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In situ hybridization is used for detection of RNA expression when conservation of tissue architecture is important. Most in situ hybridization protocols are written for tissues from animals (i.e., rat) which can be harvested and preserved rapidly. In contrast, human tissue is more difficult to obtain, hence in situ hybridization experiments must frequently be performed with less than optimal tissue preservation. This procedure details hybridization of a radiolabeled single-stranded RNA probe (riboprobe) to complementary sequences of cellular RNA in human tissue sections. This method enables detection of rare mRNA species in specific cell types of human tissue, offering distinct advantages over other in situ methods due to increased sensitivity. In particular, we have found that UV cross-linking and ribonuclease treatment protocols need to be altered for human tissues to ensure successful results, making this protocol unique to those previously described. In situ hybridization experiments can be performed using either DNA or RNA probes. RNA probes are advantageous since they form stable hybrids, are single-stranded, have little or no reannealing during hybridization, and can be synthesized to high specific activity. RNA probes can be readily created utilizing SP6, T3, or T7 promoters in both sense and antisense orientations to provide non-specific (control) and specific probes. Disadvantages of RNA riboprobes include a tendency for RNA to stick non- selectively more than DNA, and degradation by RNase (hence strict adherence to RNase-free precautions is mandatory during most of the protocol). The following protocol includes: (1) preparation of human tissues (tissue fixation and sectioning are highlighted as critical for probe penetration, preservation of tissue architecture, retention of tissue RNA, and overall success); (2) generation of radiolabeled riboprobes (total incorporation of radionucleotide is important to increase sensitivity; 35S was chosen as a compromise between excellent sensitivity, cellular resolution, and required exposure times (compared with 32p or 3H); non-isotopic methods have not been tested in a side-by-side comparison with 35S in human tissues by us, but theoretically might offer faster exposure times while maintaining high resolution); (3) hybridization conditions (stringency, temperature, washes, tissue dehydration); and (4) sample visualization (application of photographic emulsion, developing, fixing, staining, and counterstaining of individual slides).

Cleavage and polyadenylation are two necessary messenger RNA (mRNA) maturation steps for gene expression. The Cleavage and Polyadenylation Specificity Factor (CPSF) complex, which recognizes the AAUAAA polyadenylation signal and executes the cleavage reaction, is indispensable for these two processes. In this thesis, I describe my study of the regulation and functions of two non-canonical isoforms of the CPSF subunit WDR33. In addition, I provide detailed analyses on our current knowledge of CPSF subunits’ functions and their influences on a diverse collection of biological processes and conditions. In Chapter1, I provide a general introduction to cleavage and polyadenylation, WDR33, innate immune response via molecular pattern recognition, and the cGAS-STING pathway. Chapter 2 presents my original research on non-canonical WDR33 isoforms, termed WDR33v2 (V2) and WDR33v3 (V3). I determined that their mRNAs are produced by alternative polyadenylation. Both V2 and V3 proteins lack multiple WD repeats, but they can interact with and stabilize each other. This is a novel mode of protein-protein interaction, which I termed WD repeat complementation (WDRC). Unexpectedly, I found that even though V2 and V3 are isoforms of a polyadenylation factor, they are not themselves polyadenylation factors. Regulated by the NF-κB pathway, they are interestingly immune factors involved in the cGAS-STING pathway that induces immune responses against cytosolic double-stranded DNA. V2 decreases STING disulfide oligomerization and suppresses STING-mediated interferon β induction, but facilitates STING-mediated autophagy. Binding of V3 to V2 via WDRC prevents V2’s regulation of STING, suggesting that V3 is a V2 inhibitor. My findings thus further our understanding of STING-mediated immune responses. More broadly, these findings also demonstrate that isoforms produced by alternative mRNA processing can be functionally unrelated. In light of the versatility of the WDR33 gene, I performed a literature review in Chapter 3 on both the canonical and non-canonical functions of CPSF. I first summarize the general functions of CPSF subunits. Subsequently, I discuss their involvements in a variety of biological processes and conditions. This discussion reveals that different processes involve different CPSF subunits. Although CPSF is responsible for only two simple biochemical reactions, it has profound influences on cellular homeostasis. Together, my thesis studies reveal new insights into the molecular mechanism of the cGAS-STING pathway, underscore the importance of alternative mRNA processing, and provide the latest analyses of the functional significances of CPSF.

This dissertation focuses on pre-mRNA 3'-end processing in eukaryotes, a crucial step in defining the 3'end of most protein-coding mRNAs. In vertebrates, two distinct molecular machines are involved: the canonical machinery, consisting of a Cleavage Factor (CF) module, Polyadenylation Specificity (PSF) module, Cleavage Stimulation Factor, and other complexes, and the U7 snRNP machinery (U7 machinery), which consist of a core U7 snRNP complex and the Histone Cleavage Complex (HCC). U7 snRNP is involved in replication-dependent histone pre-mRNA 3'-end processing. Interestingly, the cleavage modules of the canonical and U7 machinery share an endonuclease, CPSF73, that catalyzes the cleavage reaction for 3’-end processing of pre-mRNAs. CPSF73 also possesses 5’-3’ exonuclease activity in the U7 machinery. CPSF73 has been identified as a potential target for anticancer and antimalarial small-molecule inhibitors. Traditionally, CPSF73 nuclease activity has been demonstrated using a gel-based end-point assay, using radio-labeled or fluorescently labeled RNA substrates. In Chapter Two (Ch. 2) of this dissertation introduces a novel, real-time fluorescence assay to investigate CPSF73 nuclease activity. This efficient and high-throughput assay holds potential for identifying new CPSF73 inhibitors. Chapter Three (Ch. 3) of this dissertation delves into the structural characterization of the mammalian PSF (mPSF) module in complex with the second most frequent PAS variants, AUUAAA. Structure studies have revealed the molecular mechanism underlying mPSF recognition of the most common PAS sequence, AAUAAA. This study presents a cryo-EM structure of mPSF in complex with AUUAAA. While the binding modes remain highly similar between the two PAS variants, we observed conformational differences in the A1 and U2 nucleotides in AUUAAA compared to the A1 and A2 of AAUAAA. Furthermore, CPSF30 displayed conformational changes near the U2 nucleotide of AUUAAA. Attempts to explore the binding modes of two rare PAS sequences, AAGAAA and GAUAAA, were inconclusive due to a lack of RNA density in the EM maps. An atomic model of the ternary structure (CPSF160, WDR33, CPSF30) was produced using the EM map of the AAGAAA sample. The ternary structure revealed PAS recognizing residues to be disordered in CPSF30 (ZF2 and ZF3) and WDR33. Overall, this dissertation provides insights into the intricate mechanisms of pre-mRNA 3'-end processing in mammals, laying the groundwork for future studies and potentially leading to the development of novel inhibitors targeting CPSF73.

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Orientador: Cesar Martins / Resumo: Os cromossomos B ocorrem em cerca de 2.828 espécies de diferentes táxons, sendo basicamente heterocromáticos e compostos de DNAs repetitivos. Recentemente, análises genômicas em larga escala estão sendo utilizadas para elucidar questões acerca dos cromossomos supranumerários. Os peixes ciclídeos recebem grande interesse científico, uma vez que muitas espécies passaram por um rápido e extenso processo de radiação adaptativa. Em algumas espécies do grupo, como Astatotilapia latifasciata, foi descrita a presença de cromossomos B. Neste trabalho foi caracterizado o perfil de expressão proteico em tecidos específicos na A. latifasciata e realizada análise funcional da presença do cromossomo B nesta espécie de teleósteo, elucidando a influência que este pode acarretar em vias metabólicas específicas. Além disso, esses dados foram integrados com os resultados de RNA-Seq dessa espécie, e construídas sub redes de co-expressão e interação proteína-proteína. Também foi calculada a entropia de Shannon, a qual não apresentou diversidade na expressão dos transcritos em cada biblioteca comparada. Além disso, foi analisada a expressão diferencial de RNAm em cada tecido em relação a presença do cromossomo B e ao sexo. Dentre os transcritos diferencialmente expressos, a análise de enriquecimento funcional apresentou processos relacionados ao ciclo celular, resposta imune e resposta ao estresse. Na maioria dos casos analisados entre a expressão de proteínas, os transcritos up regulated e as sub... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The B chromosomes occur in about 2,828 species of different taxa, being heterochromatic and composed of repetitive DNA.Recently, large-scale genomic analyzes are being used to elucidate questions about supernumerary chromosomes.Cichlid fish are of great scientific interest, since many species have gone through a rapid and extensive process of adaptive radiation.In some species of the group, such as Astatotilapia latifasciata, the presence of B chromosomes was described.In this work, we characterize the profile of protein expression in specific tissues in A. latifasciata and performed a functional analysis of the presence of the B chromosome in this species of teleost, elucidating the influence that it can cause in specific metabolic pathways.In addition, we integrate these data with the RNA-Seq results of this species, and construct sub-networks of co-expression and protein-protein interaction.The Shannon entropy was also calculated, which did not show diversity in the expression of the transcripts in each library. In addition, differential expression analysis was performed on each tissue separately and the relationship between the presence of B chromosome and sex chromosome was analyzed. Among the differentially expressed transcripts, functional enrichment analysis presented processes related to the cell cycle, immune response and stress response. In relation to abundance of proteins, the up regulated transcripts and the sub-networks we identified genes like the Aurora kinas... (Complete abstract click electronic access below) / Doutor

A resposta imune desencadeada em pacientes com leishmaniose tegumentar Americana (LTA) é predominantemente celular. Assim, o estudo dos mecanismos imunológicos responsáveis pela formação, progressão e cura desta infecção é de grande importância, considerando a deformação que ela pode causar em indivíduos infectados. Este estudo investigou a expressão de mRNA de IFN-?, IL-10, IL-27, TNF-?, TGF-? e IL-6 em 40 biopsias de parafina (FFPE) fixadas em formalina de pacientes com diagnóstico clínico e laboratorial para LTA. Um grupo de 10 biópsias FFPE coletadas de pacientes com diagnóstico clínico de leishmaniose mucosa (LM) foi utilizado para o controle da evolução da infecção. Inicialmente, foi padronizado a extração de RNA, a síntese de cDNA e a escolha do gene endógeno em 9 biopsias frescas e 8 FFPE. Após essas padronizações, as moléculas de RNA de fragmentos de biopsias parafinadas, foram extraídas com kit específico, tratadas com DNase, quantificadas por fluorimetria e sintetizadas para cDNA. A expressão relativa de cada citocina foi determinada (em duplicata) por PCR em tempo real. As reações foram normalizadas frente ao padrão de expressão do gene endógeno GAPDH, que foi previamente padronizado. O padrão de expressão de cada gene alvo foram determinados segundo a fórmula do \"CT comparativo\" (2-??CT), na qual calcula-se quantas vezes mais ocorre a expressão do gene alvo em relação a indivíduos normais. Também incluído nesse estudo 5 amostras de biopsia de pele de indivíduos normais (grupo IV). Os resultados foram expressos em média de quantificação relativa, a análise estatística foi realizada utilizando foi determinada por Teste t não pareado e teste F para comparação de variâncias, onde três grupos de pacientes foram formados. Grupo I, 35 pacientes com LTA, cuja expressão relativa de IFN-? foi inferior a 100. Grupo II, 5 pacientes com LTA cuja expressão relativa de IFN-? foi superior a 100. Grupo III, 10 pacientes com leishmaniose mucosa. Os níveis de expressão para IFN-? foram 33,34; 214,1 e 129,6 vezes maiores para os Grupos I, II e III, respectivamente do que as amostras do controle normal. Para o TNF-? foram 11,22; 2,37 e 4,59 vezes superiores ao normal nos Grupos I, II e III, respectivamente. Para IL-10 foram 5,14; 11,54 e 2,51 vezes superiores aos valores normais nos Grupos I, II e III, respectivamente. Para IL-27 foram 15,81; 85,29 e 17,29 vezes superiores ao normal nos Grupos I, II e III, respectivamente. Para o TGF-? foram 5,38; 4,33 e 4,72 vezes superiores ao normal nos Grupos I, II e III, respectivamente. Para a IL-6 foram 5,92; 5,69 e 2,09 vezes superiores aos valores normais nos Grupos I, II e III, respectivamente. Estes resultados foram semelhantes a resultados de estudos que analisam a expressão de citocinas já sintetizadas. No geral, os resultados de expressão gênica das citocinas estudadas sugerem que a exacerbação da resposta imune do perfil Th1 (IFN-?) poderia levar ao surgimento de lesões mais graves nos pacientes dos Grupos II e III. Os níveis elevados de mRNA de IL-10, observado nos pacientes do Grupo II podem promover baixa ativação macrofágica, colaborando com a replicação do parasita e assim culminar com a progressão da doença. A expressão de mRNA de IL-27 não interferiu na expressão de mRNA de IFN-? nas células dos pacientes com leishmaiose tegumentar ou leishmaniose mucocutânea. Em conclusão, estes resultados podem sugerir que os pacientes do Grupo II podem desenvolver formas mais graves de LTA quando comparados aos do Grupo I. / The immune response triggered in patients with American cutaneous leishmaniasis (ACL) is predominantly cellular. Thus, the study of the immunological mechanisms responsible for the formation, progression and cure of this infection is of great importance, considering the deformation that it can cause in infected individuals. This study investigated the expression of IFN-?, IL-10, IL-27, TNF-?, TGF-? and IL-6 mRNA in 40 formalin-fixed paraffin-embedded paraffin biopsies (FFPE) from patients with clinical and laboratory diagnosis for LTA. A group of 10 FFPE biopsies collected from patients with clinical diagnosis of mucosal leishmaniasis (MCL) was used to control the evolution of the infection. Initially, RNA extraction, cDNA synthesis and the choice of the endogenous gene were standardized in 9 fresh biopsies and 8 FFPE. After these standardizations, the RNA molecules of fragments of paraffin-shaped biopsies were extracted with a specific kit, treated with DNase, quantified by fluorimetry and synthesized for cDNA. The relative expression of each cytokine was determined (in duplicate) by real-time PCR. Reactions were normalized against the expression pattern of the endogenous GAPDH gene, which was previously standardized. The expression pattern results for each target gene were determined by the \"comparative CT\" (2-??CT) formula, in which it is calculated how many times more the expression of the target gene occurs in relation to normal individuals. Also included in this study 5 skin biopsy samples from normal subjects (group IV). The results were expressed as mean relative quantification, statistical analysis was performed using was determined by unpaired t test and F test for comparison of variances, where three groups of patients were formed. Group I, 35 patients with ACL, whose relative expression of IFN-? was less than 100. Group II, 5 patients with ACL whose relative expression of IFN-? was greater than 100. Group III, 10 patients with mucosal leishmaniasis. The expression levels for IFN-? were 33,34; 214,1 and 129,6 times higher for Groups I, II and III, respectively than the samples from the normal control. For TNF-? were 11,22; 2,37 and 4,59 times higher than normal in Groups I, II and III, respectively. For IL-10 were 5,14; 11,54 and 2,51 times higher than the normal values in Groups I, II and III, respectively. For IL-27, 15,81; 85,29 and 17,29 times higher than normal in Groups I, II and III, respectively. For TGF-? were 5,38; 4,33 and 4,72 times higher than normal in Groups I, II and III, respectively. For IL-6 were 5,92; 5,69 and 2,09 times higher than the normal values in Groups I, II and III, respectively. These results were similar to the results of studies that analyze the expression of cytokines already synthesized. In general, the gene expression results of the cytokines studied suggest that the exacerbation of the Th1 profile (IFN-?) immune response could lead to more serious lesions in patients in Groups II and III. Elevated levels of IL-10 mRNA observed in Group II patients may promote low macrophage activation, assisting in the replication of the parasite and thus culminating in the progression of the disease. Expression of IL-27 mRNA did not interfere in the expression of IFN-? mRNA in the cells of patients with tegumentary leishmaiosis or mucocutaneous leishmaniasis. In conclusion, these results may suggest that patients in Group II may develop more severe forms of ACL when compared to those in Group I.

Na fase ativa da leishmaniose visceral (LV) ocorrem alterações no metabolismo de lipoproteínas com redução dos níveis de HDL e aumento de triglicérides. A partir desses dados, focamos neste projeto essas alterações na progressão da infecção e apontamos alguns elementos como seus possíveis desencadeantes. Como essas alterações poderiam resultar de redução de atividade e expressão da lipoproteína lipase (LPL), do receptor alfa do proliferador ativado de peroxissoma (PPAR?) e da proteína transferidora de ésteres de colesteril (CETP), a sua expressão foi avaliada durante a progressão da LV em hamster. Em hamsteres infectados com 2 x 107 amastigotas de L. (L.) infantum observamos aumento de triglicérides nos hamsteres com 55 dias (mediana = 294,0 mg/dL) e 90 dias (303,0 mg/dL ) de infecção comparados aos controles de 55 dias (119,0 mg/dL) e de 90 dias (117,0 mg/dL) (p <= 0,05). Os níveis de colesterol total e de HDL não apresentaram diferença significante entre controles e infectados com 30, 55 e 90 dias de infecção. A expressão de mRNA de PPAR? no fígado com 55 e 90 dias de infecção apresentou tendência de redução nos infectados. Já de CETP no fígado dos hamsteres com 55 dias de infecção, a expressão relativa (CT) estava reduzida nos infectados (0,08) comparados aos controles (1,69) (p <= 0,05) e de LPL no coração dos hamsteres com 90 dias de infecção também estava reduzida (1,43) com relação aos controles (2,61) (p <= 0,05). Há dados na literatura sugerindo a importância de lipídios para o desenvolvimento de amastigotas no hospedeiro vertebrado e é possível que as alterações dos níveis de lipoproteínas contribuam na progressão da infecção. Assim, avaliamos neste estudo o efeito da droga hipolipemiante ciprofibrato no controle do parasitismo na LV em hamster, sabendo-se que ciprofibratos atuam aumentando a expressão de PPAR? e a produção e atividade de LPL. O tratamento com ciprofibrato nos hamsteres com 55 dias de infecção gerou redução de triglicérides (123,0 mg/dL) em relação aos infectados não tratados (294,0 g/dL) (p <= 0,05), além dos níveis de triglicérides nos animais infectados não tratados terem aumentado quando comparados aos controles não tratados (119,0 mg/dL) (p <= 0,05). Houve também, redução de triglicérides nos animais não infectados tratados com ciprofibrato (89,0 mg/dL) comparando-se aos infectados não tratados (p <= 0,05). Os níveis de colesterol nos hamsteres não infectados tratados com ciprofibrato reduziram (53,5 mg/dL) em comparação aos infectados não tratados (93,0 mg/dL) (p <= 0,05). Já naqueles que foram infectados e tratados com ciprofibrato, constatamos redução de colesterol (53,5 mg/dL) quando comparados aos infectados não tratados (p <= 0,05). Os níveis de HDL não aumentaram com ciprofibrato e foram similares entre os hamsteres infectados não tratados e os controles não tratados. A carga parasitária no baço e no fígado não foi reduzida com ciprofibrato. Na leishmaniose visceral em hamster ocorrem alterações do metabolismo lipídico com aumento de triglicérides e redução da expressão da mRNA de LPL e CETP. O tratamento com ciprofibrato foi eficaz no controle das alterações de níveis de lipoproteínas. / In the active phase of visceral leishmaniasis (VL) changes occur in lipoprotein me-tabolism with reduction in HDL and increase in triglyceride (TG) levels. From these data, in this project we focused these changes during the progression of the infection and we approached some elements as their underlying factors. Since these changes may result from the reduction of the activity and the expression of the lipoprotein lipase (LPL), of the peroxisome proliferator-activated receptor alpha (PPAR?) and of the cholesteryl ester transfer protein (CETP), their expression were evaluated during VL progression in hamster. In 2 x 107 L. (L.) infantum amastigote-infected hamsters we observed an increase in the triglycerides in hamsters with 55 days (median = 294.0 mg/dL) and 90 days (303.0 mg/dL) of infection compared with controls of 55 days (119.0 mg/dL) and of 90 days (117.0 mg/dL) (p <= 0.05). The total cholesterol and the HDL levels did not present significant differences between control and in-fected groups at 30, 55 and 90 days of infection. The expression of mRNA of the PPAR in the liver with 55 and 90 days of infection tended to be reduced in infected animals. However the relative expression (CT) of CETP in the liver of hamsters with 55 days of infection was signicantly reduced in infected (0.08) compared with control animals (1.69) (p <= 0.05). The relative expression (CT) of LPL in the heart of hamsters with 90 days of infection was also reduced (1.43) in relation to controls (2.61) (p <= 0.05). There are data in the literature suggesting the importance of lipids for the development of amastigotes in vertebrate host and it is possible that the changes in the lipoprotein levels contribute for the infection progression. Therefore, we evaluated in this study the effect of the lipid-lowering drug ciprofibrate in the control of parasitism in VL in the hamster, knowing that ciprofibrate acts increasing the expression of the PPAR? and of the LPL production and activity. The treatment with ciprofibrate in infected hamsters at 55 days lead to the reduction of triglyceride level (123.0 mg/dL) in relation to non-treated infected animals (294.0 g/dL) (p <= 0.05). Further the triglyceride levels in the non-treated infected animals were in-creased when compared with untreated controls (119.0 mg/dL) (p <= 0.05). There was also reduction of triglyceride in ciprofibrate treated-non infected animals (89.0 mg/dL) compared with non-treated infected animals (p <= 0.05). The cholesterol lev-els were reduced in the ciprofibrate-treated non-infected hamsters (53.5 mg/dL) in comparison to the non-treated infected ones (93.0 mg/dL) (p <= 0.05). In the ciprofibrate-treated infected ones we found a reduction of cholesterol level (53.5 mg/dL) when compared with non treated infected animals (p <= 0.05). The HDL lev-els did not increase with ciprofibrate and they were similar between the non-treated infected hamsters and non-treated controls. The parasite load in the spleen and liver were not reduced with ciprofibrate. In the visceral leishmaniasis in hamster changes occur in the lipid metabolism with increase in the triglyceride level and the reduction of expression of mRNA of LPL and CETP. The treatment with ciprofibrate was ef-fective in the control of changes in the lipoprotein levels.

Circulating RNA was previously demonstrated through the identification of tumour-derived transcripts in the plasma and serum of cancer patients. This finding inspired the detection of cell-free fetal mRNA in maternal plasma which in turn facilitated the development of promising non-invasive prenatal assessment strategies applicable to pregnancies regardless of fetal sex. / Finally, in the last study, I implemented what I have learnt from the analysis of circulating fetal RNA into the development of brain-derived RNA transcripts for detection in the plasma of patients who had sustained brain injuries. A systematic approach based on gene expression microarray analysis was adopted to search for circulating brain-specific mRNA markers. Transcripts showing high expression levels in brain tissues but low expression levels in peripheral blood were identified. However, the detectability of these brain-derived mRNA markers in both peripheral and jugular plasma was found to be low. Instead, concentrations of these mRNA markers were found to be higher in cerebral spinal fluid (CSF) from brain-injured than non-brain-injured patients. / In section IV of this thesis, I reviewed and modified the blood sample processing and plasma RNA extraction protocols that were previously practised, in an attempt to enrich circulating fetal RNA in maternal plasma. Besides mRNA, extraction protocols for microRNAs (miRNAs), a new class of circulating nucleic acid markers, were also evaluated. The modifications in the protocols that I introduced led to significant improvements in RNA yield and enhanced the accuracy and reliability of circulating RNA detection in plasma, especially for those marginally detectable transcripts. / Recently, in addition to maternal plasma, there have been studies by other research groups reporting the presence of placental/fetal mRNA in maternal whole blood. In the first part of this thesis, I studied a list of previously identified placental mRNA transcripts, including chorionic somatomammotropin hormone 1 (CSH1), KiSS-1 metastasis-suppressor (KISS1), placenta-specific 4 (PLAC4) and placenta-specific 1 (PLAC1) in maternal whole blood and determined if this whole blood-based approach offered advantages over maternal plasma analysis. The presence of KISS1, PLAC4 and PLAC1 in non-pregnant and post-partum blood samples as well as the confirmed maternal contribution of PLAC4 mRNA in maternal blood proved that most of the detected 'placental' mRNA molecules in maternal whole blood were of maternal origin. To explore if more pregnancy-associated circulating mRNA markers could be developed for maternal whole blood analysis, candidates were mined after performing gene expression microarray comparison of whole blood samples from pregnant and non-pregnant individuals. The pregnancy-specificity of the identified gene candidates was further investigated. However, their presence in non-pregnant whole blood and lack of clearance after pregnancy indicated that they were not "pregnancy-specific" markers. These data suggested that pregnancy specific transcripts could be more readily identified from maternal plasma than whole blood. / The results presented in this thesis have not only provided a foundation facilitating the precise and accurate detection of fetal-specific RNA markers but have also improved the current understanding of the biology of circulating RNA. / Heung, May Sze. / Advisers: Dennis Lo; Rossa Wk Chiu. / Source: Dissertation Abstracts International, Volume: 72-11, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 212-239). / 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.

Aortic valve (AV) disease is a major cause of cardiovascular-linked deaths globally. In addition, AV disease is a strong risk factor for additional cardiovascular events; however, the mechanism by which it initiates and progresses is not well-understood. We hypothesize that low and oscillatory flow is present on the fibrosa side of the AV and stimulates ECs to differentially regulate microRNA (miRNA) and mRNAs and influence AV disease progression. This hypothesis was tested employing both in vitro and in vivo approaches, high throughput microarray and pathway analyses, as well as a variety of functional assays. First, we isolated and characterized side-dependent, human aortic valvular endothelial cells (HAVECs). We found that HAVECs express both endothelial cell markers (VE-Cadherin, vWF, and PECAM) as well as smooth muscle cell markers (SMA and basic calponin). Using microarray analysis on sheared, side-specific HAVECs, we identified side- and shear-induced changes in miRNA and mRNA expression profiles. More specifically, we identified over 1000 shear-responsive mRNAs which showed robust validation (93% of those tested). We then used Ingenuity Pathway Analysis to identify key miRNAs, including those with many relationships to other genes (for example, thrombospondin and I&B) and those that are members of over-represented pathways and processes (for example, sulfur metabolism). Furthermore, we validated five shear-sensitive miRNAs: miR-139-3p, miR-148a, miR-187, miR-192, and miR-486-5p and one side-dependent miRNA, miR-370. To prioritize these miRNAs, we performed in silico analysis to group these key miRNAs by cellular functions related to AV disease (including tissue remodeling, inflammation, and calcification). Next, to compare our in vitro HAVEC results in vivo, we developed a method to isolate endothelial-enriched, side-dependent total RNA and identify and validate side-dependent (fibrosa vs. ventricularis) miRNAs in porcine aortic valvular endothelium. From this analysis, we discovered and validated eight side-dependent miRNAs in porcine endothelial-enriched AV RNA, including one miRNA previously identified in vitro, miR-486-5p. Lastly, we determined the relationship between important miRNAs (specifically miR-187 and miR-486-5p) and AV disease by modulating levels of miRNAs and performing functional assays. Preliminary studies overexpressing miR-187 in HAVECs have shown a reduction in inflammatory state through monocyte adhesion (p<0.05). Further, miR-486-5p overexpression reveals an increase in migration (p<0.05) and a trend for a decrease in early apoptosis, linking miR-486-5p to tissue remodeling in the AV. Better understanding of AV biology and disease in terms of gene-regulation under different hemodynamic conditions will facilitate the design of a tissue-engineered valve and provide alternative treatment options.