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The gastrointestinal mucosal immune system has the daunting task of maintaining immune homeostasis by eliminating potentially harmful microorganisms and limiting tissue injury while inducing tolerogenic responses to luminal antigens including innocuous food, commensal bacteria and self-antigens. This carefully orchestrated system depends on elaborate down-regulating mechanisms that mediate and maintain a state of tolerance under normal conditions. Changes in such delicate balance are linked to the development of gastrointestinal pathology as well as systemic disease states. Despite the rapid increase in our appreciation of the gastrointestinal immune system, there is still a major disconnect between the description of how mucosal immune responses are organized and controlled and an insufficient mechanistic understanding of how such responses shape and influence disease outcome and pathogenesis. By using model enteric microorganisms Helicobacter pylori and Clostridium difficile, this dissertation presents a systematic effort to generate novel mechanistic hypothesis based on computational predictions and experimentally elucidate the mechanisms of action underlying mucosal immune responses and pathology in the gut. In this thesis I present i) an overview on mucosal immunology and the need to develop novel therapeutics that limit the pathogenic effects of invading bacteria while maintaining their protective functions, ii) the role of miRNAs in the modulation of immune responses to enteric pathogens, iii) the mechanisms by which Helicobacter pylori is able to limit effector inflammatory responses required for bacterial clearance thus favoring tolerance over immunity, iv) intracellular mechanisms of immune evasion that contribute to bacterial persistence and chronic infection. The knowledge generated throughout this dissertation exemplifies how a combination of computational modeling, immunoinformatics and experimental immunology holds enormous potential for discovering unforeseen targets and developing novel vaccines and cures for infectious, allergic and immune-mediated diseases. / Ph. D.

Acute myeloid leukemia (AML) the most common acute leukemia in adults is characterized by various cytogenetic and molecular abnormalities. However, the genetic etiology of the disease is not yet fully understood. MicroRNAs (miRNAs) are small single- stranded noncoding RNAs that are negative regulators of gene expression. miRNAs influence processes of proliferation, differentiation and apoptosis. Deregulation of miRNAs expression can contribute to human disease. Circulating miRNAs are emerging biomarkers in many diseases and cancers such as breast cancer, colorectal cancer and lung cancer. However, defining a plasma miRNA signature in AML that could serve as a biomarker for diagnosis has been conducted only once. We studied miRNA expression in plasma of 8 AML patients in first detection of the disease and repeatedly after achieving remission using TaqMan miRNA microarray for 750 human miRNA. The plasma expression level of 25 miRNA was down-regulated whilst that of 20 miRNA was up-regulated in the AML group at diagnosis when compared to healthy controls. The plasma expression level of 21 miRNA was down-regulated whilst that of 13 miRNA was up-regulated in the AML group in remission compared to healthy controls. Keywords acute myeloid leukemia (AML), biomarker, microRNA (miRNA), plasma, TaqMan Low...

A terapia antiagregante é comumente indicada na prevenção e tratamento de doenças cardiovasculares. A dupla antiagregação com clopidrogrel e ácido acetilsalicílico (AAS) tem sido frequentemente adotada em pacientes com Doença Arterial Coronariana (DAC), mas apresenta ineficácia em uma parcela significativa da população com genótipo de respondedores. Essa falha terapêutica nos leva a questionar se outros mecanismos moleculares podem estar influenciando na resposta a esses fármacos. Recentes estudos sugerem que pequenas sequências de RNA não codificantes denominadas microRNAs (miRNAs) podem estar fortemente relacionadas com resposta ao tratamento fármaco-terapêutico, controlando as proteínas envolvidas na farmacocinética e farmacodinâmica. Entretanto, os principais miRNAs que atuam na dinâmica da resposta medicamentosa ainda não foram bem definidos. O objetivo deste estudo foi avaliar o perfil de miRNAs no sangue total periférico, procurando melhor esclarecer os mecanismos envolvidos na resposta aos antiagregantes plaquetários AAS e clopidogrel. Para isso, selecionou-se pacientes com DAC, os quais apresentavam diferentes respostas à dupla terapia de antiagregação determinadas pelo teste de agregação plaquetária. Baseados nos fenótipos, os perfis de expressão de miRNAs foram comparados entre os valores da taxa de agregação categorizados em tercis (T) de resposta. O grupo T1 foi constituído de pacientes respondedores, o T2 de respondedores intermediários e o T3 de não respondedores. Os perfis de miRNAs foram obtidos após sequenciamento de última geração e os dados obtidos foram analisados pelo pacote Deseq2. Os resultados mostraram 18 miRNAs diferentemente expressos entre os dois tercis extremos. Dentre esses miRNAs, 10 deles apresentaram importantes alvos relacionados com vias de ativação e agregação plaquetária quando analisados pelo software Ingenuity®. Dos 10 miRNAs, 4 deles, os quais apresentaram-se menos expressos no sequenciamento, demonstraram os mesmos perfis de expressão quando analisados pela reação em cadeia pela polimerase quantitativa (qPCR): hsa-miR-423-3p, hsa-miR-744-5p, hsa-miR- 30a-5p e hsa-let-7g-5p. A partir das análises de predição de alvos, pôde-se observar que os quatro miRNAs, quando menos expressos simultaneamente, predizem ativação da agregação plaquetária. Além disso, os miRNAs hsa-miR- 423-5p, hsa-miR-744-5p e hsa-let-7g-5p mostraram correlação com o perfil lipídico dos pacientes que, por sua vez, apresentou influência nos valores de agregação compreendidos no T3 de resposta a ambos os medicamentos. Sendo assim, conclui-se que maiores taxas de agregação plaquetária podem estar indiretamente relacionadas com os padrões de expressão de hsa-miR- 423-3p, hsa-miR-744-5p e hsa-let-7g-5p. Sugere-se que a avaliação do perfil de expressão destes 3 miRNAs no sangue periférico de pacientes com DAC possa predizer resposta terapêutica inadequada ao AAS e ao clopidogrel / The antiplatelet therapy is often indicated for the prevention and treatment of cardiovascular diseases. Dual antiplatelet therapy with clopidogrel and acetylsalicylic acid (ASA) has often been adopted in patients with coronary artery disease (CAD), but it has been ineffective in a significant portion of the population with genotype of responders. This fact leads us to question whether other molecular mechanisms may be influencing the response to these drugs. Recent studies suggest that small non-coding RNA sequences known as microRNAs (miRNAs) may be closely related to response to drug-therapeutic treatment, controlling proteins involved in pharmacokinetics and pharmacodynamics. The aim of this study was to evaluate the profile of miRNAs in whole blood, looking to better clarify mechanisms involved in ASA and clopidogrel response. For this purpose, we selected CAD patients who showed different responses to dual antiplatelet therapy determined by aggregation test. Based on the phenotypes, the miRNA expression profiles were compared between the platelet aggregation values categorized into tertiles (T) of response. The T1 group consisted of responding patients, the T2 consisted of intermediate responders and the T3 consisted of non-responders. The miRNA profiles were obtained after next-generation sequencing and data were analyzed by Deseq2 package. Results showed that 18 miRNAs were differentially expressed between the two extreme tertiles. By Ingenuity® software prediction analysis, 10 miRNAs showed important targets related with activation and aggregation of blood platelets. Of the 10 miRNAs, 4 of them, which were down-expressed on sequencing, showed the same fold-regulation when expression profiles were analyzed by quantitative polymerase chain reaction (qPCR): hsa-miR-423-3p, hsa-miR-744-5p, hsa-miR-30a-5p and has-hsa- let-7g-5p. By target prediction analysis, it was observed that, when the four miRNAs are simultaneously down-expressed, they predict activation of platelet aggregation. Furthermore, hsa-miR-423-5p, hsa-miR-744-5p, and hsa-let-7g-5p showed correlation with the lipid profile of patients which, in turn, demonstrated influence in aggregation values reaching T3 of response to both drugs. Therefore, we concluded that increased platelet aggregation rates may be indirectly related to the expression profiles of hsa-miR-423-3p, hsa-miR-744-5p and hsa-let-7g-5p. It is suggested that the evaluation of the expression profile of these three miRNAs in the peripheral blood of patients with CAD may predict inadequate therapeutic response to aspirin and clopidogrel.

Doenças cardiovasculares são a principal causa de morte no Brasil. Em contraste a esse cenário, o treinamento físico aeróbio (TFA) figura como importante ferramenta de combate a esse quadro, atuando, dentre diversos mecanismos, na expressão de microRNAs (miRNA) e na hipertrofia cardíaca (HC). Nesse sentido, seria o TFA capaz de reduzir expressão do miR-34c-3p em cardiopatologias, gerando melhora aos danos causados? Na tentativa de elucidar essa lacuna, ratos Wistar machos (2 meses) passaram por oclusão da coronária gerando infarto do miocárdio (IMS; IMT) ou indução de diabetes tipo I por STZ (DS;DT: 45mg/kg). Após a verificação do dano, estes foram submetidos a TFA (10 sem), e os ventrículos esquerdos (VE) foram dissecados a posteriori. Ao analisar a expressão gênica por RT-PCR, o TFA diminuiu (39%; p<0,02) a expressão do miRNA-34c-3p no VE de IMT vs. IMS. Em consonância, houve diminuição (27%; p<0,04) do miRNA em DT vs. DS. Investigando o potencial terapêutico dessa inibição, ao proceder análise in vitro em cultura primária de cardiomiócitos tratados com inibidor do miRNA (amiR-34c-3p), houve diminuição de sua expressão em 71% (50nM; p<0,01) vs. grupo controle (C), enquanto houve aumento de 41000% (50nM; p<0,01) entre o superexpressor (mmiR) e C. Analisando os marcadores de HC patológica entre amiR e C, verificou-se redução na a-actina esquelética (6,1%;p<0,02), aumento no ANF (84,4%; p<0,05) e redução na b-MHC (66%; p<0,007), demonstrando HC fisiológica em cardiomiócitos tratados com amiR. Buscando alvos do miR-34c-3p responsáveis por essas alterações, analisou-se o eIF4E, último fator iniciador de tradução ativado por via não canônica da PI3K-AKTmTOR. Sua direta ativação pode representar importante avanço na busca por terapias que induzam a HC fisiológica. O amiR aumentou o eIF4E (64%; p<0,003), com validação por diminuição da atividade da luciferase (48%; p<0,02) através de ensaio com vetor 3\'UTR do gene e miR-34c-3p. Outro alvo predito analisado, o KLF11, tem homologia com gene da insulina e pode estar relacionado com melhora metabólica do VE. O tratamento com amiR aumentou (192%; p<0,006) a expressão gênica do KLF11, validado com redução de 55% na atividade da luciferase, e aumento da captação de glicose (13%; p<0,04). Com os dados expostos, o TFA foi eficiente em inibir a expressão do miR-34c-3p em VE de animais cardiopatas que, atuando em seus alvos agora validados, foi capaz de promover HC fisiológica e melhora metabólica em cardiomiócitos. Considerando os efeitos cardioprotetores da inibição desse miRNA, vislumbra-se o potencial terapêutico dessa inibição em paciente na perspectiva de atenuação de prejuízos causados por cardiopatias / Cardiovascular diseases are the leading cause of death in Brazil. In contrast to this scenario, aerobic exercise training (AET) is an important tool to combat this condition, acting, among several mechanisms, on the expression of microRNAs (miRNA) and cardiac hypertrophy (CH). In this sense, would AET be able to reduce expression of miR-34c-3p in cardiac pathologies, generating improvement to the damages caused by them? In an attempt to elucidate this knowledge gap, male Wistar rats (2 months) underwent coronary occlusion, causing myocardial infarction (IMS; IMT) or induction of type I diabetes by STZ (DS: DT: 45mg kg). After proof that cardiac damage was caused, they were submitted to AET (10 wk), and left ventricles (LV) were dissected post mortem. Analyzing gene expression by RT-PCR, AET decreased (39%; p <0.02) miRNA-34c-3p expression in the LV of IMT vs. IMS. In agreement, there was a decrease (27%; p <0.04) of the miRNA in DT vs. DS. Investigating the therapeutic potential of this inhibition, through in vitro analysis in primary culture of neonatal rat cardiomyocytes treated with miRNA inhibitor (amiR-34c-3p), expression of miRNA- 34c-3p was decreased in 71% (50nM; p <0.01) vs. (C), whereas there was an increase of 41000% (50nM; p <0.01) between the superexpressor (mmiR) and C. Analyzing the pathologic HC markers between amiR and C, there was a reduction in skeletal a-actin (P <0.05), an increase in ANF (84.4%, p <0.05) and reduction in b-MHC (66%; p <0.007), demonstrating physiological HC in cardiomyocytes treated with amiR. Searching for miRNA-34c-3p targets responsible for these changes, eIF4E, the last non-canonically activated translation initiator of PI3K-AKT-mTOR, was analyzed. Its direct activation may represent an important advance in the search for therapies that induce physiological CH. The amiR increased eIF4E expression (64%, p <0.003), with validation for decreased luciferase activity (48%; p <0.02) by assaying 3\'UTR vector of the gene and miR-34c-3p. Another predicted target analyzed, KLF11, has homology with insulin gene and may be related to LV metabolic improvement. The treatment with amiR increased KLF 11 gene expression (192%; p <0.006), with a 55% reduction in luciferase activity and an increase in glucose uptake (13%; p <0.04). With the data presented, AET was efficient in inhibiting the expression of miRNA-34c-3p in LV of cardiopathic animals that, acting on their now validated targets, was able to promote physiological CH and metabolic improvement in cardiomyocytes. Considering the cardioprotective effects of inhibition of this miRNA, we can speculate a therapeutic potential of the inhibition of miR-34c-3p in patients in prospect of mitigating the damage caused by cardiopathies

Orientador: Marcelo Menossi Teixeira, Andréa Akemi Hoshino / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-18T07:51:32Z (GMT). No. of bitstreams: 1 Mattos_RaphaeldeSouza_M.pdf: 5526068 bytes, checksum: 090cb9f141a5a7dd324df049c18bf9c1 (MD5) Previous issue date: 2011 / Resumo: A cana-de-açúcar é uma das mais importantes espécies vegetais cultiváveis do mundo, sendo o Brasil o principal produtor. É uma fonte eficiente e de baixo custo para a obtenção de açúcar e etanol, que é considerado o mais promissor substituto do petróleo como fonte de energia a médio prazo, especialmente nos transportes. A seca é um dos principais estresses que reduzem a produtividade da cana e a produção de variedades tolerantes não só representa ganhos econômicos como contribui para a sustentabilidade dos canaviais. Embora a base genética da tolerância à seca ainda seja pouco conhecida, variedades desenvolvidas em programas de melhoramento tem apresentado progresso, apesar do ritmo ser mais lento que o desejado. Genômica funcional e desenvolvimento de marcadores colaboram aumentando a eficiência do melhoramento tradicional, mas ainda existem elementos do genoma que podem ser aproveitados de novas formas. Foram descobertos recentemente genes de função regulatória chamados microRNAs (miRNA) que também desempenham um papel na adaptação de plantas a diferentes estresses. Utilizando ESTs de cana-de-açúcar, sequenciamento de nova geração e microarranjos para avaliar a expressão de miRNAs sobre estresse hídrico foram descobertos novos miRNAs associados à seca e possíveis genes de miRNAs ligados à tolerância a este tipo de estresse / Abstract: Sugarcane (Saccharum spp.) is amongst the most relevant crops in the world and Brazil is the most prominent producer. It is an inexpensive and efficient source for commodities such as sugar and ethanol, the latter being increasingly considered the most promising immediate energy source substitute for oil, mainly in transportation. Apart from being very productive, it is largely affected by stress-related yield losses, notably from abiotic triggers. Drought stress is determinant for every crop field, and this is true for sugarcane as well. Although the molecular basis drought stress tolerance lacks further elucidation, newly developed cultivars have successfully reduced yield loss due to water shortage, albeit not at the desirable pace. Functional genomics and molecular markers development assist new cultivar selection programs by identifying and locating agronomically relevant alleles and QTL's, but there are other elements in the genome which can provide new ways to approach crop field improvement. The recently discovered microRNAs (miRNA) are regulatory genes found to have an important role in plants adaptation under different kinds of stresses. By using sugarcane ESTs, deep-sequencing and microarray technology to access stress induced miRNA expression, we have found novel sugarcane miRNA participating in the drought stress response and identified possible tolerance related miRNA genes / Mestrado / Genetica Vegetal e Melhoramento / Mestre em Genética e Biologia Molecular

A terapia antiagregante é comumente indicada na prevenção e tratamento de doenças cardiovasculares. A dupla antiagregação com clopidrogrel e ácido acetilsalicílico (AAS) tem sido frequentemente adotada em pacientes com Doença Arterial Coronariana (DAC), mas apresenta ineficácia em uma parcela significativa da população com genótipo de respondedores. Essa falha terapêutica nos leva a questionar se outros mecanismos moleculares podem estar influenciando na resposta a esses fármacos. Recentes estudos sugerem que pequenas sequências de RNA não codificantes denominadas microRNAs (miRNAs) podem estar fortemente relacionadas com resposta ao tratamento fármaco-terapêutico, controlando as proteínas envolvidas na farmacocinética e farmacodinâmica. Entretanto, os principais miRNAs que atuam na dinâmica da resposta medicamentosa ainda não foram bem definidos. O objetivo deste estudo foi avaliar o perfil de miRNAs no sangue total periférico, procurando melhor esclarecer os mecanismos envolvidos na resposta aos antiagregantes plaquetários AAS e clopidogrel. Para isso, selecionou-se pacientes com DAC, os quais apresentavam diferentes respostas à dupla terapia de antiagregação determinadas pelo teste de agregação plaquetária. Baseados nos fenótipos, os perfis de expressão de miRNAs foram comparados entre os valores da taxa de agregação categorizados em tercis (T) de resposta. O grupo T1 foi constituído de pacientes respondedores, o T2 de respondedores intermediários e o T3 de não respondedores. Os perfis de miRNAs foram obtidos após sequenciamento de última geração e os dados obtidos foram analisados pelo pacote Deseq2. Os resultados mostraram 18 miRNAs diferentemente expressos entre os dois tercis extremos. Dentre esses miRNAs, 10 deles apresentaram importantes alvos relacionados com vias de ativação e agregação plaquetária quando analisados pelo software Ingenuity®. Dos 10 miRNAs, 4 deles, os quais apresentaram-se menos expressos no sequenciamento, demonstraram os mesmos perfis de expressão quando analisados pela reação em cadeia pela polimerase quantitativa (qPCR): hsa-miR-423-3p, hsa-miR-744-5p, hsa-miR- 30a-5p e hsa-let-7g-5p. A partir das análises de predição de alvos, pôde-se observar que os quatro miRNAs, quando menos expressos simultaneamente, predizem ativação da agregação plaquetária. Além disso, os miRNAs hsa-miR- 423-5p, hsa-miR-744-5p e hsa-let-7g-5p mostraram correlação com o perfil lipídico dos pacientes que, por sua vez, apresentou influência nos valores de agregação compreendidos no T3 de resposta a ambos os medicamentos. Sendo assim, conclui-se que maiores taxas de agregação plaquetária podem estar indiretamente relacionadas com os padrões de expressão de hsa-miR- 423-3p, hsa-miR-744-5p e hsa-let-7g-5p. Sugere-se que a avaliação do perfil de expressão destes 3 miRNAs no sangue periférico de pacientes com DAC possa predizer resposta terapêutica inadequada ao AAS e ao clopidogrel / The antiplatelet therapy is often indicated for the prevention and treatment of cardiovascular diseases. Dual antiplatelet therapy with clopidogrel and acetylsalicylic acid (ASA) has often been adopted in patients with coronary artery disease (CAD), but it has been ineffective in a significant portion of the population with genotype of responders. This fact leads us to question whether other molecular mechanisms may be influencing the response to these drugs. Recent studies suggest that small non-coding RNA sequences known as microRNAs (miRNAs) may be closely related to response to drug-therapeutic treatment, controlling proteins involved in pharmacokinetics and pharmacodynamics. The aim of this study was to evaluate the profile of miRNAs in whole blood, looking to better clarify mechanisms involved in ASA and clopidogrel response. For this purpose, we selected CAD patients who showed different responses to dual antiplatelet therapy determined by aggregation test. Based on the phenotypes, the miRNA expression profiles were compared between the platelet aggregation values categorized into tertiles (T) of response. The T1 group consisted of responding patients, the T2 consisted of intermediate responders and the T3 consisted of non-responders. The miRNA profiles were obtained after next-generation sequencing and data were analyzed by Deseq2 package. Results showed that 18 miRNAs were differentially expressed between the two extreme tertiles. By Ingenuity® software prediction analysis, 10 miRNAs showed important targets related with activation and aggregation of blood platelets. Of the 10 miRNAs, 4 of them, which were down-expressed on sequencing, showed the same fold-regulation when expression profiles were analyzed by quantitative polymerase chain reaction (qPCR): hsa-miR-423-3p, hsa-miR-744-5p, hsa-miR-30a-5p and has-hsa- let-7g-5p. By target prediction analysis, it was observed that, when the four miRNAs are simultaneously down-expressed, they predict activation of platelet aggregation. Furthermore, hsa-miR-423-5p, hsa-miR-744-5p, and hsa-let-7g-5p showed correlation with the lipid profile of patients which, in turn, demonstrated influence in aggregation values reaching T3 of response to both drugs. Therefore, we concluded that increased platelet aggregation rates may be indirectly related to the expression profiles of hsa-miR-423-3p, hsa-miR-744-5p and hsa-let-7g-5p. It is suggested that the evaluation of the expression profile of these three miRNAs in the peripheral blood of patients with CAD may predict inadequate therapeutic response to aspirin and clopidogrel.

Expression of the Tudor domain containing protein 6 (TDRD6), which is restricted to the male germ line, starts at day 16 of spermatogenesis, i.e. in pachytene spermatocytes. TDRD6 is a 250 kDa protein, which we recently found to be cleaved at the C-terminal end during germ cell development, resulting in a 230 kDa product. Neither is the process of cleavage itself nor are the functions of the two different forms known. The 230 kDa isoform is the most prominent form in round spermatids, where it localizes to the chromatoid body (CB), i.e. a single filamentous, perinuclear granule. One characteristic component of the CB is the RNA helicase MVH. CBs contain components of the microRNA (miRNA) pathway, including Piwi-interacting RNAs (piRNAs), as well as MIWI, MIWI2, and MILI, the mouse homologs of the Piwi proteins, which bind piRNAs and also act in transposon regulation. We showed that TDRD6 interacts with MIWI and MILI in vitro, and a direct interaction with MVH was shown before. To reveal the function of TDRD6, we generated Tdrd6-/- mice, which lack the protein. These mice are generally healthy but the males are sterile, due to the absence of mature spermatozoa. The most striking intracellular phenotype of Tdrd6-/- mice is the highly aberrant architecture of chromatoid bodies in round spermatids. Tdrd6-/- CBs appear as diffuse, disrupted, and less condensed structures. Their interior is largely missing, and only a “ghost”-like structure remains, expected to be significantly impaired in function. Other CB components like MVH, MIWI and MILI are expressed in Tdrd6-/- testes, but they cannot localize to the disrupted CBs. This suggests a role for TDRD6 in assembling the chromatoid body complex by recruiting other proteins. The CB is important for storage and translational regulation of mRNA, through interaction with miRNAs. In Tdrd6-deficient testes 10 % of all known murine miRNAs are differently expressed, whereas most of the mature miRNAs are up-regulated, indicating less turnover, and thus, accumulation of mature miRNAs. Since some precursor miRNAs are up-regulated as well, we assume, that TDRD6 affects miRNA transcription most likely by indirectly influencing transcriptional regulation of miRNA genes. In Tdrd6-/- mice an overall abnormal mRNA gene expression pattern was observed by microarray analyses. Of all mis-regulated genes 36 % are located to the centromer-proximal region of Chr 8, and 11 % are located to the distal end of Chr 1. This mis-regulation might be due to a common transcriptional regulation. The orthologous regions on the human chromosomes show altered chromosomal structures in many different carcinomas. If TDRD6 plays a role in carcinogenesis has to be investigated.

My PhD dissertation reports my research investigations performed on bone loss projects. 2 projects are described in this thesis. One project dealing with the effect of adipogenic DKK1 on bone loss under normal and under a high-fat-diet (HFD). The 2nd projects aimed to investigate on the potential of miRNAs to be used as potential biomarkers to predict bone fragility in T1D.

This masters's thesis contains description of designed and implemented tool for classification of plant microRNA without genome. Properties of mature and star sequences in microRNA duplexes are used. Implemented method is based on clustering of RNA sequences (with CD-HIT) to mainly reduce their count. Selected representants from each clusters are classified using support vector machine. Performance of classification is more than 96% (based on cross-validation method using the training data).