBER (Consorcio Centro de Investigación Biomédica en Red) (CB07/08/0016), Instituto de Salud Carlos III, and Ministerio de Ciencia e Innovación and Unión Europea—European Regional Development Fund. CIBERDEM (CB07/08/0016) and CIBERCV (CB16/11/00257) are Instituto de Salud Carlos III Projects. A.A.-S. is member of RETICS INVICTUS PLUS (RD16/0019/0010, the Instituto de Salud Carlos III project). N.P., S.B., N.R., and J.L.S.-Q. are members of the Quality Research Group 2017-SGR-1149 from Generalitat de Catalunya and the Group of Vascular Biology of the Spanish Society of Atherosclerosis.

Introduction: The entorhinal cortex is among the earliest areas involved in Alzheimer’s disease. Volume reduction and neural loss in this area have been widely reported. Human entorhinal cortex atrophy is, in part, due to neural loss, but microglial and/or astroglial involvement in the different layers remains unclear. Additionally, -omic approaches in the human entorhinal cortex are scarce. Methods: Herein, stereological layer-specific and proteomic analyses were carried out in the human brain. Results: Neurodegeneration, microglial reduction, and astrogliosis have been demonstrated, and proteomic data have revealed relationships with up- (S100A6, PPP1R1B, BAG3, and PRDX6) and downregulated (GSK3B, SYN1, DLG4, and RAB3A) proteins. Namely, clusters of these proteins were related to synaptic, neuroinflammatory, and oxidative stress processes. Discussion: Differential layer involvement among neural and glial populations determined by proteinopathies and identified proteins related to neurodegeneration and astrogliosis could explain how the cortical circuitry facilitates pathological spreading within the medial temporal lobe., Introducción La corteza entorrinal se encuentra entre las primeras áreas involucradas en la enfermedad de Alzheimer. La reducción de volumen y la pérdida neural en esta área han sido ampliamente reportadas. La atrofia de la corteza entorrinal humana se debe, en parte, a la pérdida neural, pero la participación microglial y/o astroglial en las diferentes capas sigue sin estar clara. Además, los enfoques -ómicos en la corteza entorrinal humana son escasos. Métodos En este documento, se llevaron a cabo análisis proteómicos y específicos de capa estereológica en el cerebro humano. Resultados Se ha demostrado la neurodegeneración, la reducción microglial y la astrogliosis, y los datos proteómicos han revelado relaciones con proteínas reguladas al alza (S100A6, PPP1R1B, BAG3 y PRDX6) y a la baja (GSK3B, SYN1, DLG4 y RAB3A). Es decir, los grupos de estas proteínas estaban relacionados con procesos de estrés oxidativo, neuroinflamatorio y sináptico. Discusión La participación de capas diferenciales entre poblaciones neurales y gliales determinada por proteinopatías y proteínas identificadas relacionadas con la neurodegeneración y la astrogliosis podría explicar cómo el circuito cortical facilita la propagación patológica dentro del lóbulo temporal medial.

Objective Administration of human growth hormone (hGH) is prohibited in competitive sport and its detection in an athlete’s sample triggers an adverse analytical finding. However, the biological processes that are modulated by recombinant hGH are not well characterized and associated blood serum proteins may constitute new biomarkers for hGH misuse. Methods Thirty-five recreational athletes were enrolled in a study to investigate the time- and dose-dependent response of serum protein levels to recombinant hGH administration. Participants were randomly assigned to 4 groups, receiving 1 of 3 different doses of recombinant hGH or a placebo. Bio samples were collected at 22 time points over a period of 13 weeks, starting 4 weeks before treatment, during 3 weeks of treatment, and at 6 weeks’ follow-up. A total of 749 serum samples were analyzed for 1305 protein markers using the SOMAscan proteomics platform. Results We identified 66 proteins that significantly associated with recombinant hGH administration and dosage, including well known hGH targets, such as IGF1, but also previously unknown hGH-related proteins (eg, protease inhibitors, WFIKKN1, and chemokines, CCL2). Network analysis revealed changes in specific biological pathways, mainly related to the immune system and glucose metabolism. Conclusion Our analysis suggests that hGH administration affects biological processes more strongly than previously acknowledged. Some of the proteins were dysregulated even after hGH treatment and could potentially be developed into biomarkers for hGH misuse. Moreover, our findings suggest new roles for hGH-associated proteins in the etiology of hGH-related diseases and may indicate new risks that may be associated with hGH misuse.

Huntington's Disease (HD) is a devastating disorder characterized by a triad of motor, psychiatric and cognitive manifestations. Psychiatric and emotional symptoms appear at early stages of the disease which are consistently described by patients and caregivers among the most disabling. Here, we show for the first time that Foxp2 is strongly associated with some psychiatric-like disturbances in the R6/1 mouse model of HD. First, 4-week-old (juvenile) R6/1 mice behavioral phenotype was characterized by an increased impulsive-like behavior and less aggressive-like behavior. In this line, we identified an early striatal downregulation of Foxp2 protein starting as soon as at postnatal day 15 that could explain such deficiencies. Interestingly, the rescue of striatal Foxp2 levels from postnatal stages completely reverted the impulsivity-phenotype and partially the social impairments concomitant with a rescue of dendritic spine pathology. A mass spectrometry study indicated that the rescue of spine loss was associated with an improvement of several altered proteins related with cytoskeleton dynamics. Finally, we reproduced and mimicked the impulsivity and social deficits in wild type mice by reducing their striatal Foxp2 expression from postnatal stages. Overall, these results imply that early postnatal reduction of Foxp2 might contribute to the appearance of some of the early psychiatric symptoms in HD.

Orientador: Marcos Antonio Machado Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia Resumo: A bactéria Xanthomonas axonopodis pv. citri (Xac) é o agente causador do cancro cítrico, doença que leva a severas perdas econômicas devido à contaminação e à erradicação de plantas de citros. Com o seqüenciamento completo do genoma de Xac, vários genes supostamente envolvidos com a patogenicidade de Xac foram identificados. Os genes ligados à resposta de hipersensibilidade (hrp) e avirulência (avr) em geral estão relacionados à patogenicidade de Xanthomonas, entretanto, poucos estudos funcionais destes genes de Xac foram feitos. Foram construídas linhagens mutantes de Xac para a perda de função dos genes hrpF e avrXacE2 e, nas análises in vivo, foi verificado que hrpF está envolvido na patogenicidade de Xac e é essencial para a manifestação dos sintomas primários da doença. A mutação de avrXacE2 não provocou alterações na capacidade de Xac em provocar os sintomas do cancro, portanto, este gene não parece ser essencial para a patogenicidade da bactéria, podendo não estar envolvido diretamente na patogenicidade de Xac. Os genes hrpF e avrXacE2 foram clonados em vetores de expressão e foram realizados testes de indução da expressão destas proteínas em sistemas heterólogos. Somente a proteína AvrXacE2 foi expressa, purificada e submetida a teste de interação com as proteínas citoplasmáticas da linhagem mutante de Xac para o gene avrXacE2. Os testes de interações não confirmaram a identificação de proteínas com afinidade específica pela proteína recombinante AvrXacE2. A proteína HrpF não foi super-expressa em sistema heterólogo. Nas análises de proteoma comparativo da linhagem de Xac selvagem versus linhagem mutante para o gene hrpF, foram detectadas alterações na expressão de proteínas citoplasmáticas e "pericelulares". Com base nas observações pode-se supor que HrpF possa influenciar processos celulares relacionados à respostas à situações de estresse e não somente atuar na translocação de moléculas efetoras via T3SS. A partir do que foi exposto neste trabalho, sugere-se que as técnicas de estudos funcionais de genes e análises proteômicas podem conjuntamente permitir que novos mecanismos relacionados a patogenicidade de Xac sejam interpretados. Com os mutantes produzidos neste estudo, espera-se criar condições para novos ensaios funcionais visando a melhor compreensão da patogenicidade de Xac e buscar novas formas de combate ao cancro cítrico Abstract: The bacterium Xanthomonas axonopodis pv. citri (Xac) is the causative agent of the citrus canker disease, which leads to economic losses due the contamination and erradication of citrus plants. The complete sequencing of its genome identified a number of genes supposedly involved with pathogenicity. Genes that code for hipersensitivity response (hrp) and avirulence (avr), in general, are related to the pathogenicity of Xanthomonas, however, only a few functional studies of these genes in Xac have been made. Here we report findings based on genomics and proteomics methods for Xac. Mutant strains of Xac for genes hrpF and avrXacE2 and in vivo assays demonstrated that hrpF is strongly involved in the pathogenicity of Xac and is essential for the manifestation of the primary symptoms of the citrus canker. On the other hand, the lack of avrXacE2 expression did not result in modifications in the capacity of Xac to elicite the symptoms of canker, therefore, this gene does not seem to be essential for the pathogenicity of the bacterium. The genes hrpF and avrXacE2 were cloned in expression vectors and tests of induction of the expression of these proteins in heterologous systems were carried out. The protein AvrXacE2 was expressed, purified and tested on interaction assays with cytoplasmic proteins of the mutant of Xac for the gene avrXacE2. The tests of interactions had not confirmed the identification of proteins with specific affinity for the recombinant protein AvrXacE2. The protein HrpF was not overexpressed in heterologous system. In the comparative proteome of the wild versus mutant strains for hrpF, modifications in the cytoplasmic protein expression and "pericellular" expression levels were detected. We postulate that, besides acting as a translocator of molecules through T3SS, HrpF may influence stress-related cellular responses. Thus, it is an opportune time to highlight the new and different ways in which HrpF serves Xac function. Moreover, we can assume that the techniques of functional genomics and proteomics analyses will clarify the mechanisms of pathogenicity used by Xac to cause citrus canker and, thus, enable the search for additional information to control the disease Mestrado Bioquímica Mestre em Biologia Funcional e Molecular

Tau hyperphosphorylation is the first step of neurofibrillary tangle (NFT) formation. In the present study, samples of the entorhinal cortex (EC) and frontal cortex area 8 (FC) of cases with NFT pathology classified as stages I–II, III–IV, and V–VI without comorbidities, and of middle‐aged (MA) individuals with no NFT pathology, were analyzed by conventional label‐free and SWATH‐MS (sequential window acquisition of all theoretical fragment ion spectra mass spectrometry) to assess the (phospho)proteomes. The total number of identified dysregulated phosphoproteins was 214 in the EC, 65 of which were dysregulated at the first stages (I–II) of NFT pathology; 167 phosphoproteins were dysregulated in the FC, 81 of them at stages I–II of NFT pathology. A large percentage of dysregulated phosphoproteins were identified in the two regions and at different stages of NFT progression. The main group of dysregulated phosphoproteins was made up of components of the membranes, cytoskeleton, synapses, proteins linked to membrane transport and ion channels, and kinases. The present results show abnormal phosphorylation of proteins at the first stages of NFT pathology in the elderly (in individuals clinically considered representative of normal aging) and sporadic Alzheimer's disease (sAD). Dysregulated protein phosphorylation in the FC precedes the formation of NFTs and SPs. The most active period of dysregulated phosphorylation is at stages III–IV when a subpopulation of individuals might be clinically categorized as suffering from mild cognitive impairment which is a preceding determinant stage in the progression to dementia. Altered phosphorylation of selected proteins, carried out by activation of several kinases, may alter membrane and cytoskeletal functions, among them synaptic transmission and membrane/cytoskeleton signaling. Besides their implications in sAD, the present observations suggest a molecular substrate for “benign” cognitive deterioration in “normal” brain aging., Dysregulated brain protein phosphorylation (DBPP) occurs at the first stages of neurofibrillary tangle (NFT) pathology (stages I‐II of Braak) in the frontal cortex (FC) and entorhinal cortex (EC). It progresses at the middle (stages III–IV), and advanced stages (V and VI) linked to cognitive impairment and dementia, respectively, in Alzheimer's disease.DBPP principally affects proteins of the cell membranes, cytoskeleton, synapses, protein, and energy metabolism, and it occurs in parallel with abnormal activation of multiple kinases. Many dysregulated phosphoproteins are shared with the FC and EC, and they are found at different stages of NFT pathology.DBPP conforms to a continuum between “normal” brain aging and Alzheimer's diseaseSince protein phosphorylation is crucial in protein signaling, DBPP implies severe dysfunction of critical molecular pathways.DBPP may contribute to progressive cell degeneration and disease progression in sAD. Since DBPP already occurs at the first stages of NFT pathology, which affects about 85% of individuals at the age of 65 years, we suggest that DBPP may contribute to “benign cognitive decline” in “normal” aging.

Orientador: Maria Elvira Pizzigatti Corrêa Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba Resumo: Apesar dos avanços, a doença do enxerto contra o hospedeiro (DECH) ainda é principal complicação pós-transplante alogênico de células-tronco hematopoéticas (alo TCTH), que limita sobrevida e induz maior morbidade nos pacientes. A descoberta de proteínas diferentemente presentes na DECH pode ser capaz de tornar o diagnóstico mais precoce e preciso, além de permitir melhor tratamento. Neste contexto, o estudo dos proteomas busca encontrar proteínas que funcionem como biomarcadores e a utilização da saliva para este fim apresenta vantagens por apresentar coleta não invasiva, indolor e de baixo custo. O objetivo deste estudo foi o de avaliar prospectivamente o proteoma salivar em pacientes submetidos ao alo TCTH e comparar a perfil das proteínas em pacientes que desenvolveram a DECH (DECH+) e os que não desenvolveram (DECH-), elaborando um painel de potenciais biomarcadores para esta doença. Foram incluídos neste trabalho 20 pacientes submetidos ao transplante alogênico de células-tronco hematopoéticas na Unidade de Transplante de Medula Óssea do Hospital de Clínicas da Unicamp. Todos os pacientes foram submetidos à avaliação odontológica e adequação do meio bucal quando necessário, além de serem avaliados para grau de mucosite, parâmetros clínicos de hipossalivação e avaliação das lesões orais de DECH. Foram coletadas amostras de saliva não estimulada em 3 períodos: pré-TCTH, no d+8-10 e d+ 80-100. Adicionalmente, foi avaliado o fluxo salivar dos pacientes em todas as coletas. Todas as amostras foram acondicionadas com inibidores de proteases para avaliação proteômica. A análise das amostras foi realizada por espectrometria de massas após digestão com tripsina e separação dos peptídeos por uma coluna reversa de nanocromatografia líquida. Os dados obtidos foram confrontados com o banco International Protein Index (IPI) human utilizando o software Mascot v.2.3.2.0 ® e os arquivos resultantes da busca foram carregados no software Scaffold ®. As proteínas que mostraram variação em razão de 100% (? 0,5 e >2) entre os grupos DECH+ e DECH- no período d+8-10 para d+80-100 foram consideradas significativas. Não houve correlação estatisticamente significativa dos parâmetros clínicos de hipossalivação com o grupo DECH+. Entretanto na avaliação longitudinal dos pacientes, a presença de saliva espessa e viscosa mostrou-se significativa (p = 0,003). Foram identificadas 69 proteínas nos pacientes avaliados, sendo as mais frequentes relacionadas à defesa e imunidade. No presente estudo, dez proteínas mostraram alteração no grupo DECH+, porém 4 estavam presentes em apenas uma amostra com a doença. Portanto, as proteínas Lactoferroxin-B, IGL@ protein, Submaxillary gland androgen-regulated protein 3B, Cystatin-SA, Cystatin-S e Prolactin-inducible protein foram consideradas como potencias biomarcadores para a doença do enxerto contra hospedeiro Abstract: The graft-versus-host disease (GVHD) remains the major clinical complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT), limiting survival and inducing major morbity. Differently expressed proteins in GVHD have the potential of improving early and accurate diagnosis, also allow a better treatment. Proteome analysis is emerging for the discovery of proteins which function as biomarkers and saliva can be useful for this purpose because offers an easy, inexpensive, safe, and non-invasive approach. The aims of this study were to evaluate salivary proteome in patients underwent allo-HSCT and compare the proteins expressions between patients with and without GVHD and elaborate a panel of possible biomarkers for this condition. Twenty patients underwent allo- HSCT at the Bone Marrow Transplantation Unit - UNICAMP were enrolled in this study. Patients underwent dental evaluation before HSCT and oral environment stabilization were performed when necessary. Also, an evaluation of grade of mucositis, clinical signs of hyposalivation and oral GVHD lesions. Unstimulated saliva samples were collected on 3 different times: pre-HSCT, d+8-10 e d+ 80-100. Additionally, the unstimulated salivary flow rate was measured. Thus, these samples were stored with protease inhibitors for proteomic evaluation. The samples were analyzed by mass spectrometry after protein digestion with trypsin and peptides separated by a Reverse Phase-nanoUltra Perfomance Liquid Chromatography. Protein search was performed using the Mascot v.2.3.2.0 ® engine against International Protein Index (IPI) human Database and the output files of the searches were loaded onto Scaffold software ®. The proteins which showed 100% fold change (? 0,5 e >2) between d+8-10 and d+80-100 were significant. There was no statistically significant correlation of clinical parameters of hyposalivation with GVHD+ group. However in the longitudinal assessment of patients, the presence of thick and viscous saliva was significant (p = 0,003). Sixty nine proteins were identified and the majority was involved in defense and immunity. The results indicated ten proteins differentially expressed in GVHD+ group, but 4 were presented only in one sample with disease. Therefore, Lactoferroxin-B, IGL @ protein, Submaxillary gland androgen-regulated protein 3B, Cystatin SA, Cystatin-S and Prolactin-inducible protein were identified as potential biomarkers for graft-versus-host disease Doutorado Patologia Doutora em Estomatopatologia

Orientador: Adriana Franco Paes Leme Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia Resumo: A espectrometria de massas aplicada à análise proteômica permite a caracterização e quantificação em larga-escala de proteínas expressas em uma determinada condição ou sistema biológico. Na presente tese, mostramos por meio de diferentes abordagens a utilidade da espectrometria de massas (1) no estudo de proteínas secretadas ou clivadas da superfície (secretoma) por células tumorigênicas como fonte de biomarcadores em câncer (2) na validação de candidatos biomarcadores em saliva humana de pacientes com câncer oral e (3) no estudo de alvos de uma metaloprotease envolvida na progressão de câncer, a ADAM17 (A disintegrin and metalloproteinase 17). Os resultados gerados pela espectrometria de massas juntamente com ferramentas estatísticas e de bioinformática para visualização de dados em agrupamento, heatmaps, rede de interação proteína-proteína, enriquecimento de vias e processos biológicos indicaram proteínas específicas direcionadas à hipótese que puderam ser validadas por métodos complementares quantitativos e funcionais. No contexto de biomarcadores em câncer, um painel de marcadores foi indicado a partir da análise do secretoma de células de carcinoma, melanoma e não cancerosas. Dentre os candidatos em carcinoma, proteínas envolvidas no sistema complemento foram validadas com expressão aumentada tanto em tecidos (proteínas C3 e CFB) quanto em saliva de pacientes com carcinoma oral de células escamosas (proteínas C3, CFB, C4B e SERPINA1). Agrina e perlecan, também observados com expressão alterada no secretoma de células de carcinoma e validados com expressão aumentada em tecidos de paciente com câncer oral, foram estudados quanto à função em processos tumorigênicos: migração, adesão, proliferação e sensibilidade à cisplatina. No estudo do degradoma da ADAM17, mimecan, perlecan e glypican-1 foram revelados como novos alvos dessa protease, e os sítios de clivagem determinados por espectrometria de massas. Além disso, funções e processos biológicos associados à clivagem de glypican-1 também foram estudados por meio da análise de seus parceiros de interação no meio extracelular seguido de ensaios funcionais. Em resumo, a espectrometria de massas esteve presente em todos os passos do método científico, desde a geração de hipóteses por meio da proteômica baseada em descoberta, até o teste e confirmação de hipóteses por meio da proteômica baseada em alvos Abstract: Mass spectrometry applied to proteomic analysis allows large-scale characterization and quantification of proteins in specific conditions or biological systems. In this thesis, we showed different mass spectrometry-based approaches for (1) the study of cell secreted or released from the cell-surface proteins (secretome) as a source of candidate biomarkers in cancer (2) validation of candidate biomarkers in human saliva of patients with oral cancer and (3) the study of the target substrates of an important metalloprotease involved in cancer progression, the ADAM17 (A disintegrin and metalloproteinase 17). Together with statistical and bioinformatics tools, such as clustering, heatmaps, network analysis and enrichment analysis, we found specific hyphothesis-driven proteins that were further validated using complementary quantitative and functional methods. In the context of cancer biomarker, a panel of candidate biomarkers was revealed from the secretome analysis of carcinoma, melanoma and non-tumorigenic cell lines. Among the carcinoma candidates, proteins involved in complement system were validated with higher expression in tissues (C3 and CFB) and saliva (C3, CFB, C4B and SERPINA1) from patients with oral squamous cell carcinoma. The function of agrin and perlecan, which were also found with increased expression in squamous cell carcinoma tissue, were studied in tumorigenic processes: migration, adhesion, proliferation and sensibility to cisplatin. In the context of ADAM17 degradome, mimecan, perlecan and glypican-1 (GPC1) were revealed as novel substrates and their cleavage sites were determined using mass spectrometry. Besides, the function and biological processes associated with GPC1 shedding were studied by identifying the binding partners of GPC1 in the extracellular media and by performing functional assays. In summary, mass spectrometry was present in all part of the scientific method, from hyphothesis generation using discovery proteomics to hyphothesis testing using targeted proteomics Doutorado Bioquímica Doutora em Biologia Funcional e Molecular FAPESP

Orientadores: Marcos Nogueira Eberlin, Selma Giorgio Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia Resumo: A leishmaniose é uma doença parasitária que apresenta várias formas clínicas (cutânea, mucocutânea e visceral) dependendo da espécie do protozoário "Leishmania" que causa a infecção e da resposta imune do hospedeiro. "Leishmania spp." necessitam da célula do hospedeiro para completar seu ciclo biológico, principalmente de fagócitos mononucleares. A forma como a interação Leishmania-célula hospedeira acontece é pouco esclarecida, porém, essencial para o desenvolvimento de medidas de controle da infecção como o tratamento e a vacinação. O presente trabalho utiliza das aplicações de imageamento, proteômica e lipidômica por espectrometria de massas (MS) para a análise da alteração de proteínas e lipídeos em hospedeiro após a infeção por "Leishmania amazonensis" e "L. major", agentes etiológicos da forma cutânea da doença e, "L. infantum"¸ agente etiológico da forma visceral. Primeiramente, foi realizada a aplicação da técnica de imageamento por MS para o estudo da distribuição espacial de proteínas em lesões de camundongos após diferentes tempos de infecção com "L. amazonensis" e "L.major". Nossos resultados revelaram o potencial da aplicação de imageamento por MS como ferramenta de diagnóstico e de descoberta de candidatos a biomarcadores para leishmaniose cutânea. Em seguida, foram realizadas as análises de proteômica por MS com o intuito de identificar e quantificar a alteração de proteínas em macrófagos e, em lesões cutâneas de camundongos após infecção por "Leishmania spp". Por fim, a lipidômica por MS foi aplicada para analisar alterações de lipídeos em macrófagos infectados por "L. amazonensis" ou "L. infantum" que possam estar envolvidos no processo de visceralização da doença. Em resumo, a quantificação relativa da alteração de proteínas e lipídeos em hospedeiro após a infecção por "Leishmania spp". revelou candidatos a seres investigados como alvos terapêuticos e biomarcadores em potencial assim como auxiliares para o entendimento sobre os mecanismos fisiopatológicos da leishmaniose Abstract: Leishmaniasis is a parasitic disease that presents itself in various clinical forms (cutaneous, mucocutaneous and visceral) depending on the "Leishmania" species and on the host¿s immune response. "Leishmania spp". need host cells, especially mononuclear phagocytes, to complete its life-cycle. Therefore, understanding the Leishmania-host interaction is fundamental to develop means to control the infection, such as new therapies and vaccination. However, the details about this interaction remain unclear. The present work applies mass spectrometry (MS) based imaging, proteomics and lipidomics to analyze protein and lipid alterations in the host after "L. amazonensis" and "L. major" infection, the causative agents of the cutaneous form of the disease, and, "L. infantum", the causative agent of the visceral form. The application of imaging MS was performed to observe the spatial distribution of proteins in murine cutaneous lesions induced by "L. amazonensis" or "L. major" at different infection timings. Our results demonstrated the application of imaging MS as a potential diagnostic and biomarker¿s discovery tool for cutaneous leishmaniasis. Then, MS-based proteomics was applied to identify and quantify protein alterations of infected macrophages and of murine cutaneous lesions induced by "Leishmania spp".. Lastly, MS-based lipidomics was applied to identify and quantify lipid alterations, possibly linked to the visceralization of the disease, in macrophages infected by "L. amazonensis" or "L. infantum". Therefore, through the relative quantification of proteins and lipids in the host after infection induced by "Leishmania spp"., our results revealed candidates to be further investigated as therapeutic targets or potential biomarkers that can contribute to a better understanding of the pathophysiology of leishmaniasis Doutorado Fármacos, Medicamentos e Insumos para Saúde Doutora em Ciências FAPESP 2016/11517-2

Orientador: Carmen Silvia Bertuzzo Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas Resumo: O câncer de laringe representa cerca de 25% dos tumores malignos que acometem essa área e 2% de todas as doenças malignas. Ocorre em sua grande maioria em homens e é um dos mais comuns entre os tumores que atingem a região da cabeça e pescoço. Pode ocorrer em uma das três porções em que se divide o órgão: supraglote, glote e subglote, sendo que aproximadamente 2/3 dos tumores surgem na corda vocal verdadeira, localizada na glote, e 1/3 acomete a supraglote que se localiza acima das cordas vocais. O tipo histológico mais comum e que atinge mais de 90% dos pacientes, é o carcinoma de células escamosas (CEC) e é classificado de acordo com o grau de diferenciação celular. Estudos de expressão gênica têm sido realizados em vários tumores a fim de possibilitar o envolvimento de algumas proteínas na evolução tumoral, bem como determinar marcadores de gravidade. O objetivo deste trabalho foi estudar o contexto proteômico em células tumorais, displásicas e normais de pacientes com de CEC de laringe. Para isso após a coleta do material, foi realizada a confirmação histológica através de lâminas coradas com Hematoxilina-Eosina. A partir daí as células foram selecionadas e capturadas no Microdissecador a Laser da Zeiss. Em seguida foi realizada a análise proteômica, utilizando o espectrômetro de massas LTQ Velos Orbitrap ETD acoplado ao cromatografo líquido nLCII ambos da Thermo. Os arquivos foram carregados no programa R para as análises subsequentes. Utilizando o pacote MSnbase foi feita a identificação e quantificação das proteínas e a identificação utilizou o arquivo uniprot_sprot.fasta, o método escolhido para quantificação foi o "SIn". Foi aplicado o teste-t pareado e como resultado final foram selecionadas as proteínas que possuíam p-valor menor que 0,01. Foram gerados clusters hierárquicos para demonstrar as proteínas hiper e hipoexpressas para cada grupo estudado. Sendo que após análise, encontramos como possíveis candidatas a marcadores biológicos do CEC de laringe as proteínas codificadas pelos genes TYMP, TNC, RPLP1, MPO, ARHGEF1, QARS, SERPINC1, ATP5A1, AHNAK e para a displasia de laringe as proteínas codificadas pelos gene ZNF544, PP2D1, VCP, RAB12, ETFA, ZNF655, TUT1, XKRX, NOP58, INTS8, SARS, DKC1, ZNF470, SND1, LAS2. Este trabalho é inédito já que buscou comparar as proteínas presentes nos tecidos tumoral, displásico e normal usando a microdissecção a laser, que permite que sejam selecionadas apenas grupos celulares de interesse, evitando a contaminação de outros grupos celulares Abstract: Laryngeal cancer represents about 25% of malignant tumors that affect this area and 2% of all malignancies. It occurs mostly in men and is the most common among tumors that affect the region of the head and neck. It can occur in one of three parts which divides the laryngeal: supraglottis, glottis and subglottis, and approximately 2/3 of the tumors arise in the true vocal cord, located in the glottis and 1/3 involve the supraglottic which is located above the vocal chords. The most common histological type and achieves more than 90% of patients, is the squamous cell carcinoma (SCC). The SCC is classified according to the degree of cell differentiation. Gene expression studies have been conducted in various tumors to allow some of the proteins involved in tumor development as well as to determine severity markers. The objective of the present study was verify the proteomic context in tumor, dysplastic and normal cells in patients with laryngeal SCC. After to collect the sample, the cancer confirmation was made by histological confirmation using slides stained with hematoxylin-eosin. There after the cells were selected and captured in Zeiss Laser Mircodissection. Then carried the proteomic analysis using the LTQ Velos mass spectrometer Orbitrap ETD liquid chromatograph coupled to nLCII both from Thermo. The files were uploaded to the R program for subsequent analysis. Using MSnbase package the identification and quantification of proteins was made and used to identify the uniprot_sprot.fasta file, the method chosen for the measurement was the "Sln" .It was applied the paired t-test and the final result proteins were selected that had p

Orientador: Adriana Franco Paes Leme Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia Resumo: Câncer de cabeça e pescoço é um grupo de câncer que envolve a cavidade oral, faringe e laringe. A maioria dos casos é de carcinoma oral de células escamosas ou epidermóide (CEC), o qual apresenta alta morbidade. Em câncer, mudanças no estroma dirigem a invasão e metástase, cujos processos são característicos da malignidade. Evidências recentes sugerem que a área do fronte invasivo dos carcinomas, ou seja, na interface do tumor-hospedeiro, apresenta perfil molecular e características morfológicas diferentes em comparação a outras áreas do mesmo, sendo considerada como uma região de interesse para a identificação de potenciais assinaturas de prognóstico. Dessa forma, o objetivo desse estudo foi identificar as proteínas presentes no estroma do fronte invasivo do tumor em comparação ao estroma do interior do tumor. Para isso, a microdissecção a laser (LMD) foi associada à espectrometria de massas (MS), técnicas consideradas de alta robustez quando combinadas para a identificação e quantificação de proteínas em tecidos tumorais de regiões específicas. Vinte amostras teciduais fixadas em formalina e parafina de CEC de língua foram utilizadas para isolar o estroma da região do fronte invasivo e o estroma do interior de tumores por LMD, seguida de extração e digestão das proteínas e análise por cromatografia líquida acoplada à espectrometria de massas in tandem por proteômica baseada em descoberta (LC-MS/MS). A partir das análises, incluindo análises estatísticas e a correlação com os dados clínico-patológicos dos pacientes, cinco proteínas foram encontradas estatisticamente significantes em relação as suas abundâncias entre as regiões do fronte invasivo e do interior do estroma tumoral. As abundâncias de COL6A1 (Collagen alpha-1(VI) chain), FSCN1 (Fascin-1), ITGAV (Integrin alpha-V), MB (Myoglobin) e THBS2 (Thrombospondin-2) foram associadas ao estadiamento clínico do paciente, à presença de metástase linfonodal, à recorrência de metástase linfonodal e ao grau de diferenciação histomorfológica do tumor (p-valor

Orientador: Marco Aurélio Zezzi Arruda Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Química Resumo: Este trabalho baseou-se no estudo comparativo entre sementes de soja transgênica (T) e não-transgênica (NT) no que diz respeito a suas proteínas e metabólitos em diferentes gerações da planta, pois acredita-se que o gene inserido possa alterar seu perfil em nível molecular, uma vez que seu genoma foi alterado. Para tal avaliação, foi necessário o plantio das sementes de soja em casa de vegetação sob as mesmas condições até a obtenção da terceira geração. Além disso, foram realizadas diversas extrações das proteínas das sementes de soja para posterior separação por eletroforese. Os géis de eletroforese obtidos mostram um perfil bastante semelhante, mostrando que a separação foi adequada, e que existem proteínas diferentes e também diferenciais entre as mesmas. A quantificação relativa das proteínas foi realizada por meio da técnica de 2-D DIGE, revelando um total de 89 proteínas diferenciais com critério de seleção que partiu de p

Orientador: Adriana Franco Paes Leme Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba Resumo: O carcinoma de células escamosas da cavidade oral (CECO) é o câncer mais comum na região da cabeça e pescoço. O CECO representa a neoplasia maligna mais importante para os cirurgiões dentistas. É detectado geralmente em estágios clínicos avançados e, apesar dos progressos da medicina, os pacientes ainda apresentam um prognóstico clínico desfavorável. Esse contexto justifica a busca de biomarcadores prognósticos que representem o estadiamento da doença, além de compreender os mecanismos moleculares que controlam sua fisiopatologia. Os objetivos desta tese de doutorado foram identificar os potenciais biomarcadores prognósticos para CECO descritos na literatura biomédica, bem como aprofundar a compreensão do papel do proteoglicano agrina. Essa proteína mostrou-se relevante em vários eventos oncogênicos em estudos prévios publicados por nosso grupo. Para alcançar nosso primeiro objetivo, realizamos uma revisão sistemática da literatura. Nesse estudo, utilizamos a base de dados MEDLINE/PubMed e palavras-chave associando o risco dos pacientes com os seguintes desfechos clínicos para CECO: sobrevida global, sobrevida livre de doença e sobrevida doença-específica. Essa abordagem resultou na publicação de um artigo que identificou 41 potenciais biomarcadores prognósticos em CECO, principalmente proteínas avaliadas por inmunohistoquímica. Esses potenciais biomarcadores devem ser avaliados em novos estudos clínicos. Para o segundo objetivo desta tese, silenciamos a expressão de agrina (utilizando a tecnologia shRNA) em diferentes linhagens celulares e identificamos a rede de proteínas associadas a agrina através de proteômica baseada em espectrometria de massas. O valor prognóstico desta rede foi avaliado utilizando ferramentas de bioinformática e bancos de dados públicos. Nossos resultados sugerem que a proteína agrina é essencial para os eventos oncogênicos associados à progressão do CECO, tanto in vivo como in vitro, o que nos leva a concluir que agrina é um forte candidato como alvo terapêutico para o CECO Abstract: Oral squamous cell carcinoma (OSCC) is the most common cancer of the head and neck. It represents the most prominent malignant neoplasm for dental surgeons. It is usually detected in advanced clinical stages, and despite the medical advances, patients still have a poor clinical prognosis. It justifies the search for prognostic biomarkers that represent the staging of the disease, as well as understand molecular mechanisms that control its physiopathology. The objective of this doctoral thesis was to identify the prognostic biomarkers provided in biomedical literature, as well as to deepen the understanding of the role of a proteoglycan named agrin. Agrin proved to be relevant in several oncogenic events in previous studies published by our group. To achieve our first objective, we performed a systematic review of the literature. In this study, we used the MEDLINE/PubMed database and keywords associated with patient risk and common OSCC clinical endpoints: overall survival, disease-free survival and cause-specific survival. This approach produced an article that identified 41 potential prognostic biomarkers, mainly proteins evaluated by immunohistochemistry. These potential biomarkers must be clinically evaluated in new clinical studies. For the second aim of this thesis, we silenced the expression of agrin (using shRNA technology) in different cell lines and identified the protein network associated with agrin through mass spectrometry-based proteomics. We assessed the prognostic value of this network using bioinformatics tools and public databases. Our results suggest that agrin is essential for the oncogenic events associated with the progression of the OSCC, both in vivo and in vitro, which led us to conclude that agrin is a strong candidate as a therapeutic target for OSCC Doutorado Estomatologia Doutor em Estomatopatologia

Orientador: Marco Aurélio Zezzi Arruda Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Química Resumo: O transtorno afetivo bipolar (TAB) é uma doença psiquiátrica caracterizada por alterações de humor que afeta milhares de pessoas no mundo. A escassez de conhecimentos científicos acerca de sua patogênese faz do TAB uma doença de complexa e de difícil diagnóstico. Alguns pacientes portadores do TAB são, por exemplo, diagnosticados equivocadamente como depressivos unipolares e, como consequência, são tratados ineficazmente. Diante desse fato, uma análise exploratória utilizando estratégias ômicas (proteômica e ionômica) comparativas no soro sanguíneo de pacientes com TAB, controles saudáveis, incluindo não familiares (CSNF) e familiares (CSF), pacientes com esquizofrenia (ESQ) e pacientes com outros transtornos mentais (OTM) foi realizado, a fim de determinar potenciais marcadores biológicos do TAB. Para o estudo proteômico, três métodos foram avaliados para simplificar o proteoma do soro sanguíneo: depleção de proteínas abundantes usando agentes químicos (acetonitrila, ACN; e ditiotreitol, DTT) em sequência; depleção de proteínas abundantes, usando nanopartículas magnéticas (MNPs) e a equalização de proteínas usando kit comercial ProteoMiner® (PM). Para o estudo ionômico, dois métodos foram testados para a decomposição da amostra: decomposição assistida por microondas (método convencional) e decomposição rápida assistida por ultrassom de alta potência (método proposto). Como resultado do estudo proteômico, o método PM apresentou-se como a melhor estratégia para remover as proteínas de alta abundância. Por meio da comparação de imagens de géis 2-D DIGE, 37 spots de proteínas foram encontrados como diferencialmente abundantes (p

RESUMEN: La información disponible referente a las características proteómicas del E. granulosus es escasa (no supera los 50 estudios publicados); y nos parece que la identificación proteómica, podría mejorar la comprensión de algunas características bioquímicas e inmunológicas de la Equinococosis Quística (EQ). De tal modo que el proteoma de E. granulosus aún no está bien descrito. Sólo existen reportes de algunas secuencias de proteínas. El objetivo de este manuscrito fue comentar algunos aspectos de la evidencia existente respecto de los estudios del perfil proteómico del E. granulosus. Se recomienda el estudio de al menos el quiste y su pared, el líquido hidatídico y la víscera del hospedero. Para ello, existen metodologías que han sido empleadas para estudiar las características proteómicas de la EQ. Entre ellas, destacan SDS-PAGE, electroforesis bidimensional combinada con Western Blot, inmunoanálisis, y espectrometría de masas mediante técnicas MALDI-TOF. Se han identificado una serie de proteínas en muestras de EQ. Algunas de ellas, asociadas a procesos inmunológicos, de gluconeogénesis, glucogenolisis y glucogénesis. Por otra parte, se ha documentado la liberación de exosomas al líquido hidatídico por parte de los protoescólex y la capa germinativa; estructuras en las que se han identificado factores de virulencia asociados con la supervivencia del quiste. No obstante lo anteriormente señalado, se requiere de múltiples estudios exhaustivos en la materia para comprender mejor la caracterización perfil proteómico del E. granulosus.

The indiscriminate administration of synthetic anthelmintics such as ivermectin contributes to the selection of subpopulations capable of resisting the drugs’ effects. To understand the mechanisms of ivermectin resistance in Caenorhabditis elegans, this study attempted to identify molecular targets. C. elegans lineages that were sensitive and resistant to ivermectin were used. Collected nematodes were added to an extraction buffer and macerated in liquid nitrogen for protein extraction. The extracted proteins were separated according to molecular weight by SDS-PAGE to verify their integrity. Subsequently, proteins from both lineages were separated using two-dimensional electrophoresis. The gels were analyzed and the relevant spots were excised and identified by mass spectrometry (NanoESI-Q-TOF and MASCOT®) and subsequently assessed by GO enrichment and STRING® analyses. The increased expression of proteins associated with high metabolic activity, such as ATP-2 and ENOL-1, which are responsible for ATP synthesis, was observed. Furthermore, proteins with involvement in mediating muscular function (MLC-1, ACT-1, and PDI-2), signaling (FAR-1 and FAR-2), and embryo development (VHA-2) were identified. Protein interaction analysis indicated that the majority of the identified proteins in the resistant lineages participated in the same reaction triggered by ivermectin. Resumo A administração indiscriminada de anti-helmínticos sintéticos, como a ivermectina, contribui para a seleção de subpopulações capazes de resistir ao efeito das drogas. Para entender os mecanismos de resistência à ivermectina em Caenorhabditis elegans, este estudo visou identificar alvos moleculares. Portanto, linhagens de C. elegans sensíveis e resistentes à ivermectina foram utilizadas. Os nematóides coletados foram adicionados ao tampão de extração e macerados em nitrogênio líquido para obtenção das proteínas. As proteínas extraídas foram separadas por peso molecular em SDS-PAGE para verificar sua integridade. Posteriormente, as proteínas de ambas as linhagens foram separadas por eletroforese bidimensional. Os géis foram analisados, os spots relevantes foram excisados e identificados por espectrometria de massa (NanoESI-Q-TOF e MASCOT®), em seguida, analisados em seus termos de GO e STRING®. A expressão aumentada de proteínas associadas à alta atividade metabólica, como as proteínas ATP-2 e ENOL-1, responsáveis pela síntese de ATP, foi observada. Além disso, foram identificadas as proteínas responsáveis pelo controle da função muscular (MLC-1, ACT-1 e PDI-2), sinalização (FAR-1 e FAR-2) e desenvolvimento embrionário (VHA-2). A análise das interações proteicas indicou que a maioria das proteínas identificadas na cepa resistente participa da mesma reação desencadeada pela ivermectina.

Brucellosis is an infectious disease caused by bacteria of the genus Brucella spp. with diagnosis based on use of serological techniques. The present study aimed to develop and standardize a western blotting (WB) test for detection of antibodies against B. abortus. Samples from two groups of cattle were analyzed: group I: 60 serum samples from true positive and true negative vaccinated animals (30 positive samples from infected animals according to rose bengal test (RBT), 2-mercaptoethanol, serum agglutination test (SAT) and complement fixation test (CFT) and 30 RBT negatives samples); group II: 383 field samples (90 positive and 293 CFT negative sera). The most reactive band in the western blotting, which properly identified and separated infected from non - infected had a molecular weight of ≤ 20kDa. The sensitivity, specificity and accuracy of the WB compared to RBT was 93%, 99%, 98%, respectively and k= 0.938. When compared to CFT, the sensitivity, specificity and accuracy of the WB was 97%, 98% and 97%, respectively and k= 0.929. The WB developed and standardized in the present study is a serological test with potential use as a confirmatory test for the diagnosis of bovine brucellosis. RESUMO A brucelose é uma doença infectocontagiosa, causada por bactérias do gênero Brucella spp., com diagnóstico baseado no emprego de técnicas sorológicas. Objetivou-se neste estudo desenvolver e padronizar um teste Western blotting (WB) para detecção de anticorpos contra B. abortus. Foram analisados dois grupos de amostras bovinas: grupo I, com 60 amostras de animais verdadeiros positivos e verdadeiros negativos vacinados (30 amostras positivas de animais infectados e positivos nos testes de antígeno acidificado tamponado (AAT), 2 - mercaptoetanol (2 - ME), soroaglutinação lenta em tubos (SAT) e fixação do complemento e de 30 amostras negativas no AAT); grupo II, com 383 amostras de campo, sendo 90 soropositivas e 293 soronegativas no TFC. O resultado da análise do WB revelou peso molecular ≤20kDa como sendo a área mais reativa e característica para identificação e separação dos animais infectados dos não infectados. A sensibilidade, a especificidade e a acurácia do WB, quando este foi comparado com o AAT, foram, respectivamente, 93%, 99% e 98%, e k= 0,938. Quando comparadas com a TFC, a sensibilidade, a especificidade e a acurácia foram 97%, 98% e 97%, respectivamente, e k= 0,929. O WB padronizado neste estudo mostrou-se um teste sorológico com potencial uso como teste confirmatório no diagnóstico da brucelose bovina.

Orientadores: Marco Aurelio Zezzi Arruda, Claudio Eduardo Muller Banzato Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Quimica Resumo: O transtorno afetivo bipolar é uma doença psiquiátrica caracterizada por alterações de humor marcantes, oscilando entre episódios de mania e depressão, que afeta entre 1 a 3% da população mundial. Os mecanismos em nível molecular deste transtorno, assim como de seu tratamento com lítio, que é o medicamento mais utilizado, ainda não são estabelecidos. Assim sendo, o objetivo deste trabalho de Tese consistiu em explorar biomarcadores potenciais (metabólitos, proteínas e íons metálicos livres ou ligados a proteínas) para o transtorno afetivo bipolar e seu tratamento com lítio. Para isso, foi realizada a comparação dos perfis metabonômicos (utilizando espectroscopia de ressonância magnética nuclear de hidrogênio e análise quimiométrica), proteômicos (utilizando eletroforese bidimensional em gel de poliacrilamida e diferentes técnicas de espectrometria de massas molecular) e metalômicos (utilizando espectrometria de massas com fonte de plasma indutivamente acoplado) de amostras de soro sangüíneo de pacientes com transtorno afetivo bipolar utilizando o lítio (n = 15) ou outras drogas excluindo o lítio (n = 10) e de indivíduos saudáveis (n = 25). A análise metabonômica indicou os lipídeos como sendo os metabólitos mais afetados na presença do transtorno afetivo bipolar e do tratamento com lítio, o que corroborou com os resultados da análise proteômica, onde a apolipoproteína A-I foi uma das proteínas que sofreu maior alteração em seus níveis, sendo subexpressa em pacientes bipolares, independentemente do tratamento, porém apresentando uma restauração ao nível do grupo controle após o tratamento com lítio. As análises ionômicas apontaram As, B, Cl, Cr, Fe, K, Li, Mg, P, S, Se, Si, Sr e Zn como os íons livres diferenciais e as análises metaloproteômicas apontaram, principalmente, Ca, Co, Fe, K, Mg, Mn, Na, Ti e Zn ligados a proteínas como sendo os metais que sofreram as maiores alterações na presença do transtorno afetivo bipolar e de seu tratamento com lítio. Dentre as proteínas ligadas a metais que apresentaram diferenças entre os grupos estudados em termos de metais ligados, destacam-se a apolipoproteína A-I, a transtiretina e a vitronectina, que haviam sido identificadas previamente nas análises proteômicas por apresentarem alterações em suas expressões. A partir destes estudos, foi possível identificar, de maneira exploratória, moléculas diferenciais que podem orientar futuros estudos envolvendo os mecanismos patofisiológicos do transtorno afetivo bipolar e de ação terapêutica de drogas como o lítio, bem como na descoberta de biomarcadores para a doença e/ou seu tratamento com lítio Abstract: Bipolar disorder is a psychiatric illness characterized by marked mood changes, oscillating between mania and depression episodes, which affects 1-3% of the worldwide population. Molecular level mechanisms of this disorder, as well as of its treatment with lithium, which is the most widely used medication, are not yet known. Thus, the aim of this work consisted in explore potential biomarkers (metabolites, proteins, metal ions free or bounded to proteins) for bipolar disorder and its treatment with lithium. For this purpose, it was performed the comparison of metabonomic (using hydrogen nuclear magnetic resonance spectroscopy and chemometric analysis), proteomic (using 2-D gel electrophoresis and different molecular mass spectrometry techniques), and metallomic (using inductively coupled plasma mass spectrometry) profiles for blood serum samples of bipolar disorder patients treated with lithium (n = 15) or other drugs than lithium (n = 10) and healthy individuals (n = 25). Metabonomic analyses indicated lipids as the most affected metabolites in the presence of bipolar disorder and of lithium treatment, which corroborated with the results of proteomic analyses, where apolipoprotein A-I was one of the proteins that showed highest alterations in its levels. It was downregulated in bipolar disorder patients, independently of the treatment, but showed a level restored to that of the control group after lithium treatment. Ionomic analyses detected As, B, Cl, Cr, Fe, K, Li, Mg, P, S, Se, Si, Sr e Zn as differential free ions, and metalloproteomic analyses detected mainly Ca, Co, Fe, K, Mg, Mn, Na, Ti and Zn bound to proteins as being the metals that presented highest alterations in the presence of bipolar disorder and lithium treatment. Among the metal-binding proteins that indicated differences between the studied groups, apolipoprotein A-I, transthyretin and vitronectin, which were previously identified in the proteomic analyses, are highlighted. With the studies described in this research work, it was possible to identify, in an exploratory way, differential molecules that can guide future studies on the patophysiological mechanisms of bipolar disorder or terapeutic action pathways of drugs like lithium, as well as on the discovery of biomarkers for the illness and/or its treatment with lithium Doutorado Química Analítica Doutor em Ciências

Orientador: Marco Aurélio Zezzi Arruda Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Química Resumo: Este trabalho de pesquisa consiste na comparação entre dois tipos de sementes de soja [Glycine max (L.) Merrill], transgênica e não-transgênica, em termos proteômicos e metaloproteômicos, de modo a avaliar possíveis diferenças existentes entre as amostras (Ex. diferença de expressão protéica, concentração das espécies metálicas e não-metálicas ligadas às biomoléculas). Para isso, inicialmente, as biomoléculas foram extraídas usando um tampão extrator específico e, após o tratamento adequado da amostra, separadas por meio da técnica de eletroforese bidimensional em gel de poliacrilamida (2D-PAGE) e caracterizadas por meio de técnicas de espectrometria de massas (MALDI-QTOF e ESI-QTOF) e identificadas por meio de buscas em banco de dados relacionados utilizando o programa Mascot, a partir dos dados gerados pelo espectrômetro de massas. Sendo assim, foram identificadas um total de 192 proteínas, sendo 13 proteínas na faixa de pH de 3 a 10 e 179 na faixa de pH de 4 a 7, sendo que esta última faixa de pH mostrou melhor resolução dinâmica na separação das proteínas. A distribuição funcional das proteínas identificadas mostrou que a maior parte (49%) destas estão envolvidas em armazenamento de nutrientes e atividade proteolítica, citando como exemplo deste grupo as proteínas glicina e b-conglicinina e suas subunidades. Para uma avaliação proteômica comparativa, com o intuito de se avaliar possíveis diferenças quanto à expressão das proteínas separadas entre as amostras avaliadas, foi utilizada a técnica de eletroforese em gel diferencial bidimensional (2D-DIGE), a partir de dados como intensidade e volume dos spots protéicos. Sendo assim, foram selecionadas um total de 04 proteínas diferenciais entre as amostras, sendo que todas foram identificadas via espectrometria de massas. Para uma avaliação metaloproteômica comparativa, um estudo para identificação de íons metálicos e semimetálicos livres ou ligados a proteínas foi realizado. Para isso, foi feita uma varredura semi-quantitativa de espécies metálicas e semimetálicas presentes nos extratos protéicos das sementes de soja transgênica e não-transgênica usando a técnica de ICP-MS, sendo que os íons Na, Mg, Si, K, Ti, Mn, Zn e Rb foram mais detectados na soja não-transgênica. Já o íon Cu foi mais detectado nas sementes de soja transgênica. Estes elementos diferenciais entre as amostras foram quantificados via ICP-MS. Além disso, os spots diferenciais obtidos com a técnica de 2D-DIGE foram avaliados quanto a presença de cobre, já que este elemento mostrou diferenças em concentrações em proteínas de soja, conforme trabalhos recentemente publicados na literatura pelo nosso grupo de pesquisa Abstract: The research compairs two types of soybean [Glycine max (L.) Merrill], transgenic and non-transgenic, in terms of proteomics and metalloproteomics in order to evaluate possible differences among the samples (E.g. difference in protein expression, concentration of metal species and non-metal bounded to biomolecules). Therefore, initially, the biomolecules were extracted using a specific extration buffer and, after proper treatment of the sample, separated by the technique of two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and characterized by mass spectrometry techniques (MALDI-QTOF and ESI-QTOF) and identified through searches related database using the Mascot program, from the data generated by mass spectrometer. Thus, we identified a total of 192 proteins, 13 proteins in the pH range 3 to 10 and 179 at pH 4-7, and this latest range of pH showed a better dynamic resolution in the separation of proteins. The functional distribution of identified proteins showed that most (49%) of these are involved in nutrient storage and proteolytic activity, citing as an example of this group glycinin and b-conglycinin and their subunits. For an evaluation of proteomics in order to evaluate possible differences in protein expression between the samples, the differential gel electrophoresis (2D-DIGE) technique was used based on data such as intensity and volume of proteins. Therefore, we selected a total of 04 differential proteins between samples, all of them were identified by mass spectrometry. For a comparative assessment metalloproteomics, a study for identification of metal ions and semimetal free or bound to proteins was performed. In this way, a semi-quantitative sweeping of semi-metal and metal species present in protein extracts of transgenic and non-transgenic soybean seeds was carried out using ICP-MS, and the ions Na, Mg, Si, K, Ti, Mn, Zn and Rb were detected in seeds of non-transgenic soybean. The ion Cu was detected in transgenic soybeans seeds. These differentials elements between the samples were quantified via ICP-MS. Moreover, the differential spots obtained with the 2D-DIGE technique were evaluated according to the presence of copper, since this element showed differences in concentrations in soybean proteins, as recently published in the literature by our research group Doutorado Química Analítica Doutor em Ciências

Orientador: Joyce Maria Annichino-Bizzacchi Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas Resumo: A trombose venosa profunda (TVP) é uma doença multifactorial, e possui uma alta taxa de morbi-mortalidade devido a complicações como embolia pulmonar e a síndrome póstrombótica, e cerca de 25 % dos pacientes apresentarão recorrência em 5 anos. A identificação de novos fatores envolvidos na fisiopatologia da TVP pode ser convertida em implicações de grande importância no manejo destes pacientes, prevenção de recorrência e no desenvolvimento de novas terapias. O interesse em avaliar as plaquetas e as amostras de plasma se deve ao fato de que as funções plaquetárias não estão completamente compreendidas, e aparentemente o papel das plaquetas poderia ir além do seu envolvimento na hemostasia. Como o plasma potencialmente fornece uma janela para a observação do indivíduo como um todo, a análise proteômica tanto das plaquetas como do plasma poderia implementar o nosso conhecimento acerca da fisiopatologia da TVP. OBJETIVO: Neste estudo foram analisados os perfis proteicos de plaquetas e amostras de plasma de três pacientes com TVP, que foram comparados com os resultados obtidos a partir de amostras de 1 irmão e 1 vizinho para cada paciente, a fim de minimizar as interferências genéticas e ambientais. Estes pacientes apresentaram episódios espontâneos e recorrentes de TVP proximal e mencionaram um histórico familiar de distúrbios da coagulação. MÉTODOS: as plaquetas necessitaram ser lavadas e lisadas, e as amostras de plasmas tiveram a albumina depletada antes de as proteínas serem alquiladas, reduzidas, precipitadas com acetona e hidrolisadas com tripsina. Os peptídeos das plaquetas e das amostras de plasma foram fracionados por cromatografia de fase reversa e troca catiônica, respectivamente. Depois disto, os peptídeos plaquetários foram direcionados ao espectrômetro de massas LTQOrbitrap e a busca das proteínas foi realizadas através do Sorcerer/Sequest. Os peptídeos plasmáticos foram encaminhados ao espectrômetro de massas ESI Q-TOF Premier e as proteínas foram analisadas pelo Mascot. RESULTADOS: Cinco proteínas estiveram presentes apenas nas plaquetas dos pacientes, estando ausentes em todos os controles: a proteína ligante da Apolipoproteína A1, a sub-unidade ?1 do Coatômero, a Desidrogenase 11-17-? do Estradiol, a Leucotrieno A-4 Hidrolase e a Sorbitol Desidrogenase. Além disso, verificou-se que outras proteínas estiveram diferencialmente expressas em amostras de plasma pacientes e controles: a protease C4-A, o inibidor C1 da Inter-?-Tripsina, o inibidor H1 de cadeia pesada, a proteína Amilóide Sérica A, a glicoproteína ?-2-HS, a isoforma 2 da inter- ?-tripsina, a apolipoproteína A-IV e o inibidor de cadeia pesada H4. CONCLUSÕES: A avaliação de plaquetas e amostras de plasma de pacientes com TVP espontânea permitiu a identificação de proteínas diferencialmente expressas quando comparados a irmãos e vizinhos, que podem desempenhar importantes papéis na fisiopatologia da doença por se relacionarem a processos inflamatórios, imunes e no de transporte de lipídeos Abstract: Deep venous thrombosis (DVT) is multi-causal disease associated to a high morbimortality due to complications as pulmonary embolism and post-flebitic syndrome, and about 25 % of the patients present recurrence in 5 years. The identification of new factors involved to the physiopathology of DVT can be translated into important implications for the management of these patients, prevention of recurrence, and for the development of new therapies. We were interested about platelets and plasma because the platelets functions are not completely understood and apparently their role goes beyond a hemostatic player, and as the plasma potentially provides a window into the individual's state of health and disease, both could improve our knowledge about the DVT physiopathology. AIM: In this study we analyzed the protein profile of platelets and samples of plasma of 3 DVT patients and compared to results obtained from 1 sibling and 1 neighbor for each patient in order to minimize the genetic and environmental interferences. These patients presented spontaneous and recurrent episodes of proximal DVT and mentioned a familiar history of coagulation disorders. METHODS: the platelets needed to be washed and lysed, and the plasmas samples required albumin depletion before the proteins being alkylated, reduced, precipitated with acetone and hydrolyzed by trypsin. The peptides were fractionated by reverse phase and cation exchange liquid chromatography for platelets and plasmas samples, respectively. After that, the platelets peptides were directed to LTQ-Orbitrap mass spectrometer and the proteins search were performed by Sorcerer/Sequest. The plasma peptides went to ESI Q-TOF Premier mass spectrometer and the proteins were searched by RESULTS: We identified 5 proteins that were present on platelets from patients and absent in all the controls: Apolipoprotein A1 Binding-Protein, Coatomer (?1 sub-unit), Estradiol 11-17-? Dehydrogenase, Leukotriene A-4 Hydrolase and Sorbitol Dehydrogenase. In addition, we verified 6 proteins that were differently expressed between patients and controls: C4-A plasma protease, C1 inhibitor Inter-alpha-trypsin, inhibitor heavy chain H1, the serum amyloid A, alpha-2-HS-glycoprotein, isoform 2 of inter-alphatrypsin, apolipoprotein A-IV and the inhibitor heavy chain H4. CONCLUSIONS: The evaluation of platelets and plasma samples from patients with spontaneous DVT allows the identification of proteins that are differently expressed when compared to siblings and neighbors, which can play important roles on the physiopathology of the disease due their relation to inflammatory, immune and lipid transportation Doutorado Biologia Estrutural, Celular, Molecular e do Desenvolvimento Doutor em Fisiopatologia Médica

Aims: Current cardiovascular (CV) risk prediction algorithms are able to quantify the individual risk of CV disease. However, CV risk in young adults is underestimated due to the high dependency of age in biomarker-based algorithms. Because oxidative stress is associated with CV disease, we sought to examine CV risk stratification in young adults based on oxidative stress to approach the discovery of new markers for early detection of pathology. Results: Young adults were stratified into (i) healthy controls, (ii) subjects with CV risk factors, and (iii) patients with a reported CV event. Plasma samples were analyzed using FASILOX, a novel approach to interrogate the dynamic thiol redox proteome. We also analyzed irreversible oxidation by targeted searches using the Uniprot database. Irreversible oxidation of cysteine (Cys) residues was greater in patients with reported CV events than in healthy subjects. These results also indicate that oxidation is progressive. Moreover, we found that glutathione reductase and glutaredoxin 1 proteins are differentially expressed between groups and are proteins involved in antioxidant response, which is in line with the impaired redox homeostasis in CV disease. Innovation: This study, for the first time, describes the oxidative stress (reversible and irreversible Cys oxidation) implication in human plasma according to CV risk stratification. Conclusion: The identification of redox targets and the quantification of protein and oxidative changes might help to better understand the role of oxidative stress in CV disease, and aid stratification for CV events beyond traditional prognostic and diagnostic markers. Instituto de Salud Carlos III PT13 / 0001/0013, PI14 / 01917, CP15 / 00129, PI17 / 01093, PI17 / 01193, FI18 / 00261, PI18 / 00995, y FI19 / 00157. Sociedad Española de Cardiología para la Investigación Básica, Beca PRB3 (IPT17 / 0019 - ISCIII-SGEFI / FEDER) 7.468 JCR (2021) Q1, 53/297 Biochemistry & Molecular Biology 1.832 SJR (2021) Q1, 42/439 Biochemistry No data IDR 2021 UEM

Motivation Graphlet adjacency extends regular node adjacency in a network by considering a pair of nodes being adjacent if they participate in a given graphlet (small, connected, induced subgraph). Graphlet adjacencies captured by different graphlets were shown to contain complementary biological functions and cancer mechanisms. To further investigate the relationships between the topological features of genes participating in molecular networks, as captured by graphlet adjacencies, and their biological functions, we build more descriptive pathway-based approaches. Contribution We introduce a new graphlet-based definition of eigencentrality of genes in a pathway, graphlet eigencentrality, to identify pathways and cancer mechanisms described by a given graphlet adjacency. We compute the centrality of genes in a pathway either from the local perspective of the pathway or from the global perspective of the entire network. Results We show that in molecular networks of human and yeast, different local graphlet adjacencies describe different pathways (i.e., all the genes that are functionally important in a pathway are also considered topologically important by their local graphlet eigencentrality). Pathways described by the same graphlet adjacency are functionally similar, suggesting that each graphlet adjacency captures different pathway topology and function relationships. Additionally, we show that different graphlet eigencentralities describe different cancer driver genes that play central roles in pathways, or in the crosstalk between them (i.e. we can predict cancer driver genes participating in a pathway by their local or global graphlet eigencentrality). This result suggests that by considering different graphlet eigencentralities, we can capture different functional roles of genes in and between pathways This study received support from the following sources: The European Research Council (ERC) Consolidator Grant 770827 (awarded to NP); The Spanish State Research Agency AEI 10.13039/501100011033 grant number PID2019-105500GB-I00 (awarded to NP); and University College London Computer Science (awarded to SW). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Proteomics research infrastructures and core facilities within the Core for Life alliance advocate for community policies for quality control to ensure high standards in proteomics services. The CRG/UPF Proteomics Unit is a member of the ProteoRed PRB3 consortium which is supported by grant PT17/0019 of the PE I + D+i 2013‐2016 from the Instituto de Salud Carlos III (ISCIII) and ERDF. We acknowledge support from the Spanish Ministry of Science, Innovation and Universities, “Centro de Excelencia Severo Ochoa 2013‐2017”, SEV‐2012‐ 0208, and “Secretaria d’Universitats i Recerca del Departament d’Economia i Coneixement de la Generalitat de Catalunya” (2017SGR595). The VIB Proteomics Core acknowledges support from the VIB Tech Watch Fund and FWO‐SBO Project S006617N. Research in the Mechtler lab was supported by the ERA‐CAPS I 3686 project of the Austrian Science Fund. We acknowledge support from the European Union's Horizon 2020 research and innovation program under grant agreement No 823839 (EPIC‐XS)

Orientadores: Adriana Franco Paes Leme, Ricardo Della Coletta Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba Resumo: O carcinoma espinocelular oral (CEC) é o tipo de neoplasia maligna mais comum em cabeça e pescoço, com alta prevalência e morbidade. O tratamento é baseado em sistemas de classificação pouco precisos e o prognóstico é ruim em muitos casos. Diferentes padrões histológicos já foram descritos na tentativa de melhor compreender o curso da doença, predizer prognóstico e auxiliar no tratamento. As diferentes áreas do tumor apresentam características morfológicas e moleculares distintas resultando em comportamentos clínicos específicos, e estudos recentes apontam a região de invasão tumoral (do inglês invasive front tumor) como área de interesse para análises de perfil molecular e identificação de possíveis marcadores de prognóstico. O padrão de invasão neoplásico tem relação com a agressividade tumoral e a presença de ilhas no fronte invasivo já foi descrita como pior padrão. Assim, o objetivo desta dissertação foi comparar a composição diferencial de proteínas totais de ilhas neoplásicas grandes e pequenas do fronte e do interior do tumor, e correlacionar essas proteínas com o prognóstico. A proteômica foi associada à microdissecção a laser (ML), consideradas juntas como ferramentas de alta robustez para identificação de proteínas em tecidos neoplásicos em regiões específicas. Vinte peças cirúrgicas de CEC oral de língua fixadas em parafina foram submetidas a ML para obtenção das amostras compostas pelas seguintes regiões do tecido: 1) ilhas neoplásicas grandes da região frontal, 2) ilhas neoplásicas pequenas da região frontal, 3) ilhas neoplásicas grandes do interior do tumor e 4) ilhas neoplásicas pequenas do interior do tumor, seguida pela extração de proteínas e análise por cromatografia líquida acoplada à espectrometria de massas (LC-MS/MS). A anotação funcional das proteínas e a correlação aos dados clínico-patológicos dos pacientes foram realizadas. Foram identificadas 1906 proteínas totais, sendo 1480 proteínas comuns entre as quatros regiões estudadas. Duas proteínas foram exclusivas nas ilhas grandes do fronte e sete nas ilhas grandes do interior. Oitenta e cinco proteínas foram diferencialmente expressas entre a região do fronte e o interior tumoral, e destas, 57 foram relacionadas a dados clínico-patológicos importantes para o prognóstico. Os processos biológicos, como desenvolvimento da epiderme, adesão celular, apoptose, ciclo celular, degradação da matriz extracelular e expressão gênica, evidenciados entre as proteínas diferencialmente expressas confirmam as mudanças moleculares associadas à progressão neoplásica. A combinação de ML, MS e bioinformática foi capaz de identificar um painel de proteínas que podem auxiliar a desvendar o curso do CEC oral, predizendo agressividade e prognóstico. Em acréscimo, essa abordagem pode ajudar ainda na compreensão das diferenças e dos mecanismos de sinalização entre diferentes áreas do tecido Abstract: Oral squamous cell carcinoma (SCC) is the most common type of malignant tumor in head and neck, with high prevalence and morbidity. Treatment is based on inaccurate classification systems and the prognosis is poor in many cases. Different histological patterns have been described in an attempt to better understand the course of the disease, predict prognosis and assist in treatment. Different areas of the tumor have different morphological and molecular characteristics resulting in specific clinical behaviors, and recent studies point to the region of tumor invasion as an area of interest for molecular profile analysis and identification of possible prognostic markers. The pattern of neoplastic invasion is related to tumor aggressiveness and the presence of islands in the invasive front has been described as worst invasion pattern. The objective of this work was to compare the protein differential expression of large and small islands neoplastic from the front and the inner tumor, and to correlate these proteins with prognosis. Proteomics was associated with laser microdissection (ML), and together they are considered robustness tools to identify proteins in neoplastic tissues in specific regions of interest. Twenty surgical specimens of oral tongue SCC fixed in paraffin were subjected to ML to obtain sample composed by the following regions of tissue: 1) large neoplastic frontal islands, 2) small neoplastic islands of the frontal region, 3) large neoplastic islands inside the tumor and 4) small islands within the neoplastic tumor, followed by extraction and analysis of proteins by liquid chromatography coupled to mass spectrometry (LC-MS/MS). The functional annotation of proteins and correlation with clinicopathological data from patients were performed. A total of 1906 proteins were identified, with 1480 common proteins between the four regions studied. Two proteins were exclusives in the large islands of the forehead and seven in large islands in the interior. Eighty-five proteins were differentially expressed between the front region and inner tumor, and of these, 57 were related to clinical and pathological data. The biological processes such as development of the epidermis, cell adhesion, apoptosis, cell cycle, disassembly of the extracellular matrix and gene expression, evidenced among the differentially expressed proteins confirmed the molecular changes associated with neoplastic progression. The combination of ML, MS, and bioinformatics was able to identify a panel of proteins that may help to unravel the course of oral SCC, predicting aggressiveness and prognosis. In addition, this approach may also help in understanding the differences and signaling mechanisms between different areas of the tumor tissue Mestrado Patologia Mestra em Estomatopatologia FAPESP 2014/02288-4 CAPES

Orientador: Fernando Moreira Simabuco Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Aplicadas Resumo: Introdução: A via de sinalização da mTOR vem sendo relacionada a várias doenças e desordens metabólicas em humanos, incluindo obesidade, diabetes, vários tipos de câncer e neurodegeneração. As proteínas S6Ks têm mostrado importante papel na sinalização da mTOR, funcionando como efetoras dessa via. A família das S6Ks é composta por dois genes, RPS6KB1 e RPS6KB2, sendo que o primeiro codifica as isoformas p85- e p70-S6K1, enquanto que o segundo codifica as isoformas denominadas p56- e p54-S6K2. Este estudo investigou as parceiras de interação das isoformas p70-S6K1 e p54-S6K2 em células HEK293, com objetivo de diferenciar essas proteínas de acordo com seus envolvimentos em processos biológicos. Além disso, foi investigada mais detalhadamente a interação das isoformas p70-S6K1 e p54-S6K2 com as proteínas eIF2? e PARP1. Justificativa: Acreditava-se que as isoformas p70-S6K1 e p54-S6K2 apresentavam funções biológicas redundantes, uma vez que possuem um elevado grau de similaridade entre si. Dessa forma, poucos estudos têm como objetivo identificar diferentes funções e envolvimentos dessas isoformas em processos biológicos. Entretanto, descobrir as diferentes funções dessas proteínas pode ter grande importância para estudos que investigam novas estratégias para desenvolver terapias para certos tipos de doenças, como o câncer. De forma geral, esse trabalho contribui para um melhor entendimento da via mTOR/S6Ks. Resultados: Nos primeiros dados obtidos desse estudo, que geraram um artigo científico publicado na revista Proteomics em 2016 (Anexo I), foi observado, por espectrometria de massas, que as isoformas p70-S6K1 e p54-S6K2 possuem diferentes proteínas de interação e que atuam em diferentes processos biológicos, indicando que tais isoformas possuem funções biológicas distintas. Algumas parceiras de interação, eIF2?, PARP1, PPM1B, FXR1, FMRP, PRMT5 e WDR77, foram escolhidas para que suas relações com as isoformas p70-S6K1 e p54-S6K2 fossem estudadas de forma mais detalhada. Alguns experimentos de imunoprecipitação foram realizados mostrando evidências da interação entre p70-S6K1 e p54-S6K2 com a proteína eIF2?, e somente p54-S6K2 com a proteína PARP1. Em uma situação de estresse celular, eIF2? é fosforilada no seu resíduo serina 52 (S52), acarretando na inibição da síntese protéica global e no estimulo da autofagia e apoptose. Identificamos que eIF2? apresenta um sítio de fosforilação predito das S6Ks (RXRXXT/S) em serina 58 (S58), sendo este conservado em uma variedade de espécies. Ensaios de imunoprecipitação revelaram que eIF2? é fosforilada em um sítio RXXT/S na presença de insulina. Além disso, foi visto que a ativação das S6K1 e S6K2 através de insulina e somente S6K2 através de FGF2 está diretamente relacionada com a redução na fosforilação de eIF2? em serina 52. Por outro lado, inibidores farmacológicos de mTOR e S6Ks, como a rapamicina e o PF4708671, respectivamente, estão associados com o aumento dos níveis de fosforilação de eIF2? (S52). Também foi realizada a superexpressão de p70-S6K1 e p54-S6K2 constitutivamente ativas e foi visto redução da fosforilação de eIF2? (S52). Por fim, foi realizado um ensaio de mutação sítio dirigida em eIF2?, mimetizando uma fosforilação no resíduo 58 de eIF2?. Como resultado, observamos que a fosforilação constante no resíduo 58 está acompanhado de uma redução da fosforilação do resíduo S52. Conclusões: Este estudo mostrou que diferentes isoformas de S6Ks possuem distintas proteínas de interação, indicando que tais isoformas possuem funções biológicas distintas na célula. Os resultados ainda sugerem que as S6Ks podem fosforilar eIF2? em serina 58, e devido a proximidade dos sítios, possa haver uma inibição da fosforilação em serina 52, regulando assim a atividade de eIF2á Abstract: Introduction: The mTOR signaling pathway has been related to several diseases and metabolic disorders in humans, including obesity, diabetes, several types of cancer and neurodegeneration. The S6Ks proteins have shown an important role in mTOR signaling, acting as effectors of this pathway. The S6Ks family is composed of two genes, RPS6KB1 and RPS6KB2. RPS6KB1 encodes two isoforms: p85- and p70-S6K1, while RPS6KB2 encodes two other isoforms, known as p56 and p54-S6K2. This study investigated the interaction partners of p70-S6K1 and p54-S6K2 isoforms in HEK293 cells, in order to differentiate these proteins according to their involvement in biological processes. Furthermore, it was investigated in more detail the interaction of p70-S6K1 and p54-S6K2 isoforms with eIF2? and PARP1 proteins. Justification: It was believed that S6K1 and S6K2 exhibited redundant biological functions, since they have a high degree of similarity. Thus, few studies aim to identify different roles and implications of these isoforms in biological processes. However, discovering different functions of these proteins may have great importance for the investigation of new strategies to develop therapies for certain types of diseases such as cancer. In general, this work contributes to a better understanding of the mTOR / S6Ks pathway. Results: The first data obtained from this study, which generated a scientific article published in the journal Proteomics in 2016 (Annex I), it was observed, by mass spectrometry, that the p70-S6K1 and p54-S6K2 isoforms have different interaction proteins and act in different biological processes, indicating that such isoforms have distinct biological functions. Some partner interaction, as eIF2?, PARP1, PPM1B, FXR1, FMRP, PRMT5 and WDR77, were chosen for its relations with the p70-S6K1 and p54-S6K2 isoforms were studied in more detail. Some immunoprecipitation experiments were performed showing evidence of the interaction between p70-S6K1 and p54-S6K2 with the eIF2? protein, and only p54-S6K2 with the PARP1 protein. In a situation of cellular stress, eIF2? is phosphorylated at its serine residue 52 (S52), leading to the inhibition of global protein synthesis and the stimulation of autophagy and apoptosis. We have identified that eIF2? has a predicted phosphorylation site of S6Ks (RXRXXT/S) at serine 58 (S58), which is conserved in a variety of species. Immunoprecipitation assays revealed that eIF2? is phosphorylated at RXXT/S site in the presence of insulin. In addition, it was shown that the activation of S6K1 and S6K2 through insulin and only S6K2 through FGF2 is directly related to the reduction in eIF2? phosphorylation in serine 52. Conversely, pharmacological inhibitors of mTOR and S6Ks, such as rapamycin and PF4708671, respectively, are associated with increased of eIF2? phosphorylation levels at S52. The overexpression of constitutively active p70-S6K1 and p54-S6K2 was also performed and reduction of the phosphorylation of eIF2? (S52) was seen. Finally, a site-directed mutation assay was performed on eIF2?, mimicking a phosphorylated residue 58 of eIF2?. As a result, we observed that constant phosphorylation at residue 58 is accompanied by a reduction of phosphorylation of residue S52. Conclusions: This study demonstrated that S6Ks isoforms present different interacting proteins, indicating that those isofroms present distinct biological functions in the cell. The results also suggest that S6Ks can phosphorylate eIF2? in serine 58, and due to the proximity of the sites may be an inhibition of the phosphorylation of serine 52, thereby regulating the activity eIF2á Mestrado Metabolismo e Biologia Molecular Mestra em Ciências da Nutrição e do Esporte e Metabolismo FAPESP 2015-00311-1

Orientadores: Iscia Teresinha Lopes Cendes, André Schwambach Vieira Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas Resumo: O uso de modelos animais para estudo de doenças humanas é importante para o entendimento dos mecanismos fisiopatológicos destas doenças. Particularmente, os modelos que reproduzem a Epilepsia do Lobo Temporal (ELT) em roedores, apresentam uma epileptogenicidade similar à encontrada em tecidos "epilépticos" humanos quando estudados ex vivo. A ELT afeta cerca de 40% dos pacientes adultos, e é caracterizada clinicamente por um desenvolvimento progressivo de crises epilépticas com foco no lobo temporal. Pacientes que apresentam ELT normalmente não respondem aos tratamentos. Destas, a Epilepsia do Lobo Temporal Mesial (ELTM) e a mais comum e se caracteriza pelo acometimento das estruturas mesiais do lobo temporal, como o caso da Esclerose Hipocampal. A proteômica dispõe de ferramentas poderosas que nos permitem elucidar mecanismos biológicos complexos, encontrar proteínas alteradas em todo o organismo e descrever padrões de expressões proteicas em diferentes condições fisiológicas e patológicas. Portanto, é relevante analisar esse padrão de expressão no hipocampo de modelos animais de ELTM usando técnicas de proteômica, a fim de gerar informações que nos auxiliem no entendimento dos mecanismos envolvidos na epileptogênese desses modelos. No presente estudo, as proteínas identificadas podem nos indicar novas vias envolvidas com a epileptogênese. Além disso, nossos dados demonstram que uma complexidade molecular adicional pode ser observada quando analisamos as diferentes sub-regiões do hipocampo separadamente. Portanto, acreditamos que a integração dos dados de proteômica com dados obtidos por outras "ômicas" podem gerar dados ainda mais informativos sobre esses processos neuronais Abstract: Studies about human diseases using animal models are really important to our understanding about the physiopathology mechanisms from those diseases. Particularly, the models that reproduce the Temporal Lobe Epilepsy (TLE) in rodents, presents epileptogenicity similar to that found in ex vivo human tissues. The TLE affects around 40% of the adult patients and it is clinic characterized by a progressive development of seizures with temporal lobe focus, caused by an unbalance between the excitatory and inhibitory neurotransmission. Patients who present that type of epilepsy normally don¿t respond well to the treatments. Of this type of epilepsy, the Mesial Temporal Lobe Epilepsy (MTLE) is the most common one and it is characterized by the commitment of the mesial temporal lobe structures, such as in the Hipocampal Sclerosis. To realize these studies the proteomics has many powerful tools that allow us to elucidate complex biological mechanisms, to find altered proteins in the whole organism and describe protein expression patterns in different physiological and pathological conditions. Therefore, it¿s relevant to study this protein expression pattern in the hippocampus of animal models of MTLE using proteomics techniques, searching for informative data that lead us to the understanding of the involved mechanisms in the epileptogenicity. In this study we identified proteins that can indicate new pathways involved in the epileptogenesis processes. Furthermore, our data demonstrate that additional molecular complexity could be observed as hippocampal subfields were analyzed separately. We believe that the further integration of the proteomic data with other "omics" approaches could generate even more informative data about those neuronal processes Mestrado Fisiopatologia Médica Mestra em Ciências

Orientador: Fábio Cesar Gozzo Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Química Resumo: A aplicação da espectrometria de massas (MS) em análises proteômicas tem evoluído extensivamente, a ponto de se tornar rotineira no seqüenciamento e identificação de proteínas e determinação de modificações pós-traducionais. Entretanto, o uso de MS na análise de estruturas tridimensionais de proteínas é restrito, apesar das inúmeras vantagens potenciais que poderia apresentar nessa área. Nesse âmbito, a metodologia de ligação cruzada acoplada a MS visa obter informações estruturais de proteínas e complexos protéicos pela determinação de restrições espaciais de distância entre resíduos de aminoácidos, mediante reação com compostos bifuncionais, proteólise enzimática e análise dos peptídeos resultantes por MS. A primeira parte deste trabalho descreve a aplicação de um novo agente de ligação cruzada (ALC) heterobifuncional e clivável, contendo um grupo fotoativável do tipo diazirina, para formar ligações cruzadas em peptídeos e proteínas. O emprego desse ALC permitiu obter ligações cruzadas únicas nos alvos estudados, e os peptídeos modificados assim formados foram caracterizados por MS/MS, possibilitando a descoberta de um íon marcador característico para peptídeos ligados por esse ALC. A segunda parte do trabalho envolveu a aplicação da técnica de mobilidade iônica TWIM acoplada a MS na análise de íons de proteínas antes e após modificação por ALC, visando avaliar o efeito dessas modificações em termos de variações de seção de choque de colisão (CCS). Análises assim feitas demonstraram a presença de estruturas mais rigidas e com menor CCS após a adição de moléculas de ALC, evidenciando a existência de conformações mais compactas em fase gasosa após a reação de ligação cruzada para proteínas. Para validar a metodologia, os valores de CCS experimentais foram comparados com valores teóricos calculados computacionalmente para estruturas disponíveis. Abstract: The use of mass spectrometry (MS) in proteomics has evolved extensively, up to the point where it became routinely employed in protein sequencing and determination of post-translational modifications. However, the use of MS in structural studies of proteins is limited, despite the many advantages it could bring to the area. In this sense, chemical cross-linking coupled to mass spectrometry for structural studies of proteins aims to obtain structural information in terms of spatial distance restrictions, determined by intra- and intermolecular cross-links between side chains of amino acid residues. This is achieved by reaction of the target protein system with bifunctional compounds, followed by proteolysis and analysis of the modified peptides by MS. The first part of this work describes the application of a novel, heterobifunctional and cleavable cross-linker, containing a diazirine photoactivatable group, for cross-linking of peptides and proteins. Reaction with this cross-linker yielded unique cross-links for peptides and proteins, and such modified peptides where characterized by MS/MS, allowing the discovery of a marker ion, that may be used to identify cross-linked peptides. The second part of the work focused on the application of traveling-wave ion mobility (TWIM) coupled to MS for a comparative analysis of protein ions both before and after cross-linking reactions, aiming to evaluate the effect of these modifications on protein structures in terms of variation of collision cross sections (CCS) of their ions. TWIM-MS analysis of these systems demonstrated the presence of compact gas phase conformations of lower CCS after the cross-linking reaction and formation of intramolecular cross-links, indicating that chemical cross-linking restricts the structural dynamics of gas phase protein ions. To validate this method, experimental CCS values were then compared with theoretical values obtained computationally for available crystallographic or NMR structures. Mestrado Química Orgânica Mestre em Química

Orientador: Marco Aurelio Zezzi Arruda Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Quimica Resumo: Neste trabalho de Dissertação, os organismos em estudo foram sementes de soja transgênica e não-transgênica, sendo que a soja geneticamente modificada é do tipo Roundup Ready® (RR), na qual a inserção de um gene tornou a planta resistente a herbicidas que possuem o glifosato como princípio ativo. Procurou-se avaliar o perfil proteomico, enzimático e metálico em sementes de soja a fim de contribuir com uma recente área do conhecimento, a Metalômica. Os resultados deste estudo indicam que não foi encontrada variação quanto ao total de proteínas entre as amostras e, também, que não houve variação quanto ao número total de spots detectados nos géis de ambas as amostras para as faixas de pH avaliadas (3-10 e 4-7) após a separação do conjunto de proteínas por 2D PAGE. Porém, por meio do estudo de análise de imagens dos géis verificou-se que 10 spots apresentaram variações significativas quanto a expressão de proteínas entre as sementes ( 90%). Deste conjunto, 08 proteínas puderam ser identificadas por meio da obtenção dos espectros de massa empregando MALDI-QTOF MS e busca em banco de dados. Aparentemente, não foi encontrada variações em termos de atividade enzimática das enzimas CAT, GR, SOD e APX, avaliadas em gel e por espectrofotometria, entre as amostras de soja transgênica e não-transgênica no estágio de semente, portanto, não verifica-se uma situação de extresse oxidativo neste estágio. O mapeamento das espécies metálicas foi feito por SR-XRF e ICP MS e, alguns elementos puderam ser detectados (Co, Nb, Cd, Se, V, La, Ce, Th, Ru, Zr e Hg). Apenas 02 spots proteicos das amostras de semente de soja transgênica apresentaram teor de V que pudesse ser quantificado, no entanto, devido a enorme variação de gel para gel e de variações que são intrínsecas de sistemas biológicos, aspectos mais conclusivos sobre suas quantificações ainda não foram possíveis. Também, não foi encontrado na literatura relatos sobre a possível presença deste elemento nas proteínas identificadas Abstract: In this work, transgenic and non-transgenic soybean seeds were studied, being the Roundup Ready® (RR) type, the soybean genectically modified, to which the insertion of the gene make the plant resistent to herbicides that present the glifosate as active principle. The proteomic, enzimatic and metal profiles were evaluated in the soybean seeds to contribute to a new area, Metallomics. The results indicated that no variation was obtained related to the amount of total proteins between both samples as well as no variation related to the number of those detected spots in the gels in the pI range evaluated (3-10 and 4-7) after 2D PAGE. However, from the gel image analysis, 10 spots presented significant variation related to the protein expression between the seeds ( 90%). From this set, 08 proteins were analyzed by MALDI-QTOF-MS and the peptide sequency of each one identified in the protein data bank. Aparently, no variation when considering CAT, GR, SOD and APX in terms of enzimatic activity for transgenic and non-transgenic soybean seeds was achieved, when spectrophotometry and gel electrophoresis were used. Apparently, there is not the detection of the oxidative stress at this stage. The metallic species mapping was carried out by SR-XRF and ICP MS, and some elements were detected (Co, Nb, Cd, Se, V, La, Ce, Th, Ru, Zr and Hg). Vanadium was quantificated in 02 spots of transgenic soybean seeds only; however, due to the high gel to gel variation and those ones commonly achieved in biological systems, conclusive aspects about their quantifications were not still possible. There is no information in the literature about a possible presence of V in those identified proteins. Mestrado Química Analítica Mestre em Química