Over the last few decades, phylogenetics has emerged as a very promising field, facilitating a comparative framework to explain the genetic relationships among all the living organisms on earth. These genetic relationships are typically represented by a bifurcating phylogenetic tree — the tree of life. Reconstructing a phylogenetic tree is one of the central tasks in evolutionary biology. The different evolutionary processes, such as gene duplications, gene losses, speciation, and lateral gene transfer events, make the phylogeny reconstruction task more difficult. However, with the rapid developments in sequencing technologies and availability of genome-scale sequencing data, give us the opportunity to understand these evolutionary processes in a more informed manner, and ultimately, enable us to reconstruct genes and species phylogenies more accurately. This thesis is an attempt to provide computational methods for phylogenetic inference and give tools to conduct genome-scale comparative evolutionary studies, such as detecting homologous sequences and inferring gene families. In the first project, we present FastPhylo as a software package containing fast tools for reconstructing distance-based phylogenies. It implements the previously published efficient algorithms for estimating a distance matrix from the input sequences and reconstructing an un-rooted Neighbour Joining tree from a given distance matrix. Results on simulated datasets reveal that FastPhylo can handles hundred of thousands of sequences in a minimum time and memory efficient manner. The easy to use, well-defined interfaces, and the modular structure of FastPhylo allows it to be used in very large Bioinformatic pipelines. In the second project, we present a synteny-aware gene homology method, called GenFamClust (GFC) that uses gene content and gene order conservation to detect homology. Results on simulated and biological datasets suggest that local synteny information combined with the sequence similarity improves the detection of homologs. In the third project, we introduce a novel phylogeny-based clustering method, PhyloGenClust, which partitions a very large gene family into smaller subfamilies. ROC (receiver operating characteristics) analysis on synthetic datasets show that PhyloGenClust identify subfamilies more accurately. PhyloGenClust can be used as a middle tier clustering method between raw clustering methods, such as sequence similarity methods, and more sophisticated Bayesian-based phylogeny methods. Finally, we introduce a novel probabilistic Bayesian method based on the DLTRS model, to sample reconciliations of a gene tree inside a species tree. The method uses MCMC framework to integrate LGTs, gene duplications, gene losses and sequence evolution under a relaxed molecular clock for substitution rates. The proposed sampling method estimates the posterior distribution of gene trees and provides the temporal information of LGT events over the lineages of a species tree. Analysis on simulated datasets reveal that our method performs well in identifying the true temporal estimates of LGT events. We applied our method to the genome-wide gene families for mollicutes and cyanobacteria, which gave an interesting insight into the potential LGTs highways. / <p>QC 20161010</p>

Student Number : 9503239E - PhD thesis - Faculty of Science / Malaria is one of the most debilitating pathogenic infections known to man, responsible for approximately three million deaths annually, primarily children in sub-Saharan Africa. The parasite has evaded multiple attempts at eradication, predominantly through the complexity of its life cycle, the ability to elude host immune response, and gametocyte formation to ensure dissemination. The recent completion of the genome sequence has opened up a multitude of avenues for exploration and identification of novel drug and vaccine targets, as well as providing a glimpse into the complex mechanisms that have contributed to the success of this pathogen. The mechanisms of gene regulation, especially those governing gametocytogenesis, have, however, not yet been elucidated. In this research, differential display has been used to identify some of the genes that are differentially expressed between the asexual parasite and gametocyte stages of P. falciparum. Numerous genes involved in diverse aspects of metabolism, protein synthesis and immune evasion were identified. A combination of BLASTN and BLASTX similarity searches was used to categorize and increase the confidence with which a transcript could be identified. Expression data for confidently identified genes were confirmed using reverse slot blot and available microarray data. PfMyb2, a novel transcription factor which may regulate genes involved in gametocytogenesis, was characterized. The DNA binding domains of the protein were cloned and expressed as a histidine fusion protein. Mobility shift assays were used to assess the in vitro binding capability of the recombinant 6xHis-PfMyb2, which bound to oligonucleotides containing the consensus Myb regulatory element. Two of the oligonucleotides represent sequences located within promoters of P. falciparum genes (Pfcrk1 and Pfmap1) known to play a role in regulating the cell cycle, a function ascribed to many members of the vertebrate Myb family. The identification of PfMyb2 as a bona fide transcription factor is a first step into gaining some insight into the many regulatory processes that occur during the life cycle of this complex organism. A better understanding of the molecular mechanisms that govern its survival is essential for the ultimate eradication of this deadly parasite.

O experimento 1 objetivou estabelecer um modelo de estudo do desvio folicular, permitindo identificar o momento exato do início do desvio. Vacas das raças Nelore (NEL; n=11) e Holandês (HPB; n=10) tiveram emergência da onda folicular ovariana sincronizada e avaliação ultrassonográfica ovariana realizada a cada 12 h. Quando o maior folículo (F1) atingiu tamanho médio de início do desvio para sua raça (NEL: 6,5; HPB: 8,5 mm), em crossover, foram aleatorizadas em dois grupos (CON: Controle; ASP: Aspiração), com aspiração do F1 ASP e nas vacas do grupo CON não houve aspiração. Avaliações ultrassonográficas foram mantidas até 48h pós-desvio. Os dados foram analisados pelo PROC MIXED (P<=0,05). No grupo CON,12h após F1 atingir tamanho de desvio, diferiu em diâmetro com os demais (F2/F3), caracterizando desvio. Não foi observada diferença entre F1 na aspiração e F2 12h após, no grupo ASP nas duas raças. Não houve diferença entre F1 CON com F2 ASP após aspiração do F1. Todavia, F2 ASP apresentou maior diâmetro em relação ao F2 CON a partir de 12h após o momento do desvio, em ambas as raças. Foi realizada colheita de sangue em 0, 3, 6, 12 e 24h após aspiração, para avaliação da concentração plasmática de FSH e aumentou em 12h após aspiração do F1 ASP. O experimento 2, objetivou verificar expressão de genes associados ao desvio, CYP19A1 (Aromatase), receptor de LH (LHr) e PAPP-A (Pappalysin 1) em vacas NEL utilizando o mesmo modelo do experimento 1, entretanto obtendo células da granulosa por meio de lavagem intrafolicular. As vacas (n=10) tiveram emergência da onda sincronizada. Foram estabelecidos três tratamentos: 0h, o maior folículo atingiu 6,5 mm, e os dois maiores folículos foram aspirados (FD0h; FS0h); 12h, os dois maiores folículos (FD12h; FS12h) foram aspirados 12h após o maior atingir 6,5 mm; e grupo desvio, o maior folículo (FD0h) foi aspirado quando atingiu 6,5 mm, e o segundo maior (FS->FD) 12h depois. A suspensão contendo células da granulosa, foi centrifugada, o sobrenadante dosado para estradiol-17&beta; (E2) por ELISA. O pellet foi analisado para expressão de RNAm. Análise estatística foi realizada pelo PROC MIXED do SAS. Concentração de E2 (ng/mL) para FS->FD diferiu do FS0h e FS12h e não diferiu de FD0h e FD12h comprovando que FS->FD na ausência de um FD0h aumenta sua concentração de E2 semelhante aos folículos dominantes. Não houve diferença entre os grupos para a expressão relativa para PAPP-A e CYP19A1. Para LHr, houve tendência de aumento na expressão nos folículos FS->FD, quando comparados ao do grupo 0h. Não houve diferença entre FS->FD e o grupo 12h. FD12h diferiu do grupo 0h (FD0h e FS0h) e FS12h. Conclui-se que, o segundo maior folículo é capaz de se tornar dominante quando se aspira o maior folículo permitindo que se tenha estimativa mais segura do momento exato da dominância; também, ocorre um aumento no FSH plasmático 12h após a aspiração do maior folículo. O aumento na expressão de LHr foi o principal fator para o estabelecimento do desvio em vacas NEL. / This study was conducted in two experiments. Experiment 1 aimed to establish a manipulative model to study follicle deviation. Nelore (NEL n=11) and Holstein (HOL n=10) cows had the emergence of the wave synchronized. Ovarian ultrasonography was performed every 12h. When largest follicle (F1) reached the average size of deviation to its breed (NEL: 6.5; HOL: 8.5 mm), cows were randomized into two groups (CON: control; ASP: Aspiration) in a crossover design. F1ASP was aspirated, and for cows of CON group there was no aspiration. Ultrasound exams were maintained through 48h post-deviation. Data were analyzed using PROC MIXED (P<=0.05). In the CON group, 12h after F1 has reached deviation size, there was difference in diameter in relation to F2 and F3, characterizing deviation. There was no difference between diameters of F1 at the time of aspiration and F2 12h after in the ASP group for both breeds. There was no difference between the F1 from CON group with F2 ASP group after F1 aspiration. However, F2ASP had a bigger diameter compared to the CON group F2 12h after the time of deviation in both breeds. Blood samples were collected at 0, 3, 6, 12 and 24h after aspiration, to evaluate plasma concentrations of FSH. FSH increased at 12h after the F1 aspiration in ASP group. Experiment 2 was conducted to verify the expression of genes associated with follicle deviation, CYP19A1 (aromatase), LH receptor (LHR) and PAPP-A (Pappalysin 1) in NEL cows using same model of experiment 1, however by obtaining granulosa cells through intrafollicular flushing. Cows (n=10) had the wave emergence synchronized. Three treatments were established: 0h, when the biggest follicle reached 6.5 mm, the two biggest follicles were aspirated (DF0h; SF0h); 12h, the two biggest follicles (DF12h; SF12h) were aspirated 12h after the biggest follicle had reached 6.5 mm; and deviation group, biggest follicle (DF0h) was aspirated when reached 6.5 mm, and the second biggest follicle (SF->DF) was aspirated 12h later. The follicular fluid granulosa cells was centrifuged and supernatant was assayed for 17&beta;-estradiol (E2) concentration by ELISA. The pellet was analyzed for mRNA expression. Statistical analysis was performed using the SAS PROC MIXED. Concentration of E2 (ng/mL) for SF->DF differed from SF0h and SF12h and did not differ from DF0h and DF12h showing that SF->DF in the absence of a DF0h had an increased concentration of E2 similar to dominant follicles. There was no difference between groups for the relative expression of mRNA for PAPP-A and CYP19A1. For LHr expression, there was a tendency to increase in SF->DF follicles when compared to the group 0h. There was no difference between SF->DF and group 12h. DF12h differed from group 0h (DF0h/SF0h) and SF12h. In conclusion, second biggest follicle can become dominant when the biggest follicle is aspirated, allowing to have more reliable estimation of the exact moment of follicle deviation, associated with an increase in plasma FSH 12h after aspiration of the biggest follicle. Increased expression of LHr was the main characteristic of establishment of deviation in NEL cows.

Genes microexônicos (MEGs) apresentam uma arquitetura bastante peculiar, sendo compostos predominantemente por quatro ou mais exons simétricos bem pe- quenos dispostos em tandem (microexons Š 36 bp, com tamanhos múltiplos de 3). Eles foram descritos pela primeira vez em 2009 no platelminto parasita Schistosoma mansoni (agente etiológico da esquistossomose). Alguns desses genes apresentam evidência (de transcritômica e proteômica) de gerar diferentes isoformas de proteínas através do mecanismo de splicing alternativo. Os MEGs não apresentam homólogos fora do gênero Schistosoma. Microexons individuais já foram reportados em genes de organismos modelo e do ser humano, normalmente atuando como um hotspot de splicing alternativo, porém nestes casos apenas um único microexon por gene fora reportado. Com este pano de fundo, fizemos o seguinte questionamento: existem genes com arquitetura similar (ou seja, múltiplos microexons em tandem) em outros organismos? Desenvolvemos uma heurística capaz de detectar genes com a arquitetura dos MEGs e a aplicamos no transcritoma de S. mansoni e, além de detectar os MEGs originais, detectamos 31 novos genes. Este pipeline foi aplicado a uma coleção de anotações de genomas e detectou 494 genes distribuídos entre 125 organismos (incluindo animais, plantas, fungos e alguns eucariotos unicelulares). / Microexon genes (MEGs) have an unusual architecture, composed predominantly by four or more very small tandemly disposed symmetric exons (microexons 36 bp, with exon sizes multiple of 3). They were first described in 2009 in the parasitic platyhelminth Schistosoma mansoni (etiologic agent of schistosomiasis). Some of those genes display evidence (by transcriptomics and proteomics) of generating variable protein isoforms through alternative splicing. MEGs have no homologs outside the Schistosoma genus. The presence of individual microexons has also been reported in genes of model organisms and humans, usually as a hotspot of alternative splicing, but in those cases only a single microexon per gene was observed. Within this background, we asked the following question: are there genes with similar architecture (i.e., with multiple internal microexons in tandem) in other organisms? We developed a pipeline to detect genes with the architecture of S. mansoni MEGs, applied it to an updated transcriptome mapping of the S. mansoni genome and successfully detected the original MEGs and 31 new genes. This pipeline could be applied to the collection of public data from genome annotations of other species to eventually detect new genes with multiple tandem microexons.

A gene dosage study was conducted on a rare complete trisomy 22 human fibroblast cell line utilizing three lysosomal enzymes, ∝-iduronidase, ∝-galactosidase B, and arylsulfatase A, whose genes are located on chromosome 22 and two control enzymes, ,β-hexosaminidase A and -- fucosidase, with genes not on chromosome 22. A gene dosage effect was clearly demonstrated for an early passage number of the fibroblasts; however, later passage numbers gave inconclusive results. This study suggests that gene dosage studies must be carefully designed to be conducted only on early, matched passage number cells. ∝-fucosidase gave anomalous results most likely due to pleiotropic effects. The present gene dosage study confirmed the trisomic nature of the cell line studied and suggests that this type of study may be a useful diagnostic tool for small deletions, additions, or unbalanced translocations.

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No. of bitstreams: 3 license_text: 21490 bytes, checksum: 7ff346999f7486617a4302c4f2f02bc7 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) 2014LuanaMariaWollinger.pdf: 6093984 bytes, checksum: 2af11bb55c21559bc90e174f86d5d0b7 (MD5) / Introdução: A Pressão Arterial (PA), bem como a Hipertensão Arterial Sistêmica (HAS), são fenótipos complexos onde fatores genéticos e ambientais influenciam na sua etiologia. Dentre os fatores genéticos, polimorfismos nos genes da Enzima Conversora de Angiotensina (ACE) e Angiotensinogêneo (AGT) vem sendo associados a estes desfechos. Em relação aos fatores ambientais, a dieta é um dos fatores de maior importância no processo evolutivo da doença, principalmente no que diz respeito ao consumo de sódio. Objetivo: Verificar se existe interação entre os polimorfismos Inserção/Deleção do gene ACE e rs699 do gene AGT e o consumo de micronutrientes (sódio, potássio, cálcio e magnésio) da dieta; e se esta interação influencia os valores de PA em uma amostra de brasileiros adultos saudáveis. Metodologia: Realizou-se um estudo do tipo transversal com 341 indivíduos brasileiros adultos de ambos os gêneros. Os valores de PA foram aferidos em equipamento digital marca Omron® modelo HEM-710INT. A análise dietética foi feita por meio do software Dietwin versão 2008 a partir do método de Recordatório Alimentar de 24 horas. A genotipagem do polimorfismo InsDel foi realizada através da Reação em Cadeia de Polimerase (PCR), com primers específicos, seguida de eletroforese em gel de agarose 1,5 %; o polimorfismo rs699 foi genotipado através do sistema de discriminação alélica TaqMan (Applied Biosystems). A Análise estatística foi realizada pelo uso do software Statistical Package for the Social Sciences (SPSS) versão 20.0. Resultados: As frequências genotípicas de ambos os polimorfismos estão em equilíbrio de Hardy-Weinberg. Em relação ao consumo dos micronutrientes, observou-se uma associação com o polimorfismo InsDel do gene ACE, onde indivíduos homozigotos Del/Del consomem menos cálcio do que os heterozigotos (Ins/Del) (p=0,007). Na comparação da PA entre os genótipos, verificou-se uma associação com o polimorfismo rs699 do gene AGT. Indivíduos homozigotos GG apresentaram maior PA Sistólica quando comparados aos indivíduos AA (p=0,028). As análises de interação indicaram uma associação entre o genótipo heterozigoto do polimorfismo rs699 do gene AGT no consumo de cálcio e magnésio na modulação dos valores de PA. Conclusão: Os polimorfismos dos genes ACE e AGT parecem estar associados com fatores do consumo alimentar e PA, assim como o gene AGT pode ter um papel importante nas relações nutrigenéticas nos desfechos relacionados a PA. / Introduction: The Blood Pressure (BP) and Hypertension are complex phenotypes, in which genetic and environment factors influence in the etiology. In the genetics factors, polymorphisms of the Angiotensin Converting Enzyme (ACE) and Angiotensinogen (AGT) gene, has been associated with on outcomes. In relation to environmental factors, the diet is a factor the most importance on evolutionary process of the disease, particularly with regard to the consumption of sodium. Objetive: To verificate if there is interaction between polymorphisms Insertion/Deletion (InsDel) of the ACE gene and rs699 of the AGT gene and the micronutrientes intake (sodium, potassium, calcium and magnesium) in the diet; and if there is interaction influences the BP values in the healthy brasilian sample. Metodology: Performed a cross-section study with 341 brazilian adult individuals of both genders. The BP values was measurement by digital equipment Omron® model HEM-710INT. The dietary analysis was assessed by the software Dietwin version 2008 from the 24-hour recall method. InsDel polymorphism genotyping was performed by Polymerase Chain Reaction (PCR), using specific primers, and analyzed on 1,5% agarose gel; the rs699 polymorphism was genotyped using the TaqMan SNP genotyping assays (Applied Biosystems). The statistical analysis was performe by the software Statistical Package for the Social Sciences (SPSS) version 20.0. Results: The genotype frequencies of both polymorphisms are balance Hardy-Weinberg. In relation micronutrients intake, our results show a association with InsDel polymorphism of the ACE gene, in which homozygotes Del/Del intake fewer calcium than heterozygotes (Ins/Del) (p=0,007). Comparing BP between genotypes, our results indicate a association with rs699 polymorphism of the AGT gene. Homozygotes GG showed greater Systolic BP than individuals AA (p=0,028). The interaction analysis indicate a association between heterozygote of the rs699 polymorphism and calcium and magnesium inatke in the modulation BP values. Conclusion: The polymorphisms of the ACE and AGT gene seem to be associated with factors of the food consumption and BP, as well as, the AGT gene may have an important role in nutrigenetics relationships on outcomes related to BP.

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No. of bitstreams: 3 license_text: 22064 bytes, checksum: ef48816a10f2d45f2e2fee2f478e2faf (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) 2013RafaelaMundstockdeAzevedoBastian.pdf: 749375 bytes, checksum: b76d59045d784f0c33faadd0accae511 (MD5) / A obesidade é uma doença crônica de origem multifatorial que pode se desenvolver a partir de fatores comportamentais, metabólicos, psicológicos, e genéticos. A nutrigenética estuda os efeitos da variação genética na interação dieta-doença, o que inclui a identificação e caracterização dos genes relacionados ou até mesmo responsáveis pelas diferentes respostas aos nutrientes. Objetivo: Investigar a relação entre a dieta e os polimorfismos rs2228145 do gene IL6R e rs2069845 do gene IL6 sobre marcadores biológicos do metabolismo (parâmetros bioquímicos e antropométricos, através da técnica da PCR-Real Time. Metodologia: A amostra foi composta por indivíduos provenientes do Ambulatório de Nutrição do Universidade do Vale do Taquari Univates. O consumo alimentar foi avaliado através do recordatório 24 horas pelo software Dietwin 2008. A análise da composição corporal foi realizada através da avaliação antropométrica e do exame de bioimpedância. Foram dosados os parâmetros bioquímicos de glicemia, colesterol total, HDL e triglicerídeos, e calculada a fração LDL. As interações dos polimorfismos rs2228145 e rs2069845 entre o consumo de macro e micronutrientes para os desfechos dos parâmetros bioquímicos e antropométricos foram analisadas através do procedimento backward stepwise pelo software SPSS 19.0. Resultados: A amostra foi composta por um total de 344 indivíduos, dos quais 77% eram do gênero feminino e 23% do gênero masculino, com idade média de 25,8 anos (±6,9). Observaram-se associações entre dieta e o polimorfismo rs2228145 do gene IL6R para os desfechos de glicemia, HDL, IMC e circunferência da cintura. Já o polimorfismo rs2069845 do gene IL6 mostrou associação entre dieta para o desfecho de circunferência da cintura. Conclusão: Após a análise do consumo de macro e micronutrientes, dos parâmetros bioquímicos e antropométricos, juntamente com a análise molecular da amostra pode-se observar diversos efeitos e associações entre esses fatores e os polimorfismos. Essas associações dos polimorfismos com a dieta nos remete ao fato de que cada indivíduo é único e pode apresentar respostas dietéticas diferentes de acordo com seu genótipo. / Obesity is a chronic disease of multifactorial origin that can develop from behavioral, metabolic, psychological, and genetic factors. Nutrigenetic study the effects of genetic variation in diet-disease interaction, which includes the identification and characterization of the genes related or even responsible for the different responses to nutrients. Objective: To investigate the relationship between diet and the polymorphisms rs2228145 of the gene IL6R and rs2069845 of the gene IL6 on biochemical and anthropometric parameters, through the technique of Real - Time PCR. Methods: The sample consisted of individuals from the nutrition clinic of the University Center Univates. Dietary intake was assessed by the 24-hour recall by software Dietwin 2008. The body composition analysis was performed using anthropometric and bioimpedance exams. The biochemical parameters measured were blood glucose, total cholesterol, HDL and triglycerides and LDL calculated. The interactions of polymorphisms rs2228145 and rs2069845 between the consumption of macro and micronutrients for the outcomes of anthropometric and biochemical parameters were analyzed by backward stepwise procedure by SPSS 19.0 software. Results: The sample comprised a total of 344 individuals, of whom 77% were female and 23% male, with mean age of 25.8 years (± 6.9). Associations were observed between diet and the polymorphism rs2228145 of IL6R gene for glucose, HDL, BMI and waist circumference outcomes. The polymorphism rs2069845 of the IL6 gene showed an association between diet for the outcome of waist circumference. Conclusion: After analyzing the consumption of macro and micronutrients, biochemical and anthropometric parameters, together with the molecular analysis of the sample different effects and associations between these factors and polymorphisms can be observed. These associations relates to the fact that each individual is unique and may have different dietary responses according to their genotype

No description available.

Wan Wing Kuen. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 114-128). / Abstracts in English and Chinese. / Thesis Abstract --- p.i / Dedication --- p.iii / Acknowledgements --- p.iv / List of Figures --- p.xii / List of Tables --- p.xiv / Abbreviations --- p.xv / Chapter CHAPTER 1: --- INTRODUCTION --- p.1 / Chapter 1.1 --- Research Initiative and Significance --- p.1 / Chapter 1.2 --- Cardiomyocyte and Terminal differentiation --- p.4 / Chapter 1.3 --- Hypertension and Myocardial Hypertrophy --- p.7 / Chapter 1.3.1 --- Hypertension --- p.7 / Chapter 1.3.2 --- Myocardial Hypertrophy --- p.8 / Chapter 1.4 --- Experimental Animal Models --- p.13 / Chapter 1.4.1 --- Spontaneously Hypertensive Rats --- p.15 / Chapter 1.4.2 --- Wistar-Kyoto Rats --- p.16 / Chapter 1.5 --- Combination of Suppression Subtractive Hybridization and cDNA Microarray Analysis --- p.17 / Chapter 1.5.1 --- Suppression Subtractive Hybridization --- p.17 / Chapter 1.5.2 --- cDNA Microarray A nalysis --- p.21 / Chapter 1.5.3 --- Combination of SSH and cDNA microarray --- p.24 / Chapter CHAPTER 2: --- MATERIALS AND METHODS --- p.25 / Chapter 2.1 --- Experimental Animal Models --- p.25 / Chapter 2.2 --- RNA Isolation from Rat Ventricle --- p.26 / Chapter 2.2.1 --- Total RNA Isolation --- p.26 / Chapter 2.2.2 --- Deoxyribonuclease I Digestion --- p.27 / Chapter 2.3 --- Suppression Subtractive Hybridization --- p.29 / Chapter 2.3.1 --- First-Strand cDNA Synthesis --- p.29 / Chapter 2.3.2 --- Second-Strand cDNA Synthesis --- p.30 / Chapter 2.3.3 --- Column Chromatography --- p.31 / Chapter 2.3.4 --- Rsa I Endonuclease Digestion --- p.32 / Chapter 2.3.5 --- Adaptor Ligation --- p.33 / Chapter 2.3.6 --- Ligation Efficiency Analysis --- p.34 / Chapter 2.3.7 --- First Hybridization --- p.35 / Chapter 2.3.8 --- Second Hybridization --- p.36 / Chapter 2.3.9 --- Primary PCR Amplification --- p.36 / Chapter 2.3.10 --- Secondary PCR Amplification --- p.38 / Chapter 2.3.11 --- Subtraction Efficiency Analysis --- p.38 / Chapter 2.4 --- Construction of Subtracted cDNA Libraries --- p.40 / Chapter 2.5 --- cDNA Microarray Analysis --- p.42 / Chapter 2.5.1 --- PCR amplification of Subtracted Clones --- p.42 / Chapter 2.5.2 --- Purification of PCR Products of Subtracted Clones --- p.42 / Chapter 2 5.3 --- Rearrangement of Subtracted Clones into cDNA Microarray Format --- p.43 / Chapter 2.5.4 --- cDNA Microarray Fabrication --- p.43 / Chapter 2.5.5 --- Probe Preparation --- p.44 / Chapter 2.5.6 --- cDNA Microarray Hybridization --- p.46 / Chapter 2.5.7 --- Scanning cDNA Microarray Image and Data Analysis --- p.46 / Chapter 2.6 --- Sequencing of Differentially Expressed Genes --- p.48 / Chapter 2.6.1 --- Dye-terminator Cycle Sequencing --- p.48 / Chapter 2.6.2 --- Post-reaction Cleanup --- p.49 / Chapter 2.6.3 --- Signal Detection and Data Collection --- p.49 / Chapter 2.6.4 --- Sequencing Analysis --- p.50 / Chapter 2.7 --- Reverse Transcription Polymerase Chain Reaction --- p.51 / Chapter 2.8 --- Northern Blot Analysis --- p.54 / Chapter 2.8.1 --- RNA Transfer --- p.54 / Chapter 2.8.2 --- Probe Labeling --- p.55 / Chapter 2.8.3 --- Hybridization --- p.55 / Chapter 2.8.4 --- Chemiluminescent Detection --- p.56 / Chapter CHAPTER 3: --- RESULTS --- p.58 / Chapter 3.1 --- Suppression Subtractive Hybridization --- p.58 / Chapter 3.1.1 --- The Optimal Cycle for SMART cDNA Synthesis --- p.58 / Chapter 3.1.2 --- Adaptor Ligation Efficiency --- p.60 / Chapter 3.1.3 --- Primary and Secondary PCR Amplification of Subtracted cDNA --- p.63 / Chapter 3.1.4 --- Subtraction Efficiency --- p.66 / Chapter 3.2 --- Subtracted cDNA Libraries --- p.68 / Chapter 3.3 --- cDNA Microarray Analysis --- p.69 / Chapter 3.3.1 --- Isolation and Amplification of Subtracted cDNA Clones --- p.69 / Chapter 3.3.2 --- Microarray Scanning and A nalysis --- p.69 / Chapter 3.4 --- Sequencing Results of Subtracted cDNA Clones --- p.81 / Chapter 3.5 --- Reverse Transcription Polymerase Chain Reaction --- p.87 / Chapter 3.6 --- Northern Blot Hybridization --- p.90 / Chapter CHAPTER 4: --- DISCUSSION --- p.93 / Chapter 4.1 --- Subtraction Quality --- p.93 / Chapter 4.2 --- Differential Screening by cDNA Microarray --- p.95 / Chapter 4.2.1 --- Elimination of False Subtracted Clones --- p.95 / Chapter 4.2.2 --- Limitations of cDNA Microarray --- p.96 / Chapter 4.3 --- Differentially Expressed Genes in Hypertensive Heart --- p.98 / Chapter 4.3.1 --- Candidate Genes Showing Up-regulation --- p.99 / Chapter 4.3.1.1 --- Voltage-dependent Anion Channel 1 --- p.99 / Chapter 4.3.1.2 --- Protein Tyrosine Phosphatase 4a 1 --- p.101 / Chapter 4.3.1.3 --- Choline Transporter-like Protein 1 Splice Variant a --- p.102 / Chapter 4.3.2 --- Candidate Genes Showing Down-regulation --- p.103 / Chapter 4.3.2.1 --- Ryanodine Receptor 2 --- p.103 / Chapter 4.3.2.2 --- Guanine Nucleotide-binding Protein β1 Subunit --- p.104 / Chapter 4.3.2.3 --- Solute Carrier Family 3 Member 1 --- p.105 / Chapter 4.4 --- The Pros and Cons of Using Ventricular Tissue but not Cardiomyocytes --- p.107 / Chapter 4.5 --- Future Prospect --- p.109 / Chapter 4.5.1 --- Expression Profiling of Candidate Genes at Different Stages --- p.109 / Chapter 4.5.2 --- In vitro Studies of Candidate Genes --- p.110 / Chapter 4.5.2.1 --- Over-expression of up-regulated genes in Normal Cardiac Cells --- p.110 / Chapter 4.5.2.2 --- Suppression of down-regulated genes in Normal Cardiac Cells --- p.111 / Chapter 4.5.3 --- In vivo Studies of Up-regulated Genes --- p.111 / Chapter 4.5.4 --- Confirmation of Other Potential Candidate Genes --- p.112 / Chapter 4.6 --- Conclusion --- p.113 / REFERENCES --- p.114

Wong Wai Yin. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references. / Abstracts in English and Chinese. / Acknowledgement --- p.i / Abstract --- p.iii / Abbreviation --- p.vii / Chapter CHAPTER 1 --- INTRODUCTION --- p.1-1 / Chapter 1.1 --- Classification of common epithelial ovarian tumors --- p.1-2 / Chapter 1.1.1 --- Serous tumors --- p.1-4 / Chapter 1.1.2 --- Mucinous tumors --- p.1-5 / Chapter 1.1.3 --- Endometrioid tumors --- p.1-6 / Chapter 1.1.4 --- Clear cell tumors --- p.1-6 / Chapter 1.1.5 --- Cancer staging --- p.1-7 / Chapter 1.1.6 --- Tumor grading --- p.1-8 / Chapter 1.2 --- Etiology --- p.1-10 / Chapter 1.2.1 --- Factors associated with increased risks --- p.1-10 / Chapter 1.2.2 --- Factors associated with decreased risks --- p.1-12 / Chapter 1.2.3 --- Other factors --- p.1-13 / Chapter 1.3 --- Understanding of progression of ovarian carcinoma --- p.1-13 / Chapter 1.4 --- Current screening test for ovarian cancer --- p.1-15 / Chapter 1.4.1 --- Transvaginal utrasound --- p.1-15 / Chapter 1.4.2 --- Serum tumor markers --- p.1-16 / Chapter 1.5 --- Molecular basis of ovarian cancer --- p.1-18 / Chapter 1.5.1 --- Loss of heterozygosity --- p.1-18 / Chapter 1.5.2 --- Microsatellite instability --- p.1-19 / Chapter 1.5.3 --- Oncogenes --- p.1-19 / Chapter 1.5.4 --- Tumor suppressor genes --- p.1-21 / Chapter 1.6 --- Microarray gene expression profiling analysis --- p.1-25 / Chapter 1.6.1 --- Princeple of DNA micorarray --- p.1-26 / Chapter 1.6.2 --- Types of microarray --- p.1-29 / Chapter 1.7 --- Gene expression profiling of ovarian cancer --- p.1-29 / Chapter 1.7.1 --- Up-regulated genes in ovarian cancer --- p.1-30 / Chapter 1.7.2 --- Down-regulated genes in ovarian cancer --- p.1-32 / Chapter 1.8 --- Project aims --- p.1-35 / Chapter CHPATER 2 --- MATERIALS AND METHODS --- p.2-1 / Chapter 2.1 --- Materials --- p.2-1 / Chapter 2.1.1 --- Patients --- p.2-1 / Chapter 2.1.2 --- Ovarian tissue specimen --- p.2-1 / Chapter 2.2 --- Methods --- p.2-2 / Chapter 2.2.1 --- Preparation of OCT-embedded Specimen Sections --- p.2-2 / Chapter 2.2.2 --- Microdissection of Tumor Cells from Specimen Sections --- p.2-3 / Chapter 2.2.3 --- Disruption of normal ovarian frozen tissue --- p.2-3 / Chapter 2.2.4 --- Total RNA Extraction --- p.2-3 / Chapter 2.2.4.1 --- RNA Isolation --- p.2-4 / Chapter 2.2.4.2 --- DNase I Digestion --- p.2-4 / Chapter 2.2.4.3 --- RNA Cleanup and Elution --- p.2-5 / Chapter 2.2.5 --- Oligonucleotide Microarray --- p.2-6 / Chapter 2.2.5.1 --- Two-Cycle cDNA Synthesis --- p.2-6 / Chapter 2.2.5.2 --- Synthesis of Biotin-Labeled cRNA --- p.2-9 / Chapter 2.2.5.3 --- Fragmenting the cRNA for Target Preparation --- p.2-9 / Chapter 2.2.5.4 --- Target Hybridization --- p.2-10 / Chapter 2.2.5.5 --- "Array Washing, Staining, and Scanning" --- p.2-11 / Chapter 2.2.5.6 --- Statistical Analysis of Microarray Data --- p.2-11 / Chapter 2.2.6 --- Quantitative Real-time Polymerase Chain Reaction --- p.2-13 / Chapter 2.2.6.1 --- Primer and Probe --- p.2-13 / Chapter 2.2.6.2 --- Reverse-transcription --- p.2-13 / Chapter 2.2.6.3 --- Plate Setup --- p.2-14 / Chapter 2.2.6.4 --- Fluocogenic PCR --- p.2-14 / Chapter 2.2.6.5 --- Statistical Analysis of Quantitative Real-time PCR Data --- p.2-15 / Chapter CHAPTER 3 --- RESULTS --- p.3-1 / Chapter 3.1 --- Microarray gene expression data analysis --- p.3-1 / Chapter 3.1.1 --- Unsupervised Gene Selection --- p.3-1 / Chapter 3.1.2 --- Supervised Gene Selection --- p.3-3 / Chapter 3.1.2.1 --- Gene expression profiles distinguish Serous Epithelial Ovarian Tumor from Normal Ovary and identifydifferentially expressed genes --- p.3-3 / Chapter 3.1.2.2 --- Gene expression profiles distinguish Advanced Stage Serous Epithelial Ovarian Tumor from Early Stage Serous Epithelial Ovarian Tumor and identify differentially expressed genes --- p.3-22 / Chapter 3.1.2.3 --- Gene expression profiles distinguish Metastatic Serous Epithelial Ovarian Tumor from Primary Serous Epithelial Ovarian Tumor and identify differentially expressed genes --- p.3-24 / Chapter 3.2 --- Validation of microarray data by quantitative Real-time PCR --- p.3-27 / Chapter 3.2.1 --- Fold change of candidate genes --- p.3-27 / Chapter 3.2.2 --- Correlation between microarray and quantitative real-time PCR results --- p.3-29 / Chapter 3.2.3 --- Comparison of the expression of candidates genes among the different histological types of epithelial ovarian tumors --- p.3-32 / Chapter CHAPTER 4 --- DISCUSSION --- p.4-1 / Chapter 4.1 --- Global gene expression profiling using oligonucleotide microarray --- p.4-1 / Chapter 4.1.1 --- "Sensitivity, specificity and reproducibility of the Affymetrix GeneChip® microarray" --- p.4-1 / Chapter 4.1.2 --- Microarray analysis software --- p.4-3 / Chapter 4.1.2.1 --- DNA-Chip Analyzer software --- p.4-3 / Chapter 4.1.2.2 --- Comparison of statistical methods for analysis of Affymetrix GeneChip® microarray data --- p.4-5 / Chapter 4.2 --- Validation of microarray data --- p.4-7 / Chapter 4.2.1 --- Advantages of using real-time PCR for mRNA quantification --- p.4-8 / Chapter 4.2.2 --- Comparison of mRNA gene expression by RT-PCR and DNA microarray --- p.4-9 / Chapter 4.3 --- Gene expression profiling in serous ovarian cancer compared with normal ovarian epithelium --- p.4-10 / Chapter 4.3.1 --- Potential biomarkers or therapeutic targets in ovarian cancer --- p.4-12 / Chapter 4.4 --- Gene expression profiling in advanced serous ovarian cancer compared with early ovarian cancer --- p.4-16 / Chapter 4.4.1 --- Potential prognostic markers or therapeutic targets in advanced ovarian cancer --- p.4-17 / Chapter 4.5 --- Gene expression profiling in metastatic cancer compared with primary ovarian cancer --- p.4-22 / Chapter 4.5.1 --- Potential predictive markers or therapeutic targets in metastatic cancer of ovary origin --- p.4-23 / Chapter CHAPTER 5 --- CONCLUSIONS --- p.5-1 / Chapter CHAPTER 6 --- FUTURE PROSPECT --- p.6-1 / REFERENCES --- p.R-1