Thèses sur le sujet « Ms polymorphism »

Epigenetics is a field of molecular biology that copes with the study of gene function regulation without variations in DNA structure or nucleotide sequences. Among the main epigenetic phenomema in eukaryotic cells there are DNA methylation and post-traslational mechanisms among which the major are histone methylation and acetylation. Epigenetic changes are potentially reversible phenomena that are controlled also by nutritional factors as the methyl-donors involved in the folate cycle. Plasma levels of B vitamins, among which “in primis” plasma folate concentrations, are implicated in epigenetic modulation so that it can be hypothesized that they may affect the modulation of gene expression through epigenetic mechanisms. Epigenetic modifications represent one of the earliest events in the genesis of some complex pathologies, therefore the study of the interaction between epigenetics and nutritional status is of great interest either to define the physiopathological mechanisms of development of some illnesses, and for possible personalized strategies of prevention. The present work has been articulated, at first, on the analysis of gene-nutritional interaction mechanisms within the folate cycle through the study of polymorphisms of enzymes involved in the metabolism of methyl-group donors; the aim was to study their possible role on the modulation of genomic DNA methylation in relationship to different plasma levels of idrosoluble B vitamins. In this regard, the most important functional polymorfisms known on the genes of one-carbon metabolism and their relationship with methylation status of polymorphonuclear cells DNA have been analyzed from a cohort of around 800 subjects within a clinical study, underlining the role of the key folate-related enzymes in the modulation of DNA methylation. Besides the function of genomic DNA methylation, the methylation status at specific sites has been also approached with the specific intent of considering a possible interrelationship between the role of promoter methylation and the co-presence of functional polymorphisms in the same genic site for a gene for which a precise functional effect is well-known. To address this issue the promoter region of coagulation factor VII gene was evaluated for both genetic and epigenetic modifications as a possible model of genetic-epigenetic interaction in the modulation of gene product regulation. The results showed the key importance of genetic-epigenetic interactions, so far unknowm, in modulating gene-expression at promoter gene sites. The role of other vitamins involved in one-carbon metabolism in major chronic diseases, and specifically the emerging role of B6 vitamin, have been also studied. Furthermore, a clinical study is now in progress to evaluate the function of gene-specific methylation in liver tissue where most of the folate cycle functions take place. The aim of this project is the evaluation of both genome-wide and gene-specific methylation status in the liver in comparison to that observed in peripheral blood mononuclear cells DNA to define whether methylation status of peripheral blood DNA may be regarded as a good systemic biomarker for this epigenetic feature of DNA in relation to B vitamins nutritional status in cancer disease. Results from this study may help to define possible functional markers of gene-nutrients interactions with effects on epigenetic modulation for future preventive or therapeutic strategies. With that purpose, a novel high-throughput array-based technique for the detection of gene-specific methylation at promoter sites has been optimized in our laboratory.

The glutathione S-transferase (GST) supergene family encodes isoenzymes that appear to be critical in protection against oxidative stress. Certain GST loci are polymorphic, demonstrating alleles that are null (GSTMI/GSTT1), encode low activity variants (GSTPI), or are associated with variable inducibility (GSTM3). Interleukin-1 (IL- 1) alpha and beta are cytokines involved in recruitment of inflammatory cells, the process of inflammation, and blood-brain barrier breakdown and nerve regeneration. Polymorphisms of both GST and of a complementary interleukin-1 receptor antagonist have been associated with severity and susceptibility to other inflammatory conditions. This thesis examines the influence of the GST and IL-1 genes on both the susceptibility to Multiple Sclerosis (MS), and the course of disease progression. The population examined consisted of four hundred patients with clinically definite MS. Disease severity was measured using the Kurtzke Expanded Disability Status Scale (EDSS), a robust established ranking scale. PCR-based genotyping was performed using DNA extracted from lymphocytes. Significant associations between genotypes and clinical outcome were corrected for known demographic factors influencing prognosis, these being; gender, onset age, and disease duration using the statistical method of logistic regression. Significant associations, withstanding multiple testing corrections, with certain IL-I genotypes and disease severity were found. There was also a significant trend with the GST isoenzymeM 3 that is expressedin nervous tissue. No robust findings suggest that these genes influence susceptibility to MS, but the results suggest that long-term prognosis is genetically influenced by the modulation of inflammatory cytokines and also by the ability to remove the toxic products of oxidative stress.

Thesis (MMedSc)--Stellenbosch University, 2013.
ENGLISH ABSTRACT: The aetiology of multiple sclerosis (MS) remains largely unknown due to the multifactorial nature of disease susceptibility determined by both environmental and genetic factors. Progress has been made in identifying the genetic component of MS , as well as the possible interactions with the environment. In this study single nucleotide polymorphisms (SNPs) in the FTO (rs9939609, Intron 1 T>A), MTR (rs1805087, 2756 A>G), MTRR (rs1801394, 66 A>G), MTHFR (rs1801133, 677 C>T and rs1801131, 1298 A>C) and COMT (rs4680, 472 G>A) genes involved in the methylation metabolic pathway were studied in the context of MS. The overall objective of this study was to elucidate the mechanism underlying raised homocysteine levels in MS patients. The specific aims were 1) to analytically validate high throughput real-time polymerase chain reaction (RT-PCR) genotyping assays for the 6 selected SNPs against direct sequencing as the gold standard for 2) possible integration into a pathology-supported genetic testing strategy aimed at improved clinical management of MS. The study population included a total of 114 unrelated Caucasian MS patients (98 females and 16 males) and 195 unrelated Caucasian control individuals without a diagnosis of neurological disease (128 females and 67 males). A novel finding of this study was that the risk-associated FTO rs9939609 A-allele was associated with raised homocysteine levels (p=0.003) in patients diagnosed with MS, but not in controls. Furthermore, homocysteine levels correlated significantly with bo dy mass index (BMI) (p=0.046) and total cholesterol levels (p=0.048). Both homocysteine (p=0.011) and BMI (p=0.017) were significantly reduced with increasing intake of folate in the diet, while high saturated/trans fat intake correlated significantly with increased BMI (p<0.001). High physical activity correlated with reduced BMI (p<0.006) in the study population, adjusted for age, gender and disease status. Daily intake of at least five fruit and vegetable portions and the COMT rs4680 (472 G>A) AA genotype had a favourable lowering effect on MS disability as assessed by the expanded disability status scale (EDSS) (p=0.035), while smoking increased MS disability significantly (p<0.001). All SNPs studied were found to be in Hardy-Weinberg equilibrium (HWE), with no significant differences detected between patients and control individuals in genotype distribution or allele frequencies. This study has shown for the first time that the underlying disease process of MS moderates the effect of the FTO rs9939609 polymorphism on homocysteine levels , which is consistent with the role of FTO in demethylation and epigenetic changes. Identification of FTO rs9939609 reinforces the importance of adequate folate intake in the diet that can be assessed accurately with use of the Medical History and Lifestyle Questionnaire applied in this study. Finally, the finding that raised homocysteine levels and BMI are significantly influenced by lifestyle factors such as diet and physical activity in our study cohort , offers a solution to counteract the detrimental effects of genetic risk factors contributing to the development of these established vascular risk factors for MS. Combining this information with FTO rs9939609 and COMT rs4680 genotyping may in future translate into a comprehensive pathology supported genetic testing strategy aimed at improved risk management and quality of life in MS patients.
AFRIKAANSE OPSOMMING: Die etiologie van meervoudige sklerose (MS) is grootliks onbekend as gevolg van die multifaktoriale aard van siekte vatbaarheid wat bepaal word deur beide genetiese en omgewingsfaktore. Vordering is reeds gemaak in die identifisering van die genetiese component van MS, asook moontlike interaksie met die omgewing. In hierdie studie is enkel nukleotied polimorfismes (SNPs) in die FTO (rs9939609, Intron 1 T > A), MTR (rs1805087, 2756 A> G), MTRR (rs1801394, 66 A> G), MTHFR (rs1801133, 677 C > T en rs1801131, 1298 A> C) en COMT (rs4680, 472 G > A) gene, wat betrokke is in die metilering metaboliese padweg, in die konteks van MS bestudeer. Die oorhoofse doel van hierdie studie was om die onderliggende meganisme betrokke by verhoogde homosisteïen vlakke in MS pasiënte uit te lig. Die spesifieke doelwitte was 1) om die analitiese geldigheid van die hoë deurvoer riëeltyd polymerase kettingreaksie (RT-PCR) genotipering metode soos toegepas vir die 6 geselekteerde SNPs te bevestig teen direkte DNA volgorde bepaling as die goue standaard, vir 2) moontlike integrasie in 'n patologie-gesteunde genetiese toetsing (PSGT) stategie wat gemik is op verbeterde kliniese hantering van MS. Die studiepopulasie bestaan uit 'n totaal van 114 nie-verwante Kaukasiese MS pasiënte (98 vroue en 16 mans) en 195 nie-verwante Kaukasiese kontroles sonder ‘n diagnose van neurologiese siektes (128 vroue en 67 mans). 'n Nuwe bevinding van hierdie studie was dat die risiko-verwante FTO rs9939609 A- alleel geassosieer was met verhoogde homosisteïen vlakke (p = 0,003) in pasiënte gediagnoseer met MS, maar nie in kontroles nie. Homosisteïen vlakke was verder beduidend geassosieer met liggaamsmassa-indeks (BMI) (p=0,046) en totale cholesterol vlakke (p=0.048). Beide homosisteïen (p=0,011) en BMI (p=0,017) het aansienlik verminder met 'n hoër inname van folaat in die dieet, terwyl 'n hoë versadigde/trans vet en koolhidrate inname beduidend gekorreleer het met 'n verhoogde BMI (p <0.001). Hoë fisiese aktiwiteit was gekorreleer met 'n verminderde BMI (p< 0.006) in die gekombineerde groep, aangepas vir die ouderdom, geslag en MS diagnose. Daaglikse inname van ten minste vyf vrugte en groente porsies en die COMT rs4680 (472 G>A) AA genotipe het 'n gunstige uitwerking op vermindering van gestremdheid gehad, soos bepaal deur die uitgebreide gestremdheid status skaal (EDSS) (p=0,035), terwyl rook MS gestremdheid beduidend verhoog het (p <0.001). Alle SNPs bestudeer was in Hardy-Weinberg ewewig (HWE), met geen beduidende verskille waargeneem in genotipe verspreiding of alleelfrekwensies tussen pasiënte en kontroles nie. Hierdie studie het vir die eeste keer aangetoon dat ‘n diagnose van MS die effek van die FTO rs9939609 polimorfisme op homosisteïen vlakke modereer, wat ooreenstem met die rol van FTO in demetilering en epigenetiese veranderinge. Identifikasie van FTO rs9939609 versterk die belangrikheid van genoegsame folaat inname in die dieet wat akkuraat gemeet kon word deur gebruik te maak van die Mediese Geskiedenis en Leefstyl Vraelys soos toegepas in hierdie studie. Ten slotte, die bevinding dat verhoogde homosisteïen vlakke en BMI statisties betekenisvol beïnvloed word deur leefstylfaktore soos dieet en fisiese aktiwiteit in ons studie populasie, verskaf 'n oplossing om die genetiese bydrae tot hierdie gevestigde vaskulêre risikofaktore vir MS teen te werk. Kombinasie van hierdie inligting met FTO rs9939609 en COMT rs4680 genotipering kan moontlik in die toekoms benut word as deel van 'n omvattende patologie- gesteunende genetiese toetsing strategie wat daarop gemik is om die risikobestuur en kwaliteit van lewe te verbeter in MS pasiënte.

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Universidade Estadual de Maringá
Genetic and chemical variability of Maytenus ilicifolia Maytenus ilicifolia is a native plant of Southern Brazil commonly used as a popular medicine for indigestion, gastritis and ulcers. The large use of this plant has increased the degradation of the species, and thus it is presently included in FAO´s list for priority species for studying and conservation in South America. In this context, this work aimed to contribute with studies that help the conservation of this species. Initially, a survey in herbaria and in the field was performed to identify the incidence and distribution of the genus Maytenus in the State of Rio Grande do Sul. Three new species (M. glaucenscens Reiss, M. gonoclada Mart. and M. robusta Reiss) were found in this State. The genetic variability study of three populations showed that the intrapopulational variation (variance from 0.102 to 0.140) is higher than the interpopulational (variance between 0.076 and 0.099%). The three populations analyzed grouped themselves with low significance level and 7.6% of the present alleles are rare. The genetic variability study involving 18 native populations of Maytenus ilicifolia, Maytenus aquifolia and Maytenus evonymoidis demonstrated that the species may be separated with 100% of confidence, and that M. ilicifolia and M. aquifolia are genetically more similar. The populations of Maytenus ilicifolia analyzed formed three groups with low confidence levels. Group I included the populations of Ponta Porã, group II the populations of Santana do Livramento, Vale Verde, Canguçu and Unistalda, and group III was formed by the other 13 populations. A similarity in the genetic and environmental grouping of such species was observed. The chemical characterization was performed by gas chromatography of the extracts obtained with high pressure CO2. An increase in the yield of extract was obtained when the temperature of extraction as well as the solvent density were increased. The compounds that presented higher yield in the first fractions were dodecanoic acid, geranyl acetone, phytol, squalene, vitamin E and stigmast-5-enol. The compounds friedelan-3-ol and friedelin were extracted in higher yields in the following fractions. The chemical variability study of the volatile and semi-volatile compounds of populations of M. ilicifolia showed a yield of extract (0,488 a 0,976 %) significantly different between the populations. In addition no relation between the chemical and environmental or between the chemical and genetic grouping could be established.
Maytenus ilicifolia (espinheira-santa) é uma planta nativa da região sul do Brasil empregada na medicina popular para tratamentos de indigestão, úlceras e gastrites. A ampla utilização de Maytenus ilicifolia na medicina popular, tem levado à perda de populações da espécie, encontrando-se atualmente na lista da FAO como uma das espécies prioritárias para estudo e conservação na América do Sul. Neste sentido, o presente trabalho teve como objetivo realizar estudos que subsidiem protejos de conservação desta espécie. Inicialmente foi realizado um levantamento em herbários e a campo, verificando a ocorrência e distribuição do gênero Maytenus no estado do Rio Grande do Sul, sendo encontradas três novas espécies: M. glaucescens Reiss, M. gonoclada Mart. e M. robusta Reiss. No estudo da variabilidade genética em três populações, a variação intrapopulacional (0,102 a 0,140) é superior à interpopulacional (0,076 a 0,099). As três populações analisadas se agruparam com baixos valores de significância e 7,6% dos alelos presentes são raros. No estudo da variabilidade genética envolvendo 18 populações nativas de Maytenus ilicifolia e plantas de Maytenus aquifolia e Maytenus evonymoidis, pode-se separar as espécies com 100% de confiança e M. ilicifolia e M. aquifolia estão mais próximas geneticamente em relação a M. evonymoidis. As populações de Maytenus ilicifolia analisadas formaram 3 grupos com valores baixos de índice de confiança baixos, sendo o grupo I a população de Ponta Porá, o grupo II as populações de Santana do Livramento, Vale Verde, Canguçu e Unistalda e o grupo III formados pelas 13 populações restantes. Observou-se semelhança no agrupamento genético e ambiental destas populações. No estudo da caracterização química, via cromatografia gasosa dos extratos obtidos por CO2 a alta pressão, observou-se um acréscimo na eficiência de extração com aumento de temperatura e densidade do solvente. Os compostos ácido dodecanóico, geranil acetona, fitol, Esqualeno, Vitamina E e Stigmast-5-enol foram extraídos em maior concentração nas primeiras frações, independente das condições de extração, enquanto que os compostos Friedelan-3-ol e Friedelin, foram extraídos em maior quantidade nas frações seguintes. Na análise da variabilidade química dos compostos voláteis e semi-voláteis das populações de Maytenus ilicifolia ). Verificou-se no rendimento de extrato (0,488 a 0,976 %) e concentração dos compostos analisados diferenças significativas entre as populações analisadas e não houve relação entre o agrupamento químico das populações com os agrupamentos ambientais e genéticos.

La consommation excessive d'alcool présente des risques élevés pour l'individu et pour la société ; elle est fréquemment associée à une augmentation du risque d'accidents, d'actes de violence, et peut également conduire à court et/ou à long terme à de graves maladies et à des problèmes sociaux. Dès lors, l'utilisation de marqueurs fiables permettant de détecter une consommation excessive d'alcool, ponctuelle ou chronique, s'avère nécessaire pour prévenir des conséquences néfastes de l'abus d'alcool. L'éthylglucuronide (EtG) est un marqueur d'alcoolisation utilisé en toxicologie clinique (alcoologie) et médicolégale. Par rapport aux marqueurs indirects d'alcoolisation (CDT, γ-GT), ce métabolite mineur de l'éthanol est très spécifique et est quantifiable dans diverses matrices biologiques. La production d'EtG est catalysée par des enzymes de la famille des UDP-glucuronosyl-transférases (UGT). Cependant, les UGT impliquées dans la glucuronoconjugaison de l'éthanol, ainsi que les sources potentielles de variabilité interindividuelle de la production d'EtG, sont encore mal connues. Nos travaux ont ainsi consisté à (1) développer et valider une méthode de dosage de l'EtG dans différentes matrices biologiques par chromatographie en phase gazeuse couplée à la spectrométrie de masse en tandem, (2) identifier les UGT humaines impliquées dans la glucuronoconjugaison de l'éthanol et étudier la contribution relative de chaque isoforme active au niveau hépatique, (3) étudier l'impact de substances fréquemment utilisées par les consommateurs d'alcool sur la production d'EtG in vitro, (4) étudier l'impact de polymorphismes génétiques fonctionnels des UGT sur la production hépatique d'EtG, et enfin (5) évaluer l'impact de la consommation de cannabis et d'autres drogues sur la production d'EtG à l'aide de prélèvements post-mortem. Ces travaux ont notamment permis de montrer que (1) l'éthanol est glucuronoconjugué principalement par le foie, puis dans une moindre mesure par les reins et par l'intestin, (2) les UGT1A9 et 2B7 sont les deux enzymes majoritairement impliquées dans la glucuronoconjugaison de l'éthanol, quel que soit l'organe considéré, (3) la morphine, la codéine, la nicotine et la cotinine n'entraînent aucune modification des taux de production d'EtG in vitro ; le lorazépam et l'oxazépam augmentent légèrement cette production (p = 0,2 et 0,065, respectivement) ; le cannabidiol inhibe la glucuronoconjugaison de l'éthanol par un mécanisme non-compétitif (CI50 = 1,17 mg/L; Ki = 3,1 mg/L), alors que le cannabinol augmente cette glucuroconjugaison de manière concentration-dépendante (p <0,05), (4) les SNP c.-900G>A affectant l'UGT2B7 et IVS1+399T>C affectant l'UGT1A9 augmentent légèrement la production d'EtG in vitro. Enfin (5) le rapport des concentrations sanguines EtG/éthanol apparaît significativement plus élevé chez des co-consommateurs de cannabis et/ou d'autres drogues que chez des consommateurs d'alcool seul. L'ensemble de ces résultats démontre l'existence de plusieurs facteurs pouvant potentiellement influencer la production d'EtG et devraient donc être pris en considération lors de l'interprétation de sa concentration in vivo.

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Universidade Estadual de Maringá
Ilex paraguariensis, known as mate tea, is processed in many ways and commercialized as tea, soluble powder, essences, and processed leaves for preparation of mate tea without sugar ( chimarrão and tererê ). Aiming the conservation of this species, this work studied the intra- and interpopulational genetic variabilities of I. paraguariensis and its volatiles and semi-volatiles composition in 20 populations inside the area of incidence of this species in Brazil. Paternity tests were also performed to identify a favorable crossing for obtaining lineages with the characteristics of interest, reducing then the impact of extractive harvest on the native plants. Genetic variability of I. paraguariensis analyzed by RAPD markers (Random Amplified Polymorphic DNA) is lower to the reported variability in other dioic arboreal species. Intrapopulational variability is higher than interpopulational in this species. In intra and interpopulational studies alleles with the same allele sequence were identified, as well as alleles with very different sequences, what evidences the incidence of genetic flux among populations that are close geographically, but with alleles specific of each population. The environmental differences taken into account were not enough to explain genetic and chemical differences found among the populations. These results show the possibility of influences of microenvironmental factors and non-analyzed genes in the analyzed chemical composition of mate tea leaves. The population of Ponta Porã, geographically separated from the others, proved to be genetically distinct. Species-specific fragments were obtained for I. paraguariensis, I. dumosa and I. theezans. However, more studies for confirmation and utilization of such fragments must be carried out. The differences in the volatiles and semivolatiles composition among the populations turned their separation possible, and may be useful for identification and inclusion of genotypes, superior in specific compounds, in germoplasm collections. For conservation of this species in germoplasm collections ex situ, material collecting must be based on genetic analysis, as well as on geographical distribution, due to the higher intrapopulational variability and occurrence of rare alleles in the species. The RAPD technique may be used for paternity identification, mainly in arboreal species, making possible to obtain genetic improvement by performing controlled crossings. Based on these results, it was possible the discussion concerning the conservation aspects of this species.
Ilex paraguariensis, mais conhecida como erva-mate, é beneficiada por diversas formas e comercializada como chá, pó solúvel, essências e erva para chimarrão e tererê. Visando sua conservação, foram estudadas sua variabilidade genética intra e interpopulacional e sua composição química em 20 populações na área de ocorrência da espécie no Brasil. Também foram realizados testes de paternidade para identificação de um cruzamento favorável à obtenção de progênies com características de interesse, objetivando reduzir o impacto sobre ervais nativos causados pelo extrativismo. A variabilidade genética analisada por marcadores moleculares RAPD (polimorfismo de DNA amplificado ao acaso) em I. paraguariensis é inferior à variabilidade reportada em outras espécies arbóreas dióicas. A variabilidade intrapopulacional é maior que a variabilidade interpopulacional nesta espécie. Em estudos intra e interpopulacionais identificaram-se alelos com mesma freqüência alélica e alelos com freqüências bastante distintas, evidenciando a ocorrência de fluxo gênico entre populações próximas geograficamente, porém com alelos característicos de cada população. As diferenças ambientais consideradas não foram suficientes para explicar as diferenças genéticas e químicas encontradas entre as populações, demonstrando a possibilidade de haver interferências micro-ambientais e de genes ainda não analisados, influenciando na composição química da erva-mate. A população de Ponta Porã, separada geograficamente das demais, mostrou-se distinta geneticamente. Fragmentos espécie específicos foram obtidos para I. paraguariensis, I. dumosa e I. theezans, porém requerendo mais estudos neste sentido para confirmação e utilização destes. As diferenças nas análises dos compostos voláteis e semi-voláteis entre as populações, permitiram a separação destas e podem ser úteis para a identificação e inclusão de genótipos superiores em determinados compostos em bancos de germoplasma. Para a conservação desta espécie em bancos de germoplasma ex situ a coleta de materiais deve ser baseada em análises genéticas, além da distribuição geográfica, devido à maior variabilidade intrapopulacional e ocorrência de alelos raros na espécie. A técnica de RAPD pode ser utilizada para a identificação de paternidade, principalmente em espécies arbóreas, podendo-se obter ganhos genéticos com a condução de cruzamentos controlados. Os resultados obtidos embasaram discussões sobre aspectos de conservação desta espécie.

A esclerose múltipla (EM), doença neurodegenerativa do sistema nervoso central (SNC) com característica auto-imune e inflamatória, com eventos iniciais, bem como a evolução da EM. É uma doença heterogênea (três principais formas clínicas) e multifatoriais. A imunidade inata demonstrou recentemente ser um fator importante na EM e as variantes genéticas dos componentes do inflamassoma têm sido associadas a doenças autoimunes e neurodegenerativas, com isso hipotetizamos que o inflamassoma e suas citocinas IL-1Beta e IL-18, podem representar importantes contribuintes na patogênese da EM e eventualmente explicar, pelo menos em parte, a heterogeneidade observada em pacientes com EM. Fizemos uma análise multivariada que foi realizada com base na forma clínica (recorrente remitente/RR, primária progressivo /PP ou secundário progressiva /SP, índice de gravidade (EDSS) e índice de progressão (IP). Os monócitos do sangue periférico (PBMC) dos pacientes foram examinados para ativação do inflamassoma (Produção de IL-1Beta e IL-18, clivagem de caspase-1). Com os objetivos de avaliar a contribuição do inflamassoma na EM, em termos de (a) efeito genético sobre o desenvolvimento, gravidade e / ou prognóstico, e (b) ativação complexa de células de sangue periférico como uma forma de avaliar a inflamação sistêmica. Para isso, utilizamos variantes genéticas funcionais em componentes do inflamassoma, que foram analisadas em uma coorte de pacientes com EM, pelo uso de ensaios específicos de alelos e qPCR. A analise multivariada resultou em associação com a variante -511C / T IL1B ganho de função, sendo essa mais frequente em formas progressivas (especialmente SP) do que em RR. A variante de ganho de função NLRP3 Q705K resultou mais frequente em pacientes com EDSS > 3 do que em pacientes com EDSS < 3 e, consequentemente, esse SNP está associado a um IP mais elevado. A análise de PBMC mostrou que as células de indivíduos EM, são mais propensas a responder a um estímulo NLRP3 clássico (isto é, LPS) do que as dos doadores saudáveis. Em conjunto, esses achados indicaram que os pacientes com EM apresentam uma desregulação no inflamassoma NLRP3, podendo ser avaliada no sangue periférico facilitando um prognóstico, e que esse perfil pode ser secundário a um mecanismo genético pró-inflamassoma
The multiple sclerosis (MS), neurodegenerative disease of the central nervous system (CNS) with autoimmune and inflammatory characteristics, with initial events, as well as the evolution of MS, are heterogeneous (three main clinical forms) and multifactorial. Innate immunity has recently been shown to be an important factor in MS and the genetic variants of the components of inflammassoma have been associated with autoimmune and neurodegenerative diseases, thereby hypothesizing that the inflammassoma and its IL-1Beta and IL-18 cytokines may represent important contributors in the pathogenesis of MS and possibly explain, at least in part, the heterogeneity observed in MS patients. We performed a multivariate analysis that was performed based on clinical form (recurrent recurrent / RR, progressive primary / PP or progressive secondary / SP, EDSS and progression index.) Peripheral blood mononuclear cells (PBMC) of patients were examined for inflammatory activation (IL-1Beta and IL-18 production, caspase-1 cleavage). With the objectives of evaluating the contribution of inflammassoma in MS in terms of (a) genetic effect on development, severity and / or prognosis, and (b) complex activation of peripheral blood cells as a way of assessing systemic inflammation. For this, we used functional genetic variants in components of the inflammassoma, which were analyzed in a cohort of MS patients, through the use of specific allele and qPCR assays. For this, we used functional genetic variants in components of the inflammassoma, which were analyzed in a cohort of MS patients, through the use of specific allele and qPCR assays. Multivariate analysis resulted in association with the -511C / T IL1B function gain, which is more frequent in progressive forms (especially SP) than in RR. The gain variant of NLRP3 Q705K function was more frequent in patients with EDSS > 3 than in patients with EDSS < 3 and, consequently, this SNP is associated with a higher PI. PBMC analysis showed that cells from MS individuals are more likely to respond to a classical NLRP3 (ie LPS) stimulus than healthy donors. Taken together, these findings indicated that patients with MS have a dysregulation in the NLRP3 inflammassoma and can be evaluated in the peripheral blood facilitating a prognosis and that this profile may be secondary to a pro-inflammatory genetic mechanism

Tese de doutoramento em Biociências, no ramo de Biologia Celular e Molecular, apresentada ao Departamento de Ciências da Vida, da Faculdade de Ciências e Tecnologia da Universidade de Coimbra
Esclerose múltipla (EM) é uma doença multifatorial, inflamatória, desmielinizante e neurodegenerativa, com interrupção da transmissão nervosa no SNC. De etiologia pouco conhecida, uma reação autoimune direcionada contra antigénios da substância branca e auto-reatividade de células imunes através da BHE, mostraram ser cruciais na formação das lesões inflamatórias. O curso clínico da doença é altamente incerto, associando formas clínicas surto-remissão (SR) com formas progressivas. A heterogeneidade clínica e fisiopatológica torna o prognóstico da EM um desafio. O objetivo deste estudo foi tentar identificar marcadores biológicos que funcionassem como indicadores de prognóstico da EM. Para tal selecionaram-se marcadores que se sabe terem um papel essencial na inflamação do SNC: MMPs e seus inibidores (TIMPs), moléculas de adesão celular (CAMs), produção intratecal de imunoglobulinas (Ig). Estudaram-se amostras emparelhadas de LCR e soro de doentes de EM e controlos neurológicos (CN: outras doenças inflamatórias-DI e não inflamatórias-DNI do SNC), onde se pesquisou a presença de bandas oligoclonais (BOC) IgG e IgM por focagem isoelétrica. Os níveis de MMP-2, MMP-9, TIMP-2, TIMP-1, sICAM-1, sVCAM-1 e sE-Seletina, foram determinados por ELISA. Recorrendo à metodologia PCR-RFLP, avaliou-se a presença do polimorfismo -1562 C/T do gene da MMP-9 em doentes de EM e controlos saudáveis (CS). As BOC IgG pesquisaram-se em 183 doentes de EM e 76 CN, revelando um predomínio de IgG restritas ao LCR em 82,4%, nos doentes de EM e em 15,8% dos controlos. Quanto à progressão da incapacidade no curso da EM, avaliada pela escala de EDSS, revelou-se significativamente superior após o primeiro ano de EM nos doentes com BOC IgG positivas. Porém, esta tendência inverteu-se ao longo da doença, e numa avaliação superior a 10 anos de EM, os valores de EDSS tornaram-se significativamente superiores nos doentes sem BOC IgG, apresentando também valores superiores e significativos no último EDSS, uma maior probabilidade de atingirem um valor de EDSS≥4 após 10 anos de EM ou de alguma vez, durante o curso da EM, atingirem um EDSS≥6. Observaram-se também diferenças significativas em relação aos subtipos, com os doentes na forma primária progressiva a mostrarem uma percentagem menor de BOC IgG positivas, e às terapêuticas instituídas, com os doentes sem BOC IgG a registarem maior necessidade de recorrer a terapia de 2ª linha. Relativamente às BOC IgM (pesquisadas em 115 doentes de EM e 69 CN), apenas 16,5% nos doentes de EM revelaram a presença de BOC IgM restritas ao LCR, sem diferença significativa face aos controlos. Os doentes de EM com BOC IgM positivas evidenciaram, à data da PL, um EDSS significativamente superior e também uma maior probabilidade de atingirem alguma vez, ao longo da doença, um valor de EDSS≥6. Os marcadores de disfunção da BHE foram avaliados num subgrupo de 51 doentes de EM, 21 DNI e 33 DI. Observou-se um aumento significativo nos níveis da MMP-2 sérica, da razão MMP-2/TIMP-2 no soro e LCR, da razão MMP-9/TIMP-1 no LCR e, ainda, uma redução significativa nos níveis do TIMP-2 sérico nos doentes de EM. Quanto às CAMs, observou-se um aumento significativo da sICAM-1 e sE-Seletina e uma redução da sVCAM-1 no LCR dos doentes de EM. Quanto à relação com a progressão da EM, o aumento da expressão da sVCAM-1 sérica revelou uma associação com níveis superiores de EDSS e formas progressivas da EM; enquanto um aumento da razão MMP-9/TIMP-1 sérica mostrou associar-se a menor incapacidade no curso da EM e ao fenótipo SR. O estudo do polimorfismo -1562 C/T no gene da MMP-9 realizou-se em 169 doentes de EM e 186 CS, sem diferenças significativas na distribuição entre doentes e controlos. Contudo, nos doentes e não nos controlos, registou-se um aumento significativo da frequência do alelo T no sexo feminino. Não se observou nenhuma associação entre a presença do polimorfismo -1562 C/T e a progressão da EM. Os níveis séricos da MMP-9 eram significativamente superiores nos doentes, não sendo influenciados pela presença do alelo T, ao contrário do observado nos CS, onde o alelo T estava associado ao aumento dos níveis da MMP-9. Registou-se ainda uma redução significativa nos níveis séricos da MMP-9 nos doentes a realizar terapia com IFNβ. Os nossos resultados sugerem que alguns dos marcadores possam ter utilidade clínica no seguimento dos doentes de EM. A síntese intratecal de IgG e IgM parece ter efeito preditivo no curso da EM, com a ausência de IgG no LCR a apontar para um pior prognóstico a longo prazo e a presença de IgM a sugerir um prognóstico menos favorável a curto prazo. Também níveis elevados de sVCAM-1 séricos parecem sugerir um fenótipo mais agressivo, enquanto o aumento da razão MMP-9/TIMP-1 aparece associado a menor incapacidade ao longo da EM e poderá ser um bom marcador de resposta à terapêutica nos doentes tratados com o IFNβ.
Multiple sclerosis (MS) is a multifactorial, inflammatory, demyelinating and neurodegenerative disease, with interruption of nerve transmission in the central nervous system (CNS). Despite unknown aetiology, an autoimmune reaction directed against white matter antigens and self-reactivity of immune cells through the blood-brain barrier (BBB), have been shown to be crucial to the formation of inflammatory lesions. The clinical course of the disease is highly unpredictable, with both relapsing-remitting (RR) and progressive forms. The clinical and pathophysiological heterogeneity make MS prognosis very challenging. The objective of this study was to identify biological markers that would act as prognostic markers of MS. Therefore, we selected markers known to play an essential role in CNS inflammation: matrix metalloproteinases (MMPs) and their inhibitors (TIMPs), cell adhesion molecules (CAMs), and the intrathecal production of immunoglobulins (Ig). Paired cerebrospinal fluid (CSF) and serum samples from MS patients and neurological controls (NC: other inflammatory diseases-ID and non-inflammatory of the CNS-NID) were studied, and the presence of IgG and IgM oligoclonal bands (OCB) was assessed by isoelectric focusing. The levels of MMP-2, MMP-9, TIMP-2, TIMP-1, sICAM-1, sVCAM-1 and sE-Seletin were determined by ELISA. The presence of the -1562 C/T polymorphism of the MMP-9 gene was also evaluated in MS patients and healthy controls (HC), through PCR-RFLP methodology. The presence of IgG-OCB was investigated in 183 MS patients and 76 controls (37-NID; 39-ID), showing a predominance of CSF restricted IgG-OCB in 82.4% of MS patients and in 15.8% of controls. Regarding the progression of disability in the course of MS, assessed through the EDSS scale, significantly increased levels were found after the first year of MS in IgG-OCB positive patients. However, this trend reversed over the disease course, and after 10 years or more of MS, EDSS values became significantly higher in patients without IgG-OCB, that also presented significantly higher values in the last EDSS, a higher probability of reaching an EDSS≥4 after 10 years of MS or of reaching an EDSS≥6 at any time during the course of MS. Significant differences were also observed regarding clinical subtypes, with patients in the progressive primary form showing a lower percentage of positive IgG-OCB, and in relation to therapy, with patients without IgG-OCB being more likely to undergo second line therapy. In relation to IgM-OCB (investigated in 115 MS patients and 69 NC), 16.5% of MS patients showed CSF restricted IgM-OCB, with no significant difference in relation to controls. MS patients with positive IgM-OCB showed, at the time of LP, a significant higher EDSS score and also an increased probability of reaching an EDSS≥6 over the course of the disease. BBB dysfunction markers were evaluated in a subgroup of 51 MS, 21 NID and 33 ID patients. A significant increase in serum MMP-2, serum and CSF MMP-2/TIMP-2 ratio, CSF MMP-9/TIMP-1 ratio, as well as a significant reduction in serum TIMP-2 levels was found in MS patients. Regarding CAMs, a significant increase of sICAM-1 and sE-Seletine and a reduction of sVCAM-1 in the CSF of MS patients was observed. Regarding the relation with the progression of MS, increased serum sVCAM-1 expression was associated with higher EDSS scores and progressive forms of MS; while an increase in the serum MMP-9/TIMP-1 ratio was associated with lower disability in the course of MS and the SR phenotype. The study of the -1562 C/T polymorphism in the MMP-9 gene was performed in 169 patients with MS and 186 CS, showing no significant difference between patients and controls. However, in patients, but not in controls, an increase in the frequency of the T allele in females was found. There was no association between the presence of the -1562 C/T polymorphism and the progression of MS. Serum levels of MMP-9 were increased in MS patients and were not influenced by the presence of the T allele, contrary to what was shown in HC, where the T allele was associated with increased levels of MMP-9. There was also a significant reduction in serum MMP-9 levels in patients undergoing IFNβ therapy. Our results suggest that some of the tested markers may have clinical utility in the follow-up of MS patients. The intrathecal synthesis of IgG and IgM seems to have a predictive value in the course of MS, with the absence of IgG-OCB in the CSF pointing towards a worse long term prognosis and the presence of IgM-OCB suggesting a less favourable prognosis in the short term. Also, elevated serum sVCAM-1 levels appear to be indicative of a more aggressive phenotype, while increased MMP-9/TIMP-1 ratio seems to be associated with decreased disability throughout MS and may be a good marker of response to therapy in patients treated with IFNβ.
FCT - SFRH/PROTEC/67690/2010
Escola Superior de Tecnologia da Saúde de Coimbra (ESTeSC)

碩士
國立陽明大學
公共衛生研究所
92
Abstract Abnormal DNA methylation may affect chromosomal stability and gene expression. Many studies have shown an association between abnormal DNA methylation and an increased risk for the development of cancer. It has been shown that genes participated in the one carbon metabolism pathway, such as glycine N-methyltransferase (GNMT), 5, 10-methylenetetrahydrofolate reductase (MTHFR) and methionine synthase (MS) are involved in the regulation of the methylation level of DNA. Therefore, the goals of this study were 1. To tudy the association and interactions of the polymorphisms of genes mentioned above and the development of hepatocellular carcinoma (HCC); 2. To analyze the genotypic and phenotypic association of the plasma level of GNMT and the polymorphisms of mentioned above; 3. To evaluate whether the genotype of GNMT can be a diagnostic marker for HCC. We analyzed the polymorphisms of the GNMT (SNP1, STRP1 and INS/DEL), MS (A2756G) and MTHFR (C677T) on the lymphocyte DNA obtained from 183 HCC patients and 384 normal controls. The results showed that the rates of each polymorphism among the above two groups were not significantly different. However, among people with GNMT SNP1-C/C + INS-DEL DEL/DEL, if their PBL MS genotype has G allele, they will have 2.33-fold risk (95% confidence interval [CI], 1.19-4.59, p =0.014) to develop HCC than subjects whose MS were A/A genotype; if their non-tumorous tissue with GNMT INS-DEL INS/DEL, MS genotype has G allele, then, they will have 5.08-fold risk than people’s non-tumorous tissue MS gene are A/A genotype (95% CI =1.27-20.32, p =0.015)。. In addition, among subjects with MTHFR C/C genotype, if their HCC non-tumorous tissues carrying MS G allele, they will have 2.65-fold risk for HCC than subjects whose non-tumorous tissues’ MS gene are A/A genotype (95% CI: 1.02-6.86, p =0.041). In addition, among 50 pairs tumorous and non-tumorous tissues from HCC patients, the somatic mutation rates for MTHFR C677T and MS A2756G were 16.3%, 10.0%, respectively. Blood samples from 452 normal adults, 195 patients with chronic hepatitis, 28 patients with cirrhosis and 156 patients with HCC were collected and their plasma level of GNMT were determined using an enzyme immunoassay. The results showed that the mean (± standard deviation) levels of GNMT were 14.86 ± 10.49，17.78 ± 5.22，16.79 ± 5.08 and 6.12 ± 4.94 μg/ml, respectively for those groups mentioned above and the differences were statistically significantly (ANOVA, p< 0.0001). In addition, among normal individuals, persons with GNMT SNP1 C/C genotype had significantly higher level of GNMT than persons with TT genotype; persons with MTHFR T allele had significantly higher levels of GNMT than persons with C/C genotype (p =0.038). In conclusion, the interactions of the genetic polymorphisms of GNMT, MTHFR and MS, both in the PBL and non-tumorous liver tissues DNA, were associated with the risk of HCC. The plasma levels of GNMT correlate with certain genotypes of both GNMT and MTHFR genes and it has potential as a diagnostic marker for HCC.

Mit der vorliegenden Studie sollte zu dem wichtigen Forschungsfeld der Pharmakogenetik beigetragen werden, indem zum einen eine einfache und sichere kombinierte Phänotypisierung der drei zuvor erwähnten CYPs (CYP2D6, CYP2C9 und CYP2C19) entwickelt, und zum anderen die Vorhersagekraft des Genotyps für den gemessenen Phänotyp näher untersucht werden sollte. Es ist uns gelungen eine sichere, einfache, schnelle und kombinierte Phänotypisierung der beiden wichtigen Monooxygenasen CYP2D6 und CYP2C9 zu etablieren. Zunächst wurden dazu Wechselwirkungsstudien mit den ausgewählten Testsubstanzen Dextromethorphan (DEX, CYP2D6), Flurbiprofen (FLB, CYP2C9) und Omeprazole (OME, CYP2C19) durchgeführt. Es konnte gezeigt werden, dass DEX und FLB als Kombination verabreicht werden können. Die Gabe von OME gemeinsam mit FLB verändert jedoch das Ergebnis der CYP2C9 Phänotypisierung. Dies ist eine neue Erkenntnis, denn noch 2004 wurde ein Phänotypisierungscocktail veröffentlicht, der die Kombination von FLB und OME enthielt. Bei der genannten Studie wurden jedoch, unseres Wissens nach, keine Wechselwirkungsstudien zu den einzelnen Testsubstanz-Kombinationen durchgeführt. Die von uns entwickelte Phänotypisierungsmethode wurde durch Wechselwirkungsstudien verifiziert. Sie ist jedoch auch in anderen Bereichen den bisher veröffentlichten phänotypisierungscocktails überlegen. Zum einen wurden nur sehr kleine Dosen sicherer Testsubstanzen verwendet. Dies wurde durch Entwicklung neuer, sensitiver LC-MS/MS Methoden ermöglicht. Zum anderen ist diese neue Prozedur schnell und nicht-invasiv durchführbar. Nach Verabreichung der Testsubstanz muss der Urin nur für zwei Stunden gesammelt werden. Zudem weisen unsere Ergebnisse darauf hin, dass die normalerweise durchgeführte, aufwendige Glucuronidspaltung des CYP2D6 abhängigen DEX-Metaboliten, Dextrorphan, vermutlich vernachlässigt werden kann. Die wichtigsten Ergebnisse dieser Studie sind jedoch die Einblicke, die in die Vorhersagekraft der CYP2D6 und CYP2C9 Genotypen für die entsprechenden Phänotypen gewonnen werden konnten. Fast 300 phänotypisierte Kaukasier wurden auch in Hinsicht auf die wichtigsten varianten Allele von CYP2D6, CYP2C9 und CYP2C19 mithilfe bekannter und neu etablierter Methoden genotypisiert. Aufgrund der parallelen Phäno- und Genotypisierung konnten Geno- und Phänotyp direkt korreliert werden. Mit linearen Modellen war es möglich, allen detektierten varianten CYP2D6- und CYP2C9-Allelen Aktivitätskoeffizienten zuzuweisen. Diese können nun verwendet werden, um den Beitrag der einzelnen Allele zur resultierenden Enzymaktivität zu bestimmen, wodurch sich die Vorhersage dieser Aktivität ausgehend vom Genotyp verbessern lassen sollte. Besonders für CYP2D6 ermöglicht das neue Korrelationsmodel präzisere Vorhersagen des Phänotyps als bisher veröffentlichte Modelle. Zusammengefasst leistet diese Studie durch die Entwicklung eines sicheren und einfachen Phänotypisierungsprozesses für CYP2D6 und CYP2C9 und durch die Bestimmung von Aktivitätskoeffizienten für alle einbezogenen CYP2D6 und CYP2C9 Allele und der damit verbundenen präziseren Vorhersage des Phänotyps ausgehend vom Genotyp einen wesentlichen Beitrag zum Forschungsfeld der Pharmakogenetik
This study should contribute to the important field of pharmacogenetics by: firstly, establishing an easy and safe phenotyping method that combines the activity determination of all three previously mentioned CYPs (CYP2D6, CYP2C9, and CYP2C19) into one phenotyping cocktail and secondly, improving the knowledge about the predictive power of the genotype for the measured phenotype. It was indeed possible to develop a save, easy-to-use, fast and simultaneous phenotyping procedure for the important genetic polymorphic enzymes CYP2D6 and CYP2C9. To accomplish that, interaction studies with the chosen probe drugs dextromethorphan (DEX, CYP2D6), flurbiprofen (FLB, CYP2C9) and omeprazole (OME, CYP2C19) were conducted. It could be proven that DEX and FLB can be administered in combination, whereas OME alters the phenotyping results of CYP2C9. This is a new finding as in 2004 a phenotyping cocktail was published that used FLB and OME in combination. However, to our knowledge, no interaction tests were carried in that study. The new phenotyping procedure is not only verified by prior probe drug interaction studies, it also has other advantages over phenotyping cocktails found in literature. Firstly, save probe drugs are used in very small doses. This is possible due to the new sensitive LC-MS/MS methods that were evaluated. Secondly, the new phenotyping procedure is very fast and on-invasive. Urine has to be collected only for 2 h and the results also suggest that the time consuming glucuronide cleavage of the CYP2D6 dependent metabolite dextrorphan, usually carried out before CYP2D6 phenotyping, may be unnecessary. Most importantly, however, new insights into the phenotype prediction from genotype for CYP2C9 and CYP2D6 could be gained within this study. Nearly 300 phenotyped Caucasian subjects were also genotyped for the most important known variant alleles for CYP2D6, CYP2C9 and CYP2C19 using several established and newly developed genoptyping methods. Therefore, a direct correlation between phenotype and genotype could be conducted for CYP2D6 and CYP2C9. Employing linear modeling, it was possible to assign activity coefficients to each of the detected CYP2D6 and CYP2C9 alleles, thereby estimating their contribution to the resulting enzyme activity. This might facilitate the prediction of the CYP2D6 and CYP2C9 metabolic status of a subject knowing only its respective genotypes. Especially the new CYP2D6 genotype phenotype correlation model might allow for more precise phenotype prediction for the included variant alleles than was possible until now. Taken together, this study substantially contributes to the important research field of pharmacogenetics by (i) developing a save and easy-to-use phenotyping combination for CYP2D6 and CYP2C9, and (ii) by establishing activity coefficients for each of the detected CYP2D6 and CYP2C9 alleles, thereby allowing for a more precise prediction of the phenotype from genotype

碩士
國立陽明大學
公共衛生研究所
93
Coke-oven workers (COWs) are occupationally exposed to high concentrations of polycyclic aromatic hydrocarbons (PAHs), especially workers near the topside of the oven. Uninary 8-hydroxy-2-deoxyquanosine (8-OH-dG) and 1-hydroxypyrene (1-OHP) are biomarkers of oxidative DNA damage and PAH metabolism, respectively. Previously, we found that glycine N-methyltransferase (GNMT) can bind benzo(a)pyrene (BaP) and inhibit DNA adducts formation. Since methionine synthase (MS), 5,10-methylene- tetrahydrofolate reductase (MTHFR) and GNMT are involved in the one-carbon metabolism pathway, we would like to study the associations of the genetic polymorphisms of these genes and the urinary biomarkers in coke-oven workers. Questionnaires and lymphocyte DNA were collected from 217 coke-oven workers which including 54 workers from the topside of the oven (high exposure group, group A) ,92 workers from both sides of the oven (medium exposure group, group B) and 71 administrative persons from the same factory (low exposure group, group C). Three genotypes of GNMT-SNP1, INT/DEL, and STRP1 were analyzed using TagMan and GeneScan methods; both A2756G genotype of MS gene and C677T genotype of MTHFR gene were analyzed using PCR-RFLP. Results：The preliminary results indicate that there is a reverse relationship between the urinary levels of 1-OHP and 8-OH-dG in the group A workers with different GNMT genotypes. Use logistic regression analysis indicated that workers with GNMT STRP1-B/B genotypes had higher 8-OHdG level than those with STRP1-A/A or A/B genotype in the group A (OR= 4.41, 95%CI =1.04-18.81, p= 0.044, respectively).For the GNMT haplotype study, workers with SNP1+INS-DEL+STRP1-C/T+INS/INS+A/A had lower 8-OHdG level than those with SNP1+INS-DEL+STRP1-C/T + INS/DEL + B/B in group A（7.6 ± 7.3 ng/ml < 32.9 ± 26.8 ng/ml, p = 0.01）. In conclusion, GNMT may have a protective effect against the exposure to carcinogens such as PAHs, and the interactions of the genetic polymorphisms of GNMT, MS, MTHFR were associated with different 1-OHP and 8-OHdG level in coke-oven workers. This study may be useful for the genetic counseling of workers who expose to environment carcinogens related to PAHs.