The studies that have been published since 1990 which are dealing with the relationship of the genetic polymorphism of the dopamine D2 receptor gene (DRD2) in relation to alcoholism or other disorders or diseases have brought contradictory results. These results have led the author of this thesis to closely investigate the relationship between the genetic polymorphism of the A1 allele gene for DRD2 and hazardous alcohol consumption in the Czech Republic with a focus on the artistic area. A total of 29 participants (15 artists and 14 "non-artists") were involved in the study. The criterion for selecting the examined sample was the type of professional focus (artistic, non-artistic). A genetic analysis of blood (PCR-RFLP method) was used to determine the genotype. Only in the "non-artistic" group the presence of the genetic polymorphism of the A1 allele for DRD2 was found (a predisposition to a risk behavior in relation to alcohol). Additional parameters were found using EEG, an AUDIT test and a temperament questionnaire. A statistical analysis confirmed an association between the appearance of the A1 allele for DRD2 and a higher amplitude of the P300 component (p = 0.0000421). Statistically proven riskier alcohol consumption was found among introverted artists (n = 9) in the AUDIT test (p = 0.02298).

Made available in DSpace on 2016-08-10T10:38:27Z (GMT). No. of bitstreams: 1 Lindomar Valentin Ferreira.pdf: 1215853 bytes, checksum: 2cb0836a827c452d5ca06c97f0d59f35 (MD5) Previous issue date: 2011-05-13 / The population genetics aims to study the allelic and genotypic frequencies in populations and the mechanisms that these frequencies change over generations. This study aims to estimate and compare the allele frequencies of 15 autosomal STR markers, sheets of 501 tests the genetic link in 986 individuals in the population of Goiânia-Goiás. The markers that showed higher numbers of alleles were D18S51 and Penta E with 20 to 19. The marker that had allele frequency was higher for the TPOX 8 allele. Other statistical parameters analyzed demonstrated high PIC, PD and PE for the marker Penta E. Analysis of allele frequencies obtained in this study were statistically compared with populations of five regions of Brazil, by Student's t test, not intra-population differences were observed (p> 0.05). In this context, the genetic database based on the values of the allele frequencies of the population of Goiânia, can be used in testing the genetic link, as well as in studies of human identification, due to the similarities observed in other Brazilian populations. / A genética de populações visa o estudo das frequências alélicas e genotípicas em populações e os mecanismos capazes de mudar estas frequências ao longo das gerações. Essa pesquisa tem como objetivo estimar e comparar as frequências alélicas de 15 marcadores autossômicos STR, de 501 planilhas de exames de vínculo genético em 986 indivíduos da população de Goiânia-Goiás. Os marcadores que apresentaram maiores números de alelos foram o D18S51 com 20 e o Penta E com 19. O marcador que teve maior frequência alélica foi o TPOX para o alelo 8. Outros parâmetros estatísticos analisados caracterizaram elevados PIC, PD e PE para o marcador Penta E. As análises das frequências alélicas obtidas neste estudo foram comparadas estatisticamente com populações de cinco regiões brasileiras, pelo teste t Student, e não foram verificadas diferenças intra populacionais significativas (p>0,05). Neste contexto, o banco de dados genéticos baseado nos valores das frequências alélicas da população de Goiânia, pode ser utilizado em testes de vínculo genético, assim como em estudos de identificação humana, devido às semelhanças verificadas com outras populações brasileiras.

Em mamíferos a inativação do cromossomo X (ICX) consiste no silenciamento gênico de um dos dois X presentes nas células somáticas normais das fêmeas, garantindo a compensação de dose transcricional em relação aos machos. Existem duas formas de ICX: aleatória, na qual a escolha do cromossomo X inativado se dá ao acaso (X paterno ou materno); e de maneira completamente desviada, na qual a atividade do cromossomo X dependerá de sua origem parental. Nas fêmeas marsupiais a inativação ocorre de forma completamente desviada, sendo o X paterno preferencialmente inativado em todas as células, já nas células embrionárias de eutérios, o que se observa é a ICX aleatória. Entretanto, naquelas células que darão origem aos tecidos extra-embrionários, de camundongos e bovinos, a ICX se dá de forma equivalente à dos marsupiais, ou seja, o X paterno é preferencialmente inativado. Há mais de 30 anos o padrão de ICX em tecidos extra-embrionários humanos tem sido alvo de intenso debate. A crítica que se faz aqui é que tais estudos foram realizados com base na expressão de apenas um ou dois genes ligados ao X com amostras de tecidos extra-embrionários em diferentes idades gestacionais e, por vezes, em poucas amostras, o que deve ter levado às contradições entre as conclusões. O diferencial deste trabalho foi a utilização de técnicas de genotipagem de SNPs presentes em regiões codificadoras, para analisar o padrão de atividade alelo-específica de um grande número de genes presentes ao longo de todo o cromossomo X, gerando um panorama mais representativo da ICX em placenta humana. Neste estudo é comprovado o padrão aleatório de ICX em placenta humana a termo e demonstrado que este órgão se apresenta como um 65 mosaico em relação à escolha do X inativo. A análise global da atividade gênica no cromossomo X indicou ainda que a manutenção do estado epigenético do X inativo parece ser heterogêneo. Em conjunto, os dados gerados são capazes de explicar as incongruências entre as conclusões previamente publicadas. Este trabalho também ilustra as diferenças nos mecanismos de ICX entre humanos e camundongos e reforça a importância de se avaliar esse tema em outras espécies de mamíferos eutérios na tentativa de se elucidar os processos evolutivos envolvidos na compensação de dose em mamíferos / Imprinted inactivation of the paternal X chromosome in marsupials is the primordial mechanism of dosage compensation for X-linked genes between females and males in Therians. In Eutherian mammals, X chromosome inactivation (XCI) evolved into a random process in cells from the embryo proper, where either the maternal or paternal X can be inactivated. However, species like mouse and bovine maintained imprinted XCI exclusively in extraembryonic tissues. The existence of imprinted XCI in humans remains controversial, with studies based on the analyses of only one or two X-linked genes in different extraembryonic tissues. Here we readdress this issue in human term placenta by performing a robust analysis of allele-specific expression of 23 X-linked genes, including XIST, using 28 SNPs in transcribed regions. We show that XCI is random in human placenta, and that this organ is arranged in relatively large patches of cells with either maternal or paternal inactive X. In addition, this chromosome-wide analysis indicated heterogeneous maintenance of the epigenetic state along the inactive X, which combined with the extensive mosaicism found in placenta, can explain the lack of agreement among previous studies. Our results illustrate the differences of XCI mechanism between humans and mice, and highlight the importance of addressing the issue of imprinted XCI in other species in order to understand the evolution of dosage compensation in placental mammals

A asma brônquica é doença inflamatória crônica complexa das vias aéreas provocada pela interação de fatores genéticos e ambientais. O gene HLA-G (Antígeno Leucocitário Humano G) foi identificado como gene de susceptibilidade à asma, codificando uma molécula não clássica do complexo principal de histocompatibilidade (MHC, do inglês Major Histocompatibility Complex) de classe I com função moduladora das células do sistema imunológico. Nesse contexto, avaliamos o papel do HLA-G na asma afim de identificar genótipos, alelos e haplótipos associados com proteção ou susceptibilidade nas diferentes formas de apresentação da doença. Investigamos os sítios polimórficos da região 3\' não traduzida (3\'UTR-untranslated region) do HLA-G (14 pb Ins/Del, + 3001 C/T, +3003 C/T, +3010 C/G, +3027 A/C, +3035 C/T, +3142 C/G, +3187 A/G e +3196 C/G) em 118 pacientes asmáticos estratificados em asma leve ou moderada e grave e 183 indivíduos brasileiros saudáveis. Testes de associação foram realizados para avaliar as frequências dos genótipos, alelos e haplótipos da 3\'UTR do HLA-G na asma brônquica, considerada como grupo total ou estratificada de acordo com a gravidade da doença. Nossos resultados demonstraram que as frequências dos alelos +3001 C, +3003 C, +3035 C e +3196 C e do genótipo 14 bp DI estavam aumentadas no grupo total e nas diversas formas de apresentação da doença. Os alelos +3010 C e +3142 G e o genótipo +3010 CC estavam mais representados em pacientes com asma leve ou moderada. Por outro lado, os genótipos +3010 GG, +3142 CG e +3187 AG e o alelo +3010 G apresentaram maior frequência nos asmáticos graves, estando fortemente associados com o desenvolvimento da forma grave da asma. Além disso, os genótipos 14 pb II, +3010 CC e +3142 GG e o alelo +3010 C conferiram proteção à asma grave. Além disso, identificamos um haplótipo da 3\'UTR do HLA-G associado ao desenvolvimento de asma brônquica, a UTR-8, e um haplótipo que conferiu proteção contra a mesma, a UTR-7. Concluindo, neste estudo, observamos frequências diferenciais de sítios polimórficos do segmento 3\'UTR do HLA-G associados com predisposição à asma brônquica e, também, com a gravidade da doença / Bronchial asthma is a complex chronic inflammatory disease of the airways caused by the interaction of genetic susceptibility and environmental factors. The HLA-G (Human Leucocyte Antigen G) gene was identified as a susceptible marker for bronchial asthma, encoding a nonclassical Major Histocompatibility Complex (MHC) class I molecule, considered to be an important immune check point modulator. In the present study, we evaluated the role of HLA-G in bronchial asthma susceptibility and disease severity, evaluating HLA-G genotypes, alleles or haplotypes. We investigated the HLA-G 3\'Untraslated region (3\'UTR) polymorphic sites (14-bp INS/DEL, +3001, +3003C/T, +3010C/G, +3027A/C, +3035C/T, +3142C/G, +3187A/G, and +3196C/G) in 118 asthmatic Brazilian patients, stratified according to disease severity into mild/moderate and severe asthma, and in 183 healthy individuals. HLA-G 3\'UTR variation sites were individually analyzed or lumped together as haplotypes. Our results showed that frequencies of +3001 C, +3003 C, +3035 C e +3196 C alleles and 14 pb ID genotype were increased in asthma group considered as a whole and in patients stratified according to disease severity. The +3010 C and .3142 G alleles and the +3010 CC genotype were overrepresented in patients with mild and moderate forms. Similarly, the +3010 GG, +3142 CG, +3187 AG genotypes and +3010 G allele presented increased frequency in severe asthmatic patients. In contrast, the 14 pb II, +3010 CC and +3142 GG genotypes and +3010 C allele conferred protection against severe asthma. In addition, we identified a 3\'UTR HLA-G haplotype that was associated with bronchial asthma development (UTR-8) and one haplotype that conferred protection against asthma (UTR-7). In conclusion, in this study, we observed differential frequencies at HLA-G 3\'UTR polymorphic sites that are associated with bronchial asthma predisposition and, also, with disease severity

Mais de 200 milhões de bovinos, em sua maioria animais da raça Nelore ou produtos de cruzamento com Nelore, compõem o rebanho do maior exportador de carne in natura do mundo, o Brasil. No entanto, o faturamento por kg de carne exportada poderia ser elevado se a qualidade do produto atendesse a mercados mais exigentes. A maciez da carne, a quantidade de gordura entremeada, a cobertura de gordura da carcaça e área do músculo Longissimus dorsi são fatores relevantes na procura por qualidade e padronização. A seleção genética assistida por marcadores moleculares poderá ter impacto positivo no melhoramento animal, principalmente em características de mensuração tardia e difícil para o pecuarista. Polimorfismos dos genes da tiroglobulina (TG), µ-calpaína (CAPN1) e calpastatina (CAST) são hoje marcadores disponíveis comercialmente, implicados na maciez e na marmorização da carne. Os genes do diacilglicerol O-aciltranferase (DGAT1) e da leptina (LEP) são candidatos pesquisados no desenvolvimento de marcadores relacionados à qualidade da carne. Leptina, tiroglobulina e diacilglicerol O-aciltranferase influenciam no metabolismo energético e na deposição de gordura. A calpastatina inibe a atividade proteolítica de µ-calpaína regulando o processo de amaciamento da carne no postmortem. Este trabalho avaliou as freqüências alélicas e genotípicas de polimorfismos dos genes CAPN1, CAST, LEP, TG e DGAT1 e relacionando os com qualidade de carne. Os polimorfismos escolhidos foram alterações de ponto (SNP) observadas pelo método da reação da polimerase em cadeia seguida de restrição enzimática (PCR-RFLP). Entre animais de raça Nelore e Canchim e cruzamentos de Nelore com Bos taurus (Rubia Gallega, Brangus, Pardo Suíço) foram genotipados 147 animais. Os mesmo foram abatidos com idade entre 15 e 19 meses, os animais foram avaliados quanto à maciez da carne (Warner-Bratzler Shear Force - SF - e Índice de Fragmentação Miofibrilar - MFI), à deposição de gordura (intramuscular e de cobertura) e à área de olho de lombo (AOL - Longissimus dorsi). O estudo de associação entre genótipos e fenótipos utilizou o procedimento General Linear Model do programa SAS e o teste F. Com o nível de significância de 5%, as freqüências alélicas de CAPN1/PsyI foram semelhantes entre os grupos genéticos estudados, mas as freqüências dos demais polimorfismos diferiram entre os grupos. Um achado inovador do trabalho foi a presença das duas variantes dos polimorfismos CAST/XmnI e DGAT1/CfrI na raça Nelore. Neste estudo o polimorfismo de TG/PsuI não foi informativo para raça Nelore, pois todos os animais apresentaram o mesmo genótipo (CC). Os resultados não mostraram associações significativas entre os polimorfismos e as análises da carne, exceto pelo efeito de CAST/XmnI cujo genótipo AA foi superior ao AB com relação ao MFI. Mais estudos semelhantes se fazem necessários para desenvolver marcadores adicionais e descartar os não informativos, adequando as novas ferramentas de seleção ao gado zebuíno brasileiro e respectivos cruzamentos. Os cinco genes estudados continuam como candidatos, apresentando diversos polimorfismos para serem pesquisados. No futuro, estudos com maior número de bovinos poderão validar o polimorfismo CAST/XmnI como indicador da maciez da carne. / Over 200 million bovines, mostly Nellore breed and its crosses, form the herd of the world biggest meat in natura exporter, Brazil. Even so, profit per kg of traded meat would improve if the product\'s quality achieved selective markets. Tenderness, marbling, backfat thickness and Longissimus dorsi área are traits to consider when pursuing meat quality and standardization. Marker assisted selection (MAS) can have positive impact over genetic improvement in livestock, mainly over traits such as the above mentioned, which are measured late in the production system and are hard to measure at the farm. Polymorphisms of thyroglobulin (TG), µ-calpain (CAPN1) and calpastatin (CAST) genes are commercially available markers assumed to predict meat tenderness and marbling. The genes encoding diacylglycerol O-acyltranferase (DGAT1) and leptin (LEP) are candidats researched to develop new markers related to meat quality. Thyroglobulin, Diacylglycerol O-acyltranferase and leptin influence energy metabolism and fat deposition. Calpastatin inhibits µ-calpain proteolysis regulating postmortem meat tenderization. This study evaluated allelic and genotype frequencies of polymorphisms located at CAPN1, CAST, LEP, TG and DGAT1 and tried to correlate the polymorphisms with meat quality. In each gene a single nucleotide polymorphism (SNP) was assessed by polymerize chain reaction followed by restriction enzyme digestion (PCR-RFLP). Including Nellore, Canchim and crosses of Nellore with Bos taurus breeds (Rubia Gallega, Brangus and Brown Swiss) a total of 147 animals were genotyped. Slaughterd aging 15 to 19 month, the animals were evaluated for meat tenderness (Warner-Bratzler Shear Force - SF - Myofibrillar fragmentation index - MFI), fat deposition (intramuscular and backfat) and rib eye area (REA - Longissimus dorsi). The assossiation study between genotypes and phenotypic traits used the linear regression model of SAS and the least square means were compared by the F test. Allelic frequencies of CAPN1/PsyI were similar in every genetic group of the study, but other polymorphisms allele frequencies differed between groups. A novel finding was the occurrence of both variants of CAST/XmnI and DGAT1/CfrI in the Nellore breed. In this study, the TG/PsuI polymorphism was uninformative for Nellore animals since only CC genotype was found. The results shwoed no significant assossiation between polymorphisms and the meat chacacteristics, except for the effect of CAST/XmnI over MFI, the AA genotype was superior to AB, predicting AA animals to have more tender meat. Further research is required to develop additional markers, disposal uninformative ones and to adequate molecular selecting tools to Brazilian zebu cattle and its crosses. The five genes here presented remain as candidates, in attendance to several polymorphisms yet to be carefully evaluated. Future studies, with bigger animal numbers, may validate CAST/XmnI as a meat tenderness indicator.

Mais de 200 milhões de bovinos, em sua maioria animais da raça Nelore ou produtos de cruzamento com Nelore, compõem o rebanho do maior exportador de carne in natura do mundo, o Brasil. No entanto, o faturamento por kg de carne exportada poderia ser elevado se a qualidade do produto atendesse a mercados mais exigentes. A maciez da carne, a quantidade de gordura entremeada, a cobertura de gordura da carcaça e área do músculo Longissimus dorsi são fatores relevantes na procura por qualidade e padronização. A seleção genética assistida por marcadores moleculares poderá ter impacto positivo no melhoramento animal, principalmente em características de mensuração tardia e difícil para o pecuarista. Polimorfismos dos genes da tiroglobulina (TG), µ-calpaína (CAPN1) e calpastatina (CAST) são hoje marcadores disponíveis comercialmente, implicados na maciez e na marmorização da carne. Os genes do diacilglicerol O-aciltranferase (DGAT1) e da leptina (LEP) são candidatos pesquisados no desenvolvimento de marcadores relacionados à qualidade da carne. Leptina, tiroglobulina e diacilglicerol O-aciltranferase influenciam no metabolismo energético e na deposição de gordura. A calpastatina inibe a atividade proteolítica de µ-calpaína regulando o processo de amaciamento da carne no postmortem. Este trabalho avaliou as freqüências alélicas e genotípicas de polimorfismos dos genes CAPN1, CAST, LEP, TG e DGAT1 e relacionando os com qualidade de carne. Os polimorfismos escolhidos foram alterações de ponto (SNP) observadas pelo método da reação da polimerase em cadeia seguida de restrição enzimática (PCR-RFLP). Entre animais de raça Nelore e Canchim e cruzamentos de Nelore com Bos taurus (Rubia Gallega, Brangus, Pardo Suíço) foram genotipados 147 animais. Os mesmo foram abatidos com idade entre 15 e 19 meses, os animais foram avaliados quanto à maciez da carne (Warner-Bratzler Shear Force - SF - e Índice de Fragmentação Miofibrilar - MFI), à deposição de gordura (intramuscular e de cobertura) e à área de olho de lombo (AOL - Longissimus dorsi). O estudo de associação entre genótipos e fenótipos utilizou o procedimento General Linear Model do programa SAS e o teste F. Com o nível de significância de 5%, as freqüências alélicas de CAPN1/PsyI foram semelhantes entre os grupos genéticos estudados, mas as freqüências dos demais polimorfismos diferiram entre os grupos. Um achado inovador do trabalho foi a presença das duas variantes dos polimorfismos CAST/XmnI e DGAT1/CfrI na raça Nelore. Neste estudo o polimorfismo de TG/PsuI não foi informativo para raça Nelore, pois todos os animais apresentaram o mesmo genótipo (CC). Os resultados não mostraram associações significativas entre os polimorfismos e as análises da carne, exceto pelo efeito de CAST/XmnI cujo genótipo AA foi superior ao AB com relação ao MFI. Mais estudos semelhantes se fazem necessários para desenvolver marcadores adicionais e descartar os não informativos, adequando as novas ferramentas de seleção ao gado zebuíno brasileiro e respectivos cruzamentos. Os cinco genes estudados continuam como candidatos, apresentando diversos polimorfismos para serem pesquisados. No futuro, estudos com maior número de bovinos poderão validar o polimorfismo CAST/XmnI como indicador da maciez da carne. / Over 200 million bovines, mostly Nellore breed and its crosses, form the herd of the world biggest meat in natura exporter, Brazil. Even so, profit per kg of traded meat would improve if the product\'s quality achieved selective markets. Tenderness, marbling, backfat thickness and Longissimus dorsi área are traits to consider when pursuing meat quality and standardization. Marker assisted selection (MAS) can have positive impact over genetic improvement in livestock, mainly over traits such as the above mentioned, which are measured late in the production system and are hard to measure at the farm. Polymorphisms of thyroglobulin (TG), µ-calpain (CAPN1) and calpastatin (CAST) genes are commercially available markers assumed to predict meat tenderness and marbling. The genes encoding diacylglycerol O-acyltranferase (DGAT1) and leptin (LEP) are candidats researched to develop new markers related to meat quality. Thyroglobulin, Diacylglycerol O-acyltranferase and leptin influence energy metabolism and fat deposition. Calpastatin inhibits µ-calpain proteolysis regulating postmortem meat tenderization. This study evaluated allelic and genotype frequencies of polymorphisms located at CAPN1, CAST, LEP, TG and DGAT1 and tried to correlate the polymorphisms with meat quality. In each gene a single nucleotide polymorphism (SNP) was assessed by polymerize chain reaction followed by restriction enzyme digestion (PCR-RFLP). Including Nellore, Canchim and crosses of Nellore with Bos taurus breeds (Rubia Gallega, Brangus and Brown Swiss) a total of 147 animals were genotyped. Slaughterd aging 15 to 19 month, the animals were evaluated for meat tenderness (Warner-Bratzler Shear Force - SF - Myofibrillar fragmentation index - MFI), fat deposition (intramuscular and backfat) and rib eye area (REA - Longissimus dorsi). The assossiation study between genotypes and phenotypic traits used the linear regression model of SAS and the least square means were compared by the F test. Allelic frequencies of CAPN1/PsyI were similar in every genetic group of the study, but other polymorphisms allele frequencies differed between groups. A novel finding was the occurrence of both variants of CAST/XmnI and DGAT1/CfrI in the Nellore breed. In this study, the TG/PsuI polymorphism was uninformative for Nellore animals since only CC genotype was found. The results shwoed no significant assossiation between polymorphisms and the meat chacacteristics, except for the effect of CAST/XmnI over MFI, the AA genotype was superior to AB, predicting AA animals to have more tender meat. Further research is required to develop additional markers, disposal uninformative ones and to adequate molecular selecting tools to Brazilian zebu cattle and its crosses. The five genes here presented remain as candidates, in attendance to several polymorphisms yet to be carefully evaluated. Future studies, with bigger animal numbers, may validate CAST/XmnI as a meat tenderness indicator.

As enzimas CYP2D6, CYP2C19 e CYP2C9 são responsáveis pelo metabolismo de aproximadamente metade dos 200 medicamentos mais prescritos nos EUA. Padronizamos ensaios de genotipagem baseados na discriminação alélica com o sistema TaqMan® em 198 indivíduos. Para o gene CYP2D6, os alelos *1 e *2 foram os mais freqüentes, seguidos pelos alelos *4, *41, *35, *17, *5, *10, *6, *29 e *9. Desenvolvemos também uma nova metodologia para a determinação do número de cópias do gene CYP2D6. Para o gene CYP2C19, o alelo *1 foi o mais frequente, seguido pelos alelos *17, *2 e *3. Nosso estudo foi o primeiro a determinar a freqüência alélica do gene CYP2C19 no Brasil. Para o gene CYP2C9, o alelo *1 foi o mais frequente, seguido pelos alelos *2 e *3. Desenvolvemos uma metodologia reprodutível e acessível para a genotipagem dos polimorfismos principais dos genes CYP2D6, CYP2C19 e CYP2C9. A identificação precoce de indivíduos suscetíveis a efeitos adversos, bem como de metabolizadores rápidos pode trazer grandes benefícios aos pacientes possibilitando assim uma medicina personalizada. / The enzymes CYP2D6, CYP2C19 and CYP2C9 metabolize approximately half of the 200 most prescribed drugs in the USA. We standardized genotyping tests based on allelic discrimination, using TaqMan® genotyping system in 198 samples. For the CYP2D6 gene, allele *1 and *2 were the most frequent, followed by alleles *4, *4, *35, *17, *5, *10, *6, *29 and *9. We have also developed a new methodology for determining the copy number variations of the CYP2D6 gene. For the CYP2C19 gene, the allele *1 was the most common, followed by the alleles *17, *2 and *3. In our concern, our study was the first to determine the allele frequency of the CYP2C19 gene in Brazil. For CYP2C9 gene, the allele *1 was the most common followed by the alleles *2 and *3. We developed a methodology reproducible and accessible for genotyping the most important polymorphisms of the genes CYP2D6, CYP2C19 and CYP2C9. The previous identification of individuals at risk to develop adverse drug reactions as well as ultrarapid-metabolizers may bring benefits to the patients, leading to a personalized therapy.

<p>Microarray technology is a promising approach for the simultaneous analysis of multiple single nucleotide polymorphisms (SNPs), which are the most abundant form of genetic variation. In this thesis enzyme-assisted microarray-based methods were developed to improve the accuracy and genotype discrimination power of the current methods for SNP genotyping. The improved technology was applied for analysing recessively inherited disease mutations, for Y-chromosomal SNPs in a population study, for an evolutionary analysis of SNPs in flycatchers and for multiplexed quantitative determination of SNP-allele frequencies in pooled DNA samples. </p><p>A robust attachment chemistry for immobilising oligonucleotides on glass surface was established, based on an evaluation of eight covalent coupling methods. A four-colour fluorescence detection strategy, which enabled a multiplexed quantitative analysis for as little as 2% of a minority allele frequency in pooled samples was generated. </p><p>Twenty-five Y-chromosomal SNPs were screened in a collection of 300 samples from five Finno-Ugric-speaking populations using minisequencing on microarrays. In these populations six distinct haplotypes were defined by the six SNPs that were polymorphic. Data from five microsatellite markers was combined with the SNP data, revealing shared Y-chromosomal haplotypes between the Finns and the Saami, indicating, in accordance with earlier data, at least two founding Y-chromosomal lineages in these populations.</p><p>Database screening and subsequent validation of 125 potential SNPs in the highly repetitive type 1 interferon genes and genes coding for proteins in the interferon-related regulatory pathways revealed 25 informative SNPs in the Finnish and Swedish populations. These SNPs were included in a panel for microarray based genotyping that should find a variety of applications in genetic studies due to the important immunoregulatory functions of the IFN family.</p><p>The significance of sex-chromosome evolution on speciation was investigated in two naturally hybridising flycatcher species (N=459) by analysing a panel of 20 SNPs using minisequencing on microarrays. A strong selection against gene flow across the species boundary of sex-linked genes was observed, as well as a sex-chromosomal influence on male plumage characteristics that have previously been shown to reinforce isolation in these birds. The results suggest a major role for sex-chromosome-mediated isolation of the two flycatcher species.</p>

<p>OVARIAN cancer research focuses on answering important questions related to the disease, determining whether new approaches are feasible to contribute towards improving current treatments or discovering new ones. This study focused on the transcriptional regulation of genes that have been implicated in ovarian cancer, based on the occurrences of single nucleotide polymorphisms (SNPs) within transcription factor binding sites (TFBSs). Through the application of several in silico tools, databases and custom programs, this research aimed to contribute toward the identification of potentially bio-medically important genes or SNPs for pre-diagnosis and subsequent treatment planning of ovarian cancer. A total of 379 candidate genes that have been experimentally associated with ovarian cancer were analyzed. This led to the identification of 121 SNPs that were found to coincide with putative TFBSs potentially influencing a total of 57 transcription factors that would normally bind to these TFBSs. These SNPs with potential phenotypic effect were then evaluated among several population groups, defined by the International HapMap consortium resulting in the identification of three SNPs present in five or more of the eleven population groups that have been sampled.</p>

Detecting mutations and genomic variations is fundamental indiagnosis, isolating disease genes, association studies,functional genomics and pharmacogenomics. The objective hasbeen to use and further develop a variety of tools andtechnologies to analyze these genetic alterations andvariations. The p53 tumor suppressor gene and short arm of chromosome 9have been used as genetic markers to investigate fundamentalquestions concerning early events preceding non-melanoma skincancers, clonal progression and timing of different mutationsand deletions. Conventional gel based DNA sequencing andfragment analysis of microsatellite markers were utilized forthis purpose. In addition, a sequence-specific PCR-mediatedartifact is discussed. Pyrosequencing, a bioluminometric technique based onsequencing-by-synthesis, has been utilized to determinemutation ratios in the p53 gene. In addition, in the case ofmultiple mutations, pyrosequencing was adopted to determineallelic distribution of mutations without the use of cloningprocedures. Exons 5 to 8 of the p53 gene were also sequenced bythis method. The possibility of typing single base variations bypyrosequencing has been evaluated. Two different nucleotidedispensation orders were investigated and data were comparedwith the predicted pattern for each alternative of the variableposition. Analysis of loss of heterozygosity was possible byutilizing single nucleotide polymorphisms. A modified allele-specific extension strategy for genotypingof single nucleotide polymorphisms has been developed. Throughthe use of a real-time bioluminometric assay, it has beendemonstrated that reaction kinetics for a mismatchedprimer-template is slower than the matched configuration,butthe end-point signals are comparable. By introduction ofapyrase, the problems associated with mismatch extensions havebeen circumvented and accurate data has been obtained. Keywords:fragment analysis, microsatellite, loss ofheterozygosity, DNA sequencing, pyrosequencing, cancer,mutation, variation, single nucleotide polymorphism,allele-specific extension, bioluminescence, apyrase. / QC 20100415