Pseudomonas aeruginosa é um importante patógeno humano oportunista capaz de causar severas infecções em pacientes imunodeprimidos e em pacientes que apresentam fibrose cística. Para desencadear uma resposta efetiva contra a infecção pela P. aeruginosa é necessário uma primeira linha de reconhecimento desta bactéria pelo sistema imunológico inato. Apesar do desencadeamento da resposta imune inata ser benéfica no controle da infecção pela P. aeruginosa, esta resposta deve ser controlada. Estudos recentes têm começado a esclarecer como os miRNAs desempenham papéis fundamentais na regulação de processos como infecção, resposta imune e inflamação participando da modulação da resposta imune. Dada a relevância da bactéria P. aeruginosa nos processos infecciosos em humanos e estimulados pela necessidade de desvendar os mecanismos de regulação da reposta imune contra esta bactéria, o objetivo deste trabalho foi avaliar o papel do mmu-miR-155-5p e mmu-miR-146b-5p expressos em camundongos infectados pelas cepas ATCC 27853 e PA14 da bactéria P. aeruginosa. Para avaliar o nível de expressão dos mmu-miR-155-5p e mmu-miR-146b-5p e das citocinas IL-1β, IL-6 e IL-12 in vitro, o RNA total foi extraído de células Raw 264.7, macrófagos derivados da medula óssea (BMDM) e de células dendríticas derivadas da medula óssea (BMDC) para análise de PCR em tempo real. Após a infecção das células Raw 264.7, BMDMs e BMDCs foi possível observar que a expressão dos miRNAs e das citocinas ocorreu de forma dependente em cada tipo celular infectado. Para as análises in vivo camundongos C57BL/6 foram infectados via intratraqueal com as cepas ATCC 27853 e PA14 de P. aeruginosa para análises de UFCs, RT-qPCR, histopatologia e estereologia. Apenas a cepa PA14 foi recuperada no pulmão e baço dos animais, onde não foi observado variação na expressão do mmu-miR-155-5p nem aumento significativo de uma série de citocinas efetoras. As análises histopatológicas demostraram intenso processo inflamatório difuso apresentando número maior de células inflamatórias, diminuição no número de alvéolos e do volume da estrela quando comparados aos animais não infectados e animais infectados pela cepa avirulenta. No pulmão dos animais infectados com a cepa ATCC 27853, observou-se aumento de expressão de mmu-miR-155-5p e mmu-miR-146b-5p, das citocinas inflamatórias. A identificação das redes de regulação dos miRNAs em estudo mostrou importantes alvos diretos e indiretos associados à resposta imune inata que podem estar comprometidas durante a expressão diferencial de mmu-miR-155-5p e mmu-miR-146b-5p em favorecimento ou em detrimento da resolução da infecção pela P. aeruginosa. Os resultados obtidos até o momento permitem sugerir que a infecção pela P. aeruginosa exerce uma modulação na expressão de miRNAs e consequentemente na resposta imune contra esta bactéria e que a virulência de diferentes cepas de P. aeruginosa influenciam na expressão dos miRNAs mmu-miR-155-5p e mmu-miR-146b-5p, de citocinas pró-inflamatórias e na patologia. Mais estudos são necessários para desvendar os mecanismos pelos quais cepas virulentas da P. aeruginosa conseguem subverter a resposta imune e garantir a sua replicação no hospedeiro. / Pseudomonas aeruginosa is an important opportunistic human pathogen capable of causing severe infections in immunocompromised patients and in patients with cystic fibrosis. To trigger an effective response against infection by P. aeruginosa is required a first line of recognition of this bacterium by the innate immune system. Although the innate immune response is beneficial in the control of P. aeruginosa infection, this response should be controlled. Recent studies have begun to clarify how miRNAs play key roles in regulating processes such as infection, immune response and inflammation by participating in the immune response modulation. Due to the importance of the P. aeruginosa bacterium in infectious processes in humans and stimulated by the need to uncover the mechanisms of regulation of the immune response against this bacterium, the objective of this study was to evaluate the role of mmu-miR-155-5p and mmu-miR-146b -5p expressed in mice infected with strains ATCC 27853 and PA14 of the bacterium P. aeruginosa. To assess the level of expression of mmu-miR-155-5p and mmu-miR-146b-5p and the cytokines IL-1β, IL-6 and IL-12 in vitro, the total RNA was extracted from Raw 264.7 cells, bone marrow derived macrophages (BMDM) and bone marrow-derived dendritic cells (BMDC) for real-time PCR analysis. After infection of the Raw 264.7, BMDMs and BMDCs cells, it was possible to observe that the expression of miRNAs and cytokines occurred in a dependent manner in each infected cell type. For the in vivo analyzes C57BL / 6 mice were infected intratracheally with the strains ATCC 27853 and PA14 of P. aeruginosa for analysis of CFU, RT-qPCR, histopathology and stereology. Only the PA14 strain was recovered in the lungs and spleens of the animals, where no variation in the expression of mmu-miR-155-5p or a significant increase in a series of effector cytokines was observed. Histopathological analyzes demonstrated an intense diffuse inflammatory process, presenting a larger number of inflammatory cells, a decrease in the number of alveoli and the volume of the star when compared to uninfected animals and animals infected by the avirulent strain. In the lung of animals infected with ATCC 27853 strain, increased expression of mmu-miR-155-5p and mmu-miR-146b-5p, of inflammatory cytokines was observed. The identification of regulatory networks of the miRNAs under study showed important direct and indirect targets associated with the innate immune response that may be compromised during the differential expression of mmu-miR-155-5p and mmu-miR-146b-5p in favor or detriment of P. aeruginosa infection. The results obtained to date suggest that the infection by P. aeruginosa exerts a modulation in the expression of miRNAs and consequently in the immune response against this bacterium and that the virulence of different strains of P. aeruginosa influence the expression of miRNAs mmu-miR- 155-5p and mmu-miR-146b-5p, of proinflammatory cytokines and in pathology. Further studies are needed to uncover the mechanisms by which virulent P. aeruginosa strains can subvert the immune response and ensure replication in its host. / Fundação de Amparo à Pesquisa do Estado de Minas Gerais - FAPEMIG

In conclusion, downregulation of miR-124 and miR-383 is a frequent event in MB. Restoration of miR-124 and miR-383 inhibited cell growth and cell cycle progression in MB, suggesting these miRNAs harbor growth suppressor function. In addition, this study demonstrates SLC16A1 and PRDX3 are the direct targets of miR-124 and miR-383, respectively. Together, these data shed new light on miR-124 and miR-383 in MB pathogenesis, and suggest that miR-124/SLC16Al and miR-383/PRDX3 pathways are potential therapeutic targets for treatment of MB. / Medulloblastoma (MB) is an invasive embryonal tumor of the cerebellum, accounting for ∼20% of all primary pediatric brain tumors. The overall survival rate is 60--70% in standard-risk MB patients, but merely ∼30% in high-risk group. Patients who survive often suffer from long-term neurologic and cognitive deficits. New therapy is needed to reduce the mortality rate and to improve the quality of life of survivors. Understanding the molecular pathogenesis of MB is critical to the development of efficacious therapeutic treatment. / MicroRNAs (miRNAs) are short non-protein-coding RNAs that function in diverse biological processes through negative regulation on gene expression at the post-transcriptional level. Accumulative evidence indicates that miRNAs play an important role in the development of human cancers, with their deregulation resulting in altered activity of downstream tumor suppressors, oncogenes and other signaling molecules. / The aim of my project is to identify and characterize deregulated miRNAs located on chromosome 8p in MB. Our group has previously identified a minimally deleted region on 8p22-23.1 and partial or interstitial deletions at 8p22-23.2 in MBs. Despite extensive investigation, no promising candidate genes were identified in these. I questioned if miRNAs (miR-124, miR-383 and miR-320) were the targets on chromosome 8p. Quantitative expression analysis of 29 MBs revealed that miR-124 and miR-383 were downregulated in 72% and 79% of tumors, respectively, compared to normal cerebella. In contrast miR-320 expression was variable. Ectopic expression of miR-124 and miR-383 in MB cell lines (DAOY and ONS-76) showed significant growth inhibition. Cell cycle profiling revealed miR-124 and miR-383 inhibited cell cycle progression and induced apoptosis. These results suggest that miR-124 and miR-383 are potential growth suppressors. / To identify gene targets of miR-124, computational analysis was carried out. Twelve candidate genes predicted as miR124 target were selected for analysis. One candidate gene, SLC16A1, showed downregulation at transcript and protein levels after miR-124 transfection. Luciferase reporter assay demonstrated that miR-124 interacted at the 3' untranslated region of SLC16A1. These results suggest that miR-124 negatively regulates SLC16A1. Expression analysis further revealed that overexpression of SLC16A1 was common in MBs. / To identify miR-383 targets, global gene expression analysis and computational approach were applied. Two genes (PRDX3 and RBMS1 ) showed downregulation upon miR-383 transfection. Reporter assay confirmed that miR-383 interacted at 3' untranslated regions of these genes, suggesting that PRDX3 and RBMS1 are targets ofmiR-383. / Li, Ka Wai Kay. / Source: Dissertation Abstracts International, Volume: 73-06, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 236-293). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Choi, Pui Wah. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 202-232). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts also in Chinese.

O adenocarcinoma ductal pancreático (ADP) é uma doença altamente letal e agressiva. Alteração no perfil de acetilação das histonas envolvendo desacetilases de histonas (HDAC), assim como modificações da expressão de miRNAs podem levar ao desenvolvimento tumoral. Neste estudo, foi avaliada a expressão das HDAC1, HDAC2, HDAC3 e HDAC7 em ADP e amostras de tecido pancreático não tumoral (TN) usando análises experimentais e de banco de dados. Os níveis de expressão foram correlacionados com características clínico-patológicas dos pacientes e foi realizada uma investigação in silico de miRNAs reguladores de efeito das HDACs. Os níveis de expressão das HDACs foram avaliadas por qRT-PCR a partir de 25 amostras de ADP e 23 amostras de TN e a análise da expressão diferencial (ED) e correlação entre HDACs e miRNAs em ADP foi realizada utilizando perfis de expressão de seis microarranjos do Gene Expression Omnibus. Potenciais relações miRNA-HDACs foram coletadas em bases de dados de interação de miRNAs. Um valor de P<0,05 foi considerado estatisticamente significativo. Encontramos expressão reduzida em ADP comparado com TN para todas as HDACs analisadas, com P<0,05 para HDAC1, 2 e 3. Entretanto, os fold-changes foram muito baixos e provavelmente sem relevância biológica, e a expressão da HDAC2 e HDAC7 foi correlacionada com a idade ao diagnóstico. Nenhuma outra correlação entre a expressão das HDACs e características clínico-patológicas foi identificada. Análises de ED sugeriram significativa superexpressão das HDAC1, 2 e 7 e subexpressão da HDAC3, contudo todas apresentaram fold-changes pequenos. As análises dos bancos de dados identificaram 728 miRNAs como reguladores das HDACs. Interseções entre os conjuntos de miRNAs (GSE41369 e GSE43796) e aqueles recuperados da análise de expressão diferencial indicaram cinco miRNAs que influenciam a HDAC1 (miR-188-5p, miR-539, miR-708, miR -4269 e miR-3616-3p) e três que influenciam a HDAC2 (miR-4307, miR-944 e miR-195). A expressão das HDACs provavelmente não é um biomarcador de prognóstico robusto para o ADP, uma vez que a expressão diferencial entre os grupos é sutil. Ainda, este e estudos anteriores indicam nenhuma ou pouca associação entre a expressão HDACs e características clínico-patológicas relacionadas com o prognóstico. Finalmente, miRNAs provavelmente não estão exercendo um papel central na regulação da HDACs no ADP. / Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal and aggressive disease. The disruption of histone acetylation through histones deacetylases (HDACs) and expression regulation by miRNAs can lead to tumor development. In this study we assessed HDAC1, HDAC2, HDAC3 and HDAC7 expression in PDAC and non-tumoral tissue (NT) samples using experimental and databases analysis, correlated their expression levels with clinical and pathological features in patients and performed in silico investigation of HDACs regulation by miRNAs. Expression levels of HDACs were measured by qRT-PCR from 25 PDAC and 23 NT. An analysis of differential expression (DE) and correlation of HDACs and miRNAs in PDAC was performed using six Gene Expression Omnibus microarray datasets. Potential miRNA-HDACs relationships were collected from miRNA interaction databases. A P<0.05 was considered statistically significant. We found reduced expression in PDAC compared with NT for HDAC1, HDAC2 and HDAC3, with P<0.05. Expression levels of HDAC7 did not significantly differ between groups. However, fold-changes were very small and probably not biologically relevant. Only HDAC2 and HDAC7 were associated with age at diagnosis and no other associations between HDAC expression and clinical features were identified. DE analysis suggested significant up-regulation of HDAC1, HDAC2 and HDAC7, and down-regulation of HDAC3, albeit all of them associated with small fold changes. Databases analysis identified 728 miRNAs that could be HDACs regulators. Intersections among the set of miRNAs found in differential expression analysis of GSE41369 and GSE43796 and those retrieved from target prediction identified five miRNAs targeting HDAC1 (miR-188-5p, miR-539, miR-708, miR-4269 and miR-3616-3p) and three targeting HDAC2 (miR-4307, miR-944 and miR-195). HDACs expression is likely not a robust prognostic biomarker in PDAC since differential expression between groups is subtle. Also, this and previous studies indicate no or only very few associations between HDACs expression and clinicopathological features related to prognosis. Finally, miRNAs are probably not exerting a central role in HDAC regulation in PDAC.

Les tumeurs de vessie suivent deux voies de progression tumorale. La voie des tumeurs papillaires qui progressent rarement vers des tumeurs invasives mais qui récidivent très fréquemment et la voie des carcinomes in situ (CIS) qui progressent pour envahir le chorion puis le muscle. Les tumeurs infiltrant le muscle sont de mauvais pronostic et les traitements par chimiothérapie restent d’efficacité limitée. Il est alors important d’en comprendre les bases moléculaires. Les microRNAs sont d’importants régulateurs de l’expression post-transcriptionnelle des gènes. Des perturbations de leur expression et/ou de leur activité contribuent au développement tumoral en dérégulant l’expression de gènes clés dans les cancers. Nos travaux ont porté sur l’étude de l’expression et des fonctions des microRNAs dans la carcinogenèse urothéliale. Dans la première partie, nous avons choisi d’étudier, par une approche de gènes candidats, miR-155, un oncomiR dont l’expression dérégulée a été rapportée dans plusieurs cancers mais pas encore dans le cancer de la vessie. J’ai identifié une surexpression significative de ce miARN dans un sous-groupe de cancers invasifs de vessie. Ensuite, j’ai montré par des analyses fonctionnelles, le rôle de miR-155 dans l’invasion et la migration tumorale mais pas dans la prolifération cellulaire. Dans la deuxième partie, nous avons utilisé une approche plus globale. J’ai d'abord effectué une revue extensive de la littérature pour rechercher les miR dont l'expression avait été montrée comme dérégulée dans les cancers de vessie et/ou les miR impliqués fonctionnellement dans ce cancer. J’ai ensuite réalisé une analyse multiparamétrique en intégrant les données d'expression de ces miR, les données anatomopathologiques et moléculaires (stade et grade, statut mutationel de TP53 et FGFR3, phénotype épigénétique MRES et signature CIS) et les données du transcriptome (puces Affymetrix U133 Plus 2.0), des altérations génomiques (puces Illumina 370.000 sondes) et de méthylation (puces Illumina 27.000 sondes). Ce travail m’a permis, d'identifier des miR associés à l'une des deux voies de progression identifiées dans les cancers de vessie et de proposer des cibles candidates de ces miR. La recherche des altérations épigénétiques pouvant affecter l’expression de ces miR a permis d’identifier une association significative entre l’expression d’un miARN (miR-17-5p) et la méthylation d’un promoteur. En revanche les altérations génétiques n’ont été associées à aucune expression de miR. Ce travail propose une liste de très bons candidats miARNs pour lesquels des études fonctionnelles pourront être envisagées au-delà de mon travail de thèse. / Bladder tumors are characterized by two progression pathways. The first pathway leads to the developpement of papillary tumors, which are at high risk of recurrence but that rarely progress to invasive tumors. Another pathway involves carcinoma in situ (CIS), which often progresses by first invading the lamina propria and then the muscle. Tumors infiltrating the muscle have a poor prognosis and chemotherapy regimens are of limited benefits. It is yet important to understand the molecular basis underlying these events. The microRNAs are important regulators of post-transcriptional gene expression. Alteration in their expression and /or activity is believed to contribute to tumor development by deregulating the expression of cancer-related genes. Our work has been focused on studying the expression and function of microRNAs in urothelial carcinogenesis. In the first part, we employed a candidate gene approach to study miR-155, a oncomiR whose dysregulated expression has been reported in many cancers, but not in bladder cancer. I identified a significant overexpression of this miRNA in a subgroup of invasive bladder cancers. Next, I demonstrated a role for miR-155 in tumor invasion and migration, without any apparent effect on cell proliferation. In the second part, we used a more comprehensive approach in which I first conducted an extensive review of the literature to search for miR whose expression was already found to be deregulated and/or miR functionally involved in bladder cancer. I then performed a multiparametric analysis by integrating expression data of miR, pathological and molecular data (stage and grade, mutational status of FGFR3 and TP53, MRES epigenetic phenotype and CIS signature) data of the transcriptome (Affymetrix U133 Plus 2.0), genomic alterations (370,000 chips Illumina probes) and methylation (Illumina chips 27,000 probes). This work allowed us to identify miRs associated with one or the other pathway linked to progression of bladder cancer and also, it revealed candidate targets for these miRs. The search for epigenetic alterations capable to affect the expression of those miRs showed significant association between expression of a particular miRNA (miR-17-5p) and the methylation of its promoter. Genetic alterations however, have failed to associate with expression of miR. Finally, this work suggests a list of good candidates miRNAs for which future functional studies should help to get insight into the role of these miRNAs

Ng, Hin Yi Winnie. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 137-140). / Abstracts in English and Chinese. / Acknowledgements --- p.ii / List of Tables --- p.iii / List of Figures --- p.iv / Abstract in English --- p.vi / Abstract in Chinese --- p.ix / Table of Contents --- p.xi / Chapter CHAPTER 1: --- INTRODUCTION --- p.1 / Chapter 1.1 --- Medulloblastoma (MB) --- p.1 / Chapter 1.1.1 --- Definition of Medulloblastoma --- p.1 / Chapter 1.1.2 --- Pathological Classification --- p.2 / Chapter 1.1.3 --- Current Treatment --- p.3 / Chapter 1.1.4 --- Molecular Pathology --- p.4 / Chapter 1.1.5 --- Molecular Classification of MB --- p.7 / Chapter 1.2 --- MicroRNAs (miRNAs) --- p.9 / Chapter 1.2.1 --- Biogenesis --- p.9 / Chapter 1.2.2 --- Functions --- p.10 / Chapter 1.2.3 --- MicroRNAs & Cancers --- p.10 / Chapter 1.2.4 --- Aberrant Expressions of MicroRNAs in Medulloblastoma --- p.12 / Chapter 1.2.5 --- MiR-106b-25 Cluster in MB --- p.13 / Chapter 1.2.6 --- miR-106b-25 Cluster in Regulating Target Genes --- p.15 / Chapter 1.2.7 --- Application of Regulatory miRNAs --- p.16 / Chapter 1.3 --- Target Gene Identification --- p.18 / Chapter 1.3.1 --- Recent Molecular Advances in Target Gene Identification --- p.18 / Chapter 1.3.2 --- Importance of Target Gene Identification --- p.19 / Chapter CHAPTER 2: --- AIMS OF STUDY --- p.21 / Chapter CHAPTER 3: --- COMPUTATIONAL TARGET PREDICTION --- p.23 / Chapter 3.1 --- Introduction- Computational Approach --- p.23 / Chapter 3.2 --- Methods --- p.27 / Chapter 3.2.1 --- Prediction Algorithms --- p.27 / Chapter 3.2.1.1 --- EIMMo2 --- p.27 / Chapter 3.2.1.2 --- miRDB --- p.27 / Chapter 3.2.1.3 --- miR-Tar-miRanda --- p.28 / Chapter 3.2.1.4 --- miR-Tar-RNAhybrid --- p.28 / Chapter 3.2.1.5 --- Diana-microT --- p.29 / Chapter 3.2.1.6 --- Pic-Tar --- p.29 / Chapter 3.2.1.7 --- TargetScan 4.2 --- p.29 / Chapter 3.2.2 --- Cell Culture --- p.30 / Chapter 3.2.2.1 --- Cell Lines --- p.30 / Chapter 3.2.2.2 --- Cell Counts --- p.31 / Chapter 3.2.3 --- Transfections --- p.31 / Chapter 3.2.3.1 --- Transfection of MicroRNA Inhibitors --- p.31 / Chapter 3.2.3.1.1 --- Transfection Efficiency of Lipofectamine2000 --- p.32 / Chapter 3.2.3.1.2 --- Transfection of MicroRNA Inhibitors for Real-time PCR --- p.32 / Chapter 3.2.3.1.3 --- Transfection of MicroRNA Inhibitors for Western Blotting --- p.33 / Chapter 3.2.3.2 --- Co-transfection of Plasmid and MicroRNA Inhibitors --- p.33 / Chapter 3.2.3.2.1 --- Blocking Efficiency of MicroRNA Inhibitors --- p.33 / Chapter 3.2.3.2.2 --- Co-transfection of Target Gene Expression Vector and MicroRNA Inhibitors --- p.34 / Chapter 3.2.4 --- Real-time PCR Amplification --- p.35 / Chapter 3.2.4.1 --- Total RNA Extraction from Cell Lines --- p.35 / Chapter 3.2.4.2 --- Stemloop miRNA Taqman qRT-PCR Analysis --- p.36 / Chapter 3.2.4.3 --- Reverse Transcription --- p.37 / Chapter 3.2.4.4 --- Real-time PCR Target Gene Expression --- p.38 / Chapter 3.2.5 --- Cloning of Potential Target Genes into pMIR Luciferase Expression Vector --- p.39 / Chapter 3.2.5.1 --- High-Fidelity PCR Amplification of yUTRs --- p.41 / Chapter 3.2.5.2 --- PCR Purification of Amplified PCR Product --- p.42 / Chapter 3.2.5.3 --- Restriction Enzyme Digestions --- p.42 / Chapter 3.2.5.4 --- Ligation of 3'UTR to Expression Vector --- p.43 / Chapter 3.2.5.5 --- Transformation --- p.43 / Chapter 3.2.5.6 --- Preparation of the Cloned Plasmid --- p.43 / Chapter 3.2.5.7 --- Sequencing of the Cloned Plasmid --- p.44 / Chapter 3.2.6 --- Site-directed Mutagenesis --- p.45 / Chapter 3.2.7 --- Dual-Luciferase Assay --- p.47 / Chapter 3.2.8 --- Western Blot Analysis --- p.47 / Chapter 3.3 --- Results --- p.49 / Chapter 3.3.1 --- Expression Levels of miR-106b-25 Cluster in MB Cell Lines --- p.49 / Chapter 3.3.2 --- Evaluation of Transfection Efficiency Using Lipofetamine2000 --- p.51 / Chapter 3.3.3 --- Blocking Efficiency of MicroRNA Inhibitors --- p.52 / Chapter 3.3.4 --- Target Prediction List --- p.53 / Chapter 3.3.5 --- Recognition Sites of Potential Targets --- p.55 / Chapter 3.3.6 --- Expression Levels of ZNFX1 in MB Cell Lines --- p.56 / Chapter 3.3.7 --- Transcriptional Regulation of ZNFXl and DNAJB12 --- p.57 / Chapter 3.3.8 --- Verification of Potential Target Genes --- p.59 / Chapter 3.3.9 --- Identification of Critical Target Sites --- p.61 / Chapter 3.3.10 --- Effects of Anti-microRNA Inhibitors on ZNFX1 Protein Levels --- p.66 / Chapter 3.4 --- Discussion --- p.67 / Chapter CHAPTER 4: --- EXPERIMENTAL APPROACH IN INDENTIFYING POTENTIAL TARGETS --- p.77 / Chapter 4.1 --- Introduction- Experimental Approach --- p.74 / Chapter 4.2 --- Methods --- p.79 / Chapter 4.2.1 --- Isolation of cDNA Clone Library --- p.79 / Chapter 4.2.1.1 --- Preparation of Cytoplasmic Extracts --- p.79 / Chapter 4.2.1.2 --- Reverse Transcription Using Endogenous miRNA as Primers --- p.81 / Chapter 4.2.1.3 --- Collection of Polynucleotides --- p.82 / Chapter 4.2.1.4 --- Synthesis of Second-strand cDNAs --- p.82 / Chapter 4.2.1.5 --- PCR Purification of Double-stranded cDNAs --- p.83 / Chapter 4.2.1.6 --- Restriction Endonuclease Digestion --- p.84 / Chapter 4.2.1.7 --- Ligation to Adaptor --- p.85 / Chapter 4.2.1.8 --- PCR Amplification with Biotin-labelled miRNA PCR Primers --- p.86 / Chapter 4.2.1.9 --- Capture of Biotin-labelled PCR Fragments --- p.88 / Chapter 4.2.1.10 --- Introducing NotI Recognition Sequences --- p.88 / Chapter 4.2.1.11 --- Cloning into the pCR2.1 Vector --- p.89 / Chapter 4.2.1.12 --- Ligation of the cDNA Fragments and the pCR2.1 Vector --- p.90 / Chapter 4.2.1.13 --- Transformation --- p.90 / Chapter 4.2.1.14 --- Preparation of Purified Plasmids --- p.91 / Chapter 4.2.1.15 --- Sequencing Analysis of the cDNA Clone Library --- p.91 / Chapter 4.2.2 --- Real-time PCR Target Gene Expression in Cell Lines --- p.92 / Chapter 4.2.3 --- Real-time PCR Target Gene Expression Upon Inhibition of miR-106b --- p.92 / Chapter 4.2.4 --- Cloning of Potential Target Genes into pMIR Luciferase Expression Vector --- p.93 / Chapter 4.2.5 --- Site-directed Mutagenesis --- p.94 / Chapter 4.2.6 --- Luciferase Reporter Assay --- p.94 / Chapter 4.3 --- Results --- p.95 / Chapter 4.3.1 --- Sequencing Analysis of the cDNA Clone Library --- p.95 / Chapter 4.3.2 --- Expression Levels of Candidate Genes in MB Cell Lines --- p.100 / Chapter 4.3.3 --- Effects of Anti-miR-106b Inhibitors on 3'UTR of Target Genes --- p.101 / Chapter 4.3.4 --- Verification of Candidate Genes --- p.103 / Chapter 4.3.5 --- Verification of Target Sites with Site-directed Mutagenesis --- p.104 / Chapter 4.4 --- Discussion --- p.107 / Chapter CHAPTER 5: --- FUNCTIONAL ASSAYS --- p.111 / Chapter 5.1 --- Introduction- Functional Investigation of miR-106b-25 Cluster --- p.111 / Chapter 5.2 --- Methods --- p.113 / Chapter 5.2.1 --- Cell Culture --- p.113 / Chapter 5.2.2 --- Over-expression of miR-106b Mimic --- p.113 / Chapter 5.2.3 --- MTT Assay --- p.114 / Chapter 5.2.4 --- IC50 of Cisplatin --- p.115 / Chapter 5.2.5 --- MTT Assay with Cisplatin Treatment --- p.115 / Chapter 5.2.6 --- Cell Cycle --- p.116 / Chapter 5.2.7 --- BrdU Cell Proliferation Assay --- p.117 / Chapter 5.2.8 --- Wound Healing Assay --- p.117 / Chapter 5.3 --- Results --- p.119 / Chapter 5.3.1 --- Effects of Inhibition of miR-106b-25 Cluster on Cell Growth. --- p.119 / Chapter 5.3.2 --- Cell Cycle Distribution Analysis --- p.121 / Chapter 5.3.3 --- Sensitivity to Cisplatin --- p.123 / Chapter 5.3.4 --- Cell Proliferation Assay --- p.124 / Chapter 5.3.5 --- Cell Motility --- p.126 / Chapter 5.3.6 --- Efficiency of Over-expression Using miR-106b Mimic --- p.129 / Chapter 5.3.7 --- Effects of miR-106b on Cell Growth --- p.130 / Chapter 5.4 --- Discussion --- p.131 / Chapter CHAPTER 6: --- CONCLUSION --- p.135 / REFERENCE --- p.137

Em bovinos, o desenvolvimento embrionário pré implantacional depende das funções do endométrio bovino que tem suas funções mediadas por uma complexa interação da ação e dos efeitos dos hormônios esteroides ovarianos E2 e P4. Estes hormônios regulam a expressão gênica e controlam o ambiente uterino modulando, entre outros, a expressão de microRNAs e a rede de citocinas relacionadas ao sistema imune. Os objetivos do presente trabalho foram abordados em dois capítulos, sendo (I) comparar os efeitos dos distintos ambientes endócrinos peri-ovulatórios sobre a expressão de microRNAs (II) e na modulação do sistema IL1/TLR no endométrio bovino nos dias 4 e 7 após a indução da ovulação. Para isso, controlou-se farmacologicamente o crescimento do folículo objetivando induzir a ovulação de folículos de maior diâmetro (grupo folículo grande-CL grande, FG-CLG) ou de menor diâmetro (grupo folículo pequeno-CL Pequeno, FP-CLP). Vinte e duas vacas multíparas nelore, foram pré-sincronizadas, metade destes animais foram destinados para o grupo FG-CLG e receberam uma dose de prostaglandina F2&#945; (PGF) e um dispositivo de progesterona, juntamente com benzoato de estradiol no D10. No momento da retirada dos dispositivos de progesterona (entre D1,75 e D2,5) todos os animas receberam uma dose de PGF. A ovulação foi induzida com acetato de buserelina (D0). O que diferiu entre os tratamentos foi que os animais do grupo FP-CLP não receberam uma dose de PGF no D10 e o momento da retirada dos dispositivos foi entre D1,25 e o D1,5. No capítulo I, o a expressão de microRNAs foi determinada por qPCR nos dias 4 e 7. Dos 90 microRNAs testados, 21 apresentaram se up-regulated e dois down-regulated no grupo FG-CLG (P<0.1) no D4. No D7, quatro microRNAs foram diferentemente expressos, sendo um up-regulated e três down-regulated no grupo FG-CLG (P<0.1) no D7. Para os microRNAs diferentemente expressos determinou-se mRNA-alvos preditos. Uma análise de ontologia demonstrou que os mRNAs-alvos apresentaram enriquecimento funcional na via dos receptores de hormônios esteroides, entre outras. No capítulo II, o sistema IL1/TLR foi avaliado quanto a abundância de transcriptos envolvidos neste sistema, do microRNA bta-mir-155 e das proteínas IL1&#946; e IL1R1. A abundância relativa de mRNA apresentou diferença (P<0.1) na abundância dos mRNAs de IL1R1, TAB1 e FOXP3, das proteínas IL1&#946; e IL1R1, sendo essas moléculas up-regulated no grupo FG-CLG. O microRNA bta-mir-155 foi down-regulated no grupo FG-CLG (P<0.1). Diante disto, pode-se concluir que o ambiente endócrino peri-ovulatório determina o perfil de expressão de microRNAs e modula o sistema IL1/TLR no endométrio bovino / In cattle, the pre implantation embryo development depends on the functions of the bovine endometrium that has its functions mediated by a complex interaction of action and the effects of ovarian steroid hormones E2 and P4. These hormones regulate gene expression and control the modulating uterine environment among others, the expression of microRNAs and the network of cytokines related to the immune system. The objectives of this study were discussed in two chapters, (I) to compare the effects of different peri-ovulatory endocrine environment on the expression of microRNAs (II) and modulation of the IL-1 system / TLR in bovine endometrium on days 4 and 7 after induction of ovulation. For this, it was controlled pharmacologically follicle growth aiming to induce ovulation of follicles larger diameter (great grand-CL follicle group, FG-CLG) or smaller in diameter (small-CL Small follicle group, FP-PLC). Twenty two nelore multiparous cows were pre-sync, half of these animals were used for the FG-NCG group and received a dose of F2á prostaglandin (PGF) and progesterone device along with oestradiol benzoate in D-10. Upon withdrawal of progesterone devices (between 1.75 and D-D-2,5) all animas received a dose of PGF. Ovulation was induced with buserelin acetate (D0). What differed between treatments was that animals FP-CLP group did not receive a dose of PGF in the D-10 and the time of removal of the devices was between D-1,25 and D-1.5. In Chapter I, the expression of microRNAs was determined by qPCR on 4 and 7. Of the 90 microRNAs tested, 21 showed was up-regulated and down-regulated in two FG-CLG group (P <0.1) in the D4. In D7 four microRNAs were differently expressed, one up-regulated and down-regulated in three FG-CLG group (P <0.1) at D7. For differently expressed microRNAs was determined predicted mRNA-target. An ontology analysis showed that the mRNA-targets had functional enrichment in via the steroid hormone receptors, among others. In Chapter II, the IL-1 / TLR system was evaluated as the abundance of transcripts involved in this system, the bta-mir-155 microRNA and IL1&#946; and IL1R1 proteins. The relative abundance of mRNA was different (P <0.1) in the abundance of mRNAs IL1R1, TAB1 and FOXP3, the IL1&#946; and IL1R1 proteins, and these up-regulated molecules in the FG-CLG group. The bta-mir-155 microRNA was down-regulated in the FG-CLG group (P <0.1). Given this, we can conclude that the peri-ovulatory endocrine milieu determines the profile of microRNA expression and modulates the IL1 / TLR system in bovine endometrium

Anemia falciforme (AF) é considerada a doença monogênica mais prevalente no Brasil, e resulta de uma mutação pontual no gene da beta-globina que leva à produção de uma molécula de hemoglobina anormal (HbS). A HbS polimeriza quando submetida a baixas tensões de oxigênio, precipitando e causando a deformação dos eritrócitos pois torna rígida sua membrana plasmática que apresentará uma série de alterações e danos. Além disso, a falcemização dos eritrócitos ocasiona anemia severa, lesão de isquemia/reperfusão, superprodução de espécies reativas de oxigênio, inflamação e vaso-oclusão (VO). A VO manifesta-se clinicamente como crises de dor, ou crises vaso-oclusivas (CVOs) que pode levar ao bloqueio de vasos e capilares e ao comprometimento de órgãos. Estes pacientes possuem alta susceptibilidade a infecções, principalmente na infância. Os mecanismos envolvidos no desenvolvimento da VO, no entanto, não estão totalmente elucidados. Estudos acerca deste tema têm sugerido que a VO seria o resultado da interação entre eritrócitos falcêmicos, leucócitos, plaquetas, células endoteliais e substâncias presentes no plasma dos indivíduos afetados. Tais observações como que AF seria o resultado de uma resposta inflamatória exacerbada que estes indivíduos desenvolvem, levaram à formulação da hipótese de que a anemia falciforme se comporta como uma condição inflamatória crônica e sugerindo que uma modulação do sistema imune poderá ser útil para a regulação da sintomatologia clínica. Salientando a carência de dados na literatura referentes à correlação de polimorfismos descritos para genes do sistema imune e a patofisiologia da AF, nosso estudo tem como objetivo principal: Analisar a correlação para polimorfismos descritos para genes do sistema imune e correlacionar-lho com a patofisiologia da AF. Sabemos que o HLA-G é uma molécula HLA não-clássica, que mostrou ser expressa em sítios de inflamação e nas doenças inflamatórias. Na região promotora além de ser altamente polimórfica, encontramos a região UTR 3' que parece desempenhar um papel importante na regulação da expressão do HLA-G. Então, dentre dos polimorfismos avaliados específicamente temos os dos genes HLA-G (14pb) e MiRNA (+3142). Nossos resultados indicam que os polimorfismos do HLA-G de 14pb e +3142 em 93 pacientes com AF, 21 pacientes apresentaram uma infecção pelo VHC e 16 pacientes com AF (22,2%) eram homozigotos para o genótipo +3142C e nenhum deles era positivo para HCV. Nenhum dos resultados obtidos indicou qualquer tipo de imunodeficiência mas pelo contrário, sugerem a existência de uma tendência inflamatória crônica na clínica da AF. Assim fica evidente a importância do polimorfismo +3142 sobre a susceptibilidade a infecções entre os pacientes com SCD.

Os Transtornos do Espectro do Autismo (TEA) reúnem um conjunto de alterações no desenvolvimento, caracterizado por dificuldades nos níveis de interação social, linguagem, comunicação, processo imaginativo e no repertório restrito de interesses e atividades. O autismo ainda possui etiologia desconhecida e até o momento nenhum tratamento ou marcador clínico para diagnóstico foi identificado. Apesar de alta herdabilidade, há uma alta heterogeneidade na sua arquitetura genética. Fatores ambientais podem contribuir com o aumento do risco do desenvolvimento do autismo, incluindo a exposição pré-natal a teratógenos como o ácido valpróico (VPA). MicroRNA são pequenos RNA não codificadores com aproximadamente 19-25 nucleotídeos que atuam como reguladores da expressão gênica, podendo agir no controle de diversos processos biológicos. O objetivo deste estudo foi avaliar os perfis de expressão de alguns miRNA no sangue total do modelo animal de autismo induzido por exposição pré-natal ao ácido valpróico (VPA). Além disso, as funções potenciais dessas moléculas foram avaliadas através da correlação com seus genes alvos, os quais poderiam, por sua vez, estar associados com os fundamentos da patofisiologia do autismo. Mudanças nos níveis de miRNA induzidas pelo VPA podem contribuir para algumas características relacionadas ao autismo. Os alvos preditos e validados dos miRNA que se mostraram alterados incluem alguns genes que estão envolvidos na síntese de proteínas, na resposta imune, inflamatória e à hipóxia, no desenvolvimento dos linfócitos e do sistema nervoso entérico e na transmissão sináptica. O presente estudo sugere que a avaliação da expressão de miRNA pode ser utilizada para a identificação potencial de rotas alteradas no autismo e podem constituir marcadores para a detecção e diagnóstico, além de auxiliar nas investigações sobre mecanismos de ação molecular algumas em vias de sinalização supostamente alterados no autismo. / The Autism Spectrum Disorders (ASD) assemble a group of developmental disorders characterized by difficulties in levels of social interaction, language, communication, imaginative process and restricted repertoire of activities and interests. The etiology of autism has still unknown and no treatment or clinical markers for diagnosis were identified. Despite its high heritability, there is a high heterogeneity in its genetic architecture. Environmental factors may contribute to the increased risk of developing autism, including prenatal exposure to teratogens such as valproic acid (VPA). MicroRNA are small noncoding RNA with approximately 19-25 nucleotides that act as regulators of gene expression and may act in the control of many biological processes. The aim of this study was to evaluate the expression profile of miRNA levels in whole blood samples from animal model of autism by prenatal exposure to VPA. In addition, some potential roles of these miRNA were analyzed through correlation with their target genes, which could, in turn, be associated with the basis of the pathophysiology of autism. Changes in the miRNA levels induced by VPA may contribute to some features related to autism. The predicted and validated targets of the altered miRNA included some genes that are involved in protein synthesis, immune, inflammatory and hypoxia responses, lymphocytes and enteric nervous system development and synaptic transmission. The present study suggests that the assessment of the expression of miRNA may be used to perform the identification of potential pathways altered in autism and may constitute markers for the detection and diagnostics, in addition to studies related therapies and mechanistic investigations on signaling pathways putatively altered in autism.

Diao, Shu. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 72-73). / Abstracts in English and Chinese. / 摘要 --- p.i / Abstract --- p.ii / Acknowledgements --- p.iv / Contents --- p.viii / Chapter Chapter 1 --- Introduction and Background / Chapter 1.1 --- General introduction to miRNA --- p.1 / Chapter 1.1.1 --- The discovery and biogenesis of miRNA --- p.1 / Chapter 1.1.2 --- The function of miRNA --- p.3 / Chapter 1.2 --- The liver-specific miRNA : miR-122a --- p.5 / Chapter 1.2.1 --- Discovery and biogenesis of miR-122a --- p.5 / Chapter 1.2.2 --- "miR-122a, a liver specific miRNA" --- p.6 / Chapter 1.2.3 --- miR-122a and Hepatocellular carcinoma (HCC) --- p.6 / Chapter 1.2.4 --- Therapeutic opportunities and challenges of miR-122a --- p.7 / Chapter 1.3 --- Techniques and approaches to study miRNAs --- p.8 / Chapter 1.3.1 --- Discovery of novel miRNA --- p.8 / Chapter 1.3.2 --- Detection of miRNA --- p.9 / Chapter 1.3.3 --- Functional study of miRNAs --- p.10 / Chapter 1.3.4 --- Prediction and validation of miRNA targets --- p.10 / Chapter 1.4 --- References --- p.12 / Chapter Chapter 2 --- Materials and methods / Chapter 2.1 --- Cell culture --- p.18 / Chapter 2.2 --- Cell transfection --- p.18 / Chapter 2.3 --- RNA extraction --- p.18 / Chapter 2.4 --- Plasmid extaction --- p.19 / Chapter 2.5 --- Competent cell preparation --- p.19 / Chapter 2.6 --- Bacterial Transformation --- p.20 / Chapter 2.7 --- Purification of DNA fragments from agarose gel --- p.21 / Chapter 2.8 --- Genomic DNA extranction from MIHA cell --- p.21 / Chapter 2.9 --- Real-time RT-PCR analysis --- p.21 / Chapter 2.10 --- Lenti-vector Construction for miRNA expression --- p.22 / Chapter 2.11 --- Lentivirus production --- p.22 / Chapter 2.12 --- Lentiviral vector titering --- p.23 / Chapter 2.13 --- Bradford protein assay --- p.23 / Chapter 2.14 --- Western Blot --- p.24 / Chapter 2.14.1 --- Sample preparation --- p.24 / Chapter 2.14.2 --- Gel electrophoresis --- p.24 / Chapter 2.14.3 --- Blocking --- p.25 / Chapter 2.14.4 --- Incubation with Primary and Secondary Antibodies --- p.25 / Chapter 2.14.5 --- Substrate Incubation --- p.26 / Chapter 2.14.6 --- Exposeto x-ray film --- p.26 / Chapter 2.15 --- MTT Cell Proliferation Assay --- p.26 / Chapter 2.16 --- Apoptosis analysis :DAPI Staining --- p.26 / Chapter 2.17 --- 2-D Protein Gel Electrophoresis and MS --- p.26 / Chapter 2.17.1 --- Materials --- p.27 / Chapter 2.17.2 --- Protein extraction --- p.27 / Chapter 2.17.3 --- 2-D Electrophoresis --- p.27 / Chapter 2.17.4 --- Gel staining and image analysis --- p.28 / Chapter 2.17.5 --- In-gel protein digestion with trypsin --- p.28 / Chapter 2.17.6 --- MALDI-TOF MS and database search --- p.28 / Chapter 2.18 --- Statistical Analysis --- p.29 / Chapter Chapter 3 --- Expression of HCC-associated miRNA in HCC cell lines / Chapter 3.1 --- Introduction --- p.30 / Chapter 3.2 --- Experimental Section --- p.31 / Chapter 3.2.1 --- Cell culture --- p.31 / Chapter 3.2.2 --- RNA extraction --- p.31 / Chapter 3.2.3 --- miRNA-specific quantitative Real-time PCR --- p.31 / Chapter 3.3 --- Results and Discussion --- p.32 / Chapter 3.3.1 --- Expression of miR-let7a in HCC cells --- p.32 / Chapter 3.3.2 --- Expression of miR-221 in HCC cells --- p.33 / Chapter 3.3.3 --- "Expression of miR-122a,a liver-specific miRNA in HCC cells" --- p.34 / Chapter 3.4 --- Conclusions --- p.35 / Chapter 3.5 --- References --- p.36 / Chapter Chapter 4 --- Ectopic overexpression of miR-122a in HCC cells / Chapter 4.1 --- Introduction --- p.38 / Chapter 4.2 --- Experimental Section --- p.38 / Chapter 4.2.1 --- Overexpression of miR-122a with mimics --- p.38 / Chapter 4.2.2 --- Lentivirus-mediated miR-122a expression --- p.40 / Chapter 4.2.3 --- RNA extraction --- p.44 / Chapter 4.2.4 --- Expression level of miR-122a after transduction --- p.44 / Chapter 4.2.5 --- Western Blot --- p.44 / Chapter 4.3 --- Result and discussion --- p.46 / Chapter 4.3.1 --- Expression level of miR-122a after transfection --- p.46 / Chapter 4.3.2 --- Lentivirus-mediated miR-122a expression --- p.47 / Chapter 4.4 --- Conclusions --- p.52 / Chapter 4.5 --- References --- p.54 / Chapter Chapter 5 --- Gain-of-function study of miR-122a in HCC cells / Chapter 5.1 --- Introduction --- p.55 / Chapter 5.2 --- Experimental Section --- p.56 / Chapter 5.2.1 --- Cell culture --- p.56 / Chapter 5.2.2 --- Cell transfection --- p.56 / Chapter 5.2.3 --- Lentiviral vector transduction --- p.56 / Chapter 5.2.4 --- MTT Cell Proliferation Assay --- p.56 / Chapter 5.2.5 --- Apoptosis analysis - DAPI Staining --- p.57 / Chapter 5.2.6 --- 2-D Protein Gel Electrophoresis and MS --- p.57 / Chapter 5.2.7 --- miRNA target prediction using bioinformatic approaches --- p.59 / Chapter 5.3 --- Result and discussion --- p.59 / Chapter 5.3.1 --- Phenotypic changes of HepG2 cells caused by ectopic overexpression of miR-122a --- p.59 / Chapter 5.3.2 --- miR-122a target prediction using bioinformatic approaches --- p.62 / Chapter 5.3.3 --- Experimental validation of miR-122a targets by proteomics approach --- p.67 / Chapter 5.4 --- Conclusion --- p.70 / Chapter 5.5 --- References --- p.71 / Appendix --- p.73