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Several perturbations in the number of peripheral blood leukocytes, such as neutrophilia and lymphopenia associated with Coronavirus disease 2019 (COVID-19) severity, point to systemic molecular cell cycle alterations during severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. However, the landscape of cell cycle alterations in COVID-19 remains primarily unexplored. Here, we performed an integrative systems immunology analysis of publicly available proteome and transcriptome data to characterize global changes in the cell cycle signature of COVID-19 patients. We found significantly enriched cell cycle-associated gene co-expression modules and an interconnected network of cell cycle-associated differentially expressed proteins (DEPs) and genes (DEGs) by integrating the molecular data of 1,469 individuals (981 SARS-CoV-2 infected patients and 488 controls [either healthy controls or individuals with other respiratory illnesses]). Among these DEPs and DEGs are several cyclins (CCNs), cell division cycles (CDCs), cyclin-dependent kinases (CDKs), and mini-chromosome maintenance (MCMs) proteins. COVID-19 patients partially shared the expression pattern of some cell cycle-associated genes with other respiratory illnesses but exhibited some specific differential features. Notably, the cell cycle signature predominated in the patients' blood leukocytes (B, T, and NK cells) and was associated with COVID-19 severity and disease trajectories. These results provide a unique global understanding of distinct alterations in cell cycle-associated molecules in COVID-19 patients, suggesting new putative pathways for therapeutic intervention. This article is protected by copyright. All rights reserved.

Combination therapies or multi-targeted drugs have been pointed out as an option to prevent the emergence of resistant clones, which could make long-term treatment more effective and translate into better clinical outcomes for cancer patients. The NT157 compound is a synthetic tyrphostin that leads to long-term inhibition of IGF1R/IRS1-2-, STAT3- and AXL-mediated signaling pathways. Given the importance of these signaling pathways for the development and progression of lung cancer, this disease becomes an interesting model for generating preclinical evidence on the cellular and molecular mechanisms underlying the antineoplastic activity of NT157. In lung cancer cells, exposure to NT157 decreased, in a dose-dependent manner, cell viability, clonogenicity, cell cycle progression and migration, and induced apoptosis (p BCL2, CCND1, MYB, and MYC and increased genes related to cellular stress and apoptosis, JUN, BBC3, CDKN1A, CDKN1B, FOS, and EGR1 (p

The constitutively active BCR-ABL1 tyrosine kinase, found in t(9;22)(q34;q11) chromosomal translocation-derived leukemia, initiates an extremely complex signaling transduction cascade that induces a strong state of resistance to chemotherapy. Targeted therapies based on tyrosine kinase inhibitors (TKIs), such as imatinib, dasatinib, nilotinib, bosutinib, and ponatinib, have revolutionized the treatment of BCR-ABL1-driven leukemia, particularly chronic myeloid leukemia (CML). However, TKIs do not cure CML patients, as some develop TKI resistance and the majority relapse upon withdrawal from treatment. Importantly, although BCR-ABL1 tyrosine kinase is necessary to initiate and establish the malignant phenotype of Ph-related leukemia, in the later advanced phase of the disease, BCR-ABL1-independent mechanisms are also in place. Here, we present an overview of the signaling pathways initiated by BCR-ABL1 and discuss the major challenges regarding immunologic/pharmacologic combined therapies.

Glioblastoma is the most common aggressive primary brain tumor. Standard care includes maximal safe surgical resection, radiation, and chemotherapy with temozolomide. However, the impact of this therapeutic approach on patient survival is disappointing and poor outcomes are frequently observed. Therefore, new therapeutic targets are needed to treat this potentially deadly tumor. Aurora kinases are one of today's most sought-after classes of therapeutic targets to glioblastoma therapy. They are a family of proteins composed of three members: Aurora-A, Aurora-B, and Aurora-C that play different roles in the cell division through regulation of chromosome segregation. Deregulation of these genes has been reported in glioblastoma and a progressive number of studies have shown that inhibition of these proteins could be a promising strategy for the treatment of this tumor. This review discusses the preclinical and early clinical findings on the potential use of the Aurora kinases as new targets for the treatment of glioblastoma. KEY MESSAGES: GBM is a very aggressive tumor with limited therapeutic options. Aurora kinases are a family of serine/threonine kinases implicated in GBM pathology. Aurora kinases are critical for glioblastoma cell growth, apoptosis, and chemoresistance. Inhibition of Aurora kinases has a synergistic or sensitizing effect with chemotherapy drugs, radiotherapy, or with other targeted molecules in GBM. Several Aurora kinase inhibitors are currently in clinical trials.

Made available in DSpace on 2019-10-06T15:36:54Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-07-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Background: Adiponectin exhibits anti-inflammatory actions and is mainly expressed in adipose tissue. However, recent studies have shown that adiponectin can also be secreted by skeletal muscle fibers with autocrine and paracrine effects. Objectives: To analyze the role of adiponectin in the metabolic and inflammatory response of skeletal muscle after acute exhaustive aerobic exercise. Methods: C57BL/6 (WT) and adiponectin knockout (AdKO) mice underwent four days of treadmill running adaptation and at the fifth day, they performed an incremental maximum test to determine the maximum speed (Vmax). Acute exercise consisted of one hour at 60% Vmax. Mice were euthanatized 2 and 24 h after acute exercise session. Results: Serum and gastrocnemius adiponectin increased after 2-hours of acute exercise. NEFA concentrations were lower in non-exercise AdKO, and decreased 2-hours after exercise only in WT. No differences were found in muscle triacylglycerol content; however, glycogen content was higher in AdKO in non-exercise (p-value = 0.005). WT showed an increase in AMP-activated protein kinase (AMPK) phosphorylation 2-hours after exercise and its level went back to normal after 24-hours. Otherwise, exercise was not able to modify AMPK in the same way as in AdKO. WT showed an increase in the phosphorylation of ACC (Ser79) 2-hours after exercise and return to normal after 24-hours of exercise (p-value < 0.05), kinects that was not observed in AdKO mice. IL-10 and IL-6 concentration was completely different among genotypes. In WT, these cytokines were increased at 2 (p-value < 0.01) and 24 h (p-value < 0.001) after exercise when compared with AdKO. NF-κBp65 protein and gene expression were not different between genotypes. Conclusion: Adiponectin influences muscle metabolism, mainly by the decrease in exercise-induced AMPK phosphorylation, inflammatory profile and IL-6 in the muscle. Department of Cell and Developmental Biology Institute of Biomedical Sciences University of São Paulo (USP) Exercise and Immunometabolism Research Group Department of Physical Education Universidade Estadual Paulista (UNESP) Exercise and Immunometabolism Research Group Department of Physical Education Universidade Estadual Paulista (UNESP) FAPESP: 2016/01409-8 FAPESP: 2016/02696-0

Antenatal corticosteroid therapy is used to reduce neonatal mortality in preterm infants but it is currently unknown whether this intervention affects lipid metabolism at the peripartum. This study aimed to evaluate if antenatal corticosteroid therapy in pregnant rats and women affects lipid metabolism during early lactation. We evaluated women at risk of preterm delivery that received corticosteroid therapy (CASE) and women that were not exposed to corticosteroid and were not at risk of preterm delivery (CONTROL). Samples were collected to measure serum and milk triacylglycerol (TAG) three days after delivery. Rats were treated with dexamethasone (DEX) between the 15th and the 20th days of pregnancy. Samples were collected at different days after delivery (L3, L8 and L14). TAG was measured in serum, liver and mammary gland (MG). TAG appearance rates were measured after tyloxapol injection and gavage with olive oil. We also evaluated the expression of key genes related to lipid metabolism in the liver and in the MG and hepatic phosphorylation of AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC). CASE volunteers delivered earlier than CONTROL but presented unaltered milk and serum TAG concentrations. Early lactating DEX rats exhibited increased TAG in serum, MG and milk. No changes in CD36 and LPL were detected in the MG and liver. Early lactating DEX rats displayed increased TAG appearance rate and reduced hepatic AMPK/ACC phosphorylation. Our data revealed that antenatal corticosteroid therapy reduces hepatic AMPK/ACC phosphorylation during early lactation that reflects in increased TAG concentration in serum, MG and milk.

Glycogen synthase kinase-3 beta (GSK-3β) is an enzyme pertinently linked to neurodegenerative diseases since it is associated with the regulation of key neuropathological features in the central nervous system. Among the different kinds of inhibitors of this kinase, the allosteric ones stand out due to their selective and subtle modulation, lowering the chance of producing side effects. The mechanism of GSK-3β allosteric modulators may be considered still vague in terms of elucidating a well-defined binding pocket and a bioactive pose for them. In this context, we propose to reinvestigate and reinforce such knowledge by the application of an extensive set of in silico methodologies, such as cavity detection, ligand 3D shape analysis and docking (with robust validation of corresponding protocols), and molecular dynamics. The results here obtained were consensually consistent in furnishing new structural data, in particular by providing a solid bioactive pose of one of the most representative GSK-3β allosteric modulators. We further applied this to the prospect for new compounds by ligand-based virtual screening and analyzed the potential of the two obtained virtual hits by quantum chemical calculations. All potential hits achieved will be subsequently tested by in vitro assays in order to validate our approaches as well as to unveil novel chemical entities as GSK-3β allosteric modulators.

Orientadores: Maria de Fatima Sonati, Carolina Lanaro Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas Resumo: As fosfatidilinositol-fosfato quinases (PIPKs) são uma família de enzimas lipídio quinases responsáveis pela produção do segundo mensageiro PI4,5P2 (fosfatidilinositol 4,5 bifosfato), que tem um importante papel regulatório em uma variedade de processos celulares, inclusive na expressão gênica. As PIPKs são classificadas em 3 subfamílias -PIPK I (isoformas a, P e y), PIPK II (a, P e y) e PIPK III - que apresentam distintas funções e localização celular. Recentemente, em estudo desenvolvido em nosso laboratório, o gene da PIPKIIa apresentou-se diferencialmente expresso em reticulócitos de dois irmãos com Doença da Hb H. Sua maior expressão, bem como do gene da globina P, foi encontrada no paciente com concentração de Hb H mais elevada, sugerindo uma relação entre a PIPKIIa e a produção de globinas, particularmente da globina p. No presente trabalho, avaliamos, por PCR Quantitativa em Tempo Real (qRT-PCR), os perfis de expressão dos genes das PIPKs (I e II, isoformas a, P, y, e III) durante a eritropoese em cultura de células CD34+ do sangue periférico de 11 indivíduos sadios e de 6 pacientes com hemoglobinopatias (2 a-talassêmicos, 2 P-talassêmicos e 2 com Anemia Falciforme), comparando-os com os perfis de expressão dos genes das globinas a, P e y, nos dias 7, 10 e 13 da cultura eritróide. Na cultura de células dos indivíduos controles, os resultados revelaram que a expressão de todos os genes PIPKs aumentam durante a diferenciação eritróide e que o perfil de expressão do gene da PIPKIIa coincide com o perfil dos genes de globinas. Nas culturas de células dos pacientes, a expressão do gene da PIPKIIa tornou-se maior durante a diferenciação, enquanto os genes das outras PIPKs apresentaram resultados heterogêneos. O gene PIPKIIa esteve altamente expresso na cultura de células de um dos pacientes a-talassêmicos, enquanto na cultura de um dos P-talassêmicos apresentou expressão reduzida em relação ao controle. Os resultados também foram diferentes entre as culturas de células dos pacientes falciformes. Este é o primeiro estudo sobre o perfil de expressão dos genes dessas PIP quinases durante a eritropoese humana in vitro. Um padrão de normalidade foi estabelecido. Embora os resultados tenham sido heterogêneos entre os pacientes, o que enfatiza a complexidade dos sistemas regulatórios que atuam na formação da hemoglobina, eles fortalecem a hipótese da existência de uma relação entre PIPKIIa e produção de globina ß Abstract: Phosphatidylinositol-phosphate-kinases (PIPKs), a family of lipid kinases, produce the second messenger PI4,5P2 (phosphatidylinositol 4,5-biphosphate), which regulates various cellular activities, incluing the gene expression. PIPKs are divided into three subfamilies [PIPK I (a, p, y), PIPK II (a, p, y) and PIPK III], which are functionally distinct and located in different subcellular compartments. In a recent study in our laboratory, the PIPKIIa gene was differentially expressed in reticulocytes from two siblings with Hb H disease. Expression of both the PIPKIIa and p globin genes were higher in the patient with the higher Hb H level, suggesting a relationship between PIPKIIa and the production of globins, particularly P-globin. The aim of this study was to determine the gene expression profiles of PIPKs (I and II - with their isoforms - and III) during erythropoiesis in peripheral blood haematopoietic CD34+ cell culture from eleven healthy volunteers and six patients with haemoglobinopathies (2 with a-thalassemia, 2 with P-thalassemia and 2 with sickle cell anaemia) using quantitative real time PCR (qRT-PCR) and to compare these profiles with the gene expression profiles of a, P and y globins on the 7th, 10th and 13th days of culture. The results for the cell cultures from healthy individuals showed that the expression of PIPKs increase during erythroid differentiation and that the PIPKIIa and globin expression profiles are similar. Expression of the PIPKIIa gene increased in the patients' cell cultures during erythroid differentiation, whereas expression of the other PIPK genes varied. PIPKIIa was overexpressed in the cell culture of an a-thalassemic patient, while its expression was reduced in the culture of a P-thalassemic patient. The results also differed between the cultures from sickle cell patients. This is the first study of the gene expression profiles of PIPKs during in vitro human erythroid differentiation. We identified a standard pattern of gene expression in the healthy group. Although the results varied between patients, our findings strengthen the hypothesis of a relationship between PIPKIIa and production of P-globin Doutorado Biologia Estrutural, Celular, Molecular e do Desenvolvimento Doutor em Fisiopatologia Médica

Orientador: Paulo Mazzafera Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia Resumo: A via de biossíntese de lignina é intensamente estudada. Em grande parte, o interesse neste polímero é vinculado à manipulação do teor de lignina em determinadas espécies de plantas, tais como nas gramíneas utilizadas como forrageiras e em espécies arbóreas utilizadas para a produção de papel e, mais recentemente, em produção de biocombustíveis. Muitos componentes desta via como as enzimas e os fatores de transcrição que participam de sua regulação vêm sendo alvo de pesquisas, porém proteínas quinases reguladoras especificamente para esta via são pouco conhecidas em plantas. Para induzir modificações no metabolismo e no nível de expressão da via de biossíntese da lignina, eucaliptos foram submetidos ao tensionamento do caule. Este tipo de tratamento resulta na mudança da composição e redução da lignificação na região de tensão da madeira (TW) e no aumento de lignificação da região oposta (OW). Alterando o metabolismo da lignina, quinases potencialmente relacionadas a sinalização da via durante o tratamento de tensão foram mobilizadas e identificadas. Verificamos que as alterações anatômicas, o aumento da lignificação e a razão S/G apresentaram nas espécies de eucalipto estudadas um padrão similar. A análise de expressão gênica demonstrou alterações nas espécies E. globulus e E. grandis em genes da via de biossíntese da lignina, fatores de transcrição e proteínas quinases; proporcionado a identificação de genes potencialmente relacionadas com a regulação e formação da parede celular por apresentarem aumento de expressão na OW. O gene HCT4 demonstrou ser altamente correlacionado com as quinases analisadas na espécie E. globulus. As lacases EglLAC38, EglLAC44 e as peroxidases EglPRX11 e EglPRX12 se destacam pela possível atuação na polimerização da lignina, enquanto as quinases EglCAMK_CDPK12, EglMAPK3 e EglMAPK9-1 estariam relacionadas a vascularização e lignificação do caule. As quinases receptoras EglWAK_LR1, EgrWAK5 e EgrWAK8 são potenciais candidatas a sinalização do estresse gravitacional em eucalipto. Em E. grandis, o tensionamento do caule provocou maior expressão de enzimas relacionadas a via de biossíntese da lignina, pectina e celulose. Os resultados deste estudo confirmam dados já apresentados na literatura e identificam genes de quinases candidatos para futuras análises funcionais Abstract: The lignin biosynthesis pathway is intensively studied. Largely, the interest in this polymer linked to the manipulation of lineage content in specific plant species, such as grasses, used as fodder and tree species used in paper production, more recently, in biofuels production. Many components of this pathway, such as enzymes and transcription factors (FTs) that participate in its regulation, have been the target of research, but regulatory protein kinases specifically for this pathway are poor known in plants. To induce changes in the metabolism and expression level of lignin biosynthesis pathway, eucalyptus was subjected to tension wood. This type of treatment results on composition altering and lignin reducing in the tension wood (TW) and lignin increasing in opposite wood (OW). Kinases potentially related to pathway signaling during tension treatment were mobilized and identified after lignin metabolism altering. We found that anatomical changes, lignin increase and the S/G ratio show a similar pattern in the studied eucalyptus species. Gene expression analysis showed changes in E. globulus and E. grandis species on lignin biosynthesis pathway, transcription factors and protein kinases genes expression; identifying genes potentially related to cell wall regulation as they present increased expression in OW. The HCT4 gene was shown to be highly correlated to Eucalyptus globulus kinases. Laccases EglLAC38, EglLAC44 and peroxidases EglPRX11 and EglPRX12 were indicated by their potential to act in lignin polymerization, while kinases EglCAMK_CDPK12, EglMAPK3 and EglMAPK9-1 would be related to stem vascularization and lignification. Receptor-like kinases EglWAK_LR1, EgrWAK5 and EgrWAK8 are potential candidates for signaling gravitational stress in eucalyptus. In E. grandis, tension wood caused greater protein expression related to lignin, pectin and cellulose biosynthesis pathway. The results presented confirm literature data and identify kinases candidates for further functional analysis Doutorado Biologia Vegetal Doutora em Biologia Vegetal CNPQ 140428/2015-1

Adjustments in renal K+ excretion constitute a central mechanism for K+ homeostasis. The renal outer medullary potassium (ROMK) channel accounts for the major K+ secretory route in collecting ducts during basal conditions. Activation of the angiotensin II (Ang II) type 1 receptor (AT1R) by Ang II is known to inhibit ROMK activity under the setting of K+ dietary restriction, underscoring the role of the AT1R in K+ conservation. The present study aimed to investigate whether an AT1R binding partner, the AT1R-associated protein (ATRAP), impacts Ang II-mediated ROMK regulation in collecting duct cells and, if so, to gain insight into the potential underlying mechanisms. To this end, we overexpressed either ATRAP or β-galactosidase (LacZ; used as a control), in M-1 cells, a model line of cortical collecting duct cells. We then assessed ROMK channel activity by employing a novel fluorescence-based microplate assay. Experiments were performed in the presence of 10−10 M Ang II or vehicle for 40 min. We observed that Ang II-induced a significant inhibition of ROMK in LacZ, but not in ATRAP-overexpressed M-1 cells. Inhibition of ROMK-mediated K+ secretion by Ang II was accompanied by lower ROMK cell surface expression. Conversely, Ang II did not affect the ROMK-cell surface abundance in M-1 cells transfected with ATRAP. Additionally, diminished response to Ang II in M-1 cells overexpressing ATRAP was accompanied by decreased c-Src phosphorylation at the tyrosine 416. Unexpectedly, reduced phospho-c-Src levels were also found in M-1 cells, overexpressing ATRAP treated with vehicle, suggesting that ATRAP can also downregulate this kinase independently of Ang II-AT1R activation. Collectively, our data support that ATRAP attenuates inhibition of ROMK by Ang II in collecting duct cells, presumably by reducing c-Src activation and blocking ROMK internalization. The potential role of ATRAP in K+ homeostasis and/or disorders awaits further investigation.

Melanoma is a type of skin cancer with low survival rates after it has metastasized. In order to find molecular differences that could represent targets of quercetin in anti-melanoma activity, we have chosen SKMEL-103 and SKMEL-28 melanoma cells and human melanocytes as models. Firstly, we observed that quercetin was able in reducing SKMEL-103 cell viability, but not in SKMEL-28. Besides that, quercetin treatment caused inhibition of AXL in both cell lines, but upregulation of PIM-1 in SKMEL-28 and downregulation in SKMEL-103. Moreover, HIF-1 alpha expression decreased in both cell lines. Interestingly, quercetin was more effective against SKMEL-103 than kinases inhibitors, such as Imatinib, Temsirolimus, U0126, and Erlotinib. Interestingly, we observed that while the levels of succinate dehydrogenase and voltage-dependent anion channel increased in SKMEL-103, both proteins were downregulated in SKMEL-28 after quercetin’s treatment. Furthermore, AKT, AXL, PIM-1, ABL kinases were much more active and chaperones HSP90, HSP70 and GAPDH were highly expressed in SKMEL-103 cells in comparison with melanocytes. Our findings indicate, for the first time, that the efficacy of quercetin to kill melanoma cells depends on its ability in inhibiting tyrosine kinase and upregulating mitochondrial proteins, at least when SKMEL-103 and SKMEL-28 cells response were compared.

Orientador: Nilson Ivo Tonin Zanchin Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia Resumo: A quinase de 70 kDa da proteína ribossomal S6, isoforma 1 (S6K1), é uma fosfoproteína implicada na regulação de genes relacionados ao controle da tradução em mamíferos e possui uma forma nuclear (a1) e uma citoplasmática (a2). A fosforilação do seu principal alvo, a proteína RPS6, tem sido comumente associada ao recrutamento seletivo dos 5'-TOP (5' tract of oligopyrimidine) mRNAs pela maquinaria de tradução, embora haja estudos contrariando esta hipótese. Devido às funções de seus demais alvos, S6K1 tem sido implicada na sobrevivência celular e em diversos outros processos, como crescimento, câncer e resistência à insulina. S6K1 é ativada por um mecanismo que envolve fosforilação seqüencial através da ativação das vias mTORC1 (complexo 1 do alvo da rapamicina em mamíferos) e PI3K (fosfoinositol-3 quinase). Como uma quinase da família AGC, S6K1 deve ser fosforilada por mTORC1 no resíduo Thr389 do domínio hidrofóbico e, em seguida, por PDPK1 (proteína quinase 1 dependente de fosfoinositol) no resíduo Thr229 da alça T do domínio catalítico. Estes eventos ocorrem somente após a fosforilação em diversos sítios do domínio auto-inibitório carboxiterminal, por mTORC1. O objetivo deste trabalho foi desenvolver um ensaio modelo para análise da função da S6K1 in vitro e utilizá-lo como ferramenta na elucidação do papel de proteínas adaptadoras da via de mTOR em interações com a S6K1. Para isso foi necessário produzir as proteínas recombinantes para ensaios de interação e para realização de um ensaio de atividade para a S6K1. Foram testados vários sistemas de expressão para Escherichia coli para produção das construções GST-S6K1a1-His6, GST-S6K1a2-His6 e GST-S6K1a2T389E?CT (forma a2 de S6K1 com a substituição T389E e o carboxiterminal truncado), GST-PDPK1 e GST-CDPDPK1 (domínio catalítico de PDPK1 fusionado a GST). A expressão das formas truncadas de S6K1 e PDPK1 foi mais eficiente em E. coli. Embora o rendimento tenha ficado muito aquém do esperado, foi suficiente para os ensaios de interação in vitro. Também foi feita a expressão em E. coli da região C-terminal da proteína RPS6, que é o substrato da S6K1, em fusão com a proteína D do fago ?. Posteriormente, foram montados sistemas de expressão das construções His6-S6K1a2T389E?CT e His6-CDPDPK1 em células de inseto, a partir de vetor de baculovírus. Constatou-se que essas construções são expressas na forma de fosfoproteínas em células de inseto. Ensaios de GST pull-down com GST-S6K1a2-His6 e GST-S6K1a2T389E?CT contra as duas isoformas da subunidade catalítica da PP2AC, His6-PP2ACa(maior) e His6-PP2ACa(menor), revelaram que His6-PP2ACa(maior) não interage com GST-S6K1a2-His6, embora interaja fortemente com GST-S6K1a2T389E?CT. Já a construção His6-PP2ACa(menor) interage fracamente com as construções GST-S6K1a2-His6 e GST-S6K1a2T389E?CT. Tomados em conjunto, os resultados sugerem que a presença do C-terminal não fosforilado de S6K1a2 impede a interação com PP2ACa(maior). PP2ACa(menor) comporta-se de forma completamente diferente da isoforma maior, pois a interação entre PP2ACa(menor) e S6K1a2 parece ser independente do carboxiterminal da quinase, visto que as quantidades de S6K1a2T389E?CT e de S6K1a2 inteira que interagem com PP2ACa(menor) são semelhantes. Esses resultados necessitam ainda serem confirmados in vivo. Outros experimentos de GST pull-down confirmaram que as construções de S6K1 não interagem com a4, embora interajam com TIPRL1. Se confirmado in vivo, esse resultado compõe um novo quadro na regulação coordenada entre mTOR1 e PP2A, do qual TIPRL1 parece participar. As construções genéticas e os sistemas de expressão gerados neste trabalho possibilitaram a obtenção dos reagentes necessários para analisar o mecanismo de regulação da quinase S6K1, mediado por proteínas regulatórias. Permitem também desenvolver uma série de experimentos, como busca de inibidores específicos para a S6K1, que dependem da reconstituição de ensaios de atividade in vitro com a S6K1 ativada. Contudo, o ensaio de atividade realizado não apresentou resultados satisfatórios e precisa ser desenvolvido. Abstract: The 70kDa ribosomal S6 protein kinase 1 (S6K1) is a phosphoprotein involved in the regulation of genes related to translational control in mammals. S6K1 shows distinct nuclear (a1) and cytoplasmic (a2) forms. Phosphorylation of the S6K1 best characterized target, the protein of the small ribosomal subunit (RPS6), has been generally associated to the selective recruitment of the 5'-TOP mRNAs (5' tract of oligopyrimidine) by the translational machinery, although there is still some controversy on this issue. Due to the function of its targets, S6K1 has been implicated in several cellular processes including cell growth, cancer and insulin resistance. S6K1 is activated by a mechanism of sequential phosphorylation following activation of the mTORC1 (mammalian target of rapamycin complex 1) and PI3K (phosphoinositide-3-kinase) pathways. As a kinase of the AGC family, S6K1 activation requires mTORC1 phosphorylation of residue Thr389 of the hydrophobic domain followed by PDPK1 (phosphoinositide dependent protein kinase 1) phosphorylation of residue Thr229 at the T loop of the catalytic domain. These take place only after phosphorylation by mTORC1 of several residues of the autoinhibitory C-terminal domain. The objective of this work was to develop an assay to analyze the function of S6K1 in vitro and use it as a tool in the discovering of the functions of regulators proteins of the mTOR cascade in interactions with S6K1. For these purposes, expression systems were constructed to produce the various recombinant proteins to be used in the interaction and activity assays. Several genetic constructions were tested in Escherichia coli for the production of GST-S6K1a1-His6, GST-S6K1a2-His6 and GST-S6K1a2T389E?CT (a2 form of S6K1 with the T389E substitution and truncated carboxiterminus), GST-PDPK1 and GST-CDPDPK1 (GST fusion protein of the catalytic domain of PDPK1). The truncated forms were expressed more efficiently in E. coli. Although the yield in E. coli was lower than expected, it was sufficient to perform interaction assays. The C-terminal domain of RPS6, a substrate for S6K1, was successfully expressed in E. coli as a fusion protein with the phage ? protein D. Subsequently, expression systems for production of His6-S6K1a2T389E?CT and His6-CDPDPK1 in insect cells were constructed using baculovirus vectors. It was found that these constructs are expressed in the form of phosphoproteins in insect cells. GST pull-down assays using GST-S6K1a2-His6 e GST-S6K1a2T389E?CT to test interaction with the PP2AC isoforms His6-PP2ACa(major) and His6-PP2ACa(minor) revealed that His6-PP2ACa(major) does not interact with GST-S6K1a2-His6, although it interacts strongly with GST-S6K1a2T389E?CT. On the other hand, His6-PP2ACa(minor) interacts weakly with both GST- S6K1a2-His6 and GST-S6K1a2T389E?CT. This finding suggests that the unphosphorylated C-terminal of S6K1a2 inhibits interaction with PP2ACa(major). His6-PP2ACa(minor) behaves differently form His6-PP2ACa(major). Its interaction with S6K1a2 seems to be independent of the C-terminal since the amounts of S6K1a2T389E?CT and S6K1a2 that interact with His6-PP2ACa(minor) are similar. Future work in vivo is required to confirm these results. GST pull-down assays confirmed that a4 does not interact with the constructions of S6K1, while TIPRL1 interacts with them. If confirmed in vivo, these results provides a new perspective for the coordinated regulation between mTOR1 and PP2A, which apparently involves also TIPRL1. The genetic constructions and expression systems established in this work allow the production of the reagents required to study the mechanism of S6K1 regulation mediated by adaptor proteins. They will also allow the development of experiments such as screening for specific S6K1 inhibitors, which depend on reconstitution of S6K1 activity assays using activated S6K1. Nevertheless, the activity assay performed did not yield satisfactory outcomes and must be improved. Mestrado Bioquímica Mestre em Biologia Funcional e Molecular

The objective of this research was to evaluate the effect of Zilpaterol Hydrochloride (ZH) on myogenic or adipogenic gene and protein expression in skeletal muscle. Two feeding trials and one cell culture experiments were conducted. Semimembranosus muscle tissue was collected from steers that had been fed a diet containing 8.3 mg of ZH/kg DM for the last 0 or 20 d of the finishing period with a 3 d withdrawal period. To test the mode of action an in vitromodel was used with, isolated bovine satellite cells isolated from muscle tissue. Real Time-QPCR (RTQPCR) was used to measure the relative mRNA abundance of Adenosine Monophosphate Protein Kinase α (AMPK), Myosin Heavy Chain (MHC) I, MHCIIA, MHCIIX, Insulin-like Growth Factor I (IGF-I), β-adrenergic receptor (βAR) 1 and 2, peroxisome proliferator-activated receptor gamma (PPARγ), and Stearoyl-CoA Desaturase (SCD). Western blotting was used to measure the relative protein abundance of AMPK and Phosphorylated-AMPK (pAMPK). No differences were detected in relative mRNA abundance of AMPK, MHCIIA, IGF-I, βAR1 and βAR2. However, MHCI, SCD, and PPARγ mRNA expression was decreased and MHCIIX mRNA increased from ZH fed cattle compared to non-ZH. For one experiment, AMPK protein expression increased, while in another experiment, AMPK phosphorylation increased with ZH fed animals. The increase in MHCIIX mRNA with ZH fed cattle indicated the start of a fiber type shift towards larger diameter fibers. This shift may have been due to increased expression and phosphorylation of AMPK. These data suggest that the shift increase in MHCIIX was likely due to the ZH administration.

Nitrate and nitrite supplement deficient endogenous nitric oxide (NO) formation. While these anions may generate NO, recent studies have shown that circulating nitrite levels do not necessarily correlate with the antihypertensive effect of oral nitrite administration and that formation of nitrosylated species (RXNO) in the stomach is critically involved in this effect. This study examined the possibility that RXNO formed in the stomach after oral nitrite administration promotes target protein nitrosylation in the vasculature, inhibits vasoconstriction and the hypertensive responses to angiotensin II. Our results show that oral nitrite treatment enhances circulating RXNO concentrations (measured by ozone-based chemiluminescence methods), increases aortic protein kinase C (PKC) nitrosylation (measured by resin-assisted capture SNO-RAC method), and reduces both angiotensin II-induced vasoconstriction (isolated aortic ring preparation) and hypertensive (in vivo invasive blood pressure measurements) effects implicating PKC nitrosylation as a key mechanism for the responses to oral nitrite. Treatment of rats with the nitrosylating compound S-nitrosoglutathione (GSNO) resulted in the same effects described for oral nitrite. Moreover, partial depletion of thiols with buthionine sulfoximine prevented PKC nitrosylation and the blood pressure effects of oral nitrite. Further confirming a role for PKC nitrosylation, preincubation of aortas with GSNO attenuated the responses to both angiotensin II and to a direct PKC activator, and this effect was attenuated by ascorbate (reverses GSNO-induced nitrosylation). GSNO-induced nitrosylation also inhibited the increases in Ca2+ mobilization in angiotensin II-stimulated HEK293T cells expressing angiotensin type 1 receptor. Together, these results are consistent with the idea that PKC nitrosylation in the vasculature may underlie oral nitrite treatment-induced reduction in the vascular and hypertensive responses to angiotensin II., Graphical abstract Image 1, Highlights • Oral nitrite treatment exerts antihypertensive effects. • The mechanisms explaining such effects are not entirely known. • Oral nitrite treatment increases circulating concentrations of nitrosylating species. • Vascular PKC nitrosylation attenuates the vascular responses to angiotensin II.

Orientador: Michelle Franz Montan Braga Leite Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba Resumo: Este estudo avaliou se a hiperglicemia mantida no diabetes tipo 1 e tipo 2, apesar de etiologias diferentes, induz a uma hiponocicepção na articulação temporomandibular de ratos associado a alteração no nível proteico das isoformas de proteinoquinase PKC-'alfa', PKC-ßI e PKC-ßII. Para isto, ratos Wistar foram tratados com uma injeção peritoneal de estreptozotocina (75 mg/Kg) para indução de diabetes tipo 1 ou tratados com uma dieta hiperlipídica durante 84 dias para indução de diabetes tipo 2. Após os tratamentos, grupos separados de animais foram submetidos ao teste comportamental nociceptivo induzido por uma injeção intra-articular de formalina 1.5% nos dias 7, 14, 21, 28, 35 e 42 após os tratamentos. Imediatamente após a análise comportamental os animais foram mortos por decapitação e amostras do tecido periarticular foram removidas para análise do nível proteico das proteinoquinases PKC-'alfa', PKC-ßI e PKC-ßII. Os resultados demonstraram que os animais com diabetes tipo 1 apresentaram perda significativa de peso corporal e aumento da glicose plasmática (> 300 mg/dl) quando comparado aos animais normoglicêmicos (p 0.3, índice de Lee) e com aumento significativo da glicose plasmática (? 200 mg/dl) (p 300 mg / dl) when compared to normoglycemic animals (p 0.3, Lee index) and with a significant increase in plasma glucose (maior ou mnor que 200 mg / dl) (p

Aging is one of the risk factors for the development of low-grade inflammation morbidities, such as several types of cancer and neurodegenerative diseases, due to changes in the metabolism, hormonal secretion, and immunosenescence. The senescence of the immune system leads to improper control of infections and tissue damage increasing age-related diseases. One of the mechanisms that maintain cellular homeostasis is autophagy, a cell-survival mechanism, and it has been proposed as one of the most powerful antiaging therapies. Regular exercise can reestablish autophagy, probably through AMP-activated protein kinase activation, and help in reducing the age-related senescence diseases. Therefore, in this study, we discuss the effects of exercise training in immunosenescence and autophagy, preventing the two main age-related disease, cancer and neurodegeneration.

O Machine learning (ML) é um campo de inteligência artificial que emergiu rapidamente na biologia molecular de plantas, permitindo assim a exploração de grandes conjuntos de dados. Os principais desafios são analisar esses dados e extrair novos conhecimentos de sistemas celulares. Nesta investigação, considera-se que uma revisão sistemática para desembaraçar as abordagens de ML seria relevante para os cientistas de plantas (capítulo 1). As principais etapas do desenvolvimento do ML, como seleção de dados, extração de recursos, algoritmos de treinamento e avaliação de modelos de classificação/previsão. Além disso, discute-se o papel do ML na era pós-genômica. Além disso, com base na revisão sistemática, foi desenvolvido um método de aprendizado de máquina para classificação de receptores de superfície celular (Capítulo 2). Duas classes de receptores da superfície celular, designados receptores do tipo cinase (RLK) e receptores do tipo proteína (RLPs), são essenciais para perceber e processar sinais externos e internos em plantas e animais. Ambos estão envolvidos no desenvolvimento da planta e na resposta a patógenos e compartilham um domínio extracelular similar, capaz de detectar o sinal ambiental externo do evento de detecção inicial. No entanto, as RLPs têm regiões C-terminais curtas e divergentes, não associadas com característica de domínio das quinases conservadas das RLKs. A ausência de relações filogenéticas C-terminais entre RLK e RLPs impede o uso de algoritmos de comparação de sequência para previsões de alto rendimento da família RLP. Assim, foi desenvolvido o primeiro preditor de RLP em plantas designadas como RLPredictiOme. O RLPredictiOme foi implementado com base em modelos de aprendizado de máquina em conjunto com a inferência bayesiana. Os modelos de ML incorporam em três estágios para distinguir RLPs de noRLPs, RLPs de RLKs e classificar novas subfamílias de RLPs em plantas. Os resultados da avaliação dos modelos mostram alta precisão, sensibilidade, especificidade e probabilidade relativamente alta variando de 0,79 a 0,99 para RLPs. Além disso, uma validação completa do RLPredictiOme foi realizada com LRR-RLPs de Arabidopsis e arroz previamente caracterizados e mais de 90% dos RLPs conhecidos foram previstos corretamente. Além de prever RLPs previamente caracterizados, o RLPredictiOme descobriu novas subfamílias de RLP no genoma de Arabidopsis. Isso inclui probable lipid transfer (PLT)- RLP, plastocyanin-like-RLP, ring finger-RLP, glycosyl-hydrolase-RLP, e glycerophosphoryl diester phosphodiesterase (GDPDL)-RLP, ainda a serem caracterizadas. Em comparação com o único GDPDL-RLK, os estudos de evolução molecular confirmaram que o ectodomínio dos GDPDL-RLPs pode ter sido submetido a uma seleção purificadora com predominância de substituições sinônimas. As análises de expressão revelaram que o GDPGL-RLPs previstos exibem um nível basal de expressão e responde a sinais de desenvolvimento e sinais bióticos. Os resultados desses ensaios biológicos substanciam a noção de que os membros desta subfamília mantiveram domínios funcionais durante a evolução e podem desempenhar papéis relevantes no desenvolvimento e na defesa das plantas. Portanto, o RLPredictiOme pode fornecer novas idéias sobre o papel funcional dos receptores de superfície e suas relações com diferentes processos biológicos. Palavras-chave: Aprendizagem de máquina. Receptores proteínas-like. RLPredictiOme. Machine learning (ML) is a field of artificial intelligence that has rapidly emerged in plant molecular biology, thus allowing the exploitation of massive data. The main challenges are to analyze massive datasets and extract new knowledge of cellular systems. Here, we just presented a systematic review to disentangle ML approaches is relevant for plant scientists (Chapter 1). We presented the main steps for ML development, including data selection, features extraction, training algorithms and evaluation of classification/prediction models, indicating role ML algorithm in the post-genomic era. Additionally, based on the systematic review we also developed a framework machine learning method for cell surface receptors prediction (Chapter 2). Two classes of cell surface receptors designated receptor-like protein kinase (RLK) and receptor-like protein (RLPs) are essential for perceiving and processing external and internal signals in plants and animal. Both are involved in plant development and pathogen responses and share a similar extracellular domain, capable of initial sensing environmental signal. However, RLPs have short divergent C-terminal regions not associated with conserved kinase domain characteristic of RLKs. The absence of C-terminal phylogenetic relationships between RLK and RLPs precludes the use of sequence comparison algorithms for high-throughput predictions of the RLP family. Thus, we developed the first RLP predictor in plants designated RLPredictiOme. The RLPredictiOme was implemented based on machine learning models associated with Bayesian inference. The ML models were developed in three stages to distinguish RLPs from noRLPs, RLPs from RLKs and classify new subfamilies of RLPs in plants. The evaluation of the models resulted in a high accuracy, precision, sensitivity, and specificity and relatively high probability ranging from 0.79 to 0.99 for RLPs predictions. In addition, a complete validate the of RLPredictiOme was performed with LRR-RLPs of previously characterized Arabidopsis RLPs, Arabidopsis and rice and more than 90% of known RLPs were correctly predicted. In addition to predicting previously characterized RLPs, RLPredictiOme uncovered new RLP subfamilies in the Arabidopsis genome. These include a probable lipid transfer (PLT)-RLP, plastocyanin-like-RLP, ring finger-RLP, glycosyl-hydrolase-RLP, and glycerophosphoryl diester phosphodiesterase (GDPDL)-RLP subfamilies, yet to be characterized. In comparison with the only Arabidopsis GDPDL-RLK, molecular evolution studies confirmed that the ectodomain of GDPDL-RLPs from Arabidopsis might have undergone purifying selection with a predominance of synonymous substitutions. Expression analyses revealed that predicted GDPGL-RLPs display a basal level of expression and respond to developmental and biotic signals. The results of these biological assays substantiate the notion that the members of this subfamily have maintained functional domains during evolution and may play relevant roles in development and plant defense. Therefore, RLPredictiOme can provide new insights into the functional role of surface receptors and their relationships with different biological processes. Keywords: Machine learning. Receptor-like protein. RLPredictiOme.

Orientadora: Prof. Dra. Cristina Aparecida Jark Stern Coorientador: Prof. Dr. Roberto Andreatini Tese (doutorado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Farmacologia. Defesa : Curitiba, 22/05/2020 Inclui referências: p. 88-102 Resumo: A formação e manutenção da memória aversiva tem grande impacto na qualidade de vida dos indivíduos. Muita atenção é dada aos mecanismos subjacentes à reconsolidação da memória, uma vez que podem servir como alvo para o tratamento de transtornos psiquiátricos como o transtorno do estresse pós-traumático (TEPT). Porém, os mecanismos envolvidos na persistência da memória após a evocação e reconsolidação são pouco conhecidos. Sugere-se que a proteína quinase C (PKC) participa da persistência da memória reativada e que o córtex cingulado anterior dorsal (CCAd) em humanos com TEPT está hiperativado, e isso estaria relacionado aos sintomas da doença. Assim, o objetivo desse estudo foi investigar o papel da PKC e da PKM? (isoforma atípica da PKC) no córtex PL, na reconsolidação e na persistência da memória de medo contextual de ratos. O protocolo de condicionamento foi composto principalmente dos dias 1- familiarização; dia 2- condicionamento; dia 3- reativação; dia 4- Teste A1; dia 7- Teste A2 e dia 21- Teste A3. Os animais foram submetidos a esse protocolo e receberam a administração de tamoxifeno (antagonista de receptor de estrógeno e inibidor de PKC), queleritrina (inibidor seletivo de PKC) ou ZIP (inibidor da PKM?) em diferentes tempos após a reativação. O tamoxifeno infundido no córtex PL imediatamente após a reativação da memória de medo reduziu a porcentagem de tempo de congelamento no Teste A1 e Teste A2. Essa mesma droga reduziu o congelamento dos animais no Teste A2, mas não no Teste A1, quando administrado no córtex PL 6 h após a reativação. Esses dados sugerem a participação do córtex PL e da PKC na reconsolidação e persistência da memória reativada. Então, a expressão da proteína de citoesqueleto Arc foi avaliada por western blot em diferentes momentos após a reativação no córtex PL. Houve um aumento significativo da expressão da Arc imediatamente, 90 min, e 6 horas após a reativação. Em seguida, a queleritrina foi administrada no córtex PL imediatamente, 6, 9, 12 ou 18 h após a evocação. Imediatamente após, a queleritrina reduziu o congelamento dos animais quando expostos ao Teste A1. Esse efeito foi duradouro e dependeu da reativação da memória, sugerindo um prejuízo de reconsolidação. Quando administrada 6, 9 ou 12 h após a reativação, a queleritrina reduziu o tempo de congelamento quando os animais foram expostos ao Teste A2 após 7 dias, mas não, no Teste A1, sugerindo um efeito sobre a persistência da memória. Esse efeito dependeu da reativação da memória. Para investigar o papel da PKM?, os animais receberam o ZIP no córtex PL imediatamente, 1 ou 6 h após a reativação da memória. Em nenhum dos momentos escolhidos o ZIP reduziu a porcentagem de congelamento no Teste A1, sugerindo que a PKM? no córtex PL não participa da reconsolidação da memória. No entanto, 1 e 6 h após, o ZIP reduziu o congelamento dos animais quando testados após 7 dias no Teste A2, sugerindo que a PKM? participa da persistência da memória reativada. Ainda, o efeito do ZIP administrado no córtex PL 6 h após a reativação durou até 21 dias, depende da reativação da memória e não depende da exposição prévia ao Teste A1. Os resultados sugerem a participação de PKC's típicas, mas não atípica, na reconsolidação da memória de medo ao contexto no córtex PL e sugere que a isoforma atípica, PKM?, é necessária para a persistência da 10 memória reativada. Por fim, o presente trabalho propõe que a evocação da memória gera mecanismos independentes que levam à reconsolidação e persistência da memória, ampliando assim, a janela temporal para interferências nessa memória. Palavras-chave: PKC; PKM?; Reconsolidação da memória; Persistência da memória; TEPT. Abstract: The formation and maintenance of aversive memories have a great impact on individuals life quality. The mechanisms underlying memory reconsolidation since have received much attention, since they can serve as target for the treatment of psychiatric disorders such as post-traumatic stress disorder (PTSD). However, the mechanisms involved in memory persistence after retrieval and reconsolidation are poorly understood. It is suggested that protein kinase C (PKC) participates in the reactivated memory persistence and that the dorsal anterior cingulate cortex (dACC) in human dACC is overactivated in PTSD, and this would be related to the symptoms of disease. Thus, this study aimed to investigate the role of PKC and PKM? (atypical PKC isoform) in the PL cortex, in contextual fear memory reconsolidation and persistence. The conditioning protocol was composed mainly of days 1 - familiarization; day 2 - conditioning; day 3 - reactivation; day 4 - Test A1; day 7 - Test A2 and day 21 - Test A3. The animals were submitted to this protocol and received the administration of tamoxifen (estrogen receptor antagonist and PKC inhibitor), chelerythrine (selective PKC inhibitor) or ZIP (PKM? inhibitor) at different moments after reactivation. TMX infused into the PL cortex immediately after reactivation of fear memory reduced the percentage of freezing time in Test A1 and Test A2. When administered in the PL cortex 6 h after reactivation, this drug reduced the freezing in Test A2, but not in Test A1. These data suggest the participation of the PL cortex and PKC in fear memory reconsolidation and persistence. Then, the expression of the cytoskeleton protein Arc in the PL cortex was evaluated by Western blot at different times after reactivation. There was a significant increase in Arc expression immediately, 90 min, and 6 hours after reactivation. Thereafter, chelerythrine was administered in the PL cortex immediately, 6, 9, 12 or 18 h after reactivation. Immediately afterward, chelerythrine reduced the freezing time of animals that were exposed to Test A1. This effect was longlasting and depended on memory reactivation, suggesting a reconsolidation disruption. When administered 6, 9 or 12 h after reactivation, chelerythrine reduced the freezing time when animals were exposed to Test A2 after 7 days, but not in Test A1, suggesting an effect on memory persistence. This effect depended on memory reactivation. To investigate the role of PKM?, the animals received ZIP in the PL cortex immediately, 1 or 6 h after memory reactivation. ZIP didn't reduce the freezing percentage in Test A1 in none of the choosen moments, suggesting that PKM? in the PL cortex is not required for memory reconsolidation. However, 1 and 6 h after, ZIP reduced freezing when animals were tested 7 days later in Test A2, suggesting that PKM? participates in the reactivated memory persistence. In addition, the effect of ZIP administered in the PL cortex 6 h after reactivation lasted up to 21 days, depended on memory reactivation and did not depend on previous exposure to Test A1. The results suggest the participation of typical, but not atypical, PKC's in the reconsolidation of contextual fear memory in the PL cortex and suggest that the atypical isoform, PKM?, is necessary for reactivated memory persistence. Finally, the present work proposes that memory retrieval generates independent mechanisms that lead to memory reconsolidation and persistence, thus expanding the time window for interferences in memory.

Orientadores: Susan Elisabeth Domingues Costa Jorge, Maria de Fátima Sonati Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas Resumo: As osfatidilinositol-fosfato-quinases (PIPKins) são enzimas responsáveis pela fosforilação do anel de fosfatidilinositol, cujo produto gerado são sete diferentes tipos de segundos mensageiros, dentre eles o fosfatidilinositol-4,5-bifosfato, responsável pela regulação de diversas atividades celulares. As PIPKins são divididas em três tipos (I, II e III) com diferentes isoformas (a, b e g). Em estudo anterior, encontrou-se que os genes da enzima fosfatidilinositol-4-fosfato-5-quinase-II-alfa (PIPKIIa) e da globina beta estavam diferencialmente expressos em dois pacientes, irmãos, com o mesmo genótipo da Doença da Hemoglobina H, sendo a maior expressão observada no paciente com maiores níveis da hemoglobina anômala. Análises em cultura de células precursoras eritroides humanas (CD34+) reveleram que o aumento de expressão do gene da enzima fosfatidilinositol-4-fosfato-5-quinase-II-alfa (PIPKIIa) durante a diferenciação/maturação celular, coincide com o perfil de expressão dos genes de globinas. O papel das PIPKins no processo hematopoiético, entretanto, tem sido pouco explorado, a despeito da PIPKIIa ser abundantemente encontrada nos eritrócitos. O objetivo deste estudo foi superexpressar o gene ("PIP4K2A") da enzima PIPKIIa através do sistema de reedição genômica CRISPR (Clustered regularly interspaced short palindromic repeats), em cultura de células eritroleucêmicas KU812 e avaliar a expressão das globinas alfa, beta e gama. Foi também avaliada a expressão das demais subfamílias de PIPKins. Para validação, foi utilizado o substrato sintético PI5P ¿ alvo da fosforilação da enzima PIPKIIa. Os resultados após a ativação gênica sugerem que houve tendência ao aumento da expressão de globinas, resultados corroborados a nível proteico, com grau de significância para as globinas beta. Houve, ainda, regulação positiva da enzima PIPKIII. A validação revelou redução na expressão dos genes da fosfatidilinositol-4-fosfato-5-quinase-II-alfa ("PIP4K2A"), fosfatidilinositol-5-fosfato-4-quinase-I-alfa ("PIP5K1A") e fosfatidilinositol-4-fosfato-5-quinase-II-beta ("PIP4K2B") com concomitante aumento da síntese de globinas alfa e gama e diminuição das globinas beta. Estes achados suportam a hipótese de relação entre as enzimas PIPKins e os genes de globinas, particularmente entre a PIPKIIa e o gene da globina beta. Sugerem, ainda, possível envolvimento das outras subfamílias de PIPKins na produção de globinas Abstract: Phosphatidylinositol-phosphate-kinases (PIPKins) are enzymes responsible for phosphorylation of the phosphatidylinositol ring, whose product is generated by seven different types of second messengers, among them the phosphatidylinositol-4.5-biphosphate, responsible for the regulation of several cellular activities. PIPKins are divided into three types (I, II and III) with different isoforms (a, b and g ). In a previous study, it was found that the phosphatidylinositol-4-phosphate-5-kinase-II-alpha (PIPKIIa) and beta globin genes were differentially expressed in two patients, siblings, with the same genotype for Hemoglobin H Disease, whose the largest expression was observed in the patient with higher levels of the abnormal hemoglobin. Culture analyses of human erythroid precursor cells (CD34+) revealed that the increased expression of the phosphatidylinositol-4-phosphate-5-kinase-II-alpha (PIPKIIa) gene during cell differentiation/maturation coincides with the expression profile of globin genes. The role of PIPKins in the hematopoietic process, however, has been little explored, despite PIP4KIIs being abundantly found in red blood cells. The aim of this study was to overexpress the gene (PIP4K2A) of the PIPKIIa enzyme through the CRISPR (Clustered regularly interspaced short palindromic repeats) genomic re-edition system in erythroleukemic cell culture KU812 and to evaluate the expression of globins. The expression of the other subfamilies of PIPKins was also evaluated. For validation, the synthetic substrate PI5P - the target of PIPKIIa enzyme phosphorylation - was used. The results of gene activation suggest that there was a tendency of increasing expression of globins, results corroborated at the protein level, with a degree of significance for beta globins. There was also a positive regulation of PIPKIII enzyme. The validation revealed a reduction in the expression of phosphatidylinositol-4-phosphate-5-kinase-II-alpha (PIP4K2A), phosphatidylinositol-5-phosphate-4-kinase-I-alpha (PIP5K1A) and phosphatidylinositol-4-phosphate-5-kinase-II-beta (PIP4K2B) genes with a concomitant increase in globins alpha and gamma synthesis and decrease in beta globins. These findings support the hypothesis of the relationship between PIPKins enzymes and globin genes, particularly between PIPKIIa and beta globin gene. They also suggest the possible involvement of the other PIPKins subfamilies in globin production Mestrado Patologia Clínica Mestra em Ciências CAPES 88882.434959/2019-01 FAPESP 2014/00984-3

Orientador: Andrea Balan Fernandes Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia Resumo: A tuberculose, é uma doença infecciosa que geralmente afeta os pulmões e é causada pelo bacilo Mycobacterium tuberculosis (Mtb). Sua complexidade, duração do tratamento e o surgimento de cepas resistentes e multirresistentes, destacam a importância dos estudos sobre os mecanismos de resistência da bactéria, alvos para o desenvolvimento de novos medicamentos e inibidores de crescimento e infecção mais eficazes. Transportadores do tipo ABC (do inglês ATP-Binding Cassette) apresentam papel fundamental durante a infecção pois, estão relacionados aos mecanismos de aquisição de nutrientes (importadores) como também à extrusão de drogas (exportadores ou bombas de efluxo). O transportador ABC Rv1747 deMtb não tem função conhecida, mas sua presença é requerida para o crescimento da bactéria in vivo. Estudos prévios sugerem seu papel na reciclagem de compostos hidrofóbicos, como drogas e lipídios. Adicionalmente, sua estrutura apresenta dois domínios do tipo Forkhead Associated (FHA-1 e FHA-2), fosforilados pela proteína PknF, uma serina/treonina quinase, possível reguladora da atividade do transportador. Neste contexto, esta pesquisa teve como objetivo caracterizar estruturalmente o transportador ABC Rv1747, especificamente, a sua interação com a proteína PknF (cPknF) e avaliar os possíveis efeitos estruturais e funcionais da fosforilação nos domínios FHA. Nesse sentido, produzimos o domínio FHA e três mutantes fosfomiméticos (FHA_T152E, e FHA_T210E e FHA_T152E/T210E) e o domínio catalítico de PknF (cPknF) na forma solúvel e estável. As alterações estruturais induzidas no domínio FHA como consequência da emulação da fosforilação, bem como da mistura FHA-cPknF, foram avaliadas por modelagem molecular, docking molecular, dicroísmo circular (CD), fluorimetria de varredura diferencial (DFS), fluorêscencia intrínseca de triptofanos, espalhamento de raios X a baixo ângulo (SAXS), cristalografia de raios-X e calorimetria de titulação isotérmica (ITC). Os resultados mostraram que os mutantes fosfomiméticos não sofrem alterações no conteúdo de estrutura secundária, mas diminuição (quenching) da fluorescência intrínseca dos triptofanos, similarmente ao que ocorreu com cPknF fosforilada (cPknF*). Tais alterações foram corroboradas pelas medidas de SAXS, que evidenciaram compactação do domínio FHA nos estados fosfomiméticos, tanto nos mutantes quanto na presença de cPknF*. As mutações nos domínios FHA também afetaram a afinidade pela cPknF, a qual exibiu maior afinidade pelo domínio selvagem (Kd = 0,4 µM) e menor pelo duplo mutante FHA_T152E/T210E (Kd = 9,4 µM).Finalmente, foi possível determinar a estrutura tridimensional de cPknF em complexo com o inibidor de quinases humanas IKK16 a 2.6 Å de resolução, a qual é caracterizada por arquitetura e topologia das quinases do tipo Hanks com os lobos amino (N) e carboxi (C) terminais, entre os quais forma-se a fenda que contém o sítio ativo. As análises de docking molecular com o ATP e a estrutura com o inibidor mostraram conservação em relação àsoutras quinases. O conjunto dos resultados permitiu-nos estabelecer um modelo para aativação do transportador pela quinase. Neste modelo, cPknF* teria como primeiro alvo a T152 próxima ao domínio FHA-1, desencadeando alterações estruturais que levariam à aproximação dos domínios FHA-1 e FHA-2 e, consequente fosforilação da T210. As mudanças conformacionais nos domínios FHA teriam como consequência, alterações nas ATPases, permeases e ativação do transportador. A estrutura da quinase e sua relevância na ativação do transportador abre perspectivas muito interessantes para o estudo destas proteínas comoalvos para o desenvolvimento de inibidores e papel funcional do transportador. Abstract: Tuberculosis is an infectious disease that usually affects the lungs and is caused by the bacillus Mycobacterium tuberculosis (Mtb). Its complexity, duration of treatment and the emergence of resistant and multiresistant strains, highlight the importance of studies on the bacteria'sresistance mechanisms, targets for the development of new drugs and more effective growth and infection inhibitors. ATP-Binding Cassette (ABC) transporters play a fundamental role during infection because they are related to the mechanisms of nutrient acquisition (importers) as well as to the extrusion of drugs (exporters or efflux pumps). The ABC Rv1747transporter from Mtb has no known function, but its presence is required for the growth of the bacteria in vivo. Previous studies suggest its role in recycling hydrophobic compounds, such as drugs and lipids. Additionally, its structure presents two Forkhead-Associated domains (FHA-1 and FHA-2), phosphorylated by the protein PknF, a serine/threonine kinase, a possible regulator of the activity of the transporter. In this context, this research aimed to structurally characterize the ABC transporter Rv1747, specifically, its interaction with the PknF protein and to evaluate the possible structural and functional effects of phosphorylation in the FHA domains. In this sense, we produced the FHA domain and three phosphomimetic mutants (FHA_T152E, and FHA_T210E and FHA_T152E / T210E) and the catalytic PknF domain (cPknF) in soluble and stable form. The structural changes induced in the FHA domain as a consequence of the emulation of phosphorylation, as well as the FHA-cPknF mixture, were evaluated by molecular modeling, molecular docking, circular dichroism (CD), differential scanning fluorimetry (DFS), intrinsic tryptophan fluoroscopy, scattering of X-rays at low angle (SAXS), X-ray crystallography and isothermal titration calorimetry (ITC). The results showed that the phosphomimetic mutants do not undergo changes in the content of secondary structure but decrease (quenching) of the intrinsic fluorescence of tryptophan, similarly to what occurred with phosphorylated cPknF (cPknF *). Such changes were corroborated by the SAXS measurements, which showed compaction of the FHA domain in phosphomimetic states and an increase in stiffness, both in the mutants and in the presence of cPknF*. Mutations in the FHA domains also affected the affinity for cPknF, which exhibited greater affinity for the wild domain (Kd = 0.4 µM) and lower for the double mutant FHA_T152E / T210E (Kd = 9.4 µM). Finally, it was possible to determine the three-dimensional structure of cPknF in complex with the human kinase inhibitor IKK16 at 2.6 Å resolution, which is characterized by the architecture and topology of Hanks-type kinases with the amino (N) and carboxy (C) lobe termini, including the cleft that contains the active site. The molecular docking analyzes with ATP and the structure with the inhibitor showed conservation in relation to other kinases. The set of results allowed us to establish a model for the activation of the transporter by the kinase. In this model, cPknF* would first target T152 close to the FHA-1 domain, triggering structural changes that would lead to the approximation of the FHA-1 domains of FHA-2 and, consequently, phosphorylation of T210. Conformational changes in the FHA domains would result in changes in ATPases, permeases and transporter activation. The structure of the kinase and its relevance in the activation of the transporter opens very interesting perspectives for the study of these proteins as targets for the development of inhibitors and the functional role of the transporter Doutorado Genética de Microorganismos Doutora em Genética e Biologia Molecular COLCIENCIAS 678

We have previously reported that angiotensin II receptor type 1 (AT1R) contributes to the hypertrophic effects of thyroid hormones (TH) in cardiac cells. Even though evidence indicates crosstalks between TH and AT1R, the underlying mechanisms are poorly understood. Beta-arrestin (ARRB) signaling has been described as noncanonical signal transduction pathway that exerts important effects in the cardiovascular system through G-protein-coupled receptors, as AT1R. Herein, we investigated the contribution of ARRB signaling in TH-induced cardiomyocyte hypertrophy. Primary cardiomyocyte cultures were treated with Triiodothyronine (T3) to induce cell hypertrophy. T3 rapidly activates extracellular signal-regulated kinase 1/2 (ERK1/2) signaling, which was partially inhibited by AT1R blockade. Also, ERK1/2 inhibition attenuated the hypertrophic effects of T3. ARRB2 was upregulated by T3, and small interfering RNA assays revealed the role of ARRB2-but not ARRB1-on ERK1/2 activation and cardiomyocyte hypertrophy. Corroborating these findings, the ARRB2-overexpressed cells showed increased expression of hypertrophic markers, which were attenuated by ERK1/2 inhibition. Immunocytochemistry and immunoprecipitation assays revealed the increased expression of nuclear AT1R after T3 stimulation and the increased interaction of AT1R/ARRB2. The inhibition of endocytosis also attenuated the T3 effects on cardiac cells. Our results evidence the contribution of ARRB2 on ERK1/2 activation and cardiomyocyte hypertrophy induced by T3 via AT1R.

The molecular mechanisms underlying skeletal muscle mitochondrial adaptations induced by aerobic exercise (AE) are not fully understood. We have previously shown that AE induces mitochondrial adaptations in cardiac muscle, mediated by sympathetic stimulation. Since direct sympathetic innervation of neuromuscular junctions influences skeletal muscle homeostasis, we tested the hypothesis that &beta, 2-adrenergic receptor (&beta, 2-AR)-mediated sympathetic activation induces mitochondrial adaptations to AE in skeletal muscle. Male FVB mice were subjected to a single bout of AE on a treadmill (80% Vmax, 60 min) under &beta, 2-AR blockade with ICI 118,551 (ICI) or vehicle, and parameters of mitochondrial function and morphology/dynamics were evaluated. An acute bout of AE significantly increased maximal mitochondrial respiration in tibialis anterior (TA) isolated fiber bundles, which was prevented by &beta, 2-AR blockade. This increased mitochondrial function after AE was accompanied by a change in mitochondrial morphology towards fusion, associated with increased Mfn1 protein expression and activity. &beta, 2-AR blockade fully prevented the increase in Mfn1 activity and reduced mitochondrial elongation. To determine the mechanisms involved in mitochondrial modulation by &beta, 2-AR activation in skeletal muscle during AE, we used C2C12 myotubes, treated with the non-selective &beta, AR agonist isoproterenol (ISO) in the presence of the specific &beta, 2-AR antagonist ICI or during protein kinase A (PKA) and G&alpha, i protein blockade. Our in vitro data show that &beta, AR activation significantly increases mitochondrial respiration in myotubes, and this response was dependent on &beta, 2-AR activation through a G&alpha, s-PKA signaling cascade. In conclusion, we provide evidence for AE-induced &beta, 2-AR activation as a major mechanism leading to alterations in mitochondria function and morphology/dynamics. &beta, 2-AR signaling is thus a key-signaling pathway that contributes to skeletal muscle plasticity in response to exercise.

Aspergillus fumigatus is the most important airborne human pathogenic fungus, causing thousands of deaths per year. Its lethality is due to late and often inaccurate diagnosis and the lack of efficient therapeutics. The failure of efficient prophylaxis and therapy is based on the ability of this pathogen to activate numerous salvage pathways that are capable of overcoming the different drug-derived stresses. A major role in the protection of A. fumigatus is played by melanins. Melanins are cell wall-associated macromolecules classified as virulence determinants. The understanding of the various signaling pathways acting in this organism can be used to elucidate the mechanism beyond melanin production and help to identify ideal drug targets., The pathogenic fungus Aspergillus fumigatus is able to adapt to extremely variable environmental conditions. The A. fumigatus genome contains four genes coding for mitogen-activated protein kinases (MAPKs), which are important regulatory knots involved in diverse cellular responses. From a clinical perspective, MAPK activity has been connected to salvage pathways, which can determine the failure of effective treatment of invasive mycoses using antifungal drugs. Here, we report the characterization of the Saccharomyces cerevisiae Fus3 ortholog in A. fumigatus, designated MpkB. We demonstrate that MpkB is important for conidiation and that its deletion induces a copious increase of dihydroxynaphthalene (DHN)-melanin production. Simultaneous deletion of mpkB and mpkA, the latter related to maintenance of the cell wall integrity, normalized DHN-melanin production. Localization studies revealed that MpkB translocates into the nuclei when A. fumigatus germlings are exposed to caspofungin stress, and this is dependent on the cross-talk interaction with MpkA. Additionally, DHN-melanin formation was also increased after deletion of genes coding for the Gα protein GpaA and for the G protein-coupled receptor GprM. Yeast two-hybrid and coimmunoprecipitation assays confirmed that GpaA and GprM interact, suggesting their role in the MpkB signaling cascade.

Orientador: Ronaldo Aloise Pilli Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Química Resumo: Nos ultimos 30 anos, grandes companhias farmacêuticas vem explorando intensivamente quinases como alvos terapeuticos para o tratamento de câncer. Um grande desafio associado à essa abordagem é atingir a seletividade entre as mais de 500 quinases do "quinoma humano", uma vez que todas passuem o sitio de ligação do ATP (adenosina trifosfato) bem conservado. Além disto, das mais de 500 quinases dependentes de ATP, cerca de 40 são bem exploradas como alvos pois possuem cascatas de sinalização e modo de ação bem caracterizados. Com o intuito de promover a pesquisa de base necessária e focar os esforços da academia e da indústria, o SGC (Strucutral Genomics Consortium) vem promovendo o estudo de quinases sub-estudadas visando o desenvolvimento de novos inibidores de quinases. Especificamente, a quinase sub-estudade STK10/LOK foi escolhida como alvo uma vez que em uma triagem preliminar o composto SB-633825 foi identificado como hit com boa potencia e seletividade. A modificação do fragmento naftílico é de grande interesse, no entanto, a rota sintética original coloca esta fragmento na primeira etapa de cinco subsequentes. Uma nova abordagem sintética curta, modular e divergente foi prosposta e se baseia na síntese do imidazol 2,4(5)-diarilado via reação sequencial da oxidação de Kornblum seguida da condensação imidazolica de Debus-Radziszewski. Essa nova metodologia de síntese de imidazois foi explorada para verificar a abrangência do método. O intermediário chave bromoimidazol é obtido atraves da bromação do imidazol 2,4(5)-diarilado que é então submetido a um acoplamento de Suzuki-Miyaura, o que fornece o imidazol 2,4,5-triarilado. Uma pequena biblioteca de possiveis inibidores foi planejada baseando-se nas difrações de raio-X dos co-cristais da quinase STK10/LOK com o ligante SB-633825. Os análogos obtidos foram avaliados por ensaios de inibição enzimaticos visando atingir os criterios de sonda química Abstract: Over the last 30 years, pharma companies are intensively exploiting kinases as targets for the treatment of cancer. A major challenge associated with this approach is selectivity over more than 500 kinases of the `human kinome¿. Moreover, from more than 500 human ATP-dependent kinases, only around 40 are being explored as targets. In order to bridge the scientific knowledge gap and focus the efforts from both academia and pharma companies, the SGC (Structural Genomics Consortium) have been addressing the study of untargeted kinases. Specifically, the understudied kinase STK10/LOK have been chosen as target since in a preliminary screening the compound SB-633825 has been identified as promising hit compound with good potency and selectivity and can be further optimized. It is most desired to modify the naphthalene moiety but it has not been possible to properly explore these possibilities due to restrictions in the original synthetic route which introduced the naphthalene in the first step of a five-step route. A short, modular and divergent synthetic approach was then envisaged for the lead optimization and it is based in the synthesis of the 2,4(5)-disubstituted imidazole via sequential Kornblum oxidation followed by Debus-Radziszewski imidazole condensation. This new method for the synthesis of substituted imidazoles has been optimized and explored to verify the method abrangency. The key intermediate bromoimidazole is obtained by bromination od the 2,4(5)-disubstituted imidazole which is then subjected to Suzuki-Miyaura cross coupling to yield the 2,4,5-trisubstituted imidazole. A small library of new inhibitors has been designed based on the SGC crystal structures of STK10 with SB-633825. The analogues obtained were evaluated by binding displacement assys aiming to fulfill chemical probe criteria Mestrado Química Orgânica CNPQ 131263/2017-0

Orientador: Katlin Brauer Massirer Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia Resumo: SRPKs são serina arginina proteínas quinases que reconhecem e fosforilam de forma específica repetições de dipeptídeos arginina (R) e serina (S). Seus principais substratos estão envolvidos na regulação de splicing de pre-mRNA, como por exemplo SRSF1. As SR proteínas são necessárias à formação dos componentes do spliceossoma, sendo SRSF1 especificamente associada com a snRNP U1, que se liga à porção 5¿SS do éxon e que leva a formação do pre-spliceossoma. SRPK1 tem sido mostrado super regulado em diversos tipos de canceres, inclusive em tumores do sistema nervoso, como por exemplo gliomas. Já foi demonstrado na literatura que a redução da expressão de SRPK1 em glioblastomas inibe o crescimento, invasão e migração celular. Com objetivo de pesquisar novos alvos de splicing alternativo ligados a SRPK1 foi usada a estratégia de modular geneticamente essa quinase, em linhagens de glioblastoma, tanto por super-expressão quanto por knockdown com short-hairpin. Após diversos tratamentos dos dados por análises de bioinformática foi gerada uma lista de eventos de splicing alternativo relevantes. Dentre os eventos validados por real time PCR destacam-se os genes MBNL1, RBM39, TAU e MYL6, que tiveram alteração regulada pela modulação de SRPK1 Com este trabalho foi possível achar e validar novos eventos de splicing alternativo relacionados com glioblastoma que são regulados por SRPK1. Dentre eles houve o achado de uma isoforma inédita na literatura, da miosina de cadeia leve MYL6. Foi demonstrado com esta pesquisa que este alvo teve alteração no splicing de forma específica pela quinase SRPK1. Também colhemos evidências que indicam que o éxon alternativamente usado por SRPK1 em MYL6 poder estar relacionado à capacidade de migração celular desta miosina. Em paralelo, através de ensaios de fosfoproteômica foi realizada uma busca inicial por novos substratos de fosforilação em células da linhagem HEK sob tratamento com inibidor químico específico para SRPK1 Abstract: SRPKs are serine arginine protein kinases that recognize and phosphorylate repeats of dipeptides arginine (R) and serine (S). The major substrates are involved in the regulation of pre-mRNA splicing, such as SRSF1. The neurological proteins are related to the formation of spliceosome components, SRSF1 being associated with a snRNP U1, which binds to the 5'SS portion of the exon and leads to pre-spliceosome formation. SRPK1 has been shown to be over-regulated in cancers, including nervous system tumors, such as gliomas. It has already been demonstrated in the literature the reduction of SRPK1 expression in glioblastomas can inhibit cell growth, invasion and migration. In order to investigate new splicing targets linked to SRPK1, the strategy of genetically modulating this kinase in glioblastoma lines was used both for superexpression and knockdown. After several data treatments by bioinformatics analyzes, a list of relevant alternative splicing events was generated. Among the events validated by real time PCR, the genes MBNL1, RBM39, TAU and MYL6 were regulated by the modulation of SRPK1. In this present research it was possible to find and validate new alternative splicing events related to glioblastoma that are regulated by SRPK1. Among them it was possible to find an isoform unpublished in the literature of MYL6, a light chain myosin. It has been demonstrated by this research that this target had alternative splicing specifically by the SRPK1 kinase. We also gather evidence indicating that the exon alternatively used by SRPK1 in MYL6 may be related to the cell migration capacity of this myosin. In parallel, an initial search for new phosphorylation substrates was carried out in phosphoprotein assays in HEK cells under treatment with SRPK1-specific chemical inhibitor Doutorado Genética Animal e Evolução Doutora em Genética e Biologia Molecular CAPES 400906/2014-7 CNPQ 141297/2017-4

Innate immune response against Brucella abortus involves activation of Toll-like receptors (TLRs) and NOD-like receptors (NLRs). Among the NLRs involved in the recognition of B. abortus are NLRP3 and AIM2. Here, we demonstrate that B. abortus triggers non-canonical inflammasome activation dependent on caspase-11 and gasdermin-D (GSDMD). Additionally, we identify that Brucella-LPS is the ligand for caspase-11 activation. Interestingly, we determine that B. abortus is able to trigger pyroptosis leading to pore formation and cell death, and this process is dependent on caspase-11 and GSDMD but independently of caspase-1 protease activity and NLRP3. Mice lacking either caspase-11 or GSDMD were significantly more susceptible to infection with B. abortus than caspase-1 knockout or wild-type animals. Additionally, guanylate-binding proteins (GBPs) present in mouse chromosome 3 participate in the recognition of LPS by caspase-11 contributing to non-canonical inflammasome activation as observed by the response of Gbpchr3-/- BMDMs to bacterial stimulation. We further determined by siRNA knockdown that among the GBPs contained in mouse chromosome 3, GBP5 is the most important for Brucella LPS to be recognized by caspase-11 triggering IL-1β secretion and LDH release. Additionally, we observed a reduction in neutrophil, dendritic cell and macrophage influx in spleens of Casp11-/- and Gsdmd-/- compared to wild-type mice, indicating that caspase-11 and GSDMD are implicated in the recruitment and activation of immune cells during Brucella infection. Finally, depletion of neutrophils renders wild-type mice more susceptible to Brucella infection. Taken together, these data suggest that caspase-11/GSDMD-dependent pyroptosis triggered by B. abortus is important to infection restriction in vivo and contributes to immune cell recruitment and activation., Author summary Brucella abortus is the causative agent of brucellosis, a zoonotic disease that affects both humans and cattle. In humans, it is characterized by undulant fever and chronic symptoms as arthritis, endocarditis, and meningitis, while in cattle it causes abortion and infertility. Due to its difficult diagnosis and treatment, it leads to severe economic losses and human suffering. Recently, a novel non-canonical inflammasome pathway was described that involves sensing of bacterial LPS by an intracellular receptor termed caspase-11 and leads to pyroptosis and non-canonical NLRP3 inflammasome activation. Here, we show that B. abortus or its purified LPS is able to activate the non-canonical inflammasome. In this process, activated caspase-11 leads to GSDMD-dependent pyroptosis. Moreover, this pathway was dependent of IFN-induced GBP proteins, mainly GBP5. To analyze the role of caspase-1, caspase-11 and GSDMD in controlling B. abortus infection, we infected knockout (KO) mice for these molecules and we observed that caspase-11 and GSDMD KO animals were more susceptible to infection compared to wild-type animals. Casp11-/- and Gsdmd-/- animals also recruited less immune cells in mouse spleens compared to wild-type animals in response to B. abortus. Thus, caspase-11 and GSDMD are major components of the innate immune system to restrict B. abortus in vivo. This pathway of bacterial sensing by the host immune system is important to future development of drugs and vaccines that may contribute to the control of brucellosis worldwide.

FACEPE As semicarbazonas e tiossemicarbazonas apresentam um amplo perfil de atividades biológicas, dentre as quais as atividades citotóxica e antitumoral se destacam. Nesse contexto, o presente estudo avaliou o potencial anticâncer de quatorze novos derivados aril-semicarbazônicos e onze novos derivados aril-tiossemicarbazônicos. O screening citotóxico foi realizado pelo teste do MTT frente a linhagens de câncer humano e células normais. Os compostos mais promissores foram selecionados para o estudo do mecanismo de morte celular por citometria de fluxo. Os testes realizados analisaram a viabilidade das células, despolarização mitocondrial, ciclo celular, fragmentação do DNA e indução de morte. O teste de Toxicidade Oral Aguda foi realizado segundo o protocolo da OECD 423. A avaliação da atividade antitumoral foi realizada em modelo de tumor sólido de Carcinoma de Ehrlich e a atividade mutagênica foi realizada em sangue periférico de camundongos. Nos resultados apresentados pelos derivados aril-semicarbazônicos, os compostos 3c e 4a se destacaram e foram escolhidos para a continuidade dos testes frente à linhagem HL-60. Esses compostos foram capazes de reduzir a viabilidade das células e induzir a despolarização mitocondrial. Além disso, o derivado 3c inibiu o complexo CDK2/Ciclina A impedindo a progressão da fase S para fase G2/M do ciclo celular. O derivado 4a provocou parada de ciclo na fase G1 e induziu fragmentação do DNA. Os resultados obtidos demonstraram processo apoptótico nas células HL-60 e foi confirmado pelo teste da anexina. Diante dos resultados apresentados pelos derivados aril-tiossemicarbazônicos, o composto TS04 foi escolhido para o estudo do mecanismo de morte celular frente às linhagens K562 e NCI-H292. Na análise morfológica o composto TS04 provocou alterações típicas de apoptose nas duas linhagens. Na linhagem K562, provocou parada de ciclo celular na fase G1 e promoveu a fragmentação do DNA. Na linhagem NCI-H292 na menor concentração, provocou bloqueio do ciclo celular na fase G2/M e na maior concentração, provocou parada em G1 e G2/M. A indução de morte por apoptose foi confirmada pelo teste da anexina nas duas linhagens. Os resultados encontrados in vitro para o composto TS04 foram promissores, o que motivou a continuidade dos estudos em modelo in vivo. No ensaio de Toxicidade Oral Aguda ficou classificado na categoria 3 (50 mg/kg < DL₅₀ < 300 mg/kg). Na avaliação da atividade antitumoral foi capaz de inibir o crescimento tumoral em 82% na maior dose testada (30 mg/kg) e não provocou efeito mutagênico nos animais tratados. Os resultados apresentados demonstram que os derivados aril-semicarbazônicos e aril-tiossemicarbazônicos estudados são agentes com atividade anticâncer promissora e que podem ser eleitos para a continuidade dos testes pré-clínicos necessários visando novos medicamentos para tratamento do câncer. The semicarbazones and thiosemicarbazones present a broad profile of biological activities, among which cytotoxic and antitumoral activities stand out. The present study evaluated the anticancer potential of fourteen new aryl-semicarbazones derivatives and eleven new aryl-thiosemicarbazones derivatives. Cytotoxic screening was performed by the MTT test against human cancer lines and normal cell. The most promising compounds were chosen for study of the mechanism of death by flow cytometry. The tests performed analyzed cell viability, mitochondrial depolarization, cell cycle, DNA fragmentation and induction of cell death. The Acute Oral Toxicity test was performed following the OECD Test Guideline 423. The evaluation of the antitumor activity was performed in a solid tumor model of Ehrlich's Carcinoma and the mutagenic activity was performed in peripheral blood of mice. In the results presented by the aryl-semicarbazones derivatives, the compounds 3c and 4a stand out and were chosen for the continuity of the tests against the HL-60 line. The compounds were able to reduce cell viability and induce mitochondrial depolarization. In addition, derivative 3c inhibited CDK2/Cyclin A complex by preventing the progression of S phase to G2/M phase of the cell cycle. Derivative 4a caused arrest in the G1 phase and induced DNA fragmentation. The results obtained demonstrated an apoptotic process in HL-60 cells and was confirmed by the annexin test. In view of the results presented by the aryl-thiosemicarbazones derivatives, the compound TS04 was chosen for the study of the mechanism of cell death against the lines K562 and NCI-H292. In the morphological analysis the compound TS04 caused typical alterations of apoptotic processes in the two lines. In the K562 line, it caused cell cycle arrest in the G1 phase and promoted DNA fragmentation. In NCI-H292 line in lower concentration, caused cell cycle blockade in G2/M phase and the highest concentration, caused arrest in G1 and G2/M. The induction of death by apoptosis was confirmed by the annexin test in the two lines. The in vitro results for the compound TS04 were promising, which motivated the continuity of the studies in vivo model. In the acute oral toxicity test, it was classified in category 3 (50 mg/kg

The pyruvate dehydrogenase complex (PDH), that converts pyruvate to acetyl-coA, is regulated by pyruvate dehydrogenase kinases (PDHK) and phosphatases (PDHP) that have been shown to be important for morphology, pathogenicity and carbon source utilization in different fungal species. The aim of this study was to investigate the role played by the three PDHKs PkpA, PkpB and PkpC in carbon source utilization in the reference filamentous fungus Aspergillus nidulans, in order to unravel regulatory mechanisms which could prove useful for fungal biotechnological and biomedical applications. PkpA and PkpB were shown to be mitochondrial whereas PkpC localized to the mitochondria in a carbon source-dependent manner. Only PkpA was shown to regulate PDH activity. In the presence of glucose, deletion of pkpA and pkpC resulted in reduced glucose utilization, which affected carbon catabolite repression (CCR) and hydrolytic enzyme secretion, due to de-regulated glycolysis and TCA cycle enzyme activities. Furthermore, PkpC was shown to be required for the correct metabolic utilization of cellulose and acetate. PkpC negatively regulated the activity of the glyoxylate cycle enzyme isocitrate lyase (ICL), required for acetate metabolism. In summary, this study identified PDHKs important for the regulation of central carbon metabolism in the presence of different carbon sources, with effects on the secretion of biotechnologically important enzymes and carbon source-related growth. This work demonstrates how central carbon metabolism can affect a variety of fungal traits and lays a basis for further investigation into these characteristics with potential interest for different applications., We would like to thank the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, grant numbers 2016/07870-9 for GHG and 2014/00789-6 for LJA), the Science Foundation Ireland (SCI, grant number 13/CDA/2142 for OB) for providing financial support., info:eu-repo/semantics/publishedVersion

Nucleoside diphosphate kinase (NDK) is a housekeeping enzyme that plays key roles in nucleotide recycling and homeostasis in trypanosomatids. Moreover, it is secreted by the intracellular parasite Leishmania to modulate the host response. These functions make NDK an attractive target for drug design and for studies aiming at a better understanding of the mechanisms mediating host–pathogen interactions. Here, we report the crystal structures of three mutants of the NDK from Leishmania major (LmNDK) that affects the stability of the hexameric biological assembly including P95S, Δ5Ct (lacking the last five residues) and the double mutant P100S/Δ5Ct. Although P95S and Δ5Ct variants conserve the hexameric structure of the wild-type protein, the double mutant becomes a dimer as shown by in solution studies. Free energy calculation of dimer–dimer interfaces and enzymatic assays indicate that P95S, Δ5Ct and P100S/Δ5Ct mutations progressively decrease the hexamer stability and enzyme activity. These results demonstrate that the mutated regions play a role in protein function through stabilizing the quaternary arrangement.

Orientador: Prof. Dr. Aleksander Roberto Zamprônio Dissertação (mestrado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Farmacologia. Defesa : Curitiba, 20/12/2018 Inclui referências: p. 59-69 Resumo: A síndrome de doença é uma resposta adaptativa caracterizada por um grupo de sinais e sintomas que incluem febre e hiperalgesia generalizada, além de outras alterações comportamentais, autonômicas e endócrinas que permitem que o hospedeiro tente eliminar infecções. Embora a resposta febril seja uma resposta bem estudada, os mecanismos envolvidos na hiperalgesia observada durante a síndrome de doença são pouco compreendidos. Ainda, o dimorfismo sexual na dor é bem conhecido, embora as diferenças relacionadas ao sexo na hiperalgesia observada durante a síndrome de doença não tenham sido ainda bem exploradas. Este estudo teve como objetivo investigar a hiperalgesia durante a síndrome de doença em ratas e possíveis diferenças relacionadas às flutuações dos hormônios sexuais femininos. Foram utilizados ratos Wistar fêmeas, falso-operadas e ovariectomizadas (OVX). A hiperalgesia mecânica e térmica foi avaliada nos mesmos animais, três semanas após a cirurgia de ovariectomia ou sham, utilizando um analgesímetro eletrônico na pata traseira direita e placa aquecida para avaliar comportamento nocifensivo, respectivamente. A hiperalgesia foi induzida por injeção intraperitoneal (i.p.) de lipopolissacarídeo (LPS) ou por prostaglandina E2 (PGE2) intracerebroventricular (i.c.v.) e o limiar mecânico ou o tempo de latência na placa aquecida foi avaliado após a injeção de LPS ou PGE2. Não foram encontradas diferenças nos limiares mecânico e térmico basal entre animais falso-operados, nas fases diestro ou proestro do ciclo estral e nos animais OVX. Da mesma maneira, não se observou diferença entre a hiperalgesia térmica ou mecânica produzida pela injecção i.p. de LPS em ratos OVX e falso-operados. No entanto, enquanto a injeção i.c.v. de PGE2 produziu uma hiperalgesia mecânica rápida (30 min) e intensa em ratas falso-operadas, a resposta em animais OVX se estabeleceu mais lentamente (60-90 min). Resultados semelhantes foram observados na hiperalgesia térmica. As ratas foram então tratadas com o inibidor de proteína quinase A (PKA), H-89 ou com inibidor de proteína trocadora ativada por AMPc (Epac), ESI-09 ou os respectivos veículos, por via i.c.v. 15 min antes da injeção de PGE2 ou LPS. O tratamento com H-89 reduziu a hiperalgesia térmica e mecânica induzida por PGE2 e LPS em animais OVX, embora não tenha sido observado qualquer efeito em animais falso-operados. De modo oposto, o tratamento de animais falsooperados com ESI-09, reduziu tanto a hiperalgesia térmica quanto mecânica induzida por PGE2 e LPS, enquanto nenhum efeito foi observado em animais OVX. Estes resultados sugerem que a hiperalgesia induzida por LPS, observada durante a síndrome de doença tem diferentes mecanismos de sinalização, particularmente relacionados à PGE2, em animais falso-operados e OVX. Enquanto em fêmeas com ciclo estral preservado há uma resposta mediada pela ativação da via de sinalização AMPc-Epac, a hiperalgesia induzida pela injeção de LPS ou PGE2 em ratas OVX ativa a via de sinalização de AMPc-PKA. O conhecimento destas vias de sinalização em fêmeas em diferentes fases hormonais pode contribuir para terapêuticas diferenciadas em cada fase. Palavras-chave: hiperalgesia, prostaglandina, proteína quinase A, Epac. Abstract: The sickness syndrome is an adaptive response characterized by a group of signs and symptoms that include fever and generalized hyperalgesia, as well as other behavioral, autonomic and endocrine changes that allow the host to eliminate infections. Although the febrile response is a well-studied response, the mechanisms involved in the hyperalgesia observed during the sickness syndrome are poorly understood. Furthermore, sexual dimorphism in pain is well known, although the sex-related differences in hyperalgesia observed during the sickness syndrome have not yet been well explored. This study aims to investigate hyperalgesia during the sickness syndrome in rats and possible differences related to fluctuations in female sex hormones. Female, shamoperated and ovariectomized Wistar rats (OVX) were used. The mechanical and thermal hyperalgesia was evaluated in the same animals using an electronic analgesimeter in the right hind paw and hot plate apparatus to evaluate nocifensive behavior, respectively. Hyperalgesia was induced by intraperitoneal (i.p.) injection of lipopolysaccharide (LPS) or by intracerebroventricular (i.c.v.) prostaglandin E2 (PGE2) and the mechanical threshold or the time of latency on the hot plate was evaluated after the injection of LPS or PGE2. No differences were found in baseline mechanical and thermal thresholds between shamoperated animals, in the diestrus or proestrus phases of the estrous cycle and in OVX animals. Likewise, no difference was observed between the thermal or mechanical hyperalgesia produced by the i.p. of LPS in OVX and sham-operated rats. However, while the i.c.v. injection of PGE2 produced rapid (30 min) and intense mechanical hyperalgesia in sham-operated rats, the response in OVX animals was established later (60-90 min). Similar results were observed in thermal hyperalgesia. The rats were then treated with the protein kinase A (PKA) inhibitor, H-89 or with cAMP activated exchange protein inhibitor (Epac), ESI-09 or the respective vehicles, by i.c.v. route 15 min before injection of PGE2 or LPS. Treatment with the PKA inhibitor, H-89 reduced thermal and mechanical hyperalgesia induced by PGE2 and LPS in OVX animals, although no effect was observed in sham-operated animals. Oppositely, the treatment of sham-operated animals with the Epac ESI-09 inhibitor reduced both thermal and mechanical hyperalgesia induced by PGE2 and LPS, whereas no effect was observed in OVX animals. These results suggest that the LPS-induced hyperalgesia observed during the sickness syndrome has different signaling mechanisms, particularly related to PGE2, in sham-operated and OVX animals. While in rats with preserved estrous cycle there is a response mediated by the activation of the cAMP-Epac signaling pathway, the hyperalgesia induced by the injection of LPS or PGE2 in OVX rats activates the cAMP-PKA signaling pathway. The knowledge of these signaling pathways in rats in different hormonal phases can contribute to differentiated therapies in each phase. Key words: sickness syndrome hyperalgesia, prostaglandin, protein kinase A, Epac

Base teórica: Normalmente, a ativação dos receptores de tropomiosina relacionados à quinase (TRKs) pelas neurotrofinas (NTs) estimulam as vias intracelulares envolvidas na sobrevida e na proliferação celular. A desregulação da sinalização NT/TRK pode afetar o prognóstico de várias neoplasias. Os dados sobre a expressão de NTs e TRKs no melanoma cutâneo são limitados e não está claro se as vias de sinalização NT/TRK estão envolvidas na origem e progressão dessa neoplasia. Objetivo: Nós examinamos se a expressão de NT/TRK difere em relação aos estágios e subtipos de melanoma cutâneo, e se está associada com o prognóstico e a sobrevida dos pacientes com essa neoplasia. Métodos: Foi realizado um estudo transversal, no qual foi analisado através de imunohistoquímica, as expressões de TrkB e BDNF, além da expressão de outro TRK, o TrkA, e de seu coefetor, o NGF, em 154 amostras de melanoma. Nós investigamos as associações da expressão de NT/TRK com diferentes fatores prognósticos para melanoma, sobrevida livre de recidiva (SLR) e sobrevida global (SG). Também foram avaliadas 48 amostras de pele normal e lesões cutâneas pigmentares benignas. Resultados: Das 154 amostras de melanoma, 81 (58,3%) foram imunopositivas para TrkB, 113 (81,3%) foram imunopositivas para BDNF, 77 (55,4%) foram imunopositivas para TrkA, e 104 (75,4%) foram imunopositivas para NGF. Encontramos forte associação entre a expressão de NT/TRK e vários fatores prognósticos, incluindo estágio TNM (p

Ewing's sarcoma (EWS) is a highly aggressive bone cancer that affects children and adolescents. Despite advances in multimodal management, 5-year event-free survival rates for patients presenting with metastases at diagnosis remain at 25%. As key regulators of actin organization, the Rho-associated coiled-coil containing protein kinases, ROCK1 and ROCK2, have been associated with cancer dissemination and poorer prognosis. Recently, in vitro data indicating ROCK2 as a molecular target for the treatment of EWS has been presented. Nonetheless, a deeper exploration of the contribution of this kinase dysregulation in EWS is still necessary. In this regard, the present study aimed to evaluate the expression of ROCK1 and ROCK2 in 23 pediatric tumor samples and to verify the prospect of using their pharmacological inhibition through functional assays. Our results showed positive immunostaining for ROCK1 and ROCK2 in the majority samples (75 and 65%, respectively). A significantly increased risk of incomplete remission in patients with positive immunostaining for ROCK2 was found (P=0.026), though no correlations with other prognostic features (huvos classification, FLI1/EWS status, relapse, metastasis or death) were observed. Associations with survival were merely suggestive. Apparent protein expression of both kinases was also found in EWS cell lines (SK-ES-1 and RD-ES). Treatments with selective ROCK inhibitors did not alter cell viability or migration in vitro. However, a significant increase in invasion was observed after treatment with SR3677 (ROCK2 inhibitor) and hydroxyfasudil (pan-inhibitor). Consequently, even though the majority of EWS samples included in our study showed positivity for ROCK1 and ROCK2, the lack of significant associations with prognosis and absence of appropriate responses to their inhibition in vitro does not support their prospective use as therapeutic targets for the treatment of this metastatic tumor. Larger cohort studies might provide more evidence on whether there is a specific role of ROCK kinases in EWS physiopathology.

Aim Most studies developed to investigate the effects of glucocorticoids chronic treatment on white adipose tissue uses high doses of these hormones. This study analyzes some effects of a chronic, continuous and steady infusion of low-dose hydrocortisone and the relationship with lipid accumulation in white adipose depots in rats. Main methods Nineteen male Wistar rats were divided into control (CON) and cortisol (CORT) groups. Along six weeks CORT group received continuous infusion of 0.6 mg/kg/day of hydrocortisone, while CON group received saline. After euthanasia, subcutaneous and visceral (retroperitoneal and mesenteric) fat pads were excised, weighted and analyzed for: lipogenic enzymes activity; molecular changes of 11-hydroxysteroid dehydrogenase type 1 (11βHSD1) enzyme; enzymes involved in lipid uptake, incorporation, and metabolism and in fatty acids esterification. Besides, morphometric cell analysis was performed. Key findings CORT group showed increased triglycerides, changes in lipoprotein profile and 26,8% increment in central subcutaneous (SC) mass, while visceral fat pads masses remained unchanged. Adipocytes from SC, only, presented increased fatty acid synthase, ATP-citrate lyase and glucose-6-phosphate dehydrogenase activity, in addition to reduced AMP-activated protein kinase and 11βHSD1 enzymes content. Significance Chronic low-dose hydrocortisone treatment consequences seem to be different from those commonly seen in long term hypercortisolism. While high doses promote lipid accumulation in visceral depots, a low dose showed an increase in central SC depot only. This appears to involve an increment in lipid storage and in de novo lipogenesis enzymes activity.

Mastocytosis are myeloproliferative neoplasms commonly related to gain-of-function mutations involving the tyrosine kinase domain of KIT. We herein report a case of familial systemic mastocytosis with the rare KIT K509I germ line mutation affecting two family members: mother and daughter. In vitro treatment with imatinib, dasatinib and PKC412 reduced cell viability of primary mast cells harboring KIT K509I mutation. However, imatinib was more effective in inducing apoptosis of neoplastic mast cells. Both patients with familial systemic mastocytosis had remarkable hematological and skin improvement after three months of imatinib treatment, suggesting that it may be an effective front line therapy for patients harboring KIT K509I mutation.

Orientador: Cristina Elisa Alvarez Martinez Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia Resumo: Xanthomonas citri subsp. citri (Xac) é uma bactéria Gram-negativa responsável pela doença do cancro cítrico em uma grande variedade de plantas cítricas, causando grandes perdas econômicas. Xac sobrevive como epífita nas superfícies foliares e causa a doença após a penetração e crescimento dentro do parênquima mesofílico de plantas suscetíveis. Como uma bactéria associada a plantas, Xac precisa se adaptar às condições ambientais em constante mudança. Fatores sigma alternativos da família extracitoplásmica (ECF) estão envolvidos na regulação da expressão gênica em resposta ao estresse ambiental. O genoma de Xac306 codifica 9 fatores sigmas ECF, o que provavelmente reflete seu estilo de vida e sugere um papel importante desta família de reguladores transcricionais na adaptação de Xac. O objetivo deste estudo é caracterizar a função de um fator sigma ECF encontrado no genoma de Xac, denominado sigF. Para isso, foram obtidas duas linhagens com níveis alterados de sigF: uma linhagem mutante (?sigF) por deleção em fase do gene e uma linhagem com cópia extra do gene em vetor multicópia, o que promove a super-expressão deste sigma. Ensaios de virulência em hospedeiro suscetível demonstraram que sigF não é necessário para a virulência em Xac. A sensibilidade da linhagem ?sigF a diferentes tipos de estresse ambiental também foi analisada, incluindo estresse oxidativo, por radiação UV, deficiência de ferro e tratamento com o agente quelante de metais ácido fítico, não sendo observadas diferenças na viabilidade quando comparada com a linhagem selvagem. Além disso, as linhagens com níveis alterados de sigF não apresentaram alterações na capacidade de formar biofilmes e aderir às folhas da planta. Curiosamente, observamos que SigF é essencial para a sobrevivência de Xac a predação pela ameba de vida livre Dictyostelium discoideum. Resultados de ensaios de RT-PCR quantitativo e RNA-Seq demonstram que a super-expressão de uma versão que mimetiza um estado fosforilado de SigF causa indução da expressão de genes que codificam componentes de um sistema de secreção do tipo VI (SSVI) de Xac,que também tem papel essencial na sobrevivência a D.discoideum. Ensaios preliminares de fosforilação in vitro pela incubação de SigF purificada com a proteína quinase PknB, codificada por um gene localizado em provável operon com sigF, reforçam o modelo proposto, no qual SigF é regulado em nível pós-traducional por fosforilação. Além disto, nossos resultados demonstram que SigF regula positivamente a expressão do SSVI de Xac Abstract: Xanthomonas citri subsp. citri (Xac) is a Gram-negative bacterium responsible for citrus canker disease in a wide variety of citrus plants, causing major economic losses. Xac survives as epiphyte on leaf surfaces and causes the disease after penetration and growth inside mesophyll parenchyma of susceptible plants. As a plant-associated bacterium, Xac needs to adapt to constantly changing environmental conditions. Alternative sigma factors of the extracytoplasmic family (ECF) are involved in the regulation of gene expression in response to environmental stress. The genome of Xac strain 306 encodes 9 sigma factors of the ECF family, which probably reflects its lifestyle and suggests an important role of this family of transcriptional regulators in Xac adaptation. The aim of this study is to characterize the function of one ECF sigma factor found in Xac genome, named sigF. For that, we have constructed a sigF mutant strain (?sigF) by in frame deletion of the gene and an overexpression strain, which contains an extra copy of sigF in a multicopy plasmid. Results from virulence assays in a susceptible host have shown that sigF is not required for Xac virulence. We've also tested sensitivity of the ?sigF strain to distinct types of environmental stress, including oxidative stress, UV radiation, iron deprivation and treatment with the metal chelating agent phytic acid and no difference in viability was observed when compared to the wild type strain, indicating the SigF is not required under these conditions. In addition, we have not observed differences in biofilm formation and adhesion to plant leaves caused by deletion or overexpression of sigF. Interestingly, our results show that SigF is required for Xac survival to predation by the amoeba Dictiostelium discoideum. In addition, results from qRT-PCR analysis and RNA-seq experiments have shown that overexpression of a SigF phospho-mimicking mutant version promotes induction of genes from a Xac type VI secretion system required for survival to D. discoideum. Prelimary in vitro phosphorylation assays using purified SigF and PknB Ser/thr kinase encoded in the same operon as sigF corroborates with a model in which SigF is post-translational regulated by phosphorylation. Our results also suggest that SigF positively regulates Xac SSVI Mestrado Microbiologia Mestra em Genética e Biologia Molecular FAPESP 2014/19720-6

The FBXO25 mediates degradation of ELK-1 and thus inhibits transcriptional activation of immediate early genes (iEG). Here we show that FBXO25 regulates yet another node of this signaling pathway, by decreasing MAPK/ERK activity. We show that induction of FBXO25 reduced ERK1/2 phosphorylation independently of MEK1/2. Accordingly, in HAP1 FBXO25 knockout cells (FBXO25KO), we observed that upon PMA treatment ERK1/2 was more active than in parental cells. An increase in cell proliferation under receptor mediated activation of the ERK signaling pathway in FBXO25KO cells was also observed. Taken together we show that FBXO25 functions as a negative regulator of MAPK signaling though the reduction of ERK1/2 activation.

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas, Programa de Pós-Graduação em Biotecnologia e Biociências, Florianópolis, 2016. O Trypanosoma rangeli (Kinetoplastida; Trypanosomatidae) é um parasito hemoflagelado que compartilha reservatórios e vetores com o Trypanosoma cruzi, sendo capaz de infectar mamíferos silvestres e domésticos, assim como o ser humano. Dentre as proteínas envolvidas na regulação do metabolismo energético do T. rangeli, avaliamos neste trabalho a expressão e a atividade enzimática da arginina quinase (AK), enzima que possui atividade de fosfotransferase e cataliza a interconversão entre a fosfoarginina e ADP, assim como arginina e ATP, sendo essencial para diferentes processos celulares em tripanosomatídeos, como por exemplo, infecção, multiplicação celular e diferenciação celular. O crescimento dos parasitos em meio LIT foi acompanhado durante nove dias, sendo observada uma menor expressão da AK somente no dia 1, aumentando no dia 2 e mantendo-se estável nos dias subsequentes. A atividade enzimática da AK aumentou progressivamente até o 5º dia de cultivo, decaindo gradativamente nos dias subsequentes. Durante a diferenciação celular de formas epimastigotas para tripomastigotas realizada ao longo de oito dias em meio DMEM, a maior expressão da AK foi detectada nas formas epimastigotas (dia 0) e a maior atividade enzimática nas formas de transição (dia 4). Durante a diferenciação de tripomastigotas em epimastigotas foi observada uma menor expressão da AK somente nos dias 3 e 4, sendo aumentada no dia 5 e mantendo-se estável nos dias subsequentes. A atividade enzimática da AK se manteve constante nos tripomastigotas recém isolados e no primeiro dia de cultivo, aumentando no segundo dia e se mantendo constante até o sexto dia, quando teve um aumento da atividade, que novamente se manteve constante até o nono dia e então decaindo nos últimos dias. Embora estudo prévio tenha demonstrado uma localização flagelar da TrAK a maior parte da atividade da AK foi encontrada na fração citosólica após purificação do citoesqueleto e flagelo. Na comparação entre diferentes espécies e cepas de tripanosomatídeos, a cepa SC 58 de T. rangeli apresentou uma atividade quase duas vezes maior do que a cepa Choachí. A atividade nas cepas de T. cruzi foi praticamente constante quando comparadas entre si e também com a cepa Choachí. Através destes resultados é possível inferir que a atividade da AK parece estar relacionada à multiplicação celular e a adaptação à condições metabólicas adversas (ex. diferenciação celular), sendo influenciada pela demanda energética do parasito. Abstract : Trypanosoma rangeli (Kinetoplastida; Trypanosomatidae) is a hemoflagellate parasite that infects wild and domestic mammals, as well as humans, sharing reservoirs and vectors with Trypanosoma cruzi. In this work we evaluate the expression and the enzymatic activity of the T. rangeli arginine kinase (AK), an enzyme with phosphotransferase activity that catalyzes the interconversion between phosphoarginine and ATP¸ as the arginine and ATP. AK is essential in trypanosomatids for diferent cellular processes, like infection, cellular multiplication and cellular diferentiation. The AK expression was monitored during the T. rangeli epimastigotes growth in LIT medium for nine days, revealing an increasing expression of AK during the first couple of days and then stabilizing. The enzymatic AK activity have increased up to the fifth day of in vitro culture and then gradually decreased during the subsequent days. During the cellular differentiation in vitro, higher AK expression levels were observed for the epimastigote forms (day 0) but the higher enzymatic activity was detected for the transition forms (day 4). During the in vitro differentiation of bloodstream forms to replicative forms the AK expression have increased up to the fifth day and remained stable up to the ninth day. During this process the AK enzymatic activity revealed a slight increase up to the second day and the stabilised up to the sixth day when a second peak of activity was observed followed by decay up to the ninth day. Although previous studies have demonstrated a flagellar localization for the TrAK, higher AK activity was found on the cytosolic fraction. The AK activity revealed to be distinct among T. rangeli strains, being two times higher for the SC58 strain when compared to the Choachí strain, which revealed similar AK activity as observed for T. cruzi. We conclude that AK activity might be related to the T. rangeli cellular multiplication, differentiation and metabolic adaptation to adverse conditions, being influenced by the parasite?s energetic demands.

Orientador: Jörg Kobarg Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia Resumo: NEKs (NIMA-related kinases) são um grupo de proteínas cinases que compartilham alta identidade entre suas sequências de aminoácidos na região do domínio cinase com a proteína NIMA (Never in mitosis gene A) e estão envolvidas na regulação do ciclo celular. Dentre as NEKs já estudadas, funções frequentemente encontradas incluem: funcionamento dos cílios, progressão do ciclo celular ¿ principalmente durante os "checkpoints" ¿ e a resposta a danos no DNA. Até o presente momento, 11 NEKs humanas foram descobertas no genoma, com os seus ortólogos presentes em camundongos. NEK3 é uma serina/treonina cinase de 506 aminoácidos, presente no citoplasma e no núcleo, com o seu gene humano localizado no cromossomo 13, região q14.2. Em função das correlações entre NEK3 e diversas patologias e processos, tais como o câncer de mama, degeneração axonal, e por seu gene estar localizado em uma região de freqüentes mutações, tornam-se necessários maiores esclarecimentos de como ocorre a sua participação, através de estudos funcionais, moleculares e estruturais, buscando mapear a rede de interações na qual ela está envolvida. Utilizando o Matchmaker Gold Yeast Two-Hybrid system, uma triagem para a busca de parceiros de interação foi realizada e 65 clones encontrados, que codificam 27 proteínas diferentes foram identificados. Os interatores identificados estão envolvidos em diversos processos celulares tais como sumoilação, ubiquitinação, regulação da transcrição, reparo de DNA, processamento de RNA e a regulação da proliferação celular, invasividade e metástase. Alguns parceiros de interação da NEK3 estão localizados no núcleo e membrana plasmática, mas a maioria está presente no citoplasma. Um dos interatores citoplasmáticos, RhoGDI2, é um regulador das RhoGTPases e inibe a ativação de ambas Rac1 e RhoA. Em nosso ensaio de pull down, a superexpressão de NEK3 aumenta os níveis de Rac1-GTP enquanto a superexpressão concomitante com RhoGDI2 reduz Rac1-GTP a níveis não detectáveis. Nossos dados sugerem que a interação de NEK3 com RhoGDI2 é um importante elemento regulador da sinalização de Rac1. Ademais, o papel da NEK3 no ciclo celular também foi investigado em células depletadas para NEK3 que apresentaram um atraso da citocinese causada por pontes citoplasmáticas e de DNA, que corrobora com o papel de outros interatores de NEK3 e confirma a importância dos resultados apresentados neste trabalho Abstract: NEKs (NIMA-related kinases) are a group of kinase proteins that share high identity of their amino acid sequences with NIMA (Never in mitosis gene A) and are involved in cell cycle regulation. Among mammalian NEKs already studied, frequently found functions include: cilia function, cell cycle progression - especially during checkpoints- and the DNA damage response. Eleven NEKs have been discovered, with all their orthologous present in mice. NEK3 is a 506-amino acid serine/threonine kinase, present in the cytoplasm and nucleus, and its gene is located at chromosome 13, bands q14.2 According to the association of NEK3 to several disease processes, including breast cancer and axonal degeneration, as well frequent mutations in its chromosomal site, and since it has characteristics different from other more studied NEKs, it becomes necessary to further clarify its role, through functional, molecular and structural studies, aiming to map the interaction network in which it is involved. Using the Matchmaker Gold Yeast Two-Hybrid system, a screening for interaction partners was performed and 65 clones were obtained, which cDNAs encode 27 different proteins. The identified candidate interacting proteins are functionally involved in sumoylation, ubiquitination, transcriptional regulation, DNA repair, RNA processing, and the regulation of cell proliferation, invasiveness and metastasis. Some interaction partners for NEK3 are located in the nucleus and plasma membrane but most of them localize to the cytoplasm. One of the cytoplasmatic interactors for NEK3, RhoGDI2, is a regulator of RhoGTPases and inhibits Rac1 and RhoA activation levels. In our pull down assay, NEK3 overexpression increases Rac1-GTP while concomitant overexpression of RhoGDI2 reduces it to non-detectable levels. Our data suggest that NEK3 interaction with RhoGDI2 are important new regulatory elements in Rac1 signaling pathways. Moreover, the role for NEK3 in cell cycle was also investigated. ShRNA constructs for NEK3 were responsible for cytokinesis delay induced by cytoplasmic and DNA bridges, which corroborate with some NEK3 interactors role and supports the implications of the results from this work Doutorado Bioquímica Doutora em Biologia Funcional e Molecular FAPESP 2011/06730-5

Made available in DSpace on 2021-06-25T10:32:46Z (GMT). No. of bitstreams: 0 Previous issue date: 2015-02-01 The myeloid differentiation factor 88 (MyD88) plays a pivotal role in Toll-like receptor (TLR)- and interleukin-1 receptor (IL-1R)-induced osteoclastogenesis. We examined the role of MyD88 on p38 mitogen-activated protein kinase (MAPK) and nuclear factor kappa-light-chain-enhancer of activated B cell (NF-κB) activation and nucleotide-binding oligomerization domain (Nod) induction by lipopolysaccharide (LPS) and IL-1 beta, and their effect on receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG) production in bone marrow stromal cell (BMSC). RANKL, Nod1, Nod2, NF-κB, and p38 protein levels were determined by Western blot. Nod2 was stimulated with muramyl dipeptide (MDP) prior to TLR4 stimulation with LPS. MyD88 deficiency markedly inhibited RANKL expression after LPS stimulation and increased OPG messenger RNA (mRNA) production. Also, MyD88 was necessary for NF-κB and p38 MAPK activation. MDP alone did not induce RANKL and OPG expressions; however, when combined with LPS, their expressions were significantly increased (p < 0.05). Our results support that MyD88 signaling has a pivotal role in osteoclastogenesis thought NF-κB and p38 activation. Nod2 and especially Nod1 levels were influenced by MyD88. School of Dentistry Federal University at Pelotas (UFPel) Department of Diagnosis and Surgery School of Dentistry at Araraquara State University of Sao Paulo (UNESP), Rua Humaitá, 1680, Centro Department of Cell Biology and Microbial Pathogenesis School of Medicine USP Department of Biochemistry and Immunology School of Medicine USP Department of Diagnosis and Surgery School of Dentistry at Araraquara State University of Sao Paulo (UNESP), Rua Humaitá, 1680, Centro

Orientador: Jörg Kobarg Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia Resumo: Membros da família das Neks são cruciais para o início da mitose em eucariotos. Têm sido funcionalmente atribuídas a todas as 11 Neks humanas uma das três principais funções estabelecidas para esta família em mamíferos: (1) centríolos / divisão celular; (2) funções no cílio primário / ciliopatias; e (3) resposta à danos no DNA (DDR). No artigo de revisão (artigo I), relatamos uma análise detalhada atual sobre cada uma das 11 Neks. A hipótese é que as Neks possam conectar elementos reguladores que permitem o refinamento e a sincronização de eventos celulares. Dentre os membros desta família, Nek5 é a cinase mais negligenciada. Ensaios de duplo híbrido em leveduras (Y2H) foram realizados para identificar e caracterizar parceiros de interação Nek5; e proteínas mitocondriais foram observadas (artigo II). Ensaios de apoptose mostraram efeitos protetores na morte celular após tratamento com tapsigargina (2 ?M) de células HEK293T que superexpressam a hNek5, bem como a diminuição na formação de Espécies Reativas de Oxigênio após 4 horas de tratamento. A atividade da cadeia respiratória mitocondrial estava diminuída após superexpressão de hNek5, especialmente nas etapas de transferência de elétrons do TMPD para o citocromo c e no complexo II. O Y2H permitiu também a identificação da poliglutamilase de proteínas TTLL4 como um parceiro de Nek5 (artigo III). Células silenciadas para a Nek5, assim como células que expressam a versão "kinase dead" de Nek5, apresentaram por western blot e ensaio in vitro de atividade poliglutamilação um aumento na poliglutamilação de proteínas após transfecção com TTLL4. Em conclusão, nossos dados sugerem pela primeira vez a localização mitocondrial e a participação de Nek5 na morte celular e no processo poliglutamilação diminuindo a atividade de TTLL4 através de sua fosforilação inibitória Abstract: Members of the Nek Family are crucial for the initiation of mitosis eukaryotes. All 11 human Neks have been functionally assigned to one of the three core functions established for this family in mammals: (1) centrioles/mitosis; (2) primary ciliary function/ciliopathies; and (3) DNA damage response (DDR). In the core section of the review (article I), we report the current detailed functional knowledge on each of the 11 Neks. We raise the hypothesis that Neks may be the connecting regulatory elements that allow the cell to fine tune and synchronize cellular events. Nek5 is the most neglected among members of the Nek kinases family. A yeast two-hybrid (Y2H) screen was performed to identify and characterize Nek5 interaction partners and mitochondrial proteins were retrieved (article 2). Apoptosis assay showed protective effects of hNek5 over-expression from Hek293-T¿s cell death after thapsigargin treatment (2 ?M) as well as an increase in ROS formation after 4 hours of treatment. Mitochondrial respiratory chain activity was found decreased upon hNek5 over-expression especially at the electrons transfer steps from TMPD to cytochrome c and at the complex II. The yeast two-hybrid allowed also the identification o TTLL4 as a Nek5 partner (article 3). Nek5 silenced cells as well as cells expressing a "kinase dead" version of Nek5, displayed an increase in polyglutamylation of proteins after TTLL4 transfection by western blot and in vitro polyglutamylation activity assay. In conclusion, our data suggest for the first time mitochondrial localization and functions for Nek5 and its participation in cell death and cell respiration regulation. This work also showed the function of Nek5 in the polyglutamylation process decreasing the role of TTLL4 through inhibitory phosphorylation by Nek5 Doutorado Bioquímica Doutora em Biologia Funcional e Molecular

Hematopoietic stem and progenitor cells (HSPCs) are predominantly quiescent in adults, but proliferate in response to bone marrow (BM) injury. Here, we show that deletion of Ca2+/calmodulin (CaM)-dependent protein kinase kinase 2 (CaMKK2) promotes HSPC regeneration and hematopoietic recovery following radiation injury. Using Camkk2-enhanced green fluorescent protein (EGFP) reporter mice, we found that Camkk2 expression is developmentally regulated in HSPC. Deletion of Camkk2 in HSPC results in a significant downregulation of genes affiliated with the quiescent signature. Accordingly, HSPC from Camkk2 null mice have a high proliferative capability when stimulated in vitro in the presence of BM-derived endothelial cells. In addition, Camkk2 null mice are more resistant to radiation injury and show accelerated hematopoietic recovery, enhanced HSPC regeneration and ultimately a prolonged survival following sublethal or lethal total body irradiation. Mechanistically, we propose that CaMKK2 regulates the HSPC response to hematopoietic damage by coupling radiation signaling to activation of the anti-proliferative AMP-activated protein kinase. Finally, we demonstrated that systemic administration of the small molecule CaMKK2 inhibitor, STO-609, to irradiated mice enhanced HSPC recovery and improved survival. These findings identify CaMKK2 as an important regulator of HSPC regeneration and demonstrate CaMKK2 inhibition is a novel approach to promoting hematopoietic recovery after BM injury.