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Genome-wide association studies (GWAS) and exome sequencing have identified single nucleotide polymorphisms (SNPs) associated with genetic generalized epilepsy (GGE). However, these studies were based on European and Chinese populations, and the SNPs found could not be useful for polygenetic risk assessment in underrepresented populations as admixed Americans. Therefore, the purpose of this study is to evaluate whether these candidate SNPs for susceptibility to GGE are also found to be associated with the phenotype in a Brazilian GGE cohort.

According to the World Health Organization, there are more than 50 million individuals with epilepsy worldwide; approximately 10% of these patients have juvenile myoclonic epilepsy (JME). JME is considered one of the most common forms of the genetic generalized epilepsies. Currently, it is believed that most patients with JME have a complex inheritance with different genetic and non-genetic factors involved. Therefore, we hypothesize that there may be epigenetic factors that influence the pattern of DNA methylation, which could be involved in the predisposition of JME. To test our hypothesis, we will analyze the pattern of DNA methylation in patients with JME and compared it to control individuals. We will use the whole-genome bisulfite sequencing (WGBS) technique. This is the first study accessing differentially methylated regions throughout the genome in JME patients. Our results may lead to a better understanding of the different etiological factors which may be predisposing to JME.

Ischemic stroke (IS) is one of the most common causes of mortality worldwide and represents 80% of all stroke cases. Previous studies have shown that some metabolites can be markers of oxidative injury to vascular endothelial cells and neuromodulation after brain ischemia. Biomarkers are small molecules that are dysregulated during diseases and that can be used to improve diagnosis and to determine prognosis, including responses to treatment. This study aims to identify if there are specific metabolites in the plasma of patients with IS in the acute and chronic stages. Currently, we have evaluated plasma samples of 21 individuals, using proton Nuclear Magnetic Resonance (1H NMR). The samples were divided into 4 groups: 6 samples from patients in the acute phase of IS (less than 24 h after the event), 6 samples from patients in the chronic phase of IS (more than 3 months after the acure event), 5 samples from healthy and agematched individuals and 4 samples from asymptomatic patients with severe internal carotid artery stenosis. Samples were evaluated using 1 H NMR spectra, recorded under the same conditions in a 600 MHz spectrometer.

RNA interference (RNAi) is a powerful post-transcriptional gene silencing (PTGS) induced by small double-stranded RNA (dsRNA). The method allows silencing of genes of interest by translation inhibition or by mRNA degradation. In this chapter, we provide a brief overview of the mechanisms involved in each step of gene silencing. A nonviral infusion of short siRNA into ventricular system of rats was used to study purinoceptor in the rat brain.

A doença de Machado-Joseph (MJD) é uma ataxia espinocerebelar (SCA) neurodegenerativa até o momento sem tratamento específico. Nos últimos anos, diversos resultados promissores na busca por tratamentos farmacológicos para a doença tem sido descritos. Contudo, a limitada disponibilidade de biomarcadores associada à evolução lentamente progressiva da MJD/SCA3 pode impedir ou dificultar muito a detecção dos efeitos terapêuticos em ensaios clínicos. Nesse sentido, os exames de neuroimagem pela possibilidade de realização in vivo, pela segurança e pelo fato de não serem invasivos, constituem ferramentas ideais. Neste trabalho, utilizamos a técnica CERES, ainda não utilizada na MJD/SCA3, para a segmentação do cerebelo de pacientes com MJD/SCA3. A análise estatística foi realizada pelo teste GLM (Generalized Linear Model) com a correção de Bonferroni, sendo p≤0,05 considerado significativo. Porém, os resultados foram discrepantes do que é observado nos estudos neuroanatômicos pois observamos um aumento da espessura e do volume do cerebelo dos pacientes quando comparados aos controles saudáveis. Uma das principais limitação da CERES é o tamanho da biblioteca de imagens segmentadas manualmente (apenas 10 imagens atlas). Outra limitação, por sua vez, consiste na imprecisão da ferramenta CERES em delimitar líquor-substância cinzenta e substância cinzenta-substância branca e realizar a normalização da intensidade de cinza das imagens de ressonância magnética.

This project aims to analyze the HLA alleles frequency in the Brazilian population. We used DNA samples of 36 healthy Brazilians subjects to sequence classic genes of the HLA class I (HLA-A, -B and -C) and class II (HLA-DQA1, -DQB1, -DPA1, -DPB1 and -DRB1, 3, 4 and 5) using next-generation sequencing (NGS). We used the HLA TruSight v2 Illumina kit to prepare HLA amplicons of these genes and the libraries generated were subsequently sequencied in an Illumina equipament, The FASTQ data generated by sequencing were analyzed by the TruSight Assign HLA (Illumina) software. The data generated in this study will be made publicly available in the BIPMed project (www.bipmed.org), so it can be used as a reference for further studies in the Brazilian population.

Mesial temporal lobe epilepsy (MTLE) is the most frequent type of epilepsy in adults and it is usually refractory to clinical treatments. In most patients with MTLE a characteristic histopathological lesion is observed, including hippocampal sclerosis (HS). The subiculum is an important area which connects the hippocampus with the enthorrinal cortex. In this study aims to understand the molecular role of the subiculum in MTLE in the classical model of pilocarpine using RNA-seq (RNA sequencing)

Much of the studies related to the hippocampus are aimed to elucidate the diseases which affect this structure, such as Mesial Temporal Lobe Epilepsy (MTLE) and Alzheimer’s disease (AD). However, to better understand the pathophysiology of these diseases, which are not fully elucidated, we must focus in other structures from the Hippocampal Formation, that can be involved with these mechanisms, such as the DG¹. The DG itself is also subdivided into dorsal and ventral in rodents and posterior and anterior in humans². Moreover, the neurodegenerative diseases, as others, are usually investigated using animal models, mainly rodents, due to its possibility of study the development of the diseases and not only its final stage³. However, despite its remarkable similarities with humans, it is necessary to evaluate the differences between the two species to validate the rodents as a suitable model for human pathologies. Therefore, here we present the comparative multi-OMICs analysis of the laser microdissected DG from rat and humans, intending to characterize and describe both species, validating the rat as a good animal model for human pathologies. We also present the comparative analysis of dorsal and ventral DG isolated from the rats.

Spinocerebellar ataxias (SCA) are a clinically and genetically heterogeneous group of monogenic diseases that share ataxia and autosomal dominant inheritance as the core features. An important proportion of SCAs are caused by CAG trinucleotide repeat expansions in the coding region of different genes. In addition to genetic heterogeneity, clinical features transcend motor symptoms, including cognitive, electrophysiological and imaging aspects. Despite all the progress in the past 25 years, the mechanisms that determine how neuronal death is mediated by these unstable expansions are still unclear. The aim of this article is to review, from an historical point of view, the first CAG-related ataxia to be genetically described: SCA 1.

Introduction Differences between left and right hemispheres regarding susceptibility to disease process is not entirely clear. In Temporal lobe epilepsy (TLE), left hippocampal sclerosis (HS; LTLE) is associated with bilateral structural damage, while in right HS (RTLE) there is more ipsilateral pattern of abnormalities. The reasons for such differences are unclear. Here we investigate the impact of neurodegenerative diseases [Amyotrophic Lateral Sclerosis (ALS) and Parkinson Disease (PD)] on patterns of structural grey matter alterations, comparing abnormalities on left and right hemispheres. Methods We performed Voxel Based Morphometry analyses on T1 weighted images with MATLAB2014b/SPM12, comparing patients and paired controls (age and gender) for each separate disease. We included 99 patients with ALS, 113 patients with PD, 45 with RTLE and 50 with LTLE and 265 healthy controls. Basically, images were segmented into grey and white matter individual maps, modulated and smoothed. Posteriorly, we performed T-tests for ALS and PD, and a full factorial model for RTLE/LTLE. All statistical analyses were performed on SPM12. Results As expected, we observed a more bilateral pattern of GM atrophy on LTLE, while RTLE present more ipsilateral GM atrophy. Interestingly, the analysis of other neurodegenerative diseases (which are not usually associated with specific MRI lesions such as hippocampal sclerosis) revealed a more left sided pattern of GM atrophy for both ALS and PD. Conclusion The results show an intriguing pattern of GM alterations on both ALS and PD, as visual analysis is usually unremarkable and mostly because neurological deficits are usually bilateral, especially at late stages. On TLE we speculate that left HS causes more widespread damage probably because left hemisphere is highly connected with right hemisphere, with more unidirectional pathways. The present work suggests that left hemisphere is somehow more susceptible to damage, regardless to the pathogenic process (i.e. seizures, neurodegeneration). Further analyses including hand dominance as covariate may help us to understand better these left/right side differences.

Background: Despite resulting from homozygosity of a single mutation at position 6 of the beta-globin locus, the clinical aspects of sickle cell anemia (SCA) are very heterogeneous. Leg ulcer is one of the many resulting complications, exerting a quite negative impact in the quality of life of patients and is related to the severity of the disease. The pathogenesis of such complication is complex and still not well explained. Objective: Seeking for novel genes associated with leg ulcer in SCA, we proposed exome sequencing of extreme phenotypes in a sample of the Brazilian population. Methods: Our sample consisted of 40 unrelated patients with SCA from the Hematology and Hemotherapy Center of the University of Campinas (Unicamp), Campinas, SP, Brazil. The cohort was composed by 20 patients who didn't have leg ulcer, with ages varying from 40 to 61 years (it is unusual the appearance of this disorder at such age) and 20 patients with chronic leg ulcer. DNA was collected from peripheral blood leukocytes and submitted to exome sequencing. Capture and enrichment were performed with the Nextera Rapid Capture Exome kit (Illumina) and samples were loaded in the Illumina HiSeq2500. The bioinformatics process was based on the GATK Pipeline and manual filters in vcftools and excel. Briefly, we prioritized the variants according to some conditions, like score quality, depth, Hardy-Weinberg equilibrium and minor allele frequency greater than 0.1. Subsequently, through PLINK, we selected the variants with p value less than 0.05 from Fisher association test. The annotation was made through wAnnovar. We excluded the synonymous variants, remaining with 244 variants at this point. Finally, the deleterious variants were selected, in addition to those not classified by SIFT, PolyPhen2 and FATHMM. Twenty-one variants remained, all of them in genes beyond the usual set of a priori biological candidate genes for this phenotype. We picked up 6 variants with the lowest p values: rs4857302 (CRYBG3), rs3782489 (KRT77), rs11800462 (TNFRSF25), rs13428956 (FOXD4L1), rs201853154 (UBTFL1) and rs11454536 (VWDE). We suggest that these variants and their genes could potentially be related to the development of leg ulcer in SCA. However, they should all be validated in other populations for confirmation. Disclosures No relevant conflicts of interest to declare.

A Síndrome Ablefaria Macrostomia (SAM) é uma condição rara, onde os pacientes apresentam características clínicas marcantes como o encurtamento ou ausência das pálpebras superiores e inferiores, ausência de sobrancelhas e cílios, macrostomia por defeitos na fusão dos lábios, entre outros. O padrão de herança da síndrome não está elucidado, tendo a herança autossômica dominante com expressividade variável sido sugerida. SAM possui sobreposição fenotípica com a Síndrome de Barber-Say e com a Síndrome de Fraser, porém nenhum gene já descrito apresentou mutação nos pacientes portadores da SAM. A abordagem genômica no estudo de doenças raras tem sido amplamente utilizada, devido principalmente ao surgimento da Nova Geração de Sequenciamento, que possui alto poder de descriminar as seqüencias nucleotídicas com grande cobertura, em um curto período de tempo. No presente estudo o sequenciamento completo do exoma foi realizado, com cinco indivíduos de uma mesma família, três membros afetados e dois não, e permitiu a análise das regiões codificantes nestes indivíduos. A base molecular da Síndrome Ablefaria Macrostomia é aqui sugerida como autossômica dominante, e decorrente da mutação nova não sinônima c.223G>A (p.E75K) no gene TWIST2. Essa mutação patogênica ocasiona a troca de um aminoácido pequeno de carga negativa, o ácido glutâmico, para um aminoácido de cadeia maior carregado positivamente, a lisina. A modelagem in silico da proteína Twist2 mostrou que a estrutura geral tridimensional da proteína não foi alterada, mas a troca do aminoácido ocorre na posição 75 dentro do domínio básico HLH, e pode impedir a formação de dímeros, ou a própria ligação ao DNA. Sugere-se ainda que a heterogeneidade de fenótipos associados a mutações no gene TWIST2, pode ser atribuída às interações que essa proteína é capaz de formar, e a ampla ação regulatória que ela desempenha em diversos genes do desenvolvimento. Ablepharon-Macrostomia Syndrome (AMS) is a rare condition characterised by absent or hypoplastic eyelids, absent eyebrows and eyelashes, macrostomia caused by fusion defects of the mouth with unfused lateral commissures, as well as other clinical features. The inheritance pattern has not been confirmed and while autosomal dominant inheritance with variable expressivity has been suggested, recessive inheritance has not been ruled out. The phenotype of AMS overlaps that of Barber-Say and Fraser Syndrome, but any reported gene for these syndromes is mutated on AMS patients. The genomic approach for rare disease studies has been widely used mainly due to the emergence of Next Generation Sequencing, which is very effective at determining nucleotide sequences with large coverage in a short period of time. The whole exome sequencing of five family members was undertaken, with three affected and two unaffected, and the coding regions of the individuals were subsequently analysed. The molecular basis of AMS is suggested here as autosomal dominant, and due to a novel non-synonymous mutation c.223G>A (p.E75K), in TWIST2 gene. This pathogenic mutation causes glutamic acid, a small negatively charged amino acid, to be substituted for a larger and positively charged lysine. The in silico protein modeling of Twist2 shows that the general 3D-structure of the protein is not affected, but the amino acid change is located inside the basic Helix-Loop-Helix domain which could disrupt dimerization and DNA binding. It has also been suggested that the phenotype heterogeneity associated with mutations on TWIST2 gene can be attributed to the interactions that this protein is capable of, and the role that it plays in the regulation of several developmental genes.

A leucemia mielóide crônica (LMC) é uma doença mieloproliferativa resultante da expansão clonal da célula hematopoética precursora. Sua fisiopatologia está associada ao cromossomo (cr) Philadelphia (Ph) originado da t(9;22) e ao oncogene bcr-abl1 que codifica a proteína Bcr-Abl1 com constitutiva atividade de tirosinoquinase (TK). A expressão de Bcr-Abl determina a leucemogênese por meio da alteração da adesão das células progenitoras leucêmicas ao estroma medular e resistência à apoptose. Os inibidores de TK, o mesilato de imatinibe, dasatinibe e nilotinibe são utilizados no tratamento da LMC, entretanto, casos de resistência têm sido relacionados à presença de mutações em Bcr-Abl1, duplicação do cr Ph e superexpressão do gene bcr-abl1. A resistência ou refratariedade de alguns pacientes ao tratamento com inibidores de TK impulsiona a realização de estudos para melhor conhecimento da fisiopatologia da LMC e descrição de novos alvos terapêuticos. Nesse contexto, o presente estudo investigou a participação de microRNAs na modulação da expressão de genes que regulam a apoptose. O objetivo geral deste trabalho foi investigar o efeito de bcr-abl1 e da atividade tirosinoquinase de Bcr-abl na expressão desses miRNAs em linhagens celulares e pacientes com LMC. O RNA das linhagens celulares, de pacientes e controles foram obtidos por meio da extração com Trizol® e o cDNA sintetizado com o kit High Capacity cDNA reverse transcription. A expressão dos microRNAs e dos genes alvoss foi quantificada por PCR em tempo real utilizando o kit SYBR Green PCR Master Mix® e TaqMan Universal PCR Master Mix®. A inibição de Bcr-Abl1 na linhagem HL-60.Bcr-Abl1 tratada com o mesilato de imatinibe aumentou a expressão de miR-let-7d, miR-15a, miR-130a e miR-145 e diminuiu os níveis de miR-21. O tratamento com dasatinibe aumentou a expressão de miR-let-7e, miR-15a, miR-16, miR-21, miR-30e, miR-130a e miR-142-3p. O nilotinibe aumentou a expressão de miR-let-7e, miR-15a, miR-16, miR-130a e miR-145 e, diminuiu os níveis de miRlet- 7d e miR-21. Os resultados obtidos da análise entre os de pacientes com LMC em diferentes fases da doença mostraram elevados níveis de miR-15a, miR-130b e miR-145 em pacientes na fase crônica versus controles e baixos níveis de miR-16, miR-26a e miR-146a. Pacientes em fases avançadas versus controles apresentaram baixa expressão de miR-let-7d, miR-16, miR-142-3p, miR-145 e miR-146a. Baixos níveis de miR-let-7d, miR-15a, miR-16, miR-29c, miR-142-3p, miR-145 e miR-146a foram observados nas fases avançadas da LMC em relação a fase crônica. Os genes anti-apoptóticos a1, bcl-2, c-flip, ciap-1 e ciap-2 estavam mais elevados na fase crônica do que nos controles. A expressão do gene c-flip estava diminuída e dos genes a1, ciap-1 e mcl-1 aumentada nas fases avançadas em relação aos controles e a fase crônica. Pacientes com LMC resistentes ao MI apresentaram menores níveis de miR-26a, miR-29c, miR-130b, miR-146a e dos genes anti-apoptóticos ciap-1 e mcl-1. Os dados obtidos sugerem que a TK Bcr-Abl modula a expressão de microRNAs que possuem como alvos genes que regulam a apoptose celular Chronic myeloid leukemia (CML) is a myeloproliferative disease resulting from clonal expasion of hematopoietic precursor cells. Its physiopathology is associated to Philadelphia (Ph) chromosome (cr) originated from the t(9;22) and bcr-abl1 oncogene that encodes the Bcr-Abl protein with constitutive tyrosine kinase activity (TK). The Bcr-Abl1 expression determines leukemogenesis by altering the leukemic progenitor cells´ adhesion by bone marrow stroma and apoptosis resistance. TK inhibitors imatinib mesylate, dasatinib and nilotinib are used to treat CML, however, cases of resistance have been linked to mutation in Bcr-Abl1, duplication of the cr Ph and overexpression of the bcr-abl1. The resistance or refractoriness of some patients to treatment with TK inhibitors drives the studies to better understand the CML physiopathology and description of new therapeutic targets. In this context, this study investigated the participation of microRNAs in modulating expression of the genes that regulate apoptosis. The aim of this study was to investigate the effect of Bcr- Abl1 and its kinase activity in the expression of miRNAs in cell lines and CML patients. The RNA from cell lines, patients and controls were obtained by extraction with Trizol® and cDNA was synthesized with the kit High Capacity cDNA reverse transcription. The expression of miRNAs and target genes was quantified by real time PCR using SYBR Green PCR Master Mix® kit and TaqMan Universal PCR Master Mix®. The Bcr-Abl1 inhibition in the cell line HL-60.Bcr-Abl1 treated with imatinib mesylate increased the expression of miR-let- 7d, miR-15a, miR-130a and miR-145 and decreased miR-21 levels. Treatment with dasatinib increased the expression of miR-let-7e, miR-15a, miR-16, miR-21, miR-30e, miR-130a and miR- 142-3p. Nilotinib increased the expression of miR-let-7e, miR-15a, miR-16, miR-130a and miR- 145 and, decreased miR-let-7d and miR-21 levels. The results of the analysis among patients with CML in different stages of disease showed high levels of miR-15a, miR-130b and miR-145 in chronic phase versus controls and low levels of miR-16, miR-26a and miR-146a. Patients in advanced phases versus controls showed low expression of miR-let-7d, miR-16, miR-142-3p, miR-145 and miR-146a. Low levels of miR-let-7d, miR-15a, miR-16, miR-29c, miR-142-3p, miR-145 and miR-146a were observed in CML advanced phases when compared with chronic phase. The antiapoptotic genes a1, bcl-2, c-flip, ciap-1 and ciap-2 were higher in chronic phase than in controls. The c-flip expression was decreased and a1, ciap-1 and mcl-1 expression was increased in advanced phases when compared to controls and chronic phase. CML patients resistant to imatinib mesylate presented low levels of miR-26a, miR-29c, miR-130b, miR-146a and ciap-1 and mcl-1 antiapoptotic genes. The data obtained suggest that Bcr-Abl1 TK modulates the miRNA expression which has target genes involved in the apoptosis´ regulation.

Os insetos desenvolveram um sistema imune eficiente contra parasitas e patógenos, que compreende a resposta celular e a humoral. Os mecanismos celulares envolvem a fagocitose e a encapsulação pelos hemócitos, enquanto que as respostas humorais incluem a ativação da Profenoloxidase, e a síntese pelo corpo gorduroso dos peptídeos antimicrobianos, que são liberados na hemolinfa. Duas vias de sinalização intracelular, Toll e Imd, controlam a expressão dos genes codificadores dos peptídeos antimicrobianos. A análise do Genoma da abelha Apis mellifera permitiu a identificação dos genes dessas vias. No entanto, pouco se conhece do mecanismo de resposta imune nessas abelhas. Desta maneira, nos propusemos analisar a transcrição de genes efetores da resposta imune (abaecina, hymenoptaecina, defensina, transferrina, profenoloxidase), assim como os genes integrantes das vias de sinalização, tais como os genes de reconhecimento de microorganismos (PGRP, GNBP) e ainda, os de sinalização (cactus, relish, dorsal 1-B). Avaliamos também possíveis proteínas implicadas na resposta imune, como as proteínas de estocagem Vitelogenina, Hexamerina 70a, Lipoforina I/II e Lipoforina III. Finalmente, analisamos o efeito da nutrição e do envelhecimento sobre a imunidade em abelhas. Para análise da expressão dos genes das vias de sinalização, as abelhas foram infectadas com bactérias Serratia marcescens ou Micrococcus luteus por injeção ou via alimentação. A infecção com esses microorganismos provocou a transcrição de peptídeos antimicrobianos e de transferrina em altas quantidades após 3 e 12 horas de tratamento, além da alteração na quantidade de transcritos de outros genes. O papel dos genes profenoloxidase e dorsal na imunidade, descritos como codificadores de importantes proteínas em outros insetos, foi avaliado através da metodologia de silenciamento gênico por RNA de interferência. Observamos a diminuição da transcrição do gene alvo, mostrando a eficiência da metodologia. No entanto, a simples injeção de um RNA de fita dupla foi capaz de ativar o sistema imune de abelhas. Este efeito contribuiu para a dificuldade de atribuição do papel da Profenoloxidase na imunidade de abelhas. Contudo, os resultados de silenciamento de dorsal e suas isoformas, nos levaram a considerar que dorsal 1-A ou dorsal 2 participam da via de sinalização intracelular para produção de peptídeos antimicrobianos, principalmente de defensina. Em relação às proteínas de estocagem, tanto a quantidade de transcritos quanto de proteínas diminui após infecção com bactérias, indicando que estas proteínas estão envolvidas de alguma forma no processo de imunidade em abelhas. Além disso, consumo de alimentos ricos em proteína aumentou os níveis de transcritos das proteínas de estocagem, o que muito provavelmente favorece a manutenção da capacidade de resposta imune de abelhas. O efeito do envelhecimento no declínio da imunidade foi analisado em abelhas nutridoras (novas) e forrageiras (velhas) de uma colônia típica. Além disso, foram utilizadas abelhas de uma colônia single-cohort, que eram de uma mesma idade, mas algumas eram nutridoras, enquanto outras eram forrageiras. Todas as abelhas, independentemente da idade ou comportamento, foram capazes de ativar o sistema imune após infecção pela bactéria S. marcescens. No entanto, as abelhas com o comportamento de forrageira, independentemente da idade, sempre foram mais susceptíveis a infecções que as nutridoras. Este fato se deve, muito provavelmente, às diferenças fisiológicas entre essas abelhas, que proporciona às nutridoras maior competência à sobrevivência. Insects have developed an efficient immune system against parasites and pathogens, which is comprised of both cellular and humoral responses. The cellular mechanisms involve phagocytosis and encapsulation by hemocytes, whereas the humoral responses include activation of prophenoloxidase and synthesis of antimicrobial peptides by the fat body, which are released into the hemolymph. Two signaling pathways, Toll and Imd, control the expression of genes encoding antimicrobial peptides. Genome-wide analyses of the honey bee, Apis mellifera, have identified predicted genes for these signaling pathways. However, immune response mechanisms in honey bees were not yet in depth studied. We analyzed the transcription of effector genes (abaecin, hymenoptaecin, defensin, transferin, prophenoloxidase), as well as other immune genes, such as pathogen recognition genes (PGRP, GNBP) and signaling genes (cactus, relish, dorsal 1- B). We also investigated the role of the storage proteins Vitellogenin, Hexamerin 70a, Lipophorin I/II and Lipophorin III in the honey bee immunity. Finally, we analyzed the effect of nutrition and aging on honey bee immunity. Gene expression of signaling pathway components was assessed in honey bees that had been infected with the bacteria Serratia marcescens or Micrococcus luteus through injection or oral challenge. Honey bees infected with these microorganisms had strong up-regulation of antimicrobial peptide genes and of transferin, and also other changes in transcript abundance after 3 and 12 hours of challenge. The roles of prophenoloxidase and dorsal in the immune response, described as genes encoding important proteins in other insects, were also investigated. In this case we used RNA interference (RNAi) to silence the expression of these genes. RNAi efficiently silenced the target genes. However, injection of doublestranded RNA in honey bees induced a reaction by the immune system. This made it difficult to determine the role of prophenoloxidase in honey bee immunity. Yet, silencing of dorsal and its isoforms led us to consider dorsal 1-A or dorsal 2 as members of the signaling pathways that produce antimicrobial peptides, especially defensin. The abundance of storage proteins transcripts and proteins was lower in infected bees than in controls, giving evidence that these proteins participate in the immune process in honey bees. Moreover, protein consumption caused up-regulation of genes encoding storage proteins, which may favor the maintenace of the immune response capacity. The effect of aging on decline in immunity was analyzed in (young) nurse bees and (old) foragers from normal free-flying colony. We also examined bees from a single-cohort colony, in which all individuals were at the same age; but some were nursing, while others were foraging. All the bees, independent of age or behavior, were able to activate the immune system after infection with S. marcescens. However, foragers, independent of age, were always more susceptible to infections than were nurse bees. This is probably due to physiological differences between bees, which confers to the nurses more competence to survivorship.

Introdução: A demência frontotemporal (DFT) inclui a variante comportamental da demência frontotemporal (vcDFT), a variante semântica da afasia progressiva primária (vsAPP), e a variante não fluente da APP (vnfAPP). Os genes em que são encontradas mutações causadoras de DFT mais frequentemente são: GRN (que codifica a progranulina), MAPT (que codifica a proteína tau) e C9orf72. Métodos: Foram incluídos probandos diagnosticados com vcDFT, vsAPP ou vnfAPP, com base com os critérios diagnósticos mais recentes, e um grupo de indivíduos cognitivamente normais. Os éxons 2-12 de GRN e os éxons 1, 9-13 de MAPT foram sequenciados pelo método de Sanger, e foi realizada dosagem de progranulina no plasma. Resultados: foram incluídos 62 probandos, sendo 44 com vcDFT, 9 com vsAPP, e 9 com vnfAPP. Antecedente familiar de demência foi positivo em 45,1% dos probandos, e de DFT, em 24,1%. Os 60 indivíduos do grupo controle tinham idade média de 60,8±8,5 anos. Foram identificadas seis mutações nulas em GRN (p.Q130X, p.V200Gfs*18, p.Q257Pfs*26, p.Q300X, p.S301Cfs*60 e p.D317Afs*11) e uma mutação patogênica em MAPT (p.N279K). A dosagem média de progranulina plasmática nos pacientes com mutações de GRN foi de 29,8±11,9ng/ml Conclusões: A frequência de mutações patogênicas em GRN nesta casuística foi de 9,6%, e a de mutações em MAPT foi de 1,6%. Entre casos familiais de DFT, a frequência de mutações em GRN foi de 33,3%, e em MAPT foi de 6,7%. Duas das mutações encontradas em GRN (p.Q130X e p.D317Afs*11) ainda não foram descritas em casos de DFT. O valor de corte de 70ng/ml identificou as mutações nulas de GRN com sensibilidade e especificidade de 100% Introduction: Frontotemporal dementia (FTD) encompasses behavioral variant of frontotemporal dementia (bvFTD), semantic variant of primary progressive aphasia (svPPA), and nonfluent variant PPA (nfvPPA). The genes in which FTD-causing mutations are most frequently found are: GRN (which encodes progranulin), MAPT (which encodes tau protein) and C9orf72. Methods: We included probands diagnosed with bvFTD, svPPA or nfvPPA, based on the most recent diagnostic criteria, and a group of cognitively normal individuals. GRN exons 2-12 and MAPT exons 1, 9-13 were sequenced by the Sanger method, and plasma progranulin levels were measured. Results: we included 62 probands (44 with bvFTD, 9 with svPPA, and 9 with nfvPPA). Family history of dementia was positive in 45.1% of probands, and of DFT, in 24.1%. The control group of 60 individuals had a mean age of 60.8±8.5 years. Six null GRN mutations were identified in (p.Q130X, p.V200Gfs*18, p.Q257Pfs*26, p.Q300X, p.S301Cfs*60 e p.D317Afs*11) and one MAPT pathogenic mutation (p.N279K). The mean plasma progranulin level in patients with GRN mutations was 29.8±11,9ng/ml. Conclusions: The frequency of pathogenic mutations in GRN was 9.6%, and of MAPT mutations was 1.6%. Among cases of familial FTD, the frequency of GRN mutations was 33.3%, and of MAPT mutations was 6.7%. Two of the mutations found in GRN (p.Q130X and p.D317Afs*11) are novel. The cutoff value of 70ng/ml identified null GRN mutations with sensitivity and specificity of 100%

O presente estudo discute o papel dos miRNAs na fisiopatologia molecular de Esclerose Múltipla Recorrente Remitente (EMRR). O estudo demonstrou que em linfócitos T CD4+ de pacientes com EMRR em surto ocorre a diminuição da expressão do miR-15a e do miR16-1 em contraposição ao aumento de seu gene alvo BCL-2, um importante gene regulador da apoptose. Esses achados sugerem a participação desses miRNAs no controle da apoptose na EM. Para explorar essa associação, foi analisado a expressão global de miRNAs nas subpopulações de linfócitos T de pacientes com EMRR no estágio de remissão. O resultado dessa análise determinou de forma inédita o aumento significativo da expressão de 9 miRNAs (miRNAs-16, miRNAs-20a, miRNAs-21, miRNAs-24, miRNAs-155, miRNAs-221, miRNAs-222, miRNAs-720 e miRNAs-1281) nos linfócitos T CD8+ de memória central. A análise in silico dos alvos desses miRNAs indicou que três vias canônicas, relacionadas à ativação da apoptose, eram enriquecidas com alvos preditos e validados experimentalmente desses miRNAs. Desse modo sugerimos a forte relação desses miRNAs no controle da apoptose nos linfócitos T dos pacientes com EMRR. A fim de aprofundar nossos estudos, selecionamos os miRNAs miR-21 e miR-24, para a realização de experimentos funcionais in vivo. Foi verificada a indução da expressão miR-21 somente nos linfócitos T CD4+ de modelo experimental da EM. Adicionalmente, experimentos in vitro demonstraram que a expressão do miR-21 e restrita as populações de células Th2 e Th17. Nesse caso, miR-21 parece ser regulado pelo fator de transcrição STAT3, sugerindo assim que o aumento da expressão do miR-21 verificada no modelo animal possa estar relacionada com a presença de linfócitos T CD4+ de perfil Th17 nesse tecido. Em resumo, o conjunto desses resultados demonstra a relevância dos miRNAs na fisiopatologia da EMRR, principalmente no controle da apoptose. This study discusses the role of miRNA in the Molecular Pathophysiology of Relapse Remitting Multiple Sclerosis (RRMS). The study has shown that CD4+ lymphocytes from relapsed RRMS patients had lower expression of miR15a and miR-16-1 in contraposition of higher expression of the target gene BCL-2, a key regulator of apoptosis. These findings suggest the role of those on the control of apoptosis in MS. In order to explore this association, the global expression of miRNAs was analysed in T lymphocyte subpopulations from remission RRMS patients. The result of this analysis has demonstrated for the first time a significant higher expression of 9 miRNAs (miRNAs-16, miRNAs-20a, miRNAs-21, miRNAs-24, miRNAs-155, miRNAs-221, miRNAs-222, miRNAs-720 e miRNAs-1281) in central memory T CD8+ lymphocytes. In silico analysis of the miRNAs targets indicates that three canonical pathways related to the activation of apoptosis were enriched with predicted and experimental validated gene targets for those miRNAs. In this way, we suggest the strong relation of these miRNAs in the control of apoptosis in the lymphocytes from RRMS patients. In order to intensify our studies we selected miR-21 and miR-24 to perform in vivo functional experiments. It was verified miR-21 induction only in T CD4+ lymphocytes from MS animal model. Additionally, in vitro experiments have demonstrated that miR-21 expression was restricted to Th2 and Th17 cell populations. In this way, miR-21 seems to be regulated by the STAT3 transcription factor, thereby suggesting that the increase of miR-21 expression observed in vivo could be related with Th17 CD4+ present in this tissue. In summary, this set of results showed the relevance of miRNAs in the RRMS pathophysiology, mainly in the control of apoptosis.

O objetivo do trabalho foi de investigar os mecanismos genéticos relacionados à resposta humana à malária em duas populações Amazônicas. Cerca de 180 indivíduos de Portuchuelo e mais de 800 indivíduos de Monte Negro foram incluídos na amostra. Os indivíduos amostrados passaram por um levantamento epidemiológico e caracterização de marcadores genéticos. O número de episódios de malárias relatado pelos indivíduos, após correção para idade e sexo, foi submetido a testes de associação. A associação esperada entre indivíduos Fy- e a característica estudada foi encontrada nas duas populações, sem heterogeneidade significante. Análises de segregação complexa indicaram a presença de um gene principal relacionado ao acometimento de malária. Utilizando 108 STRs foi realizada uma varredura genômica na população de Portuchuelo onde foi encontrado um pico de lod score, sugestivo de ligação no braço curto do cromossomo 4. Os resultados obtidos em Portuchuelo não foram confirmados em 9 STRs estudados na população de Monte Negro. With the aim of investigate the genetic mechanisms related to the human response to malaria infection, two Amazonian populations were studied. About 180 individuals from Portuchuelo population and more than 800 individuals from Monte Negro were included in the sample. Those individuals where subjected to an epidemiological survey, followed by characterization of genetic markers. Among the characteristics surveyed, the number of malaria episodes reported by the subjects was tested for association with classic blood groups markers after correction for age and sex. The expected association with Fy- individuals was observed in both populations, without significant heterogeneity. Complex segregation analyses indicated the presence of a major gene related to the number of malaria episodes. A genomic scan using STRs markers was conducted at the Portuchuelo sample. The multipoint linkage scan showed a lod score suggestive of linkage at the short arm of chromosome 4. The results found at Portuchuelo were not confirmed at the Monte Negro sample using 9 STRs.

Abelhas adultas produzem vitelogenina, a principal proteína da hemolinfa. Ela está envolvida na reprodução, comportamento, imunidade, longevidade e regulação da organização social. A interferência por RNA interference é a mais promissora ferramenta para estudos de função gênica, baseada na introdução de duplex de RNA (dsRNA) que induz a degradação de transcritos alvo-específicos. Injeção de dsRNA altera a transcrição de vitelogenina, mas evidências apontam que a ativação do sistema imune em abelhas seja um efeito colateral destaa manipulação. Desenvolvemos um método para o silenciamento do gene codificador de vitelogenina no desenvolvimento pós-embrionário, que minimiza os efeitos da manipulação, onde 0,5 ?g de dsRNA de vitelogenina (dsVg) ou de GFP (controle exógeno, dsGFP) foi oferecido na dieta natural de larvas de segundo estágio, as quais foram mantidas na colônia. Nosso enfoque principal foi a compreensão dos efeitos do silenciamento pós-transcricional de rainhas e operárias de A. mellifera, em especial na fase larval. Operárias adultas reconhecem larvas tratadas e as remove. Mantemos certa distância entre as células de cria que recebiam o tratamento e a remoção de larvas tratadas diminuiu consideravelmente. A expressão de transcritos de vitelogenina em indivíduos sem tratamento e tratados foi analisada no quinto estágio larval de ambas as castas, bem como em operárias adultas de 7 dias e rainhas recémnascidas, utilizando-se PCR em tempo real e a expressão do gene codificador de actina como controle endógeno. Em adultos, controles sem tratamento e dsGFP expressaram quantidades similares de transcritos de vitelogenina. Os grupos alimentados com dsVg tiveram expressão reduzida de vitelogenina, a saber: quinto estágio larval de operárias (91%) e de rainhas (71%), operárias de 7 dias (88%) e rainhas recém-nascidas (70%). O silenciamento da vitelogenina não afetou a morfologia dos adultos, mas sim a fisiologia de larvas de ambas as castas, como nos títulos de hormônio juvenil e concentração de proteínas circulantes na hemolinfa. Concluímos que a ingestão de dsRNA é um método não-invasivo que induz silenciamento gênico e, assim, uma ferramenta eficiente para estudos funcionais pós-genoma. Os mecanismos regulatórios do gene codificador de vitelogenina e seu papel na diferenciação de castas estão em discussão. Adult bees produce vitellogenin (Vg), the main protein in hemolymph; it is involved in honey bee (Apis mellifera) reproduction, behavior, immunity, longevity and regulation of social organization. Genetic interference mediated by injection of double-stranded RNA (dsRNA) is a powerful tool for the analysis of gene function in Apis mellifera. Injection of dsRNA effectively alters vitellogenin transcription; however, evidence has been found of immune system activation in treated bees, which could be a collateral effect of treatment. Consequently, we developed a non-invasive protocol for disruption of the A. mellifera genes exemplified by vitellogenin mRNA silencing, to understand it, mainly, in the female larval context. Second instar larvae were treated as follows: the treatment group received 0,5 ?g of double-stranded vitellogenin RNA (dsVg) mixed with larval food deposited in the worker brood cells; control group 1 was left to develop without treatment, while control group 2 received dsGFP (Green Fluorescent Protein), as an exogenous control. Treated and control larvae were maintained in the colony until adult emergence. Workers recognized dsRNAtreated larvae and frequently removed them. To circumvent this problem we increased the distance between the treatment groups. Vg gene expression were determined for fifth instar larvae of both castes and for 7 day-old workers and newly-emerged queens, evaluated by quantitative real time PCR, using actin as an endogenous control. For adults, we found that controls, dsGFP- and non-treated bees expressed similar amounts of Vg transcripts. The dsVg-fed groups had significantly reduced Vg gene expression in fifth instar larvae of workers (91%) and queens (71%) and, also, in 7 day-old workers (88%) and newly-emerged queens (70%). Disruption of the Vg gene did not affect adults morphology but physiological larval traits of both castes, as juvenile hormone titre and protein concentration. We conclude that dsRNA ingestion is an effective non-invasive method for inducing knockdown and an efficient approach for post-genome functional studies. The regulatory mechanisms of vitellogenin gene and its rules during caste differentiation are discussed.