Dissertations / Theses on the topic 'Hepatocyte nuclear factor'

Heterozygous mutations and deletions of the gene that encodes the transcription factor hepatocyte nuclear factor 1β (HNF1B) are the commonest known monogenic cause of developmental kidney disease. However, diagnosis remains challenging due to phenotypic variability and frequent absence of a family history. There is also no consensus as to when HNF1B genetic testing should be performed. This thesis includes work looking at the identification of HNF1B-associated disease. An HNF1B score was developed in 2014 to help select appropriate patients for genetic testing. The aim in chapter 2 was to test the clinical utility of this score in a large number of referrals for HNF1B genetic testing to the UK diagnostic testing service for the HNF1B gene. An HNF1B score was assigned for 686 referrals using clinical information available at the time of testing; performance of the score was evaluated by receiver-operating characteristic curve analysis. Although the HNF1B score discriminated between patients with and without a mutation/deletion reasonably well, the negative predictive value of 85% reduces its clinical utility. HNF1B-associated disease is due to an approximate 1.3 Mb deletion of chromosome 17q12 in about 50% of individuals. This deletion includes HNF1B plus 14 additional genes and has been linked to an increased risk of neurodevelopmental disorders, such as autism. The aim in chapter 3 was to compare the neurodevelopmental phenotype of patients with either an HNF1B intragenic mutation or 17q12 deletion to determine whether haploinsufficiency of the HNF1B gene is responsible for this aspect of the phenotype. Brief behavioural screening showed high levels of psychopathology and impact in children with a deletion. 8/20 (40%) patients with a deletion had a clinical diagnosis of a neurodevelopmental disorder compared to 0/18 with a mutation, P=0.004. 17q12 deletions were also associated with more autistic traits. Two independent clinical geneticists were able to predict the presence of a deletion with a sensitivity of 83% and specificity of 79% when assessing facial dysmorphic features as a whole. These results demonstrate that the 17q12 deletion but not HNF1B intragenic mutations are associated with neurodevelopmental disorders; we conclude that the HNF1B gene is not involved in the neurodevelopmental phenotype of these patients. Extra-renal phenotypes frequently occur in HNF1B-associated disease, including diabetes mellitus and pancreatic hypoplasia. Faecal elastase-1 levels have only been reported in a small number of individuals, the majority of which have diabetes. In chapter 4 we measured faecal elastase-1 in patients with an HNF1B mutation or deletion regardless of diabetes status and assessed the degree of symptoms associated with pancreatic exocrine deficiency. We found that faecal elastase-1 deficiency is a common feature of HNF1B-associated renal disease even when diabetes is not present and pancreatic exocrine deficiency may be more symptomatic than previously suggested. Faecal elastase-1 should be measured in all patients with a known HNF1B molecular abnormality complaining of chronic abdominal pain, loose stools or unintentional weight loss. Hypomagnesaemia is a common feature of HNF1B-associated disease and is due to renal magnesium wasting. The aim in chapter 5 was to measure both serum and urine magnesium and calcium levels in individuals with an HNF1B molecular defect and compare to a cohort of patients followed up in a general nephrology clinic in order to assess their potential as biomarkers for HNF1B-associated disease. The results of this pilot study show that using a cut-off for serum magnesium of ≤0.75 mmol/L was 100% sensitive and 87.5% specific for the presence of an HNF1B mutation/deletion. All individuals in the HNF1B cohort had hypermagnesuria with fractional excretion of magnesium >4%; a cut-off of ≥4.1% was 100% sensitive and 71% specific. This suggests serum magnesium levels and fractional excretion of magnesium are highly sensitive biomarkers for HNF1B-associated renal disease; if these results are confirmed in a larger study of patients with congenital anomalies of the kidneys or urinary tract they could be implemented as cheap screening tests for HNF1B genetic testing in routine clinical care.

Primary human hepatocytes are a scarce resource with variable function, which diminishes with time in culture. As a consequence their use in tissue modelling and therapy is restricted. Human embryonic stem cells (hESCs) could provide a stable source of human tissue due to their properties of self-renewal and their ability to give rise to all three germ layers. hESCs have the potential to provide an unlimited supply of hepatic endoderm (HE) which could offer efficient tools for drug discovery, disease modelling and therapeutic applications. In order to create a suitable environment to enhance HE formation, hESC culture needed to be standardised. As such, a media trail was carried out to define serum free media capable of maintaining hESC in a pluripotent undifferentiated state. We also ensured hESC cultured in the various media could be directly differentiated to HE in a reproducible and efficient manner. The project then focused on the effect of post-translational modifications (PTMs), specifically SUMOylation, in hepatocyte differentiation and its subsequent manipulation to enhance HE viability. SUMOylation is a PTM known to modify a large number of proteins that play a role in various cellular processes including: cell cycle regulation, gene transcription, differentiation and cellular localisation. We hypothesised that SUMO modification may not only regulate hESC self renewal, but also maybe required for efficient hESC differentiation. We therefore interrogated the role of SUMOylation in hESC differentiation to hepatic endoderm (HE). hESC were differentiated and the cellular lysates were analysed by Western blotting for key proteins which modulate the conjugation and de conjugation of SUMO. We demonstrate that peak levels of SUMOylation were detectable in hESC populations and during cellular differentiation to definitive endoderm (DE), day 5. Following commitment to DE we observed a decrease in the level of SUMO modified proteins during cellular specialisation to a hepatic fate, corresponding with an increase in SENP 1, a SUMO deconjugation enzyme. We also detected reduced levels of hepatocyte nuclear factor 4 α (HNF4α), a critical regulator of hepatic status and metabolic function, as SUMOylation decreased. As a result, we investigated if HNF4α was SUMOylated and if this process was involved in modulating HNF4α’s critical role in HE. HNF4α is an important transcription factor involved in liver organogenesis during development and is a key regulator for efficient adult liver metabolic functions. We observed a decreasing pattern of HNF4α expression at day 17 of our differentiation protocol in conjunction with a decrease in SUMO modified proteins. In order to further investigate and validate a role of SUMOylation on HNF4α stability Immunoprecipitation (IP) was employed. HNF4α protein was pulled down and probed for SUMO 2. Results show an increase in the levels of SUMO2 modification as the levels of HNF4α decrease. Through deletion and mutation analysis we demonstrated that SUMO modification of HNF4α was restricted to the C-terminus on lysine 365. Protein degradation via the proteasome was responsible for the decrease in HNF4α, demonstrated by the use of a proteasome 26S inhibitor MG132. Additionally, a group at the University of Dundee has shown that polySUMOylation of promyelocytic leukaemia protein (PML) leads to its subsequent ubiquitination via RNF4, an ubiquitin E3 ligase, driving its degradation. Using an in vitro ubiquitination assay, we show that polySUMOylated HNF4α is preferentially ubiquitinated in the presence of RNF4. Overall polySUMOylation of HNF4α may reduce its stability by driving its degradation, hence regulating protein activity. In conclusion, polySUMOylation of HNF4α is associated with its stability. HNF4α is subsequently important for HE differentiation both driving the formation of the hepatocytes and in maintaining a mature phenotype, in agreement with a number of different laboratories. Creating the ideal environment for sustaining mature functional hepatocytes, primary and those derived from hESCs and iPSCs, is essential for further use in applications such as drug screening, disease modelling and extracorporeal devices.

Thesis (Ph. D.)--University of California, Riverside, 2009.
Includes abstract. Includes bibliographical references (leaves 293-348). Issued in print and online. Available via ProQuest Digital Dissertations.

The nuclear receptor Hepatocyte Nuclear Factor 4a (HNF4a; NR2A1) regulates the transcription of many genes involved in glucose and lipid metabolism. Genetic linkage analyses have implicated HNF4a in the disease processes leading to Type 2 Diabetes Mellitus and dyslipidaemia. The aim of this study was to investigate the regulation of target genes in the metabolic pathways of glycolysis, lipogenesis and gluconeogenesis by HNF4a. Initally, the expression of HNF4a and its splice variants was investigated in three human hepatoma cell lines, HuH7, HepG2 and Hep3B, with the latter two cell lines shown to express the same range of HNF4a splice variants as human adult liver. The regulation of specific HNF4a target genes, L-PK, PEPCK and SREBP-1c, was subsequently investigated in HepG2 cells using a reporter gene approach. HNF4a was found to induce expression of reporter genes containing L-PK, PEPCK and SREBP-1c proximal promoter sequences. Insulin (1 ?M), but not high glucose (25 mM), was found to stimulate HNF4a-driven expression of the SREBP-1c reporter gene, while co-expression of HNF4a with the nuclear receptor coactivators, PGC-1a or p300, led to a reduction in SREBP-1c reporter gene expression. The changes in expression of various HNF4a target genes in response to physiological mediators of the fasting-fed cycle were characterised in HepG2 cells using a real-time quantitative PCR approach. The role of HNF4a, p300 and PGC-1a was further investigated by plasmid overexpression. HNF4a and PGC-1a were found to positively regulate PEPCK expression under cell culture conditions simulating fasting (cAMP), whilst overexpression of HNF4a, PGC1a and p300 reduced L-PK mRNA expression under fed conditions. In conclusion, the results indicate that HNF4a is a transcriptional activator of both glucagon- stimulated gluconeogenic gene expression and insulin-stimulated glycolytic and lipogenic gene expression. It is hypothesised HNF4a forms separate multi-protein complexes to differentially regulate metabolic pathways under different metabolic states.

Final article is available at "wileyonlinelibrary.com" Takako Okamoto,Masaki Mandai,Noriomi Matsumura,Ken Yamaguchi,Hiroshi Kondoh,Yasuaki Amano,Tsukasa Baba,Junzo Hamanishi,Kaoru Abiko,Kenzo Kosaka,Susan K. Murphy,Seiichi Mori,Ikuo Konishi "Hepatocyte nuclear factor-1β (HNF-1β) promotes glucose uptake and glycolytic activity in ovarian clear cell carcinoma" Molecular Carcinogenesis 54:35–49 (2013)
Kyoto University (京都大学)
0048
新制・論文博士
博士(医学)
乙第12804号
論医博第2076号
新制||医||1001(附属図書館)
80848
京都大学大学院医学研究科医学専攻
(主査)教授 野田 亮, 教授 武藤 学, 教授 稲垣 暢也
学位規則第4条第2項該当

The UDP-glucuronosyltransferases (UGTs) are a superfamily of enzymes that glucuronidate small, lipophilic molecules, thereby altering their biological activity and excretion. In humans, important examples of UGT substrates include molecules of both endogenous and xenobiotic origin; thus, UGTs are considered essential contributors to homeostatic regulation and an important defence mechanism against chemical insult. In keeping with both roles, UGTs are most strongly expressed in the liver, a predominant organ involved in detoxification. Rates of glucuronidation in humans are neither uniform among individuals, nor constant in an individual over time. Genetic determinants and non-endogenous signals are both known to influence the expression of UGTs, which in turn may affect the efficacy of certain pharmaceutical treatments or alter long-term risk of developing disease. Thus, this thesis focuses on the transcriptional regulation of UGT genes in humans, particularly on mechanisms that are likely to be relevant to their expression and variation in the liver. Two major approaches were used: firstly, extensive studies of several UGT promoters were performed to identify and characterise transcriptional elements that are important for UGT expression; and secondly, important hepatic transcription factors were investigated as potential regulators of UGT genes. UGT1A3, UGT1A4 and UGT1A5 are a subset of highly related, but independently regulated, genes of the human UGT1 subfamily. UGT1A3 and UGT1A4 are expressed in the liver, whereas UGT1A5 is not. The presented analysis of the UGT1A3, UGT1A4 and UGT1A5 proximal promoters demonstrates that a hepatocyte nuclear factor (HNF)1-binding site common to all three promoters is important for UGT1A3 and UGT1A4 promoter activity in vitro, but is insufficient to drive UGT1A5 expression. Two additional elements required for the maximal activity of the UGT1A3 promoter were also identified that may distinguish this gene from UGT1A4. UGT1A3 was investigated further, focusing on mechanisms that may contribute to interindividual variation in UGT1A3 expression. Polymorphisms in the UGT1A3 proximal promoter were identified and their functional consequences tested. Known variants of HNF1alpha were also tested for altered activity towards the UGT1A3 gene. UGT1A9 is the only hepatic member of the UGT1A7-1A10 subgroup of UGT1 enzymes. Previous work had identified HNF1-binding sites in all four genes, and HNF4alpha as an UGT1A9-specific regulator. The work presented herein extends these findings to show that HNF1 factors and HNF4alpha synergistically regulate UGT1A9, and that HNF4alpha is not the only transcription factor responsible for the unique presence of UGT1A9 in the liver. Liver-enriched transcription factors screened as potential UGT regulators were chosen from the HNF1, HNF4, HNF6, FoxA and C/EBP protein families. Functional interactions newly identified by this work were HNF4alpha with UGT1A1 and UGT1A6, HNF6 with UGT1A4 and UGT2B11, FoxA1 and FoxA3 with UGT2B11, UGT2B15 and UGT2B28 and C/EBPalpha with UGT2B17. Observations were also made regarding different patterns of interaction between each UGT and the transcription factors tested, particularly HNF1alpha.

Vhnf1 est un facteur de transcription a homeodomaine exprime dans les reins, le foie et le pancreas. Nous avons inactive ce gene chez la souris en remplacant son premier exon par le gene lacz. La detection de l'activite -galactosidase placee sous controle du promoteur de vhnf1 nous a permis de decrire finement le profil d'expression de ce gene, chez l'adulte et au cours du developpement. Nous avons ainsi montre l'expression particuliere de ce gene dans des structures epitheliales tubulaires telles que les canaux biliaires et pancreatiques et les tubules renaux. La mutation vhnf1, letale a l'etat homozygote au jour 7,5 post-coitum, conduit a un retard de croissance et a la desorganisation des tissus. Or a ce stade, l'expression de vhnf1 est limitee au domaine extra-embryonnaire de l'endoderme visceral. Afin d'etudier l'effet possible de la mutation vhnf1 dans ce tissu, nous avons analyse la differenciation des cellules es vhnf1-/- et montre que la formation de l'endoderme visceral est alors bloquee. Nous avons ainsi identifie des genes cibles potentiels de vhnf1, egalement affectes in vivo. De plus, nous avons montre que l'endoderme visceral sauvage permet la survie du tissu embryonnaire mutant jusqu'au jour 9,5. L'ensemble de nos resultats montrent le role essentiel de vhnf1 pour la differenciation de l'endoderme visceral. L'analyse d'embryons chimeres contenant des cellules mutantes revele que vhnf1 est requis egalement au cours de la morphogenese de l'embryon. Pour rechercher des fonctions plus tardives de vhnf1, nous avons produit un allele conditionnel en utilisant le systeme cre/loxp. Cet allele, equivalent a un allele sauvage, est delete en presence de la recombinase cre. Les souris conditionnelles croisees avec une lignee exprimant la cre controlee par le promoteur de l'albumine permettent ainsi l'inactivation de vhnf1 dans le tissu hepatique. Les mutants presentent un retard de croissance et un ictere severe, suggerant un role de vhnf1 dans le foie.

Thesis (Ph. D.)--University of Kentucky, 2005.
Title from document title page (viewed on November 4, 2005). Document formatted into pages; contains viii, 188 p. : ill. Includes abstract and vita. Includes bibliographical references (p. 165-184).

My first project was to investigate the role of Hepatocyte Nuclear Factor 1 (HNF1) and Nuclear Factor I (NFI) on alpha-fetoprotein (AFP) promoter activity during liver development. AFP is highly expressed in the fetal liver, silenced at birth, and remains at very low levels in the adult liver. A GA substitution located at -119 of the human AFP promoter is associated with hereditary persistence of AFP (HPAFP) expression in the adult liver (Hum Molec Genet, 1993, 2:379). The -120 region harbors overlapping binding sites for HNF1 and NFI. While it has been shown that the GA substitution increases HNF1 binding, the role of NFI in AFP regulation has not been investigated. This overlapping HNF1/NFI site is conserved in other mammals, including mice. In this study, I used a combination of biochemical, tissue culture, and animal studies to explore further the role of this HNF1/NFI site in AFP regulation. Transient co-transfections in Hep3B hepatoma cells indicate that HNF1 activates while NFI represses the mouse AFP promoter. EMSAs indicate that HNF1 and NF1 compete for binding to this site. Transgenes regulated by the wild-type AFP promoter are expressed at low levels in the adult liver. Transgenes with a GGAA mutation (similar to the G-A human mutation) are more active in the adult liver. My data indicate that HNF1 and NFI compete for binding to the -120 region of the AFP promoter and this competition is involved in postnatal AFP repression. My second project was to study the control of Elongation of very long chain fatty acids like 3 (Elovl3) in the liver by Zinc fingers and homeoboxes 2 (Zhx2). The Zhx2 gene was originally characterized in our lab based on its ability to control the developmental repression of several hepatic genes, including AFP (PNAS, 102:401). Zhx2 is a member of a small family of proteins found only in vertebrates that also includes Zhx1 and Zhx3. These proteins all contain two zinc fingers and four homeodomains, suggesting that they function as regulators of gene expression. My study shows that Zhx2 regulates Elovl3 expression in female liver. Mouse strain-specific differences in adult liver Elovl3 mRNA levels and transgenic mouse data indicate that Zhx2 activates Elovl3 expression in the female adult liver. I also demonstrate that Elovl3 is repressed in the regenerating liver and that the level of Elovl3 repression is controlled by alpha-fetoprotein regulator 2 (Afr2). In addition, I show that Elovl3 expression is reduced in liver tumors, fibrotic livers and fatty livers, raising the possibility that Elovl3 can serve as a marker for HCC and liver damage.

Abstract Cytochrome P450 (CYP) enzymes are the major metabolizers of xenobiotics, e.g. drugs, and environmental toxins. Thus, changes in CYP expression have an important impact on drug metabolism and susceptibility to chemical toxicity. In the present study, the transcriptional mechanisms of both constitutive and inducible regulation of the Cyp2a5 gene in mouse liver were investigated. Mouse primary hepatocyte cultures were used as the main model system together with cell and molecular biology methods. The key activation regions of the Cyp2a5 5' promoter were determined using reporter gene assays. Two major transcription activation sites of the Cyp2a5 5' promoter, called the proximal and the distal, were found. Transcription factors hepatocyte nuclear factor-4 (HNF-4) and nuclear factor I were shown to bind to the proximal promoter. Aryl hydrocarbon receptor nuclear translocator (ARNT) and upstream stimulatory factor bound to a common palindromic E-box element in the distal promoter region. All three response elements were shown to be essential for constitutive expression of CYP2A5 in murine hepatocytes. ARNT appeared to control Cyp2a5 transcription without a heterodimerization partner suggesting active involvement of the ARNT homodimer in mammalian gene regulation. Aryl hydrocarbon receptor (AHR) ligands were shown to induce Cyp2a5 transcriptionally by an AHR-dependent mechanism, and established Cyp2a5 as a novel AHR-regulated gene. The AHR response element and the E-box, identified in these studies, were located near to each other and close to a separately defined nuclear factor (erythroid-derived 2)-like 2 binding site in the distal region of the Cyp2a5 promoter, suggesting cooperation between these elements. Peroxisome proliferator-activated receptor gamma coactivator-1α was shown to up-regulate Cyp2a5 transcription through coactivation of HNF-4α. This indicates that xenobiotic metabolism can be regulated by modification of co-activation. The present results show that CYP2A5 is regulated by several different cross-regulatory pathways. The regulatory mechanisms involved in the transcription of the Cyp2a5 gene may also control other CYP genes, especially the human ortholog CYP2A6, and may explain some of the individual variations in the metabolism of xenobiotics.

The overall objective of this research was to understand the mechanims by which growth hormone (GH) regulates insulin-like growth factor-I (IGF-I) gene expression in liver. Previous studies have suggested that GH regulation of IGF-I gene expression in liver is mediated by binding of the transcription factor signal transducer and activator of transcription (STAT) 5 to four binding sites located distantly from the IGF-I promoter. The first specific objective of this research was to determine whether additional STAT5 binding sites were involved in GH stimulation of IGF-I gene expression in liver. Sequence analysis of 170 kb of mouse genomic DNA revealed nineteen consensus STAT5 binding sequences corresponding to fourteen ~200 bp chromosomal regions that were conserved in the corresponding human DNA sequence. Eight of these chromosomal regions were able to mediate STAT5 activation of reporter gene expression in cotransfection experiments. Two of these chromosomal regions corresponded to those previously identified. Gel-shift assays indicated that the eight new STAT5 binding sites and three of the four previously identified STAT5 binding sites could bind GH-activated STAT5 from mouse liver. Together, these results suggest that GH stimulation of IGF-I gene transcription in the mouse liver may be mediated by at least eleven STAT5 binding sites located distantly from the IGF-I promoter. In a previous study, I found that liver expression of liver-enriched transcription factor hepatocyte nuclear factor 3γ (HNF-3γ) was increased by GH in cattle. Therefore, the second specific objective of this research was to determine how GH stimulates HNF-3γ gene expression and whether the increased HNF-3γ mediates GH stimulation of IGF-I gene expression in bovine liver. Sequence analysis of the bovine HNF-3γ promoter revealed the presence of two putative binding sites for STAT5. The proximal putative STAT5 binding site appears to be conserved in other mammals. Chromatin immunoprecipitation (ChIP) assays demonstrated that GH increased the binding of STAT5 to the HNF-3γ promoter in bovine liver and that this binding was associated with increased HNF-3γ expression. Gel-shift assays demonstrated that the proximal STAT5 binding site in the HNF-3γ promoter could bind GH-activated STAT5 from bovine liver. Cotransfection analyses showed that the proximal STAT5 binding site was necessary for the HNF-3γ promoter to be activated by GH. The promoter of the bovine IGF-I gene contains three putative HNF-3 binding sites that seem to be evolutionarily conserved. ChIP assays indicated that GH stimulated the binding of HNF-3γ to the IGF-I promoter in bovine liver. Gel-shift assays showed that one of the putative HNF-3 binding sites could bind HNF-3γ protein from bovine liver. Co-transfection analyses demonstrated that this HNF-3 binding site was necessary for HNF-3γ activation of reporter gene expression from the IGF-I promoter. In summary, the results of this dissertation research suggest that GH-activated STAT5 directly stimulates IGF-I gene transcription in liver by binding to at least eleven distantly located STAT5 binding sites in the IGF-I locus and indirectly stimulates IGF-I gene transcription by enhancing HNF-3γ gene expression in the liver.
Ph. D.

La surinfection par le Virus de l'Hépatite Delta (HDV) de patients chroniquement infectés par le virus de l'hépatite B (HBV) est la forme la plus agressive d'hépatite virale chronique avec une progression plus rapide vers la cirrhose ainsi qu’un risque accru d'insuffisance hépatique et de carcinome hépatocellulaire. Environs 15 à 20 millions de personnes sont co-infectés par ces deux virus ; il s’agit d’une des coinfections la plus répandue et difficile à combattre au monde. Les traitements disponibles pour HBV ne sont pas efficaces contre HDV. L'unique option thérapeutique pour les patients co-infectés par HBV et HDV repose sur l'utilisation d'interféron alpha pégylé avec de très nombreux effets secondaires et une efficacité très limitée. Très peu de molécules sont actuellement en R&D pour HDV et à priori aucune ne cible directement la réplication du virus. Il est donc urgent de développer de nouvelles stratégies antivirales efficaces pour traiter les patients co-infectés. Lors d’une infection virale classique, plusieurs cytokines sont produites. Plusieurs études ont montré une activation de la voie de signalisation de l’interféron par HDV in vitro et in vivo. Toutefois il n’y a aucune donnée sur l’activation de la voie « Nuclear factor κ B » (NFκB) par HDV, et même une absence d’activation de celui-ci a déjà été proposée par notre laboratoire. Notre objectif était de tester l'effet sur HDV d’immunomodulateurs activant cette voie. Après criblage de différents inducteurs des voies canoniques et non-canoniques de NFκB nous avons identifié un agoniste du récepteur "toll like" 1/2 (Pam3CSK4) et un agoniste du récepteur à la lymphotoxine β (BS1) diminuant les ARN et protéines d’HDV. Des études approfondies ont montré un effet anti-HDV dose-dépendant et stable malgré une augmentation de la quantité de particules virales utilisées pour infecter les cellules. Des expériences d'arrêt de traitement ont révélé un effet antiviral persistant et altérant l’infectivité des nouvelles particules d’HDV. Ceci suggère un effet irréversible sur la matrice de transcription et réplication d’HDV. Des analyses transcriptomiques sur des cellules infectées par HBV et HDV et traitées par Pam3CSK4, BS1 ont confirmé l’induction de cette voie NFκB et révélé l’activation de nombreux gènes impliqués dans une réponse inflammatoire. Une étude par Gene Ontology des fonctions moléculaires et processus biologiques significativement modulés lors des traitements par Pam3CSK4 et BS1 a permis d’identifier une liste de potentiels effecteurs ciblant les ARN et responsables du phénotype anti-HDV. L’identification mécanisme sous-jacent (i.e. dégradation des ARN ou régulation transcriptionnelle négative) reste toutefois à réaliser. Ce projet a montré que l’induction de la voie NFκB est une potentielle cible thérapeutique pour inhiber l’infection à HDV. Notre recherche du ou des effecteurs de l’effet anti-HDV nous permettra d’identifier à terme un nouveau facteur de restriction. Ainsi, nos efforts de recherche devraient ouvrir des pistes pour mener au développement de nouvelles stratégies antivirales pour éliminer HDV
Super-infection by Hepatitis Delta Virus (HDV) of chronically Hepatitis B Virus (HBV) infected patients leads to the most aggressive forms of chronic viral hepatitis, with a faster progression towards fibrosis/cirrhosis and an increased risk of liver failure and hepatocellular carcinoma. Around 15-20 millions of people are co-infected with both viruses, which ranks this co-infection as one of the most prevalent and clinically challenging of the world. HDV infection is not susceptible to available direct anti-HBV. The only therapeutic option for HBV/HDV co-infected patients is the pegylated-interferon-α, a drug which has many side effects and suboptimal responses. Few molecules that target HDV are currently in development but none of them is affecting the replicative step of this virus. There is an urgent need to develop efficient treatment strategies for patients. An infection produced many cytokines. Different studies showed an activation of the interferon pathway during HDV infection in vitro and in vivo. Nevertheless, there is no current data available concerning an activation of the Nuclear factor κ B » (NFκB) pathway by HDV, and more than that our laboratory has suggested a lack of activation. Our purpose was to test the effect on HDV of various immunomodulatory drugs activating this pathway. After screening of various inducers of the canonical and non-canonical NFκB pathways we identified an agonist of the “toll like” receptor 1/2 (Pam3CSK4) and an agonist of the lymphotoxin β receptor (BS1) decreasing the HDV RNA and proteins. More extensive studies have shown a dose-dependent and stable anti-HDV effect despite an increase of the number of viral particles used to infect cells. Rebound experiments have shown a persistent antiviral effect and an alteration of the HDV particles infectivity. Its suggests an irreversible effect on HDV replication and transcription template. Transcriptomic analysis on HBV/HDV infected and treated with Pam3CSK4 and BS1 cells has confirmed the induction of the NFκB pathway and revealed the activation of numerous genes involved in an inflammatory response. A Gene Ontology study of molecular functions and cellular processes significantly modulated by Pam3CSK4 or BS1 treatments had enable us to identify a list of potential effectors targeting the RNA and responsible of the anti-HDV phenotype. The underlying mechanism (i.e. RNA degradation or negative regulation of transcription) remains to be deciphered. Our project has shown that the NFκB induction is a potential target to inhibit the HDV infection. Research of effector(s) will probably enable us to identify a new restriction factor. Thus, our research efforts should pave the way for the development of novel efficient antiviral strategies to eliminate HDV

A nefropatia diabética (ND) decorre da hiperglicemia crônica, de fatores de risco como a hipertensão arterial e a dislipidemia e de uma susceptibilidade genética já evidenciada em inúmeros estudos clínicos. Uma das características histológicas da ND é o acúmulo de proteínas de matriz extracelular no mesângio, para o qual contribuem várias vias bioquímicas. O GLUT-1, codificado pelo gene SLC2A1, é o principal transportador de glucose da célula mesangial e sua expressão está aumentada no glomérulo de animais diabéticos, o que constitui uma alça de feedback positivo pela qual a glicose extracelular aumentada estimula ainda mais sua própria captação, piorando a lesão mesangial. O GLUT-2, codificado pelo gene SLC2A2, é expresso nas células tubulares e nos podócitos e sua expressão também está aumentada na ND. A expressão deste transportador de glicose é regulada pelo fator de transcrição HNF-1. Participa, ainda, da lesão renal induzida pela hiperglicemia o fator de crescimento transformante - (TGF-), que exerce vários efeitos deletérios, tais como diminuir a atividade de metaloproteinases de matriz e promover fibrose renal. Esse fator de crescimento determina a ativação transcricional de genes-alvo, mas necessita de outros ativadores e co-ativadores da transcrição, tais como a proteína SMIF, codificada pelo gene DCP1A. Tendo em vista a participação das proteínas mencionadas acima na patogênese da ND, o presente estudo teve o objetivo de avaliar a associação de polimorfismos de um único nucleotídeo (SNPs) nos genes SLC2A1, SLC2A2, HNF1A, TGFB1 e DCP1A com a doença renal em portadores de diabetes mellitus tipo 1 (DM1). Um total de 449 pacientes (56,4% do sexo feminino, idade média de 36,0±11,0 anos) com mais de 10 anos de doença foram incluídos e classificados de acordo com o estágio de ND: (1) Ausência de ND: excreção urinária de albumina (EUA) normal (< 30 mg/24h ou < 20 g/min) e creatinina plasmática < 1,7 mg/dL sem tratamento anti-hipertensivo; (2) ND incipiente: microalbuminúria (EUA de 30 299 mg/24h ou 20 199 g/min) e creatinina plasmática < 1,7 mg/dL sem tratamento anti-hipertensivo e (3) ND Franca: macroalbuminúria (EUA > 300 mg/24h ou > 200 g/min) ou proteinúria ou tratamento para reposição renal. Também foram avaliadas as associações dos SNPs com o ritmo de filtração glomerular estimado (RFGe). Os SNPs foram genotipados pela metodologia de reação em cadeia da polimerase em tempo real, com o uso de sondas fluorescentes. As associações dos SNPs com a ND foram avaliadas por análise de regressão logística e os odds ratios (OR) e respectivos intervalos de confiança (IC) de 95% foram calculados após ajuste para possíveis confundidores, que foram incluídos como co-variáveis no modelo de regressão. Valores de P < 0.05 (bicaudal) foram considerados estatisticamente significantes. As seguintes associações foram observadas: (1) gene SLC2A1: genótipos CT+TT do SNP rs841848 conferiram risco para a ND incipiente na população global (OR 1,88; CI95% 1,06-3,34; P= 0,03) e nos pacientes do sexo masculino (OR 2,67; CI95% 1,13-6,35; P=0,0247) e para a ND franca (OR 2,70; CI95% 1,18-6,31; e P= 0,0197) apenas nos pacientes do sexo masculino; genótipos GA+AA do SNP rs1385129 conferiram risco para a ND franca na população do sexo masculino (OR 3,09; CI95% 1,34-7,25; P=0,0085); genótipos AT + TT do SNP rs3820589, conferiram proteção contra a ND incipiente na população global (OR 0,36; CI95% 0,16-0,78; P=0,0132) e na população do sexo feminino (OR 0,14; CI95% 0,02-0,52; P=0,0122). (2) gene SLC2A2: genótipos GA+GG do SNP rs5396 conferiram proteção contra ND franca nos pacientes do sexo masculino (OR 0,29; CI95% 0,12-0,69; P=0,0052); os genótipos AG+GG do SNP rs6800180 conferiram proteção contra a ND franca nos pacientes do sexo masculino (OR 0,16; CI95% 0,14-0,90; P=0,0324). (3) gene HNF1A: genótipos AC + CC do SNP rs1169288 conferiram risco para ND franca na população global (OR 2,23; CI95% 1,16-4,38; P=0,0175); genótipos CG+GG do SNP rs1169289 conferiram risco para ND franca na população global (OR 3,43; CI95% 1,61-7,73; P=0,002); (4) Gene TGFB1: genótipos CT + TT do SNP 1800468 conferiram risco para ND incipiente na população total (OR 2,99; CI95% 1,26-7,02; P 0,0116) e o alelo polimórfico T do SNP rs1800469 conferiu risco para um menor RFGe (p=0,0271). (5) gene DCP1A: o alelo polimórfico A do SNP rs11925433 também se associou com um menor RFGe (p=0,0075). Em conclusão, SNPs em genes que codificam as proteínas envolvidas na patogênese da ND GLUT-1, GLUT-2, HNF-1, TGF- e SMIF conferem susceptibilidade para essa complicação crônica nos portadores de DM1 avaliados no presente estudo
Diabetic nephropathy (DN) results from chronic hyperglycemia, risk factors such as hypertension and dyslipidemia as well as from genetic susceptibility, already demonstrated in numerous clinical studies. A histological feature of DN is the accumulation of extracellular matrix proteins in the mesangium after activation of multiple biochemical pathways. GLUT-1, encoded by gene SLC2A1, is the major glucose transporter in mesangial cell and its expression is increased in the glomeruli of diabetic animals, comprising a positive feedback loop whereby high extracellular glucose stimulates its own uptake and worsening mesangial injury. GLUT-2, encoded by SLC2A2 gene, is expressed in podocytes and tubular cells and its expression is also increased in DN. The expression of this glucose transporter is regulated by the transcription factor HNF-1. Transforming growth factor - (TGF-) also participates in renal injury induced by hyperglycemia, exerting several deleterious effects, such as to decrease the activity of matrix metalloproteinases and to promote renal fibrosis. This growth factor determines the transcriptional activation of target genes, but needs other activators and co-activators, such as the protein named SMIF, encoded by the gene DCP1A. Given the involvement of the aforementioned proteins in the pathogenesis of DN, the present study aimed to evaluate the association of single nucleotide polymorphisms (SNPs) in the genes SLC2A1, SLC2A2, HNF1A, TGFB1 e DCP1A with renal disease in patients with type 1 diabetes mellitus (T1DM). A total of 449 patients (56.4% female, mean age 36.0±11.0 years) with disease duration > 10 years were included and grouped according to DN stages: (1) absence of DN: normal urinary albumin excretion (UAE) (< 30 mg/24h or < 20 g/min) and plasmatic creatinine < 1.7 mg/dL without antihypertensive treatment; (2) incipient DN: microalbuminuria (UAE 30 299 mg/24h or 20 199 g/min) and plasmatic creatinine < 1.7 mg/dL without antihypertensive treatment and (3) overt DN: macroalbuminúria (UAE > 300 mg/24h or > 200 g/min) or proteinuria or renal replacement therapy. Associations of SNPs with estimated glomerular filtration rate (eGFR) were also evaluated. All SNPs were genotyped by real time polymerase chain reaction using fluorescent-labelled probes. Associations of the SNPs with DN were assessed by logistic regression analyses and odds ratios (OR) were calculated after adjustments for possible confounders included as covariables in the regressive model. P values <0.05 (two-tails) were considered significant. The following associations were observed: (1) SLC2A1: genotypes CT+TT from rs841848 conferred risk to incipient DN in the overall population (OR 1.88; 95%IC 1.06-3.34; P= 0.03) and in the male patients (OR 2.67; CI95% 1.13-6.35; P=0.0247) and to overt DN (OR 2.70; CI95% 1.18-6.31; e P= 0.0197) only in the male patients; genotypes GA+AA from rs1385129 conferred risk to overt DN in the male population (OR 3.09; CI95% 1.34-7.25; P=0.0085); genotypes AT + TT from rs3820589 conferred protection against incipient DN in the overall population (OR 0.36; CI95% 0.16-0.78; P=0.0132) and in the female population (OR 0.14; CI95% 0.02-0.52; P=0.0122). (2) SLC2A2: genotypes GA+GG from rs5396 conferred protection against overt DN in the male patients (OR 0.29; CI95% 0.12-0.69; P=0.0052); genotypes AG+GG from rs6800180 conferred protection against overt DN in the male patients (OR 0.16; CI95% 0.14-0.90; P=0.0324). (3) HNF1A: genotypes AC + CC from rs1169288 conferred risk to overt DN in the overall population (OR 2.23; CI95% 1.16-4.38; P=0.0175); genotypes CG+GG from rs1169289 conferred risk to overt DN in the overall population (OR 3.43; CI95% 1.61-7.73; P=0.002); (4) TGFB1: genotypes CT + TT from 1800468 conferred risk to incipient DN in the overall population (OR 2.99; CI95% 1.26-7.02; P=0.0116) and the polymorphic allele T from SNP rs1800469 conferred risk to a lower eGFR (p=0.0271). (5) DCP1A: the polymorphic allele A from SNP rs11925433 was also associated with a lower eGFR (p=0.0075). In conclusion, SNPs in the genes encoding proteins GLUT-1, GLUT-2, HNF-1, TGF- e SMIF, all involved in the pathogenesis of DN, conferred susceptibility to this chronic complication in the T1DM patients evaluated in the present study

碩士
大葉大學
分子生物科技學系碩士班
100
The hepatocyte nuclear factors-3 (HNF-3) family members HNF-3α, HNF-3β and HNF-3γ are hepatocyte-enriched transcription factors, they play important roles in controlling development, differentiation, metabolism and organogenesis. In our previous study, the expression of insulin-like growth factor-I/II (IGF-I/II), HNF-1α, -1β and -3β were detected in the liver and gonads of tilapia, and expreession level of HNF-3β was higher than others and it could be regulated by 17β-estradiol. In this study, four fragments (0.5, 1.0, 1.5 and 2.0 kb) of tilapia HNF-3β promoter were constructed with green fluorescent protein (GFP) gene for biological activity assay by performing transfection into tilapia ovarian cell line (TO-2) and human hepatoma cell line (Hep3B) for western blot analysis and luciferase assay or microinjection into zebrafish eggs and assay. The GFP was mainly expressed in yolk and somites 24 h after injection, in notochord and floor plate 96 h after injection. The 0.5 kb fragment was expressed in notochord, yolk, eye and head, the expression rates were 18.3%, 1.3%, 35.6% and 26.0%, respectively; the 1.0 kb fragment was expressed in notochord, yolk, and head, the expression rates were 44.4%, 44.4% and 2.0%, respectively; the 1.5 kb fragment was expressed in notochord and yolk, the expression rates were 33.7% and 50.5% respectively, and the 2.0 kb fragment was expressed in notochord, yolk and head, the expression rates were 61.2%, 26.1% and 2.7% respectively. The results were similar to our previous studies. The supplement of add 17β-estradiol enhanced western blot analysis of eGFP expression and luciferase assay in TO-2 and Hep3B cells. Based on the present results, hypothesizing that estrogen response element (ERE) in tilapia HNF-3β promoter could promote the expression of HNF-3β in the gonads of tilapia through the action of steroids.

碩士
大葉大學
分子生物科技學系碩士班
94
Hepatocyte nuclear factor (HNF)-4 is a liver-enriched transcription factor and belongs to the highly conserved member of the nuclear receptor superfamily. HNF-4 together with other factors play a key role in the tissue specific expression of a large number of genes involved in lipid and glucose metabolism. However, its function in fish is still poorly understood. In our previous study, RNAs and proteins of HNF-1, HNF-1, and HNF-3are detected in the liver, ovary, and testis of tilapia (Oreochromis mossambicus). And the expression of HNF-3 in the gonads could be induced by 17-estradiol and hydrocortisone in vitro. Besides, HNF-4 binding site on the promoter region of HNF-3 gene has also been found. However, there are only limited literatures dealing with HNFs in bony fish. The roles and their relation of HNFs in the physiology of fish remain to be explored. Here we report on the first cloning of full-length cDNA and protein localization of HNF-4from a tilapia (O. mossambicus). A total of 2,031 bp of tilapia HNF-4fhas been cloned and its deduced amino acid sequence of the coding region (340 amino acids) of tilapia HNF-4 has a 89% identity with that of zebrafish, over 84% with mammals (human, bovine, rat, and mouse), 81% with chicken, and 79% with Xenopus. RT-PCR detected HNF-4in liver, kidney, intestine and stomach, and the identity of the PCR fragments was confirmed by sequencing analysis and PCR hybridization. Its relative expression ratio was higher in the liver than in intestine and kidney, and lowest expression ratio was observed in stomach. The same result happened in both genders. Western blotting and immunohistochemical localization also detected HNF-4 protein in the tissues mentioned before. Expression of HNF-4 in the tilapia suggests that multi-HNFs may form a cascade to regulate physiology in the bony fish.

碩士
大葉大學
分子生物科技學系碩士班
94
The hepatocyte nuclear factors-3 (HNF-3) family members HNF-3, HNF-3 and HNF-3 are hepatocyte-enriched transcription factors, they play important roles in controlling development, cell differentiation, organogenesis and in the regulation of metabolism. In our previous study, the expression of insulin-like growth factor-I (IGF-I), IGF-II, HNF-1a, -1b, and -3bwere detected in the liver and gonads of tilapia, and the expression level of HNF-3b was higher than others and it could be regulated by 17b-estradiol. Based on the results, the aim of this study was to clone and analyze the tilapia HNF-3b promoter, and to provide a foundation for further studies on the relation between HNFs and gonad/gamete development in tilapia, and on biomedical and developmental studies in the future. The 5’-flanking region of tilapia HNF-3b promoter containing 2364 bp was cloned and sequenced, and on which 373 putative transcription factor binding sites for HNFs (HNF-1, -3, -4 and -6), CCAAT/enhancer binding protein(C/EBP), cAMP responsive element binding (CREB), sterol regulatory element, steroid hormones receptor binding site, signal transducer and activator of transcription (STAT), GATA-binding factor and other factors were found. These factors are important for embryo development and gonadal function. Four deletion fragments (2, 1,5, 1 and 0.5 kb) of tilapia HNF-3b promoter were constructed with green fluorescent protein (GFP) gene for biological activity assay by cell line (TO-2 and Hep3B) transfection or microinjection into zebrafish eggs. The GFP was mainly expressed in yolk and somite 10 h after injection, and in somite, notochord and floor plate 72 h after injection, but that of 0.5 kb fragment was mainly expressed in eye and head. And the expression rates of 2 kb and 1.5 kb groups seemed better than those of 1 kb and 0.5 kb (p<0.05). Western blotting analysis of GFP expression in TO-2 and Hep3B cells after transfected with different of tilapia HNF-3b promoter proximal regions showed that 1 kb proximal region had a lower expression level than others (2, 1.5, 0.5 kb), this could result from the repressor binding site on the 1 kb region. The effect of steroid hormones (17b-estradiol and progesterone) on the expression of GFP after injection or transfection of the 4 fragments into zebrafish eggs or cells showed the level of GFP expression of these groups increased significantly. Based on the present results, hypothesizing that estrogen response element (ERE) and progesterone receptor region in tilapia HNF-3b promoter could promote the expression of HNF-3b in the gonads of tilapia through the action of steroids, however, the actual mechanism needs further study.

碩士
慈濟大學
人類遺傳研究所
94
It has been reported that in mouse hepatogenesis HNF4α is essential for the expression of E-cadherin in the organ formation. Thus if HNF4α is down-regulated the consequence would be the lost of polarity and increase in cell mobility and invasiveness of hepatocyte. Whether HNF4α can regulate metastasis of human hepatoma cell has not been determined yet. It has been reported that in most hepatocellular carcinoma (HCC) and colorectal carcinoma (CRC) SMYD3, a histone methyaltranferase, was over expressed. SMYD3 can down-regulate HNF4α. The decreased HNF4αexpression may alter carcinogenesis of HCC through decrease expression of E-cadherin, thus increasing metastatic potential of HCC. The aim of this study is to characterize several transcription factors (TFs) that are important in regulating HNF4α expression in the HCC. Here, we examined whether deletions of SMYD3 binding motif in the promoter region of HNF4αcould increase the metastasis potential of human hepatoma cell lines. The results of functional assay of SMYD3 on HNF4α regulatory sequence could not confirm the previous study. Therefore SMYD3 appeared not to be a critical factor for HNF4α expression in our study. More detailed serial deletions of HNF4αpromoter allowed us to identify three putative positive regulatory elements and one putative negative regulatory element. We want to identify which transcription factor acts as a positive modulator for the HNF4αexpression. There are two putative SP1 binding motifs in the HNF4α promoter region at (-243/154) and (-192/154), respectively. The results of functional assays indicated that SP1 binding motif in the (-192/154) promoter region may not be functional. The functional SP1 binding motif is located in the position of -243. Our results also showed that Cdx2A is a negative regulator for HNF4α, thus it may be a potential suppressor for E-cadherin expression, it may also contribute to HCC metastasis.

碩士
大葉大學
分子生物科技學系碩士班
93
Tilapia is the most general aguacultivating fish in the fresh water and has characteristics of fast growing and well disease resistance. Thus tilapia is an important animal model for relative aquaculture researches. Hepatocyte nuclear factors (HNFs) are liver-enriched transcription factors, which can activate the expressions of tissue-specific, growth and development related genes. The expression of HNFs was detected in the gonads of tilapia previously, and which could be regulated by steroid hormones. Therefore, the reproductive system in tilapia could exist a different endocrine pathway other than the traditional one. To identify this hypothesis and to find out the optimal steroid, concentration and culture period, gonads from tilapia were cultured in vitro in a time course manner (0, 6, 12, 18, 24, 32 and 36 hrs) with different kinds (-estradiol and hydrocortisone) or concentrations (0, 0.1, 1, 10, 100 and 1000 nM) of steroid hormones, which were all performed after a 6-hr preculture without hormone supplement. The total RNA isolated from previous different groups was analyzed by RT-PCR and semi-quantified with an internal control of -actin. Though the expression of HNF-1 and -1 could not be induced by -estradiol and hydrocortisone, the expression of HNF-3 could be induced by these two steroids, and showed a dose-dependent manner. Beta-estradiol exerted a better induction result, and the optimal concentration and incubation period were 10 nM and 12 hours, respectively. The detection of HNF proteins in 1-, 2-week and one month old juvenile tilapia by immunohistochemistry showed that HNFs were found mainly in liver and epithelial cells of the digestive organs. According to the above results, the expression of HNF-3β in the gonads of tilapia can be regulated by the steroid hormone and could be involved in the development and gametogenesis of gonads in tilapia. Whether the growth and development of juvenile tilapia is affected by HNFs still needs further investigation.

碩士
國立海洋大學
水產生物技術研究所
87
The liver-enriched hepatocyte nuclear factor 1 (HNF1) is an important transcription factor for IGF-I in adult liver. Only one conserved HNF1 binding site was found near the transcription initiation site of tilapia IGF-I gene. To study the role of HNF1 in the signaling pathway of GH-IGF-I axis, degenerated primers designed from known sequences of HNF1 were used to amplify HNF1 partial sequence from tilapia liver mRNA by one step RT-PCR. After gel electrophoresis, RT-PCR product revealed two major bands in size of 400 and 500 bps. Sequence comparisons of these 400 bps and 500 bps clones showed highly homologous to HNF1-alpha and HNF1-beta, respectively. Furthermore, comparison of POU and homeo-domain within HNF1-alpha (400 bps) clones, we found two types of HNF1-alpha with four amino acids variation in the C-terminal of homeo-domain. One is highly homologous to salmon HNF1 and the other is similar to mammalian HNF1-alpha in amino acid usage. This is the first evidence to show HNF1-beta existing in fish and two different types of HNF1-alpha observed in vertebrates. Both HNF1-alpha and HNF1-betacDNA clones with 5’-truncation were obtained from tilapia liver cDNA library, and 5’RACE then was applied to amplify the 5’ truncated region of HNF1-alpha cDNA. Full length HNF1-alpha cDNA was re-amplified by using 5’ and 3’primer located at the terminal of its ORF. Two HNF1-alpha full-length cDNAs in length of 1680 and 1760 bps were obtained and designated as tiHNF1-alpha-A and tiHNF1-alpha-B, respectively. Compared with other known HNF1s, the dimerization domain, POU domain and homeo-domain of tiHNF1-alpha-A consisting of 559 amino acids were highly conserved. tiHNF1-alpha-B had a 80bp insertion containing an in-frame stop codon, and this insert replaced the last 107 amino acids of the tiHNF1-alpha-A protein with 6 amino acids resulting in a 458 amino acids protein. In vitro transcription and translation was carried out to verify the different protein size of these two clones. Sequence comparisons of tiHNF1-alpha-A with human HNF1-A and B suggest that close related mechanism of alternative splicing results in the occurrence of tiHNF1-alpha-B. Different sizes of PCR amplified tilapia IGF-I promoters were constructed into luciferase reporter vector for further studies on the binding and transactivation ability of tilapia HNF1s.