Hypoxia inducible factor-1α (HIF-1α) plays a critical role in the oxygen homeostasis of all metazoans. HIF-1α is a master transcriptional regulator which coordinates the adaptive response to low oxygen tension. Through activation of a plethora of downstream target genes, HIF-1α facilitates oxygenation by promoting angiogenesis and blood vessel dilation, in addition to modulating metabolic pathways to inhibit oxidative phosphorylation and promote glycolytic energy production. Given the critical roles of hypoxia, insufficient blood supply and perturbed energetics in the pathogenesis of cardiovascular disorders, notably ischaemic heart disease, therapeutic modulation of HIF-1α is of significant clinical interest. Previous studies have demonstrated an acute cardioprotective role for both endogenous and supraphysiological HIF-1α signalling in the context of myocardial ischaemia. In contrast, chronic supraphysiological HIF-1α activation in the unstressed heart has been shown to induce cardiac dysfunction. To address the effect of chronic endogenous HIF-1α activation post-myocardial infarction (MI), the present work employed a murine coronary artery ligation (CAL) model in conjunction with temporally-inducible, cardiac-specific deletion of Hif-1α. While CAL surgery successfully modelled myocardial infarction – eliciting substantial adverse cardiac remodelling and contractile dysfunction – there was no evidence of chronic HIF-1α activation by CAL in HIF knockout or control left ventricular samples. In keeping with this, chronic ablation of Hif-1α (from 2 weeks post-CAL) had no discernible additional effect upon cardiac function. Overall, these findings do not support a potential therapeutic role for inhibition of HIF-1α signalling in the chronic phase post-MI. The fundamental tricarboxylic acid (TCA) cycle enzyme fumarate hydratase (FH) converts fumarate to malate. FH deficiency is associated with smooth muscle and kidney tumours which exhibit normoxic HIF signalling due to fumarate accumulation. To investigate the potential for fumarate accumulation to elicit protective HIF signalling, a cardiac-specific Fh1 null mouse was developed through Cre-loxP recombination. Strikingly, despite interruption of the TCA cycle in a highly metabolically demanding organ, cardiac Fh1 null mice were viable, fertile and survived into adulthood, demonstrating the remarkable metabolic plasticity of the heart. However, by 3-4 months Fh1 null mice develop a lethal cardiomyopathy characterised by cardiac hypertrophy, ventricular dilatation and contractile dysfunction. Despite lack of a pseudohypoxic response, Fh1 null hearts did exhibit another phenomenon observed in FH-deficient cancers and also attributed to fumarate accumulation – activation of the nuclear factor (erythroid-derived 2)-like 2 (NRF2) antioxidant pathway. Heterozygous, but not homozygous, somatic deletion of Nrf2 extended the life expectancy of cardiac Fh1 null mice. Exploration of redox status revealed a more reductive environment in Fh1 null hearts than controls. As a corollary, inhibition of the rate limiting enzyme of the pentose phosphate pathway – a major source of cellular reducing equivalents – with dehydroepiandrosterone conferred striking amelioration of the Fh1 null cardiomyopathy, suggesting a possible pathogenic role for reductive stress. While loss of mitochondrial Fh1 activity and subsequent TCA cycle dysfunction likely contribute to the Fh1 null phenotype, the importance of cytosolic FH was unclear. To clarify this, FH was expressed specifically in the cytosol in vivo. This was sufficient to substantially rescue the Fh1 null cardiomyopathy, supporting a role for cytosolic FH disruption in its pathogenesis. Taken together, these findings highlight the potential for reductive stress to contribute to cardiac dysfunction and suggest a function for cytosolic FH in cardiac metabolic homeostasis.

Diss. (sammanfattning) Göteborg : Göteborgs universitet, 2009. / Härtill 5 uppsatser.

Heritable ossification defects of the skull vault often present as enlarged parietal foramina (PFM), bilateral oval openings of the posterior parietal bones. Isolated PFM may originate from wider defects in infancy and usually show an autosomal dominant mode of transmission, offering unexplored genetic insights into the molecular pathways of calvarial development. Haploinsufficiency of the homeobox gene MSX2, located at 5q34-q35, underlies a fraction of PFM families but the locus can be excluded in others, indicating heterogeneity. The proximal 11p deletion syndrome (P11pDS), characterised by multiple exostoses (due to haploinsufficiency of EXT2), occasional mental retardation, and PFM, pointed to a second locus at 11p11-p12. The human orthologue of the mouse paired-like homeobox gene Alx4 was identified adjacent to EXT2. ALX4 was structurally characterised and heterozygous loss-of-function mutations were detected in association with skull vault defects in twenty-nine individuals from six families, including a new case of P11pDS. The calvarial phenotype of ALX4 mutations was almost indistinguishable from the MSX2-caused defects and ranged from a midline gap to non-penetrance, nevertheless typified by classical PFM; abnormal morphology of the dural septa was also observed. The mutation spectrum and the subtle genotype-phenotype correlations suggested haploinsufficiency as the predominant pathophysiological mechanism. Interestingly, Alx4^-/+ mice manifest polydactyly but no skull defects, illustrating species-specific dosage sensitivity. Two new MSX2 mutations were also ascertained, one of which segregated with PFM and clavicular hypoplasia. The potential contribution of ALX4 and MSX2 to premature fusion of the cranial sutures - craniosynostosis - was investigated, but no unequivocally pathogenic variants were found. To elucidate the functions of Alx4 and Msx2 in skull development, spatial expression analysis was performed in mouse embryos between embryonic days E12.5-E17. Transcripts of both Alx4 and Msx2 were seen in the early calvarial skeletogenic condensations and in later stages their expression displayed a more restricted pattern, overlapping minimally with the domains of mature bone. By assessing expression in embryonic heads of reciprocal knockout mice, activation of Alx4 was found to be independent of functional Msx2 and vice versa. Analysis of compound mutants demonstrated that the two loci exert roughly additive effects on the skull vault while protein interaction assays did not indicate any physiological interaction between Alx4 and Msx2. Hence, Alx4 and Msx2 appear to regulate proliferation, differentiation, or survival of osteoblast precursors and pre-osteoblasts through parallel pathways.

Orientadores: Mônica Barbosa de Melo, José Paulo Cabral de Vasconcellos / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-25T15:53:03Z (GMT). No. of bitstreams: 1 Oliveira_MarianaBorges_M.pdf: 6901699 bytes, checksum: b84b4ac92949fdb082625b01ad6c66a1 (MD5) Previous issue date: 2014 / Resumo: O glaucoma engloba um grupo de doenças que têm como característica comum a atrofia progressiva do disco óptico com alteração correspondente de campo visual, decorrente da perda de células ganglionares da retina. O aumento da pressão intraocular (PIO) é o principal fator de risco para o desenvolvimento do glaucoma, mas tem sido sugerida a existência de outros aspectos relevantes, tais como alterações no metabolismo do óxido nítrico, na regulação do fluxo vascular, sinais de estresse oxidativo e alterações no sistema imunológico. O glaucoma primário de ângulo aberto (GPAA), a forma mais prevalente entre os glaucomas, está associado a uma série de fatores de risco para sua instalação e desenvolvimento, além da PIO. Os demais fatores de risco são: idade (relação direta), raça (mais frequente e grave em indivíduos da raça negra), miopia e história familiar de glaucoma. A maioria dos casos de GPAA apresenta um padrão complexo de hereditariedade, em que variantes de suscetibilidade contribuem para a gravidade da doença. O objetivo deste estudo do tipo caso-controle foi avaliar o papel de polimorfismos nos genes da interleucina alfa (IL1A), interleucina beta (IL1B) e fator de necrose tumoral alfa (TNFA) em relação à suscetibilidade ao GPAA. Após avaliação oftalmológica, foram recrutados 214 indivíduos não relacionados portadores de GPAA. A análise molecular foi realizada por meio de técnicas de PCR e sequenciamento direto. Neste estudo observou-se que o alelo C do polimorfismo -31 C/T, presente na região promotora do gene IL1B, confere risco para o glaucoma (p=0,002). Este alelo está contido em um "TATA" box, influenciando na transcrição deste gene. Existe um desequilíbrio de ligação entre os SNPs -31 C/T e -511 T/C do gene IL1B e este último também está associado a um risco aumentado para o glaucoma (p=0,006). Com relação aos demais SNPs estudados (+3954 C/T do gene IL1B, -889 C/T e +4845 G/T do gene IL1A e -238 G/A e -308 G/A do gene TNFA), não houve associação com o glaucoma. Este estudo sugere que há relação das variantes "C" do SNP -31 C/T e "T" do SNP -511 T/C do gene IL1B com a etiologia do GPAA em uma amostra da população brasileira / Abstract: Glaucoma comprises a group of diseases characterized by progressive atrophy of the optic disc and corresponding visual field loss, as consequence of retinal ganglion cells death. Augmented intraocular pressure (IOP) is the main risk factor for glaucoma development, but it has been suggested that there are other relevant aspects that may lead to neuronal and trabecular meshwork (TM) damage and contribute to the development of glaucoma, including alterations in nitric oxide metabolism, oxidative stress and vascular and immune system dysregulations. Primary open angle glaucoma (POAG), the most prevalent form of glaucoma, is associated with several risk factors to its initiation and progression besides IOP. The additional risk factors are: age (direct correlation), race (more frequent in black subjects), myopia and family history of glaucoma. The majority of POAG cases presents a complex pattern of heritability, in which susceptibility variants contribute to disease severity. The aim of this case-control study was to evaluate the role of single nucleotide polymorphisms (SNPs) in the interleukin alpha (IL1A), interleukin beta (IL1B) and tumor necrosis factor alpha (TNFA) genes in relation to POAG susceptibility. After ophthalmologic evaluation, 214 unrelated subjects with POAG were recruited. The molecular analysis was performed by PCR and direct sequencing techniques. In this study it was observed that the C allele of the -31 C/T promoter polymorphism of the IL1B gene confers risk for glaucoma (p=0.002). This allele disrupts a "TATA" box, influencing the transcription of the IL1B gene. There is linkage disequilibrium between the -31 C/T e -511 T/C SNPs, and the latter is also associated with an increased risk for glaucoma (p=0.006). Regarding the others studied SNPs (+3954 C/T of the IL1B gene, -889 C/T and +4845 G/T of the IL1A gene and -238 G/A and -308 G/A of the TNFA gene), there is no association with glaucoma. This study suggests the relationship of the "C" variant of -31C/T and "T" variant of -511T/C of IL1B gene with POAG etiology in a sample of the Brazilian population / Mestrado / Oftalmologia / Mestra em Ciências Médicas

No description available.

Genetic and functional studies have demonstrated that KIR gene polymorphism, including different haplotypes, allelic polymorphisms and different expression levels of KIRs, may all play a part in the association with HIV-1 infection outcome. Currently, there are very few studies focusing on the association between KIR and HIV in the Chinese population. In this project, we started to look at the polymorphism of KIRs in a unique chronic HIV-1 infected cohort (SM cohort), evaluating the impact of KIR and KIR-HLA interactions in terms of HIV-1 infection progression. The SM cohort is unique because the major factors such as viral strain, transmission route and timing of infection, which could affect the natural history of HIV-1, have been narrowly controlled. Through comparison with a healthy control population, some genetic associations were identified. The frequency of KIR2DL3 was lower in the “slow progressors” group; the compound genotype of KIR3DS1+ Bw4 homozygotes was significantly lower in the “slow progressors” group; additionally, group B genotypes (multiple activating genes) were shown to be likely to mount a greater immune pressure on HIV-1. In terms of KIR footprints, several amino acid positions were identified for which the substitution of an amino acid may be ascribed to the immune response from KIR-modulated NK cells rather than from HLA restricted CTL immune pressure. In Chapter 4, we report a novel method to sequence the entire locus of KIR3DL1/S1. Two specific pairs of primers have been successfully designed and tested to amplify KIR3DL1 and KIR3DS1 exclusively. Using this novel sequencing method, we showed the polymorphism of this locus at a 6-digit level. 8 new KIR3DS1 alleles, 12 new KIR3DL1 alleles, 1 new KIR3DL1 gene and 1 new KIR3DS1 gene have been identified in this study. In Chapter 5, we used a valuable acute HIV-1 infection cohort to further study associations between KIRs and the clinical outcomes. It was interesting to find that the frequency of KIR3DS1 was significantly lower in the slow progressors group (31%) than in the acute group (40.7%), which implies that KIR3DS1 plays a role in HIV-1 disease progression. There are two other trends demonstrated in this study. One trend was that positive KIR2DL2 and/or KIR2DS2 (they are in strong linkage disequilibrium with each other) were associated with a higher set point viral load (at 3 month) (p=0.06). Another trend was that KIR3DS1 might have an association with disease progression (p=0.057). Overall, in this study, the role of KIRs and KIR/HLA interactions were evaluated in acute and chronic HIV-1 infection, which has provided important information for further study.

Variation at regulatory elements, identified through hypersensitivity to digestion by Deoxyribonuclease I (DNase I), is believed to contribute to variation in complex traits, but the extent and consequences of this variation are poorly characterized. To investigate the relationship between sequence variation, and the functional consequences of variation in chromatin accessibility, genome-wide DNase I hypersensitive sites (DHS) of terminally differentiated erythroblasts were studied in eight inbred strains of mice studied (A/J, AKR/J, BALBc/J, C3H/HeJ, C57BL/6J, CBA/J, DBA/2J, and LP/J). These strains were selected because of the availability of their genome sequence and quantitative trait loci (QTL) data. After confirming that next generation sequencing could identify DNase I hypersensitive sites with high sensitivity and specificity, and that differential peaks could be found, an automated peak calling pipeline was developed and optimized. 36,693 DHS peaks were identified covering 9.1 Mb (0.29%) of mouse genome. There was no indication of within strain variation. Between strains reproducible variation was observed at approximately 5% of DNase hypersensitive sites (1,397 DHSs). Variable DHSs were more likely to be enhancers than promoters and less likely to occur at conserved regions of the genome. Only 36% of such variable DHSs contain a sequence variant predictive of site variation and 12% contain at least one variant that disrupts transcription factor binding sites. The majority (86%) of variable DHSs differ in size/shape and the remaining 14% demonstrate discrete variation in single peak or cluster of peaks. Sequence variants within variable DHS are more likely to be associated with complex traits than those in non-variant DHS, and variants associated with complex traits preferentially occur in enhancer-like elements. Changes at a small proportion (7%) of discretely variable DHS are associated with changes in nearby transcriptional activity. Our results show that whilst DNA sequence variation is not the major determinant of variation in open chromatin, where such variants exist they are likely to be causal for complex traits.

In 2007, a genome wide association study identified a SNP in intron 1 of FTO with increased BMI. Homozygous risk allele carriers are on average three kg heavier than those homozygous for the protective allele. Mouse models have been made, including a conditional knockout, which is lean when globally expressed, as well as a conditional overexpression allele, which has increased body weight when globally expressed. The results from these and other studies suggest that the FTO SNPs lead to weight gain by increasing FTO activity and/or expression. Adult inactivation of Fto using the tamoxifen inducible Cre demonstrated that removal of Fto may be as deleterious as overexpression, with the adult knockout mice having increased fat mass and decreased lean mass. It also supported the role FTO plays in development as adult inactivation of Fto did not increase mortality rates as seen in the global Fto-/- pups. This study also revealed the importance of effective energy expenditure analysis in the mouse. I have confirmed a link between Fto-/- and increased mortality, which may be caused by alterations to developmental processes. Fto-/- reduces cilia formation in MEFs and results in dysregulated cilia formation in specific tissues in Fto-/- embryos. Levels of FTO also appear to affect adipogenic differentiation, which could be due to altered WNT/β-CATENIN signalling. Pharmacological inhibition of FTO was a success in vitro and a compound screen identified FG2216, which could be used in vivo to inhibit FTO. The in vivo effects of FG2216 at 60 mg/kg/2days did not affect body weight or composition in the mouse. My research suggests that there is dysregulation of gut hormones and neuronal signalling pathways in the FTO overexpression mice, which could cause the hyperphagia and increased body weight. These studies add to our current knowledge of FTO function, and suggest a role for FTO in control of body composition, development, and satiety signalling.

Lowering LDL-cholesterol with statin therapy produces substantial reductions in cardiovascular events, and larger cholesterol reductions may produce larger benefits. Rarely, myopathy occurs with statins, especially at higher doses and in combination with certain medications. Similarly strong associations might exist between myopathy with high-dose statin regimens and genetic variants, especially those affecting blood statin levels. This study aimed to find genetic variants associated with statin-induced myopathy. A feasibility study was completed to assess whether plausible effect sizes of 5 to10-fold higher risks per genetic variants could be detected among 50-100 cases with statin-induced myopathy and to consider the best study design. A genome-wide association study was then carried out using approximately 300,000 genetic markers (and additional fine-mapping) in 85 people with definite or incipient myopathy and 90 controls, who were all taking 80mg simvastatin daily in a 12,000 participant trial of 80mg vs 20mg simvastatin daily. The cases were also compared to 2,300 additional controls who had not been exposed to intensive-dose statin therapy. Replication of the myopathy result and lipid-lowering associations were tested in a 20,000 participant trial of 40mg simvastatin daily versus placebo. The genome-wide scan yielded a single strong association (p = 4x10^-9) of myopathy with the rs4363657 single nucleotide polymorphism (SNP) located within the SLCO1B1 gene on chromosome 12. This non-coding SNP was in nearly complete linkage disequilibrium (r²=0.97) with the non-synonymous rs4149056 SNP. The population prevalence of the rs4149056 C allele was 15%, and the odds ratio for myopathy was 4.5 (95% confidence interval 2.6 to 7.7) for each copy of the C allele and 16.9 (4.7 to 61.1) for CC vs TT homozygotes. Over 60% of these myopathy cases could be attributed to the C variant. The SLCO1B1 gene encodes the organic anion transport polypeptide OATP1B1, which has been shown to regulate hepatic uptake of statins. In literature reports, rs4149056 reduced statin transport and was associated with 37% (31% to 44%) higher systemic statin acid levels per C allele. The association of rs4149056 with myopathy was replicated in the trial of 40mg simvastatin daily, which also showed that it was associated with the cholesterol-lowering effects of simvastatin. No SNPs in any other region were clearly associated with myopathy (although comparison of the myopathy cases with the 2,300 controls identified a region of chromosome 1p12 that warrants further study). This study identified common variants in SLCO1B1 that influence the risks of statin-induced myopathy substantially. Genotyping these variants may be useful for tailoring both the statin dose and safety monitoring. More generally, such studies of the genetic determinants of serious adverse reactions with other drug classes may help to improve the balance between treatment efficacy and safety.

It has been demonstrated that retinal photoreception among mammals extends beyond rods and cones to include a small number of intrinsically photosensitive retinal ganglion cells (pRGCs), which are capable of responding to light due to expression of the melanopsin (OPN4) photopigment. OPN4 may have therapeutic potential if ectopically expressed in the degenerate retina in cases where photoreceptors are lost, but the other molecules involved in this light induced transduction cascade are less well characterized. Therefore I sought to probe further the mechanism of OPN4 mediated light sensitivity by siRNA mediated knock down of specific molecules in two mice models in which complete loss of rods and cones renders them almost exclusively dependent on the OPN4 pathway for light sensitivity. I generated siRNA probes against the long transcript variant of murine Opn4 mRNA and first tested these probes on the murine Neuro2A (N2a) cell line, before assessing effects in C3H/HeN rd and rodless/coneless rd/rd cl mice. siRNA was injected intravitreally into one eye and pupillometry was assessed, combined with molecular analyses at various timepoints. Reverse transcription polymerase chain reaction (RT-PCR) analysis in N2a cells confirmed Opn4 knockdown and immunolabelling techniques identified >85% silencing with siRNA. Pupil responses in the rd and rd/rd cl mice were inhibited by the siRNA injections in vivo which confirmed the functional effect of Opn4 silencing detected by molecular analysis. I therefore present a novel reproducible in vivo model in which siRNA induced silencing of the melanopsin pathway can be assessed by pupillometry and compared to levels of mRNA and protein at specific timepoints. Probes against other putative candidate genes, such as TRPC3, may unravel the molecular interactions of this pathway. This may help in future to induce light sensitivity in other retinal neurons in patients who are completely blind from photoreceptor loss.