Specific SNPs in intronic regions of the human TCF7L2 gene are associated with an elevated risk of T2D development and progression. Several investigations have suggested a role of TCF7L2 in pancreatic β-cells. Whether this transcription factor is indeed expressed in the pancreatic islets of rodent species, however, has been a controversial issue. Here, we found that TCF7L2 mRNA level was significantly lower in the pancreas compared to the gut or Ins-1 cell line. In addition, TCF7L2 mRNA abundance in the pancreas was decreased by insulin. Finally, both TCF7 and TCF7L1 but not LEF-1 could be detected in the mouse pancreas. mRNA abundance for these two transcription factors was also decreased by insulin, and the level of TCF7, TCF7L1, and TCF7L2 mRNAs could be down-regulated by HFD. We speculate that reduced expression of these TCF genes during hyperinsulinemia may alter the Wnt signalling pathway and therefore impair the function of β-cells.

The canonical Wnt pathway is a vitally important signalling pathway that plays an important role in cell proliferation, differentiation and fate decisions in embryonic development and in the maintenance of adult tissues. The twelve Armadillo (ARM) repeat-containing protein beta-catenin acts as the signal transducer in this pathway and is continuously degraded in the cytosol by the beta-catenin destruction complex (BDC). Upon receiving the Wnt signal the BDC is inactivated, allowing beta-catenin to accumulate in the cytosol and be transported to the nucleus where it binds to the TCF/LEF family of transcription factors, inducing the expression of cell cycle promotor genes. In this Thesis I describe investigations into the roles of leucine-rich repeat kinase 2 (LRRK2) and the transcription factor TCF7L2 within this signalling pathway. LRRK2 is a large multi-domain protein with strong links to Parkinson’s disease and suggested to play a role in inactivating the BDC in response to the Wnt signal. A recent paper proposed that the previously uncharacterised regions of LRRK2 contain a series of tandem repeat sub-domains. I began an investigation into these sub-domains but I was unable to produce soluble protein constructs despite the use of a range of common techniques, and so I was forced to conclude this project early. The main body of this thesis focuses on the interaction between the intrinsically disordered TCF7L2 and the repeat protein beta-catenin, a very long interface of approximately 4800 Å2 that spans from the third to the eleventh ARM repeat of beta-catenin and residues 12 to 50 of TCF7L2, as determined by X-ray crystal structures. First, a fluorescence reporter system for the binding interaction was developed and used to determine the kinetic rate constants for the association and dissociation of the wild-type construct using stopped-flow fluorescence spectroscopy and time-dependent fluorescence spectroscopy. It was found that association of TCF7L2 and beta-catenin was rapid (7.3 ± 0.1 x107 M-1s-1) with only a single phase was observed, whereas dissociation was biphasic and slow (5.7 ± 0.4 x10-4 s-1, 15.2 ± 2.8 x10-4 s-1). Using either of these two dissociation rate constants the calculated Kd value obtained is much lower than the values previously reported in the literature (8 ± 1 / 20 ± 2 pM compared with 16 nM). This reporter system was then used to investigate the striking variability between three crystal structures previously obtained for the TCF7L2-beta-catenin complex, in which different regions of TCF7L2 show different elements of secondary structure. Mutational analysis revealed that the interface residues on TCF7L2 identified in these structures make little or no contribution to the overall binding affinity, pointing to a transient nature of these contact in solution and suggesting that the observed differences between the structures are due to differences in crystal packing. Further experiments into the effect of osmolarity on the binding equilibrium and kinetics supported this conclusion and suggest a change in the association/dissociation mechanism as a function of ionic strength. Lastly, further mutational analysis of TCF7L2 revealed two regions that contribute particularly strongly to the binding kinetics, suggesting that TCF7L2-beta-catenin assembly proceeds via a two-site avidity mechanism. Some of the most destabilising variants display two additional dissociation phases, indicating the presence of an alternative dissociation pathway that is inaccessible to the wild-type. In summary, the results presented here provide insights into the kinetics of molecular recognition of a long intrinsically disordered region with an extended repeat protein surface, a process shown to involve multiple routes with multiple steps in each.

Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2015-05-28T13:37:34Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) TESE Carlos Eduardo Soares de Macedo.pdf: 1009580 bytes, checksum: b4ee26d2671e171607c2daa6d85a4824 (MD5) / Made available in DSpace on 2015-05-28T13:37:34Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) TESE Carlos Eduardo Soares de Macedo.pdf: 1009580 bytes, checksum: b4ee26d2671e171607c2daa6d85a4824 (MD5) Previous issue date: 2015-02-23 / INTRODUÇÃO: Obesidade e diabetes tipo 2 (DM 2) são um crescente problema de saúde. O fator de transcrição 7-like 2 (TCF7L2) é o gene mais associado ao DM 2 e sua potencial associação com a obesidade tem sido aventada em estudos recentes. OBJETIVO: O objetivo deste estudo foi avaliar os efeitos do tratamento cirúrgico da obesidade na expressão do TCL7F2 e suas associações com o índice de massa corpórea (IMC) e o DM 2. METODOLOGIA: Foi realizado um estudo tipo coorte, amostras de sangue periférico de 26 pacientes portadores de obesidade foram colhidas antes da cirurgia bariátrica e após um ano de seguimento, foi extraído RNA das amostras por sistema automatizado QIAsymphony® (QIAGEN©), Realizou-se PCR em tempo real, utilizando reagentes SYBR® Green PCR Kit (QIAGEN©) através do RotorGene Q® (QIAGEN©). As variáveis categóricas foram avaliadas utilizando o teste do qui-quadrado com a correção de Fisher quando necessário e as variáveis contínuas com o teste t-student, modelos de regressão linear foram criados para avaliar a relação do IMC com a expressão genética. Apenas associações com p-valor < 0,05 foram consideradas significativas. RESULTADOS: a expressão genética do TCF7L2 após a cirurgia bariátrica não se alterou na população como um todo (p-valor= 0,38), contudo, nos pacientes diabéticos, houve redução significativa (27%, p-valor= 0,021). Houve tendência à diferença (p-valor 0,051) entre a proporção de pacientes diabéticos que apresentaram alguma redução da expressão (81,88%) em relação aos não diabéticos (40%). A expressão préoperatória dos pacientes diabéticos foi significativamente maior em relação aos não diabéticos (p-valor= 0,042). Houve correlação positiva entre IMC e os valores do dCt (delta cycle threshold) do TCF7L2 antes e após a cirurgia bariátrica (p-valor= 0,037 e 0,007 respectivamente), ou seja, quanto maior o IMC do paciente menor a expressão do gene. Houve correlação positiva estatisticamente significativa entre a redução de IMC e a expressão do TCF7L2 (p-valor= 0,027), ou seja, quanto maior a redução de IMC maior o aumento de expressão genética comparada com os valores pré-operatórios. CONCLUSÃO: A expressão do TCF7L2 um ano após a cirurgia bariátrica não se alterou na população estudada, apenas nos pacientes diabéticos mostrou-se uma redução estatisticamente significativa. A expressão do TCF7L2 foi significativamente maior no grupo diabético antes da cirurgia bariátrica. Houve correlação positiva entre os valores do dCt do TCF7L2 e o IMC tanto no pré quanto no pós-operatório. A redução de IMC foi significativamente associada a aumento da expressão do TCF7L2.

For more than 20 years, T-cell specific factor 4 (Tcf4) is the most intensively studied member of the conserved Tcf/Lymphoid enhancer-binding factor (Lef) family of transcription factors. Together with β-catenin coactivator, Tcf4 represents the prominent nuclear effector of canonical Wnt signaling in the intestinal epithelium. Regulation of Wnt-β-catenin signaling in intestinal stem cells is crucial for tissue homeostasis and tumor formation initiation. Up to date, several mouse models were generated to manipulate Tcf4 abundance or activity in vivo and dissect its function. Moreover, mutational screens and expression profiling of human colorectal tumors were carried out to disclose a contribution of TCF4 to tumor progression. However, subsequent studies brought conflicting results in relation to the potential of Tcf4 to activate or repress Wnt target genes and drive or inhibit cell proliferation. Here in this study, we analyze publicly available datasets for global expression of TCF4 and its paralogs in human tissues and colorectal cancer (CRC) samples. Notably, we present newly generated Tcf4flox5 mouse with a conditional Tcf4 allele that can be used to eliminate expression of Tcf4 from two alternative promoters of the gene. Using this mouse strain we documented that Tcf4 loss led to the demise of...

No description available.

La Protéine Cellulaire du Prion (PrPc), isoforme non pathogène de la Protéine Scrapie, est une protéine ubiquitaire qui a été impliquée dans de nombreux processus cellulaires tels que la prolifération, la migration, l’adhésion, la différenciation et l’apoptose, par des mécanismes qui restent en grande partie à élucider. L’épithélium intestinal est en constant renouvellement et son homéostasie repose sur une régulation fine et coordonnée de l’ensemble de ces processus. Notre équipe s’est intéressée au rôle de la PrPc dans l’épithélium intestinal et a mis en évidence son expression dans le type cellulaire majoritaire de cet épithélium, les entérocytes, et sa double localisation selon leur état de différenciation. En effet, dans les cellules différenciées, la PrPc est majoritairement présente au niveau des desmosomes, alors que dans les cellules prolifératives, elle est principalement nucléaire. Nous mettons en évidence que la PrPc desmosomale est impliquée dans le maintien et l’intégrité de l’ensemble des jonctions intercellulaires (jonctions serrées, adhérentes et desmosomes) et contribue à la fonction de barrière de l’épithélium intestinal. La PrPc nucléaire, quant à elle, interagit avec plusieurs effecteurs de la voie de signalisation Wnt : la -caténine, la -caténine et le facteur de transcription TCF7L2. Dans ce contexte, nous révélons la capacité de la PrPc nucléaire à moduler l’expression de gènes cibles de la voie Wnt canonique. L’ensemble de ces travaux permet de révéler la PrPc comme un nouvel élément clé de l’homéostasie de l’épithélium intestinal – du maintien de la fonction de barrière jusqu’à la régulation de l’expression de gènes – et de définir la PrPc comme un nouveau membre de la famille des protéines NACos. / The cellular Prion Protein (PrPc), the normal conformer of the Scrapie protein, is a ubiquitous protein, which has been involved in several cellular processes such as proliferation, migration, adhesion, differentiation and apoptosis, through mechanisms that are not fully characterized. Intestinal epithelium is renewing constantly and its homeostasis requires a fine and coordinated regulation of all these processes. Our team has focused on PrPc functions in this tissue and has demonstrated that it is expressed in enterocytes, the major cell type in the intestinal epithelium, with a dual localization depending on the differentiation state of the cells. Indeed, in differentiated cells PrPc is localized in desmosomes while being mostly in the nucleus in proliferative cells. We demonstrated the involvement of desmosomal PrPc in the maintenance and integrity of all the intercellular junctions (tight, adherens junctions and desmosomes) and its requirement for the intestinal barrier function. PrPc in the nucleus interacts with key effectors of the Wnt pathway: -catenin, -catenin and the transcription factor TCF4/TCF7L2. In this context, we revealed the ability of nuclear PrPc to modulate the expression of a subset of Wnt target genes. Altogether, this work highlights the role of PrPc as a new key element of the intestinal epithelial homeostasis – from the barrier function to gene regulation – and allows considering PrPc as a new member of the NACos family proteins (proteins associated with the Nucleus and Adhesion Complexes).

Type 2 Diabetes (T2D) disproportionally affects ethnic minorities in the United States. The development of T2D involves complex interaction between environmental factors and genetic predisposition. The genetic associations of six single nucleotide polymorphisms (SNPs) in TCF7L2 gene with the risk of T2D were evaluated in three high risk minority populations: Cuban Americans, Haitian Americans, and African Americans. For Cuban Americans, four SNPs (rs7901695, rs4506565, rs7903146 and rs11225537) were significantly associated with the risk of T2D after multivariable adjustment (p=0.018, p=0.016, p=0.014, and p=0.0008, respectively). Among controls, risk allele carriers of SNPs rs7901695, rs4506565 and rs7903146 had significantly higher fasting glucose level, compared to non-risk allele carriers. Additionally, a significant interaction between dietary fiber intake and SNP rs7903146 for the risk of T2D (p= 0.044) was found in Cuban Americans. Similarly, for SNP rs7901695, significant interaction was also found for fiber intake (p=0.014) as well as glycemic load (p=0.040). Subgroup analysis revealed that significant associations were only found within higher intake groups of dietary factors for both SNPs. For Haitian Americans, SNPs rs11196205 (p=0.059) and rs7895340 (p=0.069) showed marginal significance for the risk of T2D. After stratification by gender, SNPs with marginal significance from the gender-combined analysis became statistically significant with the same trend for the risk of T2D when analysis were done in males: rs11196205 (p=0.034) and rs7895340 (p=0.024). For African Americans, SNP rs7903146 (p=0.065) showed a marginal significance with the risk of T2D in gender-combined analysis and a statistical significance (p=0.013) in males. Two additional SNPs rs7901695 and rs4506565 were found to be significantly associated with the risk of T2D in males. Risk allele carriers of these two SNPs had significantly higher mean level of the fasting glucose level, compared to non-risk allele carriers in controls. T2D related quantitative trait analysis also demonstrated that in controls, compared to non-minor allele carriers of SNP rs12255372, minor allele carriers had significantly higher means of BMI, diastolic blood pressure, numbers of components of Metabolic Syndrome, significantly lower mean values of HDL-cholesterol and adiponectin. Taken together, our studies demonstrated ethno-specific genetic associations between TCF7L2 gene and the risk of T2D and related phenotypes.

Submitted by Haroudo Xavier Filho (haroudo.xavierfo@ufpe.br) on 2016-10-07T19:14:19Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Dissertação - Andriu dos Santos Catena.pdf: 3090562 bytes, checksum: de170aa11897a6b398c8ebdfe1731d36 (MD5) / Made available in DSpace on 2016-10-07T19:14:19Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Dissertação - Andriu dos Santos Catena.pdf: 3090562 bytes, checksum: de170aa11897a6b398c8ebdfe1731d36 (MD5) Previous issue date: 2016-02-03 / Segundo a hipótese da programação fetal, alterações metabólicas in utero estabelecem padrões fisiológicos que modulam a saúde do ser humano, contribuindo ao desenvolvimento de síndrome metabólica (SMet), obesidade e diabetes tipo 2 (DT2) na vida adulta. O risco de estresse oxidativo é uma condição metabólica que se eleva durante a gravidez, desencadeando a expressão de genes cruciais ao desenvolvimento dessas patologias, como o TCF7L2. Portanto, torna-se importante identificar a frequência dos principais polimorfismos desse gene (49080T>C, 103894G>T e 53341C>T) assim como analisar sua expressão em amostras de RNs com peso ao nascer alterado e de pacientes adultos obesos. Desta forma, foram analisadas amostras de 149 indivíduos, subdividas em duas coortes: 98 recém-nascidos da cidade da Paraíba - JP, sendo 11 pequenos para idade gestacional (PIG), 41 grandes para idade gestacional (GIG) e 46 apropriados para idade gestacional (AIG); e 51 adultos atendidos na cidade do Recife - PE, sendo 12 obesos com DT2, 17 obesos sem DT2 e 22 saudáveis (não obesos). Em segundo momento, foram utilizadas ferramentas de bioinformática para compreender as interações biomoleculares envolvendo TCF7L2 na via de sinalização Wnt. O polimorfismo 49080T>C foi o mais prevalente na população estudada (38,9%) comparado ao 103894G>T (27,7%) e 53341C>T (31,9%). Níveis de mRNA entre as coortes analisadas demonstraram significância estatística (p=0,001). Recém-nascidos PIG apresentaram expressão de TCF7L2 maior que GIG (1,751 e 1,229, respectivamente) (p=0,017), além de expressão relativa similar com adultos obesos com DT2. Não houve diferença estatística entre a coorte dos adultos (p=0,115). GIG e obesos revelaram forte similaridade (p=0,922). Adicionalmente, análises in silico demonstraram que a resposta inflamatória condicionada ao estresse oxidativo durante a gravidez contribui para o aumento de IL-6 e TNFa. Essas citocinas estimulam o aumento de ß-catenina, que é translocada ao núcleo para ativar fatores de transcrição como TCF7L2. A rede metabólica da TCF7L2 envolve genes e produtos relacionados à via Wnt, como DKK1, CTNNB1, GCG, APOE, APOC1 e FTO. Estas moléculas participam da regulação via Wnt e do metabolismo de carboidratos e lipídeos. Dessa forma, TCF7L2 parece influenciar o peso ao nascer, o que contribui ao desenvolvimento de SMet e obesidade na vida adulta. / According to fetal programming hypothesis, metabolic exchange in utero establishes physiological standards that modulate the human health, contributing to common diseases in adulthood development, like metabolic syndrome (MetS), obesity, and type 2 diabetes (T2D). The risk of oxidative stress is a metabolic condition that rises during pregnancy, triggering the expression of critical genes to the development of these pathologies, such as TCF7L2. Therefore, it becomes necessary to frequencies identify the main polymorphisms this gene (49080T>C, 103894G>T, and 53341C>T) and analyze their expression in samples of newborns with abnormal birth weight and obese individuals. Thus, 149 subjects of Northeast Brazilian were enrolled in this study, performed in two cohorts: 98 newborns, being 11 with small for gestational age (SGA); 41 large for gestational age (LGA); and 46 appropriate for gestational age (AGA); and 51 adults, being 12 obese with type 2 diabetes (T2D); 17 non-T2D obese; and 22 healthy adults. In the second step, were used bioinformatics tools to understand the molecular interactions involving TCF7L2 in the Wnt signaling pathway. 49080T>C polymorphism was more prevalent in the population (38.9%) compared to 103894G>T (27.7%) and 53341C>T (31.9%). mRNA levels were showing a statistical difference between newborns and adults cohorts (p=0.001). SGA neonates presented a TCF7L2 expression higher than LGA (1.751 and 1.229, respectively) (p=0.017), beyond a similar relative expression compared to adults obese DT2. There was no statistical significance in the adult cohort (p=0.115). LGA and obese adult groups revealed high similarity (p=0.922). 53341C>T, 103894G>T, and 49080T>C allelic frequencies were similar to the findings of other studies. Additionally, the in silico analysis demonstrated that inflammatory response due oxidative stress during pregnancy contributes to IL-6 and TNFa increases. This cytokine allowed to ß-catenin increase, with is translocated to the nucleus for activating of transcription factors such TCF7L2. Furthermore, this gene interacts with other genes and products related to Wnt signaling pathway, like GCG, DKK1, CTNNB1, APOE, APOC1, and FTO. Thus, TCF7L2 may influence the birth weight, therefore contributing for MetS and obesity in adulthood.

No description available.

<p>Most human traits and common diseases have a complex genetic makeup involving more than one gene. The work presented in this thesis investigates standing body height and the common disease type 2 diabetes mellitus (T2DM). In study I we analyzed two single nucleotide polymorphisms (SNPs) in the TCF7L2 gene that had been shown to be associated with T2DM. Analysis was performed in the ULSAM population cohort of ~1500 males. We were able to replicate the association to type 2 diabetes and in addition to that we made a novel find, showing association between the risk alleles and increased proinsulin levels. In study II we analyzed four genes identified to be associated with T2DM in a genome-wide association study. We analyzed SNPs in these genes in the ULSAM population cohort and found an association between SNPs in the HHEX gene and insulin responses and insulin levels. </p><p>The aim of studies III-V was to identify genes affecting normal variation in standing body height. Using a candidate gene approach in study III, 17 genes were screened in the ULSAM population cohort using SNPs. A suggestive association of the ESR1 gene with height was found and confirmed as significant in males from the PIVUS population cohort. In study IV, as a part of the GenomEUtwin project, we performed genetic fine mapping of a linked locus for body height on the X-chromosome. By analyzing 1377 SNPs in 780 Finnish twins, we mapped a region spanning 65kb of this locus with linkage to body height in males. This region contains the GPC3 and PHF6 genes that have known connections to syndromes were standing body height is affected. In study V significant linkage and association to standing body height in males was found for the COL1A11 gene, using population cohorts from Finland and Iceland. </p>