Dissertations / Theses on the topic 'VDR polymorphism'

Colorectal cancer is estimated to cause approximately 50,000 deaths each year in the United States. Epidemiological studies have demonstrated an inverse association between sunlight exposure, which stimulates the formation of vitamin D in the skin, and colorectal carcinoma. Laboratory studies report that metabolites of vitamin D, acting through the vitamin D receptor (VDR), regulate cellular proliferation, differentiation and apoptosis. In addition, VDR contains a polymorphic variant, FokI, which results in two different isoforms of VDR. We have demonstrated a differential suppression of β-catenin transcriptional activity by these isoforms in the presence of 1,25(OH)₂D₃ (1,25D). Epidemiological evaluation of metachronous colorectal adenoma formation indicates that VDR includes several single nucleotide polymorphisms (SNPs) which influence the odds of developing colorectal adenoma. In addition, we have found full length Adenomatous Polyposis Coli (APC), a frequently mutated tumor suppressor gene in colorectal cancer, augments both the interaction of VDR and β-catenin as well as the suppression of β-catenin transcriptional activity in the presence of 1,25D. We have also demonstrated in epidemiological studies that the presence of a T-A haplotype in APC codons 486 and 1822, respectively, reduces the odds of any metachronous adenoma by 27% [odds ratio (OR), 0.73; 95% confidence interval (95% CI), 0.59 – 0.91]. Taken together, these data support not only a protective role for vitamin D acting through the VDR, but also for an important role of heritable polymorphic variation in VDR and APC in carcinogenesis.

Today, we have ample evidence that hormonal system of vitamin D, the main component of which is calcitriol (1α,25(OH)2D3) and receptor of vitamin D (VDR) plays an important role not only in the regulation of functional and metabolic processes in the body, but also in the development of many diseases, including cardiovascular disease. The risk factors for ischemic stroke can be divided into unregulated (age, gender, race) and susceptible (malnutrition, lack of physical activity, smoking, alcohol abuse). Smoking doubles the risk of stroke. Once you stop smoking, the risk of a stroke in you will start to decrease immediately, after five years, the risk of developing a stroke will be the same as that of non-smokers.

In the overwhelming majority of cases, cerebral stroke is a multifactor disease, in the development of which, along with other factors, an undoubted role is played by changes in the system of hemostasis. In the last decade considerable attention has been paid to the study of the influence of genetic predisposition on the hemostatic system. In general, the risk of stroke in men is 30% higher than that of women. However, this is typical only for the age group of the population from 45 to 64 years. At the age of more than 65 years, the risk of stroke in men and women is practically the same.

Актуальність. Інсульт є однією з основних причин смерті в розвинених країнах і тим більше в Україні. Поширеність інсульту і тягар інвалідності, як очікується, зростуть в майбутньому, оскільки відбувається процес старіння населення. Крім віку до чинників ризику розвитку інсульту відносяться артеріальна гіпертензія, паління, цукровий діабет, гіпертрофія лівого шлуночка і фібриляція передсердь. Ожиріння, як правило, передує розвитку артеріальної гіпертензії, цукрового діабету та їх ускладнень, що грає важливу непряму роль в епідеміології ішемічного інсульту. Метою нашої роботи було провести аналіз зв’язку BsmI поліморфізму гена рецептора вітаміну D (VDR) у двох груп, утворених за показником ІМТ (<25 кг/м2 і ≥25 кг/м2) хворих на ішемічний атеротромботичний інсульт (ІАІ).

Introdução: A Neurofibromatose tipo 1 (NF1) é uma doença genética autossômica dominante causada por mutação no gene NF1 (17q11.2; proteína neurofibromina). A incidência é de aproximadamente 1:3500 recém-nascidos e o diagnóstico clínico é possível na maioria dos casos. Alguns estudos sugeriram que pacientes com NF1 são mais suscetíveis a apresentar deficiência de vitamina D quando comparados à população geral. Objetivo: Determinar os níveis de 25(OH)D em indivíduos com NF1 e em controles saudáveis. Nos pacientes com NF1 nos também avaliamos o fenótipo clínico e analisamos polimorfismos comuns do gene VDR (BsmI e FokI) para avaliar se estavam correlacionados com os níveis de 25(OH)D. Métodos: Estudo de caso-controle que incluiu 45 pacientes com NF1 do Sul do Brasil e 45 controles saudáveis pareados por idade, sexo e tipo de pele de acordo com a classificação de Fitzpatrick. Resultados e Conclusões: Setenta e sete vírgula oito por cento dos 90 sujeitos de pesquisa apresentaram níveis de 25(OH)D abaixo de 30ng/ml. Deficiência de 25(OH)D (<20ng/mL) foi observada em 28 (31,1%) e insuficiência (de 20 a 30ng/mL) em 42 (46,6%). Deficiência ou insuficiência de 25(OH)D não foi mais frequente em pacientes com NF1 do que em controles (p=0,074). Nos não observamos qualquer associação entre os polimorfismos do gene VDR e os níveis de vitamina D sugerindo que o fenótipo da insuficiência ou deficiência bioquímica de 25(OH)D nos pacientes estudados não está associado com essas variantes genéticas.<br>Introduction: Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder caused by mutations in the NF1 gene (17q11.2, neurofibromin). The estimated incidence is approximately 1:3500 newborns and clinical diagnosis is possible in the majority cases. A few studies suggest that patients with NF1 are more likely to have vitamin D deficiency when compared to the general population. Objectives: The goal of the study was to determine the levels of 25-OH-vitamin D [25(OH)D] in individuals with NF1 and disease-unaffected controls. In NF1 patients we also assessed clinical phenotype and analyzed common VDR gene polymorphisms (BsmI and FokI) to verify whether they were associated with lower vitamin D levels. Methods: Case-control study that included 45 NF1 patients from Southern Brazil and 45 healthy controls matched by sex, skin type and age to the cases. Results and conclusions: Overall, 70 (77.8%) of the individuals studied had levels of 25(OH)D below 30ng/ml: vitamin D deficiency was observed in 28 (31.1%) and vitamin D insufficiency in 42 (46.6%) subjects. 25(OH)D deficiency or insufficiency were not more frequent in NF1 patients than in controls (p=0,074). We did not observe an association of VDR gene polymorphisms and vitamin D levels suggesting that the insufficient or deficient biochemical phenotypes in the patients studied here are not associated with these genetic variants.

Made available in DSpace on 2015-04-17T15:03:04Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 2451134 bytes, checksum: cd1a58c82bfe6936ed40ff974a4a3740 (MD5) Previous issue date: 2014-03-13<br>Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES<br>The high prevalence of hypovitaminosis D in the elderly population is identified as a potential risk factor for the development of cardiovascular diseases. The vitamin D receptor (VDR) is present in various body cells and polymorphisms of the gene encoding it can affect the cell responses to vitamin D. Therefore, this study aimed to evaluate the relationship between cardiometabolic profile, vitamin D status and BsmI polymorphism of the VDR gene in non-institutionalized elderly subjects. An observational cross-sectional study was conducted with a random and representative sample of 142 elderly subjects selected by conglomerate and recruited from a municipal assistance program. Clinical and nutritional, biochemical and inflammatory profiles, oxidative stress and genotyping for BsmI polymorphism were evaluated. For statistical analysis of data, significance level of p < 0.05 was adopted. Participants had mean age of 69.9 (7.0) years, BMI of 28.3 (4.4) kg / m² and 80.3 % were women. The prevalence of levels of 25 (OH) D < 30 ng / ml was 40.8 % (n = 58), three of them were deficient (25(OH) D < 20 ng / ml) and 55 were insufficient (25(OH) D between 21-29 ng/mL). The influencing factors associated with hypovitaminosis D were gender and fish consumption. The INSUF/DEF group showed fasting blood glucose and MDA values significantly higher when compared to the SUF group. The BsmI polymorphism analysis results showed allelic frequency for the SUF group of B = 0.49 and b = 0.51 and for INSUF/DEF group of B = 0.38 and b = 0.62. The frequency of homozygous bb was significantly associated with lower serum total cholesterol and LDL cholesterol concentrations compared to Bb both in the general population as in the SUF group, whose association was not observed in the INSUF/DEF group. Among individuals with bb, INSUF/DEF group showed higher levels of triglycerides and VLDL cholesterol. It was concluded that blood glucose levels and oxidative stress are increased in elderly subjects with hypovitaminosis D. The presence of genotype bb with adequate vitamin D levels resulted in lower total and LDL cholesterol levels, which benefit is lost when hypovitaminosis D is present. Therefore, low levels of vitamin D can be considered a risk factor for the development of cardiovascular diseases.<br>A alta prevalência de hipovitaminose D em idosos é identificada como um fator de risco potencial para o desenvolvimento de doenças cardiovasculares. O receptor de vitamina D (VDR) está presente em diversas células do corpo e polimorfismos no gene que o codifica podem influenciar nas respostas celulares à vit. D. Portanto esse estudo teve como objetivo avaliar a relação entre perfil cardiometabólico, status de vitamina D e polimorfismo BSMI do gene VDR em idosos não institucionalizados. Foi realizado um estudo transversal observacional com amostra aleatória e representativa de 142 idosos, selecionada por conglomerado e recrutada em um programa assistencial municipal. Foram avaliados: perfis clínico-nutricional, bioquímico e inflamatório, estresse oxidativo e genotipagem para o polimorfismo BsmI. Para análise estatística dos dados, foi considerado nível de significância p<0,05. Os participantes tinham idade média de 69,9 (7,0) anos, de IMC 28,3(4,4) Kg/m² e 80,3% eram mulheres. A prevalência de níveis de 25(OH)D < 30 ng/mL foi de 40,8% (n= 58), estando três deles deficientes (25(OH)D <20 ng/mL) e 55 com insuficiência (25(OH)D entre 21-29 ng/mL). A média de 25(OH)D foi 36,5(5,8) ng/dL entre os SUF e 25,7 ± 3,3ng/dL nos INSUF/DEF. Indivíduos brancos tiveram percentual significativamente maior de inadequação de níveis de vit. D em relação aos pardos e negros (p = 0,024). Entre o consumo dos principais alimentos fonte de vit. D foi encontrada relação positiva entre consumo de peixe e níveis de vit. D (p = 0,000). O grupo INSUF/DEF apresentou valores de glicemia de jejum significativamente mais elevados (p = 0,022) comparado ao SUF. O estresse oxidativo foi significativamente mais elevado no grupo INSUF/DEF em relação ao grupo SUF (p = 0,003). Os resultados da análise do polimorfismo BsmI do gene do VDR mostraram frequência alélica para o grupo SUF: B= 0,49 e b=0,51 e INSUF/DEF: B=0,38 e b=0,62 com p=0,051. A frequência de homozigoto bb foi significativamente associada com menores concentrações séricas de colesterol total e colesterol LDL, em relação ao BB e Bb, tanto na população em geral como no grupo SUF, associação não observada no grupo INSUF/DEF. Conclui-se que glicemia e estresse oxidativo estão aumentados em idosos com hipovitaminose D. A presença do genótipo bb, em condições adequadas de vit D sérica, resultou em níveis menores de colesterol total e LDL, este benefício se perde quando há insuficiência/deficiência de D. Portanto, baixo nível sérico de 25(OH)D pode ser considerado fator de risco cardiovascular.

У розвитку атеросклерозу вагоме значення належить запальним процесам у судинній стінці. Враховуючи імуномоделюючі властивості вітаміну D, його вплив на регуляцію атеросклеротичного процесу може мати важливе значення у розвитку серцево-судинних захворювань, включаючи клапанні кальцифікації, гіпертонію, ішемічний атеротромботичний інсульт.

A doença cardiovascular (DCV) é a principal causa de mortalidade e morbidade em pacientes portadores de diabetes mellitus tipo 2 (DM 2), estando associada com mais de 80% das mortes nesses pacientes. Portadores de DM 2 têm um risco três vezes maior em relação à indivíduos não diabéticos de desenvolver aterosclerose e suas complicações clínicas como infarto agudo do miocárdio (IAM), acidente vascular cerebral (AVC) e doença vascular periférica. O sistema endócrino da vitamina D regula a diferenciação e a proliferação de vários tipos celulares, além de possuir propriedades antiinflamatórias e antiangiogênicas. Assim, a vitamina D poderia ter um papel protetor contra as doenças degenerativas crônicas como a DCV. Estudos epidemiológicos sugerem que a deficiência de vitamina D está associada com doença arterial coronariana (DAC). As ações da 1,25(OH)2D3 são mediadas pela sua ligação ao seu receptor nuclear (VDR). O objetivo do presente estudo foi investigar as associações de polimorfismos de um único nucleotídeo (SNPs) no gene VDR com DAC em duas coortes de pacientes portadores de DM 2. Um total de 3.137 pacientes provenientes do estudo prospectivo DIABHYCAR (14,8% de incidência de DAC no seguimento) foi estudado. Uma outra coorte (NECKER - COCHIN) independente composta de 713 indivíduos portadores de DM 2 (32,3% dos quais tinham DAC) também foi avaliada. Três SNPs no gene VDR foram genotipados: rs1544410 (BsmI), rs7975232 (ApaI) e rs731236 (TaqI). Uma associação do alelo A de BsmI com casos incidentes de DAC (Hazard Ratio = 1,16; IC 95% = 1,05-1,29; p = 0,002) foi observada, assim como associações do alelo A de BsmI (p = 0,01) e do alelo C de TaqI (p = 0,04) com casos de DAC no início do estudo. O haplótipo AAC (BsmI / ApaI / TaqI) foi significantemente associado com aumento da prevalência de DAC no final do estudo, em comparação com o haplótipo GCT (Odds Ratio = 1,12; IC 95% = 1,02-1,28; p = 0,04). Associações do alelo A de ApaI (p = 0,009) e do alelo C de TaqI (p = 0,05) com DAC foram observadas no estudo transversal da coorte Necker - Cochin. Os resultados obtidos com os haplótipos também foram replicados nessa coorte (Odds Ratio = 1,33; IC 95% = 1,03-1,73; p = 0,03). Em conclusão, o haplótipo composto pelo alelo raro de BsmI, pelo alelo frequente de ApaI e pelo alelo raro de TaqI (AAC) foi associado a um maior risco de DAC em pacientes portadores de DM 2. Este efeito foi independente dos efeitos de outros fatores de risco cardiovasculares<br>Cardiovascular (CVD) disease is the leading cause of mortality and morbidity in patients with type 2 diabetes being associated with up to 80% of the deaths in these patients. Diabetic patients have a 3-fold higher risk than non diabetic individuals for developing atherosclerosis and its clinical complications such as myocardial infarction, stroke, and peripheral vascular disease. The vitamin D endocrine system regulates the differentiation and replication of several cell types and has antiangiogenic and antiinflammatory properties. Thus, it could have a protective role against chronic degenerative disorders such as CVD. Epidemiological studies suggested that vitamin D deficiency is associated with coronary heart disease. Actions of vitamin D are mediated by the binding of 1,25-(OH)2D3 to a specific cytosolic/nuclear vitamin D receptor (VDR). The present study investigated associations of VDR gene variants with coronary artery disease (CAD) in two cohorts of type 2 diabetic subjects. A total of 3,137 subjects participating in the 6-year prospective DIABHYCAR study (14.8% of CAD incidence at follow-up) were evaluated. An independent, hospital-based cohort (NECKER-COCHIN) of 713 diabetic subjects, 32.3% of whom had CAD, was also studied. Three single nucleotide polymorphisms (SNPs) in the VDR gene were genotyped: rs1544410 (BsmI), rs7975232 (ApaI) and rs731236 (TaqI). In the DIABHYCAR cohort, an association of the A-allele of BsmI with incident cases of CAD was found (Hazard Risk = 1.16; 95% C.I = 1.05-1.29; p = 0.002). Associations were also observed for the A- allele of BsmI (p=0.01) and C-allele of TaqI (p = 0.04) polymorphic variants with baseline cases of CAD. The AAC haplotype (BsmI/ApaI/TaqI) was significantly associated with increased CAD prevalence at the end of the study as compared with the GCT haplotype (Odds Ratio = 1.12; 95% C.I. = 1.02-1.28; p = 0.04). Associations of ApaI (p = 0.009) and TaqI (p = 0.05) alleles with CAD were observed in a cross-sectional study of the NECKER-COCHIN cohort. The haplotype results were also replicated (Odds ratio = 1.33; 95% C.I. = 1.03 - 1.73; p = 0.03). In conclusion, the haplotype composed by the minor allele of BsmI, the major allele of ApaI and the minor allele of TaqI (AAC) was associated with increased risk of CAD in type 2 diabetic patients. This effect was independent from effects of other known cardiovascular risk factors

Thyroid cancer is the most common endocrine malignancy and a complex disease with a largely unknown aetiology. Thyroid carcinomas are derived from thyroid follicular cells and parafollicular cells. The majority of thyroid cancer cases comprise both papillary (PTC) and follicular carcinomas (FTC). The evaluation of genetic susceptibility could give valuable information regarding the risk of thyroid cancer development. There are many genes associated with the thyroid function that modulates the risk of tumour development. Among them, is the vitamin D receptor gene (VDR), located on chromosome 12q12-q14, and includes eight protein coding exons (exons 2-9) and one untranslated exon (exons 1a-1f). The most common VDR polymorphisms investigated are FokI (rs10735810 C>T), located in exon 2 of VDR, BsmI (rs1544410 G>A) and ApaI (rs7975232 G>T), located in intron 8, and TaqI (rs731236 T>C), located in exon 9 of VDR. The importance of vitamin D and its receptor VDR, in many signalling pathways is well known. Therefore we aim to verify in which way VDR polymorphisms influence the predisposition for thyroid cancer development. The contribution of four well-known VDR polymorphisms (FokI, BsmI, ApaI and TaqI) for the genetic susceptibility of thyroid cancer in the Portuguese population was analysed, including haplotypes comparisons. The following parameters were studied: thyroid cancer type differences (PTC vs. FTC), age (≤45 vs. >45 years), gender (male vs. female), carcinoma size (≤10mm vs. >10mm), lymph node metastasis and distant metastasis multicentricity, and stage of cancer (I-II vs. III-IV). All the participants in the study were Caucasian Portuguese inhabitants, being subdivided into two groups: patients with thyroid cancer (N = 208) and a control group (N = 248). In conclusion, there were some statistically significant differences in some parameters assessed. However, the results considered were only those with a statistical significant p-value < 0.005, according to Bonferroni’s correction. Therefore, the results suggest that BsmI polymorphism genotype AA (p = 0.004) may influence lymph node metastasis or distant metastasis in patients with DTC. Moreover, the TT genotype (p = 0.004) of ApaI polymorphism may increase the predisposition for more aggressive phenotypes of DTC, since it is overrepresented in patients with more advanced cancer stages (III-IV).<br>O cancro da tiróide é, de todas as neoplasias endócrinas, a mais comum, revelando ser uma patologia complexa e com uma etiologia desconhecida em parte. Tem uma incidência mundial que tem tendência a aumentar, contabilizando cerca de 1.7% dos cancros diagnosticados. Adicionalmente, o cancro da tiróide é mais prevalente em pacientes de meia idade e idosos, onde mais de metade dos indivíduos diagnosticados têm uma idade superior aos 45 anos. Ademais, esta neoplasia endócrina é mais comum nas mulheres, com uma incidência de 3 a 5 vezes maior. Os nódulos que surgem na tiróide são diagnosticados em cerca de 5% da população adulta mundial, e podem ser adenomas ou lesões malignas. Os carcinomas da tiróide derivam quer das células foliculares da tiróide, bem como das células C, porém, a grande maioria deles tem origem nas células foliculares. De todas as variantes de carcinomas da tiróide, os carcinomas papilar e folicular da tiróide são os mais predominantes, sendo a variante papilar a mais comum de entre todos, seguida da variante folicular. Apesar da elevada incidência mundial de cancro da tiróide, a taxa de mortalidade associada permanece estável. O tratamento do cancro da tiróide é um processo multifatorial, envolvendo a combinação de terapias cirúrgicas, hormonais ou de medicina nuclear. Sabe-se que o cancro da tiróide, em especial os tumores diferenciados da tiróide, estão a aumentar de incidência em alguns países desenvolvidos. Existem muitos fatores de risco que aumentam a predisposição para este tipo de cancro, incluindo fatores genéticos com risco associado a esta patologia. A título de exemplo, o cancro diferenciado da tiróide está associado a uma forte hereditariedade, aumentando a suscetibilidade genética do indivíduo em desenvolver cancro de acordo com o seu historial familiar. Para além disso, a presença de polimorfismos genéticos podem determinar a suscetibilidade individual do indivíduo para o desenvolvimento de cancro da tiróide. Atualmente são conhecidos vários genes associados com a função tiroideia e que modulam o risco para a tumorigénese. O VDR é um membro da superfamília de recetores nucleares, sendo a única proteína com afinidade para a 1α,25-dihidroxivitamina D, também conhecida como calcitriol. Nos mamíferos, a expressão do VDR encontra-se aumentada em tecidos metabólicos tais como o intestino, rins, pele e glândula da tiróide. O impacto biológico do VDR surge quando este se liga aos seus elementos localizados nas regiões promotores dos genes alvo, interferindo assim em muitas ações celulares e moleculares que vão desde a regulação do metabolismo de cálcio até à regulação de péptidos antimicrobiais. Desta forma, a ação molecular da vitamina D/ VDR está envolvida na regulação mineral e homeostase óssea, modulação do crescimento, eventos cardiovasculares, prevenção de cancro e regulação de respostas imunes. Uma disfunção do VDR ou défice de vitamina D podem levar a consequências no desenvolvimento e saúde óssea assim como aumentar a predisposição do indivíduo para o desenvolvimento de algumas doenças crónicas, incluindo o cancro. Os polimorfismos associados ao gene VDR já provaram estar implicados como um fator principal de risco em vários tipos de cancro, tais como o cancro da próstata, mama ou cólon. Ao longo do tempo, estudos de associação têm sido feitos de modo a se poder correlacionar os polimorfismos genéticos e o seu impacto na saúde do indivíduo. Assim, no presente trabalho pretende-se estudar a suscetibilidade genética do cancro da tiróide associada aos polimorfismos do gene VDR. Neste trabalho, foram estudados quatro polimorfismos diferentes gene do VDR. Para tal, através do uso de enzimas de restrição, foi possível analisar áreas restritas do gene VDR, localizado no cromossoma 12q12-q14 de forma a se poder observar variações da sequência de DNA. Os quatro polimorfismos estudados no âmbito deste projeto foram o FokI (rs10735810 C>T), localizado no exão 2 do VDR, BsmI (rs1544410 G>A) e ApaI (rs7975232 G>T), localizados no intrão 8, e TaqI (rs731236 T>C), localizado no exão 9 do VDR. Estes quatro polimorfismos foram analisados com o objetivo de verificar de que forma influenciam a predisposição de um indivíduo para o desenvolvimento de cancro da tiróide. Desta forma, este estudo realizado na população Portuguesa, fez a análise destas variantes do VDR, e o seu impacto no desenvolvimento de cancro da tiróide de acordo com os seguintes parâmetros: tipo de cancro, idade de diagnóstico, sexo, dimensões do carcinoma, metástases ganglionares e à distância, multicentricidade tumoral, e estádios de cancro. Todos os participantes deste estudo foram indivíduos caucasianos de origem Portuguesa. Estes indivíduos foram divididos em dois grupos distintos. Um dos grupos foi composto por indivíduos com cancro diferenciado da tiróide (N = 208), provenientes do Instituto Português de Oncologia de Coimbra. O grupo de indivíduos saudáveis (N = 248), que constituíam o grupo controlo, consistiram em dadores voluntários de sangue Portugueses caucasianos, que não possuíam um historial clínico de cancro da tiróide. Após o recrutamento dos indivíduos e obtenção das amostras de sangue dos mesmos, procedeu-se a uma série de metodologias práticas que visaram como objetivo final genotipar as amostras recolhidas. A cada indivíduo, doente ou controlo, foi atribuído um número de código único, de forma a poder identificar e diferenciar a amostra em estudo. O processo clínico dos doentes foi registado com todos os dados necessários para este estudo. Quanto aos indivíduos saudáveis, estes permaneceram no anonimato, sendo apenas registado a idade, sexo, peso, altura e naturalidade. Após estes procedimentos de registo, o DNA genómico foi extraído das amostras de sangue recolhidas através do método de “salting-out”. De seguida o DNA extraído foi quantificado e armazenado. Para efeitos de genotipagem, o DNA de cada indivíduo participante no estudo foi submetido à técnica “polymerase chain reaction”, mais conhecida por PCR. Com este procedimento pretende-se amplificar o fragmento do gene VDR onde se encontra cada polimorfismo. Após a amplificação do fragmento do VDR que se pretendeu estudar, conforme o polimorfismo, procedeu-se à digestão enzimática utilizando a respetiva enzima. Desta forma, conseguimos determinar o genótipo do indivíduo, através da visualização desses produtos digeridos num gel de agarose de 3%. Para além deste método, a genotipagem foi também confirmada através da sequenciação de DNA, sendo utilizada uma amostra representativa de cada genótipo para cada polimorfismo. Terminada a genotipagem de todos os indivíduos participantes deste estudo, para os quatro polimorfismos do VDR, procedeu-se ao tratamento estatístico dos dados analisando os parâmetros acima referidos. Como resultados, verificaram-se, em alguns parâmetros, algumas diferenças de frequências dos polimorfismos. De entre esses resultados, nas comparações entre pacientes de sexo diferente, o genótipo GA do polimorfismo BsmI foi mais frequente no sexo masculino (p = 0.044). Na análise de metástases ganglionares e à distância, o genótipo AA (p = 0.004) do BsmI e o alelo A (p = 0.014) e o genótipo CC (p = 0.024) do TaqI foram mais frequentes no grupo de doentes com metástases. No estudo da multicentricidade tumoral, o alelo C (p = 0.041) do FokI, o genótipo AA (p = 0.013) do BsmI, e o genótipo CC (p = 0.017) do TaqI foram mais frequentes nos doentes com multicentricidade. No estudo dos estádios de cancro, os genótipos GT (p = 0.012) e TT (p = 0.004) do ApaI, e seu respetivo alelo T (p = 0.031) foram mais frequentes em doentes com estádios mais avançados. A correção estatística de Bonferroni para comparações múltiplas revelou que os resultados foram estatisticamente significativos apenas para o genótipo AA do polimorfismo BsmI, que parece estar envolvido na presença de metástases ganglionares em indivíduos com cancro diferenciado da tiróide. Para além disso, também o genótipo TT do polimorfismo ApaI revelou diferenças estatisticamente significativas, podendo estar associado a um estádio mais avançado de cancro da tiróide. Desta forma, os polimorfismos do gene do VDR podem servir como marcadores de risco úteis para pacientes com cancro diferenciado da tiróide, uma vez que estes já forma associados em outros tipos de cancro. No entanto, não é possível retirar conclusões a partir destes resultados uma vez que são necessários mais estudos que permitam compreender as ações celulares e moleculares do VDR. Para tal, estudos funcionais genómicos serão necessários, para que se possa clarificar de que forma os polimorfismos deste gene podem influenciar a suscetibilidade genética para o cancro da tiróide.

The vitamin D receptor (VDR), through the binding of 1,25 vitamin D and subsequently modulating the transcription of target genes, mediates the vitamin D endocrine system. When you are citing the document, use the following link http://essuir.sumdu.edu.ua/handle/123456789/27494

The metabolism of vitamin D commences with exposure of the skin to sunlight. The growing recognition of its role in insulin resistance, autoimmune disorders, infections, cancer, as well as the health of cells that influence physical and mental function have profound implications on how we define vitamin D requirements and why we should care whether they are met or not. Most of the actions of vitamin D are mediated by the vitamin D receptor (VDR), a protein whose gene sequence can vary, giving rise to polymorphic forms which are potent enough to affect the binding capacity of this protein to vitamin D. Some of these polymorphic forms of VDR gene may be associated with reduced effectiveness of vitamin D and hence predispose individuals to diseases such as type 2 diabetes and insulin resistance. An earlier study, the Surya Study, looked at the responsiveness of the South-Asian women living in Auckland to vitamin D. The research described here is an extension of this study and its focus was to identify the associations/linkages between certain polymorphic forms of the VDR gene and the disease conditions and intervention responsiveness in the same women. The first objective was to compare two well known techniques for genotyping single nucleotide polymorphisms (SNPs) of the VDR gene at the 3’ end, namely BsmI, ApaI and TaqI: the newer real-time polymerase chain reaction (qPCR) and the traditional restriction fragment length polymorphism polymerase chain reaction (RFLP-PCR) techniques. This comparison was performed to evaluate alternative methods for genotyping which consumed less time than RFLP-PCR. When the presence of each polymorphism by both the techniques was compared in this cohort of South-Asian women, it was found that RFLP-PCR proved to be a more reliable technique than qPCR for genotyping the VDR gene. Another objective of this project was to investigate the prevalence of the above three polymorphisms along with Cdx-2 and FokI SNPs which are present at the 5’ end of the VDR gene, in the population under study and their possible association with phenotypes such as vitamin D responsiveness and insulin resistance. These women were screened and biochemical data was collected during the earlier Surya Study. Of these, eighty-one women were then selected for intervention based on them having high insulin resistance (HOMA-IR>1.93) and serum 25(OH)D<50 nmol/L. Out of these eighty-one women, forty-two were given vitamin D supplement and thirty-nine were given a placebo for six months. Baseline and endpoint measurements included insulin resistance (HOMA-IR), insulin sensitivity (HOMA2%S) etc. How each individual responded to treatment in the intervention group was analysed in the context of the polymorphisms that they had. An association of insulin resistance with BsmI, ApaI and TaqI SNPs was observed in this cohort of 239 women. The response to insulin resistance in the vitamin D supplemented group significantly differed for FokI genotype compared to other genotypes. This explained why certain women responded to treatment better than the others. When the frequencies of the genotypes of these five SNPs of the VDR gene were compared to other studies of different ethnicities, the results of this study were consistent with few studies but contradictory to others. The possible reasons for these differences could be because of small sample size and different ethnicities under study due to which the frequency of alleles and hence the genotypes differed.

A Thesis submitted to The University of Arizona College of Medicine - Phoenix in partial fulfillment of the requirements for the Degree of Doctor of Medicine.<br>Vitamin D and resveratrol have been widely researched in recent years, especially their apparent abilities to impact a host of physiological processes. Resveratrol, a phytoalexin found in various berries, peanuts, and other vegetables, is purported to possess anti-aging, anti-inflammatory, antioxidant, anticancer, neuroprotective, and antiarthritic properties, while the classical endocrine functions of vitamin D are the control of calcium and phosphate homeostasis. The biologically active metabolite of vitamin D, 1,25-dihydroxyvitamin D (1,25D), is typically synthesized in the kidney, bound to vitamin D binding protein, and shuttled to cellular target sites. Mounting data on the effect of locally synthesized 1,25D in immune, epithelial, neural, and other tissues have led to an increased awareness of the myriad functions of vitamin D, including detoxification, cellular aging and its modulation, immune regulation, neurotransmitter activity, and metabolic control. Both endocrine and intracrine actions of vitamin D are mediated by the vitamin D receptor (VDR), a nuclear receptor that controls vitamin D-directed transcription of target genes. Importantly, the VDR has numerous polymorphisms, one of which results in two phenotypically distinct isoforms, designated M1 and M4. VDR M4 is postulated to be more active than M1 in vitamin D-dependent transactivation. Variable binding affinities between the two isoforms and VDR interacting proteins such as TFIIB and RXR have also been observed. Another protein known to interact with VDR is β-catenin, the mediator of the Wnt/β-catenin signaling pathway that can drive colorectal carcinogenesis. The goal of this study was to investigate the ability of vitamin D and resveratrol to regulate the Wnt/β-catenin system via stimulation of β-catenin-VDR (both M1 and M4) interaction and subsequent inhibition of β-catenin-mediated transcription. The current data reported herein support and extend previous work by demonstrating that VDR binds directly to β-catenin and that both vitamin D and resveratrol appear to enhance this interaction. We also present data that 1,25D-stimulated VDR is capable of inhibiting β-catenin transcriptional activity. Significantly, we have shown that the two common VDR polymorphisms M1 and M4, are functionally variable, both in their induction of vitamin D-dependent genes and in their inhibition of β-catenin-mediated transcription. VDR M4 exhibits both elevated transactivation and amplified capacity for β-catenin suppression compared to M1, and studies employing site-directed mutagenesis of VDR implicate the glutamic acid at position 2 as being responsible for the reduced activity of the M1 variant. Both polymorphic VDR variants display 1,25D-mediated enhancement of -catenin association, with the M1 SNP possessing a lower basal (-1,25D) binding to this protein partner but a higher fold stimulation in -catenin interaction in the presence of 1,25D. Taken together, these data support the notions that VDR influences pathways important for colorectal carcinoma (CRC) development, and that supplementation with vitamin D and resveratrol may reduce colon cancer risk in the general population, especially in individuals with the less active M1 VDR polymorphism. A comprehensive understanding of 1,25D and resveratrol action in VDR signaling may allow for a more personalized approach toward treating vitamin D–related disorders and evaluating risk for carcinogenesis.

Kyoto University (京都大学)<br>0048<br>新制・課程博士<br>博士(人間・環境学)<br>甲第21875号<br>人博第904号<br>新制||人||215(附属図書館)<br>2018||人博||904(吉田南総合図書館)<br>京都大学大学院人間・環境学研究科相関環境学専攻<br>(主査)教授 瀬戸口 浩彰, 教授 加藤 眞, 教授 市岡 孝朗, 准教授 西川 完途<br>学位規則第4条第1項該当

A pubarca precoce (PP) é definida como o desenvolvimento de pêlos pubianos antes dos 8 anos de idade em meninas e 9 anos de idade em meninos. Embora a PP não interfira diretamente com os eventos da puberdade, algumas evidências sugerem que estas meninas tenham maior risco para desenvolver, mais tarde, a Síndrome dos Ovários Policísticos (PCOS). A 17ß-hidroxiesteróide desidrogenase tipo 5 (17ßHSD5) é a principal responsável pela conversão de androstenediona em testosterona. Variações no gene que codifica para essa enzima, em especial os polimorfismos de nucleotídeo único (SNPs), podem estar relacionados com hiperandrogenismo e PCOS. A vitamina D, além dos efeitos sobre metabolismo ósseo, parece modular outras ações extra-esqueléticas, incluindo secreção e sensibilidade tecidual à insulina. A Vitamina D vem sendo associada com resistência insulínica e variantes do gene do receptor da vitamina D (VDR), vem sendo estudadas em populações de risco como no diabetes. No entanto, pouco se sabe sobre o envolvimento destes polimorfismos na PP. Os objetivos do presente trabalho foram: Avaliar os níveis de 25-hidroxivitamina D; determinar a frequência dos polimorfismos FokI, BsmI, ApaI e TaqI do gene do VDR e do SNP-71AG do gene da 17ßHSD5; verificar se existe associação entre esses polimorfismos com variáveis antropométricas, metabólicas e hormonais em uma amostra de pacientes com PP e controles do sul do Brasil. Foram arroladas 36 meninas com PP e 197 controles saudáveis. As genotipagens foram realizadas por PCR em tempo real para os SNPs -71AG, BsmI e FokI e por PCR-RFLP para os SNPs ApaI e TaqI. O SNP -71 AG do gene da 17ßHSD5 apresentou distribuição genotípica de 52,4% AA, 39,1% AG e 8,6% GG, sendo a frequência dos alelos A:G de 0,72:0,28. Analisando os dois grupos, verificamos uma maior freqüência do alelo variante (G) no grupo de meninas com PP quando comparadas aos controles (0,37 e 0,26, respectivamente), no entanto sem diferença estatística (p=0,054); não foram verificadas associações do polimorfismo com os dados clínicos e hormonais. As meninas com PP apresentaram níveis séricos de 25(OH)D inferiores aos das meninas controles (18,08±8,32 versus 21,27±7,03; p=0,032). Na análise dos polimorfismos, observou-se que o genótipo polimórfico GG do SNP ApaI TG, apresentou uma frequência maior em PP (30,6%) do que nas controles (16,2%) (Odds Ratio: 2,269; 95% Intervalo de Confiança: 1,015 – 5,076; p=0,042). Este genótipo foi também associado com níveis mais baixos de estradiol (35,30 (14,80 – 50,48) versus 12,22 (6,49 – 23,69); p=0,030) e testosterona total (0,52 (0,39 – 0,84) versus 0,20 (0,11 – 0,47); p=0,009) nas meninas com PP, mas não foi associado com os níveis de 25(OH)D. Por outro lado, verificou-se associação entre a presença dos polimorfismos TaqI TC (genótipo TC+CC) e BsmI GA (genótipo GA+AA) e níveis séricos de 25(OH)D mais elevados no grupo de meninas saudáveis (19,86±7,16 versus 22,55±6,69, p=0,007; 19,53±6,94 versus 22,88±6,76, p=0,001, respectivamente). Em conclusão, os dados deste estudo indicam que: 1) houve maior freqüência do alelo variante G SNP -71 AG do gene da 17βHSD5, com uma associação limítrofe desse alelo com o diagnóstico clínico de PP; 2) o polimorfismo ApaI TG associou-se com PP e parece estar modulando os processos esteroidogênicos nas meninas com PP; 3) houve uma interação entre os polimorfismos TaqI TC e BsmI GA e concentrações circulantes de vitamina D em meninas do sul do Brasil.<br>Precocious pubarche (PP) is usually defined as the development of pubic hair before the age of 8 in girls and age of 9 in boys. Although the PP does not interfere directly in puberty events, some evidence suggests that these girls have higher risk for the development of Polycystic Ovary Syndrome (PCOS) at later ages. The Type 5 17β-Hydroxysteroid Dehydrogenase (17ßHSD5) is the principal responsible for the conversion of androstenedione to testosterone. Variations in the gene encoding for this enzyme, especially Single Nucleotide polymorphisms (SNPs), may be related with hyperandrogenism, and PCOS. Besides the effects on bone metabolism, vitamin D appears to modulate other extra-skeletal actions, including secretions and tissue sensitivity to insulin. Vitamin D has been associated with insulin resistance and variants in the vitamin D receptor (VDR) gene, have been studied in populations at risk of Diabetes. However, little is known about these polymorphisms in the PP. The aims of this work were: to evaluate the levels of the 25-hydroxyvitamin D; to determine the polymorphisms FokI, BsmI, ApaI and TaqI in VDR gene and SNP -71AG in 17ßHSD5 gene frequencies; to asses if exist association between this SNPs and anthropometric, metabolic and hormonal characteristics in patients with PP and controls of the southern Brazil. Were enrolled 36 girls with PP and 197 healthy controls. Genotypic analyzes were evaluated by Real Time for the SNPs -71AG, BsmI and FokI and by PCR-RFLP for the ApaI e TaqI polymorphisms. Genotype frequency for SNP -71 AG of the 17ßHSD5 gene was 52.4% AA, 39.1% AG and 8.6% GG, A:G allelic frequency was 0.72:0.28. Analyzing both groups, higher frequency of the variant allele (G) in patient PP than controls (0.37 e 0.26, respectively) was found but without statistical difference (p=0.054); there were no associations between this polymorphism and clinical and hormonal features. PP girls have serum levels of 25(OH)D lower than those from control group (18.08±8.32 versus 21.27±7.03; p=0.032). The polymorphism analyze was observed that genotype GG of the SNP ApaI TG showed a higher frequency in PP (30.6%) than controls (16.2%) (Odds Ratio: 2.269; 95% confidence interval: 1.015 – 5.076; p=0.042). The same genotype was associated with lower estradiol (35.30 (14.80 – 50.48) versus 12.22 (6.49 – 23.69); p=0.030) and total testosterone levels (0.52 (0.39 – 0.84) versus 0.20 (0.11 – 0.47); p=0.009), in girls with PP. There were no association between this polymorphism and serum 25(OH)D. On the other hand, there was association between the presence of the polymorphisms TaqI TC (TC + CC genotype) and BsmI GA (GA + AA genotype) and higher serum 25(OH)D in the group of healthy girls (19.86 ± 7.16 versus 6.69 ± 22:55 , p = 0.007; 19:53 ± 6.94 versus 22.88 ± 6.76, p = 0.001, respectively). In conclusion, data from this study indicate that: 1) there was a higher frequency of the variant allele G of the SNP -71 AG of the 17ßHSD5 gene, with a borderline association of this allele with the clinical diagnosis of PP; 2) ApaI TG polymorphism is associated with PP and seems to modulate the processes steroidogenesis in girls with PP; 3) there was an interaction between the polymorphisms TaqI TC and BsmI GA and vitamin D concentrations in girls from southern Brazil.

Les β-arrestines jouent un rôle important dans la transduction du signal par les récepteurs couplés aux protéines G (RCPG). Nous montrons dans cette thèse que les β-arrestines exercent des régulations plus complexes et subtiles qu'on ne le pensait jusque-là sur la voie AMPc/PKA/CREB qui est activée par les RCPGs couplés à Gs. Nous montrons que les β-arrestines interagissent directement la PKAcat et contribuent à sa translocation nucléaire. De plus, nous mettons en évidence une interaction β-arrestine/CREB qui conduit à la formation d'un complexe transcriptionnellement actif sous l’action de l’agoniste. D’autre part, nous avons constaté que les β-arrestines interagissent directement avec PKAcat, p70S6K et Src via un même site et lesquelles sont donc potentiellement mutuellement exclusives. Nous avons ensuite mesuré l’impact d’une mutation et d’un polymorphisme du R-FSH sur la signalisation dépendante des β-arrestines, notamment grâce à l’utilisation de senseurs FRET et BRET<br>Β-arrestins play an important role in G protein-coupled receptor (GPCR)-induced signal transduction. In this thesis, we show here that β-arrestins exert more complex and subtle regulation than previously thought on the cAMP/PKA/CREB pathway which is activated by Gs-coupled GPCRs. We demonstrate that β-arrestins directly interact with PKAcat and promote its translocation to the nucleus. Moreover, we provide evidence that β-arrestins directly interact with CREB thereby forming a transcriptionally active complex upon agonist stimulation. We also found that PKAcat, p70S6K and Src all directly interact with β-arrestins through the same interaction site and are therefore potential mutually exclusive interactions. We then measured the impact of a point mutation and of a polymorphism in the FSH-R on β-arrestin-dependent signaling, in part using FRET and BRET sensors

Tese (doutorado) - Pontifícia Universidade Católica do Paraná, Curitiba, 2007<br>Inclui bibliografias<br>A doença renal crônica (DRC) e a doença periodontal (DP) são sérios problemas de saúde-pública, sendo esta última especialmente negligenciada na população de renais crônicos. A vitamina D é um hormônio esteróide solúvel, metabolizado no fígado e nos rins,<br>Chronic kidney disease (CKD) and periodontitis (PD) are serious public-health concerns, but the latter has been especially neglected in the CKD population. Vitamin D is a fat-soluble steroid hormone, metabolized in the liver and then in the kidney, result

碩士<br>臺灣大學<br>職業醫學與工業衛生研究所<br>96<br>Lead may produce adverse effects on the nervous system and influence cognition, memory and intelligence. Even with low dose exposure, lead may still have adverse effects on humans, especially infants and pregnant women. Genetic polymorphism may play an important role in modulating the health effects, but there have been few studies about the relation between genetic polymorphism and infant neurobehavioral development. The objective of this study was to explore the modifier effect of VDR FokI polymorphism on lead exposure and early childhood neurodevelopment. Our study subjects were pregnant women and their neonates in the pilot study of Taiwan Birth Panel Study (TBPS) conducted between April 2004 and January 2005. We collected neonatal umbilical cord blood at delivery for lead analysis. The cord blood lead concentration was detected by ICP-MS. We obtained information on relevant confounding factors by personal interview based on a structured questionnaire. The Comprehensive Developmental Inventory for Infants and Toddlers (CDIIT) and Infant/Toddler HOME of Home Observation for Measurement of the Environment Inventory (IT-HOME) were used to evaluate infant neurobehavioral development at six months of age. The second evaluation was performed when children were twenty-four months of age. VDR genetic polymorphisms were analyzed by real-time PCR method. The mean cord blood lead levels was 1.3 ug/dl and the VDR FokI genetic type rates were 21% for TT, 54% for CT and 24% for CC. Subjects with high lead exposure (> 1.64 ug/dl) had lower 5.85 scores in cognitive domain than subjects with low lead exposure (<= 1.64 ug/dl). Further, our analyses suggest that as cord blood lead increased, the scores of children with CT and CC genotype declined significantly (6.83 scores) in social domain compared with children who carried TT genotype. However, language scores were significantly lower in children with VDR FokI polymorphism regardless of lead levels. Overall, VDR FokI polymorphism may have a modifier effect on the neurobehavioral development of children who have lead toxicity.

Vitamin D deficiency and/or reduced function, as per certain polymorphisms of the vitamin D receptor (VDR) gene, have been related to several autoimmune disorders. The present study was aimed to investigate the association of Hashimoto's thyroiditis with vitamin D status and functional polymorphisms (SNPs) of the VDR gene. In this case-control study, 200 euthyroid subjects were enrolled: 100 newly diagnosed HT patients (87 F, 13 M; mean age ± SD 42 ± 15 year) and 100 healthy individuals, matched for age, sex, BMI, and month of blood sampling. Serum 25(OH)D3 was measured by HPLC. The VDR SNPs BsmI, ApaI, and TaqI, in strong linkage disequilibrium with each other, were detected by restriction fragment length polymorphism-PCR. The prevalence of vitamin D deficiency in HT patients was significantly higher than that in the control group (70 vs 18.2 %; p < 0.0001), and median serum 25(OH)D3 level was significantly lower in HT patients than controls (median value: 16.2 vs 37.4 ng/ml; p = 0.026). Moreover, there was a significant inverse correlation between serum 25(OH)D3 and TPOAb concentration (r = -0.669; p = 0.034). Contrarily, the genotype distribution of the studied SNPs was not different in the two groups (BsmI p = 0.783; ApaI p = 0.512; TaqI p = 0.471), as was the allelic frequency [f(B) p = 0.776, f(b) p = 0.887; f(A) p = 0.999, f(a) p = 0.999; f(T) p = 0.617; f(t) p = 0.617]. The present study first investigates newly diagnosed untreated HT and suggests that vitamin D deficiency may contribute to HT development and/or progression, acting as an environmental trigger, while the VDR locus does not appear to be involved in conditioning the genetic susceptibility to the disease, at least in Caucasians.

The vitamin D receptor (VDR) is a transcription factor mediating genomic responses to the biologically active form of vitamin D, 1,25(OH)2D3, a key modulator of the immune system. Knowledge on how these polymorphisms modulate the vitamin D endocrine system and confer risk of disease is hindered by the fact that several of the associated allelic variants are located in introns or are synonymous and likely serve as markers within an extended haplotype covering disease-causing alleles. The functional relevance of VDR polymorphisms need to be studied in the context of the haplotype, comparing haplotypes with the process of DNA transcription, protein levels and biological function. These functional studies should be performed using techniques reflecting the in vivo, naturally occurring milieu as close as possible. VDR has several known allelic variants including a FokI restriction fragment length polymorphism in exon II, BsmI and ApaI polymorphisms in the intron VIII, and a synonymous TaqI variant in exon IX. The aim of the current study was the identification of sequence variants in VDR, to define haplotype patterns in the Caucasian population and to understand the functional consequences of single nucleotide polymorphisms (SNPs) and haplotypes. Methods: EBV transformed B-lymphocyte cell lines, from twenty-three individuals, within the Caucasian population were established. Polymorphisms and haplotypes in VDR were identified by genotyping and sequencing. Quantification of the VDR protein level measured with flow cytometry was studied together with the genotype and haplotype data to determine possible influence of genotypes or haplotype and VDR protein levels. Biological function was analysed by the percentage inhibition that each individual experienced in the presence of 1,25(OH)2D3, measured by the Alamar Blue assay and the Trypan blue dye exclusion method. Results: The results showed thatVDR genotypes and haplotypes may not influence VDR protein level although certain genotypes and haplotypes significantly influenced biological function. It was proposed that VDR variants may account for significant influences on cellular responsiveness to 1,25(OH)2D3 as mediated by VDR. Conclusions: The findings of the current study suggest that individual SNPs and haplotypes of VDR influences quality of the repose in the presence of 1,25(OH)2D3, rather than quantity of the VDR levels. This knowledge may permit a rational choice of polymorphisms to use in epidemiology studies or improve our understanding of the significance of VDR genetic polymorphisms on biological function. Keywords: functionality, polymorphism, haplotype, vitamin D receptor, VDR, 1,25(OH)2D3, structure-function analysis, biological responsiveness.<br>Prof. L. Bornman

M.Sc.<br>Tuberculosis, of which the causative agent is Mycobacterium tuberculosis, presents itself as a serious health problem globally, especially in Africa. Susceptibility to this infectious disease is influenced by the virulence of the strain of mycobacteria, environmental factors, and genetic variation within the host. The Vitamin D Receptor gene or VDR has been identified as a candidate gene for TB susceptibility. This gene codes for the VDR protein that mediates the biological actions of the active form of vitamin D. Vitamin D has been shown to impair growth of Mycobacterium tuberculosis in human monocytes and macrophages. Vitamin D also provides a link between Toll-like receptor activation and the antibacterial responses of innate immunity in its production of cathelicidin. The VDR protein is a transcription factor that mediates the effects of the active form of vitamin D. Vitamin D has an immunomodulatory role and variations in the VDR gene may result in variations in the functioning of the VDR protein, and hence variations in response to infection. The VDR gene includes the largely non-coding 5’ regulatory region exons 1a-1f and the coding exons 2-9. As a result of increased awareness of the heritability of gene expression and reports of disease associations with VDR promoter region variants, the focus of the research described in this dissertation was the regulatory region of the VDR gene. Polymorphisms that occur within the regulatory region were viii investigated, as were the effects these polymorphisms may have on gene expression, influencing host susceptibility to tuberculosis, with an emphasis on African populations. VDR polymorphisms have been shown to be involved in susceptibility to tuberculosis, particularly the FokI SNP in exon 2, BsmI and ApaI in intron 8 and the synonomous TaqI in exon 9. However, results have been inconsistent. SNPs shown to be associated with TB may serve as markers of truly functional SNP with which they are in linkage disequilibrium (LD). The majority of these association studies involve single nucleotide polymorphisms (SNPs) found in the introns or are silent mutations in the coding exons. Variations in the 5’ regulatory region have been shown to affect gene expression, in particular if they influence the binding sites of transcription factors.

A dissertation submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, South Africa in fulfilment of the requirements for the degree of Master of Science, 2019<br>The study of flower colour is a particularly valuable approach to investigating fundamental evolutionary questions such as the maintenance of variation within species, the role of natural selection and genetic drift in maintaining polymorphisms, and how such polymorphisms contribute to biodiversity. Flower colour is a phenotype that is easily measured and it provides a strong indicator of the outcomes of selection pressures. It is both driven and maintained by either non- pollinator mediated selection agents, pollinator selection agents, or a combination of both. The overall aim of this study was to investigate the maintenance of flower colour using Rhodohypoxis baurii (Baker) Nel. var. confecta Hilliard and Burtt as a study system. This involved understanding the genetic mechanisms that regulate flower colour and examining the environmental variables that drive this trait variation across populations. Molecular and ecological methods were used in combination to understand flower colour by investigating the genes responsible, as well as the environmental selection pressures acting on this phenotype. Rhodoypoxis baurii var. confecta is a Drakensberg near-endemic species that has naturally occurring populations of magenta, pink, and white flower colour morphs. These flower colour morphs are discrete variants that co-exist in single populations and are thus considered to be true polymorphisms. Three populations of R. baurii var. confecta that occur in Sentinel Peak in the Royal Natal National Park, South Africa were studied. Non-pollinator mediated selection agents were investigated as potential drivers of flower variation in R. baurii var. confecta. The frequency of unpigmented (white) and pigmented (pink/magenta) flower colour morphs was measured over the flowering season. No pollinator visitation was observed at populations of R. baurii var. confecta and therefore pollinator-mediated selection was excluded. The extent to which non pollinator selection agents drive this intra-population variation was investigated by measuring the change in soil moisture, temperature, and precipitation over the flowering season. One population shifted from a greater proportion of unpigmented morphs at the beginning of the flowering season to a relatively greater proportion of pigmented morphs at the end of the season. In this population, a positive correlation was found between the proportion of pigmented morphs and soil moisture. This suggests that the accumulation of water in the soil promotes the production of pigmented flowers. The anthocyanin biosynthetic pathway (ABP) is responsible for the production of pigment in R. baurii var. confecta. The ABP is pleiotropic and in addition to pigment production is responsible for traits related to plant physiology and environmental stress response. Consequently, the ABP is cited as being well conserved among angiosperm species. The presence of expression in four ABP genes (CHI, F3H, F3’H, and F3’5’H) was tested for and compared between unpigmented and pigmented R. baurii var. confecta flowers. As there is no sequence data for Rhodohypoxis or any Hypoxidaceae species, primers for this molecular work were designed using closely related monocotyledon ABP gene sequences. Sequencing results showed that the amplified PCR products were not the targeted ABP genes. These results suggested that the designed primers were non-specific. The similarity of the four ABP genes within angiosperm species was investigated. All available complete sequences of the four regions of interest were aligned and sequence similarity was quantified. The results from this alignment indicate that the four investigated ABP gene sequences are polymorphic and are likely not well conserved within angiosperm species as a whole and, to some extent, within monocotyledon and dicotyledon species respectively. This suggests that the basic structure of the ABP is well conserved amongst angiosperm species however, the gene sequences that make up the pathway are polymorphic and less well conserved. The study highlights the importance of non-pollinator mediated selection on the presence and maintenance of flower colour polymorphisms. In addition, it provides some insight on the ABP and its conservation amongst angiosperm species. It also contributes to understanding how flower colour polymorphisms are maintained in natural populations both on a molecular and ecological level and is the foundational work in understanding the polymorphisms of R. baurii var. confecta.<br>TL (2020)

碩士<br>國立臺灣師範大學<br>生物研究所<br>91<br>Vesicoureteral reflux (VUR) is a common pediatric disease that may lead to severe end-stage renal disease (ESRD) in part of patients. The development of VUR is highly familial inherited and the disease progression is variable. The renin-angiotensin system (RAS) and transforming growth factor-b1 (TGF-b1) involve in the development of urinary system and may also be the potential candidate prognostic factors in the progression of ESRD. In this study, the RAS related angiotensinogen (AGT), angiotensin-converting enzyme (ACE), and angiotensin II type 1 receptor (AT1R) as well as TGF-b1 gene polymorphisms were investigated for association with VUR susceptibility and progression in 74 Taiwanese children, including 16 with ESRD, and 117 normal controls. By polymerase chain reaction and directing sequencing, restriction enzyme digestion, or single strand conformation polymorphism analysis, a total of 25 polymorphisms within the AGT, ACE, AT1R, and TGF-b1 genes were studied. The novel ACE A-3692C was not formally reported, and no TGF-b1 A-880G, G-800A, R25P and T263I polymorphisms were detected. All the polymorphisms examined were in Hardy-Weinberg equilibrium. In the AGT gene, strong linkage disequilibrium between C-532T and G-217A, A-20C and T174M, A-6G and M235T were observed. The strong non-random association among the ACE gene T-5491C, A-5466C, T-3892C, A-3692C, A-240T, and Alu I/D polymorphisms spanning promoter to intron 16 suggests low levels of intragenic recombination within the ACE gene. Similar strong linkage disequilibrium was also seen in the AT1R gene A-1138T, T-810A, T-713G, C-521T, A-214C/G-213C, and A-153G polymorphisms. A statistically significant difference between primary VUR patients and normal controls was observed at the TGF-b1 gene -509 site, with T allele associated with primary VUR development. Furthermore, a significant allele association with ESRD was observed for the ACE loci, with the linked T-A-T-A-A-I haplotype as a risk factor for primary VUR progression. To assess the effect of polymorphism on gene expression, fragments containing the polymorphic haplotypes were fused to firefly luciferase reporter construct and transiently expressed in 293 cells. Within the 1.2 kb ACE and 1.4 kb AT1R promoter fragments, no appreciable effect on the gene expression was observed for the linked ACE A-240T and T-93C as well as the linked AT1R polymorphisms. However, reporter construct containing the AGT -152 G allele drove 2.4 times transcriptional activity compared with the -152 A allele.

碩士<br>國立中興大學<br>森林學研究所<br>84<br>Schima superba var. superba is an important native and plantation species in Taiwan and S. superba var. kankaoensis is a variety of S. superba var. superba. Since there is no reli-able morphological characteristic to distinguish the two of them, we tried to use a new technique, random amplified polymorphic DNA(RAPD), to study the taxonomic identity of the two species. Twenty arbitrary sequenced primers(10 nucleotides in length) were selected to proceed RAPD-PCR. One hundred and forty seven bands were recorded and 90(%) of them were polymorphic bands. DNA markers generated by primers U2, T1, V5 and X11 have specific bands that can differentiate S. superba var. superba from S. superba var. kankaoensis. These results indicate that the two species do have some levels of genetic variation.In Nei''s similarity matrix, S. superba var. superba has higher variation within population(1－F＝0.150∼0.314) than that in S. superba var. kankaoensis(1－F＝0.110∼0.289). The cause of this variation may due to the former species spreading widely in Taiwan that may generate higher genetic variation than the latter species that is restricted in Heng-Chun peninsula. Based on the dendrogram generated by the RAPDistance program, when the genetic distance is equal to 0.400, the two species can be separated intotwo groups. However when the genetic distance reduced to 0.213, the S. superba var. superba population can be further divided into three subgroups. This may due to the samples originally collected from plots in Hue-Sun Experimental Forest Station were geographically separated long enough to diverge. On the other hand, samples collected from the S. superba var. kankaoensis population in Heng-Chun peninsula also geo-graphically separate enough to diverge into three subgroups.

碩士<br>中國醫藥大學<br>公共衛生學系碩士班<br>101<br>Background: The International Organization for Migration (IOM) has reported that there are approximately 215 million international migrants among nations at present, the number will reach 405 million by 2050. Several studies also showed that international immigrants are at a high risk of exposing to lead and cadmium. Importantly, the genetic variation of metallothionein 2A (MT2A) and vitamin D receptor (VDR) may moderate the metabolic ability for lead and cadmium in the human body. Methods: A cross-sectional study was performed to include 670 new immigrant female subjects. Participants received health examinations and questionnaire interview for information on demographic variables and life style. Peripheral blood was collected to perform extract biochemical tests and extract genomic DNA. The polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) were used to determine the genotype for the MT2A ‐5A/G and VDR ApaI A/C. Results: Significant associations were found between blood cadmium levels and tea consumption (p = 0.0125) for immigrant women. The genotypes of MT2A ‐5A/G was significantly associated with blood cadmium level (p < 0.0001). No Significant associations between blood lead and the genotypes of VDR ApaI A/C. Conclusion: There is a significant association between blood cadmium level with MT2A genotypes and environmental factors among new immigrant females in Taiwan.

In the interest of understanding the genetic basis of adaption to environment, we developed F2 lines from an F1 interspecific cross between the rare serpentine endemic, Caulanthus amplexicaulis var. barbarae and the non-serpentine Caulanthus amplexicaulis var. amplexicaulis. Using genomic DNA from Caulanthus amplexicaulis var. barbarae, we developed a suite of microsatellite markers. In addition, we developed gene specific markers for genes known in Arabidopsis to be ecologically important. Our suite of markers was used to genotype 186 F2 plants, the basis for our F2 linkage map. In order to further resolve evolutionary relationships among related taxa, we constructed a molecular phylogeny for 52 taxa within the related genera Caulanthus, Guillenia, Sibaropsis, Streptanthella, and Streptanthus, using the sequences from the ribosomal ITS region and two chloroplast regions. To create a useful system to enable comparative genomics within the related taxa of the ecologically and morphologically diverse Streptanthoid Complex, we demonstrated that our molecular tools are portable across a large group of ecologically significant taxa. To use the significant genomic resources available in Arabidopsis, we constructed a collinear comparative map of Caulanthus and the model plant Arabidopsis thaliana based on ancestral linkage blocks with the Brassicaceae family. This comparative map acted as a guide for candidate gene selection in the mapping of sepal color. We identified a region of MYB transcription factors in an orthologous region of Arabidopsis. Sequence data from Caulanthus amplexicaulis var. barbarae and Caulanthus amplexicaulis var. amplexicaulis in this MYB region showed significant sequence divergence between the two taxa. To determine the genetic basis for the tolerance of high concentrations of magnesium in Caulanthus amplexicaulis var. barbarae, we phenotyped multiple individuals from 88 F2:3 families under two nutrient treatments, differing in the ratio of calcium to magnesium. Through QTL analysis, using our F2 linkage map as a framework for the analysis, we identified one major effect QTL on Caulanthus Linkage Group 8 and another QTL on Caulanthus Linkage Group 3. We identified candidate genes for the QTLs using our collinear comparative map to Arabidopsis.

碩士<br>國立臺灣師範大學<br>生命科學研究所<br>92<br>Vesicoureteral reflux (VUR) is a common pediatric disease. The development of VUR is highly familially inherited and the disease progression is variable. Some patients may lead to severe end-stage renal disease (ESRD). In this study, polymorphisms of human renal kallikrein gene (KLK1), TGFβ-inducible early gene 1/early growth response  gene (TIEG1/EGR), tumor necrosis factor- gene (TNF-), interleukin-1 gene (IL-1), interleukin-1 gene (IL-1) were evaluated for the association with VUR development and progression in Taiwanese children. The KLK1 promoter -130 GN polymorphism was analyzed by genotyping, single-strand conformation polymorphism (SSCP) analysis, and allele-specific polymerase chain reaction (PCR). By PCR and direct sequencing, two new polymorphisms, C-1445G in the EGR promoter region and T216 (A>C) in the exon 3 of the TIEG1 gene were identified and analysed. Both polymorphisms as well as TNF- C-863A and C-857T, IL-1 C-889T, and IL-1 C-511T polymorphisms were analyzed by restriction enzyme digestion of PCR products. IL-1 3''''UTR polymorphism was analyzed by SSCP. Among these eight polymorphisms studied, EGR C-1445G showed association with VUR development, whereas KLK1 -130 GN and TNF- C-863A polymorphisms with VUR progression. The fragments containing KLK1 -130 GN and EGR C-1445G polymorphisms were fused to a firefly luciferase reporter and transiently expressed in HEK-293 cells. Significant lower transcriptional activity was observed with -130 G12 K allele and EGR -1445 G allele.

Background: This study sought to investigate the relationship of physical activity (PA), dietary calcium and 3 candidate gene (VDR, COL1A1 and TNFR2) genotypes on bone mass in children (n = 327, age 10.33 ± 0.65). The study also sought to investigate the effect of PA and genotype on bone mineral-free lean mass (BMFL). Finally, the relationships between bone mass and BMFL and PA, BMFL and genotype and PA and genotype interactions were investigated. Methods: Anthropometric data (height, sitting height and weight) was determined using standard techniques. Bone mass, and BMFL were assed using total body DXA scan using a Hologic QDR 4500. Dietary calcium, PA and maturity were assessed using previously validated questionnaires. VDR FokI and VDR BsmI genotypes were determined by standard restriction fragment length polymorphism techniques. COL1A1 genotype was determined by a novel TaqMan technique and TNFR2 genotypes and haplotypes were determined by a novel automated sequenceing protocol. Associations between PA or candidate gene genotype and either BMFL or bone mass was first controlled for inter-subject differences in body size and maturity. Results: PA was significantly associated with BMFL in boys (p = 0.038). PA score was associated with a 3-5% difference in proximal femur BMC, femoral neck BMC and femoral neck aBMD but not lumbar spine BMC in boys as well as a 4% difference in femoral neck aBMD in girls. Average dietary calcium intake was not associated with differences in bone mass in children. VDR Bsml and VDR Fokl genotype did not have a relationship to bone mass or BMFL in children. COL1A1 Ss or ss genotype is associated with 4.8% higher femoral neck BMC in boys but not BMFL in either sex. TNFR2 A593G gg genotype was associated with a 3.8% higher BMFL in boys (p =0.038) and a 3.4% higher femoral neck BMC in boys (p = 0.045). Girls with a TNFR2 T598G tg genotype had a 3.3% higher femoral neck BMC (p = 0.029). TNFR2 G593-G598/G593-T598 haplotype was associated with a 10% higher femoral neck BMC in girls. For boys, when the BMFL by PA interaction term was added to the model it explained significantly more (2.5-3.9%, p = 0.004) of the variance in femoral neck BMC, femoral neck aBMD and lumbar spine aBMD than the main effect for PA alone. When the BMFL by VDR Fokl genotype interaction was added to the model the main effect of the VDR Fokl genotype became significant where boys with the FF genotype had a 1.4% greater femoral neck aBMD than boys with the Ff or ff genotype. For girls, significant interactions between TNFR2 haplotype and BMFL changed the model such that girls with the G593-G598/G593-T598 haplotype had a 10-11% greater femoral neck BMC or aBMD than girls with other TNFR2 haplotypes. Girls with the Ss or ss genotype had a 4% greater femoral neck aBMD after a significant interaction between COL1A1 genotype and PA was accounted for. Conclusions: High levels of PA are associated with increased BMFL and bone mass. TNFR2 genotypes are associated with both lean mass and bone mass in a complex fashion suggesting that the TNFR2 genotypes and interactions between BMFL and TNFR2 genotypes affect and moderate a combined lean mass/bone mass effect. COL1A1 Ss and ss genotypes are associated with high bone mass particularly in girls with high PA.