Dissertations / Theses on the topic 'Mutational mechanisms'
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Robertson, Scott C. "Mechanisms of protein kinase activation determined by molecular modeling and mutational analysis /." Diss., Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 1999. http://wwwlib.umi.com/cr/ucsd/fullcit?p9938596.
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Legler, Patricia Marie. "Kinetic, magnetic, resonance, and mutational studies of the mechanisms of GDP-mannose mannosyl hydrolase, an unusual nudix enzyme." Available to US Hopkins community, 2002. http://wwwlib.umi.com/dissertations/dlnow/3046492.
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Kennouche, Paul. "New insights into meningococcal pathogenesis : exploring the role of the major pilin PilE in the functions of type IV pili Mechanisms of meningococcal type IV pili multiple functions revealed by deep mutational scanning." Thesis, Sorbonne Paris Cité, 2018. https://wo.app.u-paris.fr/cgi-bin/WebObjects/TheseWeb.woa/wa/show?t=1972&f=12515.
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Les pili de type IV (PT4) sont des filaments micrométriques qui exercent de multiples fonctions à la surface de nombreux procaryotes. Chez Neisseria meningitidis, les PT4 sont des homopolymères de la piline majeure PilE. Leur implication dans l'agrégation interbactérienne et l'adhésion aux cellules humaines les rend centraux dans la virulence du méningocoque. Cependant, les mécanismes permettant aux PT4 d'exercer ces diverses fonctions restent trop élusifs. Durant ce doctorat, nous avons simultanément déterminé les régions de PilE impliquées dans l'assemblage des pili, l'auto-agrégation, l'adhésion aux cellules humaines et la compétence à la transformation en utilisant la technique de deep mutational scanning. L'analyse approfondie de cette carte fonctionnelle de la séquence de la piline offre de nouvelles perspectives sur les mécanismes de fonctionnement des PT4 : tout d'abord, le domaine hyperconservé 1 de PilE est impliqué dans la régulation de la balance entre la longueur et le nombre des pili ; par ailleurs, nous avons identifié un groupe d'acides aminés électropositifs autour de la lysine 140 requis pour l'agrégation ; enfin, nous montrons l'importance de l'extrémité distale des PT4 dans l'adhésion. En résumé, ces résultats sont en faveur d'un rôle direct de PilE dans l'agrégation et l'adhésion bactérienne et identifient les domaines spécifiquement impliqués dans ces fonctions. Ces travaux ouvrent aussi de nouvelles perspectives sur la pathogénicité de Neisseria meningitidis et pourraient participer au développement de nouvelles thérapies pour combattre les pathologies provoquées par le méningocoqueType IV pili (TFP) are multifunctional micrometer-long filaments expressed at the surface of many prokaryotes. In Neisseria meningitidis, TFP are homopolymers of the major pilin PilE. They are crucial for virulence as they mediate interbacterial aggregation and adhesion to host cells although the mechanisms behind these functions remain unclear. During this doctoral work, we simultaneously determined the regions of PilE involved in pili display, auto-aggregation and adhesion to human cells by using deep mutational scanning. Mining of this extensive functional map of the pilin sequence provides new mechanistic insights: first, the hyperconserved 1-domain of PilE was found to be involved in the balance between pili length and number; moreover, we identified an electropositive cluster of residues centered around Lysine 140 necessary for aggregation; finally, we show the importance of the tip of TFP in adhesion. Overall, these results support a direct role of PilE in aggregation and adhesion to host cells and identify these specific functional domains. This doctoral work opens up new perspectives on the pathogenicity mechanisms of Neisseria meningitidis and could help design new therapies to fight meningococcal disease
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Kugelberg, Elisabeth. "Mechanisms of adaptive mutations in bacteria /." Stockholm, 2005. http://diss.kib.ki.se/2005/91-7140-446-5/.
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Kitzenmaier, Alexandra [Verfasser], Carmen [Gutachter] Villmann, and Erhard [Gutachter] Wischmeyer. "GlyT2-Mutationen als zweithäufigste Ursache bei Hyperekplexie – Pathologischer Mechanismus der Mutation P429L / Alexandra Kitzenmaier ; Gutachter: Carmen Villmann, Erhard Wischmeyer." Würzburg : Universität Würzburg, 2020. http://d-nb.info/1208629344/34.
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Sahlin, Charlotte. "Pathogenic Mechanisms of the Arctic Alzheimer Mutation." Doctoral thesis, Uppsala University, Department of Public Health and Caring Sciences, 2007. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-7582.
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Alzheimer’s disease (AD) is a progressive neurodegenerative disorder, neuropathologically characterized by neurofibrillay tangles and deposition of amyloid-β (Aβ) peptides. Several mutations in the gene for amyloid precursor protein (APP) cause familial AD and affect APP processing leading to increased levels of Aβ42. However, the Arctic Alzheimer mutation (APP E693G) reduces Aβ levels. Instead, the increased tendency of Arctic Aβ peptides to form Aβ protofibrils is thought to contribute to the pathogenesis.
In this thesis, the pathogenic mechanisms of the Arctic mutation were further investigated, specifically addressing if and how the mutation affects APP processing. Evidence of a shift towards β-secretase cleavage of Arctic APP was demonstrated. Arctic APP did not appear to be an inferior substrate for α-secretase, but the availability of Arctic APP for α-secretase cleavage was reduced, with diminished levels of cell surface APP in Arctic cells. Interestingly, administration of the fatty acid docosahexaenoic acid (DHA) stimulated α-secretase cleavage and partly reversed the effects of the Arctic mutation on APP processing.
In contrast to previous findings, the Arctic mutation generated enhanced total Aβ levels suggesting increased Aβ production. Importantly, this thesis illustrates and explains why measures of both Arctic and wild type Aβ levels are highly dependent upon the Aβ assay used, with enzyme-linked immunosorbent assay (ELISA) and Western blot generating different results. It was shown that these differences were due to inefficient detection of Aβ oligomers by ELISA leading to an underestimation of total Aβ levels.
In conclusion, the Arctic APP mutation leads to AD by multiple mechanisms. It facilitates protofibril formation, but it also alters trafficking and processing of APP which leads to increased steady state levels of total Aβ, in particular at intracellular locations. Importantly, these studies highlight mechanisms, other than enhanced production of Aβ peptide monomers, which could be implicated in sporadic AD.
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Segditsas, Stefania. "Mechanisms of intestinal tumorigenesis resulting from APC mutations." Thesis, University College London (University of London), 2008. http://discovery.ucl.ac.uk/15923/.
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Colorectal cancer typically arises through the sequential accumulation of mutations in different genes. Mutations in the adenomatous polyposis coli (APC) gene are thought to be the initiating step in this sequence of events and are found in the majority of early colorectal tumours. Investigation of these lesions has revealed that selection of 'optimal' combinations of mutations at the APC locus is in place, but the roles of such selected combinations have never been clarified. In the work presented in this thesis, I have demonstrated that similar constraints on APC mutations are active in tumours from attenuated FAP (AFAP) patients and that given the sub-optimal location of the germline APC mutation in these patients, additional somatic mutations are often required, especially in patients with germline mutations in the alternatively spliced region of exon 9. I have also shown that APC promoter hypermethylation does not appear to play a fundamental role in the selection of optimal APC genotypes. I have shown that the optimum combinations of mutations at APC are those that allow retention of some APC activity with respect to β-catenin degradation and that this has effects on the resulting activation of the Wnt signalling pathway. Optimal combinations of APC mutations result in intermediate nuclear β-catenin levels, which surprisingly highly activate a selection of Wnt targets. In an attempt to identify novel Wnt targets that are important for tumorigenesis and could serve as therapeutic targets, I have validated results from a cross-species comparison that identified a set of genes showing consistent differential expression between early tumour samples carrying APC mutations and normal tissue. In addition, I have investigated the biological function of one such-identified molecule, the serum/glucocorticoid-regulated kinase (SGK1), and I have revealed its potential role as a key regulator of intestinal cell differentiation and apoptosis.
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Williams, Louise Jane. "Recombinational mechanisms in human genetic diversity." Thesis, University of Nottingham, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.342483.
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Robinson, Alexis Anne. "Molecular Mechanisms of DJ-1 Mutations in Parkinson's Disease." Thesis, University College London (University of London), 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.487292.
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Mutations in PARK7 - the human chromosome Ip36 locus which harbours the DJ-J gene have been shown to be responsible for the onset of autosomal recessive Parkinson's disease. The exact function of DJ-I is unknown due to its diverse role in numerous biological processes including oncogenesis, transcriptional regulation and oxidative stress. This study focuses on the reported missense mutations ofDJ-1 in an attempt to elucidate their pathogenic mechanisms. Effects on DJ-I dimerisation and DJ-I interaction with known protein partners were initially assessed using the yeast two-hybrid system. Results were confinned in a mammalian cell system using af9nity purification methods. This study demonstrated that dlmerisation of DJ-I is required for all DJ-I binding protein interactions, and only mutation Ll66P had an effect on protein dimerisation. DJ-I mutations were found to have a specific disruptive effect on DJ-I interaction with DJ-I binding proteins. Mutation M261 abolished interactions with SUMO-I, a small ubiquitin-like modifier, by an unknown mechanism. Of particular interest was the finding that three distinct DJ-I missense mutations (A104T, Dl49A and E163K) selectively abolished interaction with the so-called DJ-I binding protein (DJBP). Further investigation of DJBP, involving peR amplification from human brain eDNA, revealed the existence of multiple isofonns of DJBP, generated by alternative splicing. Sequence analysis indicated the potential of DJBP to function as a mitochondrially-Iocated calcium-binding protein, due to the identification of EF-hand motifs and an N-tenninal mitochondrial targeting sequence... Disruption of the DJ-I/DJBP interaction may provide a molecular explanation for the underlying cause of DJ-I-related Parkinson's disease. The predicted role of DJBP as a mitochondrially-Iocated calcium-binding protein supports the involvement ofDJ-1 and DJBP in a pathway which exerts a protective effect on the cell under conditions of oxidative stress. Mutations which abolish DJ-I/DJBP interaction may abrogate the protective effect, resulting in increased susceptibility to cellular stress, providing a link between mitochondria and the pathogenesis of Parkinson's disease. These results also suggest that the gene encoding DJBP on human chromosome 22ql3 is a suitable candidate for genetic analysis in Parkinson's disease.
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Rutherford, Jodie. "Germline p53 mutations : characterisation and mechanisms of P53 dysfunction." Thesis, King's College London (University of London), 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.252278.
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Bartolo, Claire. "Novel mutations and molecular mechanisms in muscular dystrophy patients /." The Ohio State University, 1996. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487940665437141.
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Cheung, Jonathan Yu. "Pathogenic mechanisms of RAPSN mutations in congenital myasthenic syndromes." Thesis, University of Oxford, 2015. https://ora.ox.ac.uk/objects/uuid:c343ca03-563e-4b4a-9e35-aac09bfc5ea7.
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Rapsyn is essential for clustering acetylcholine receptors (AChR) at the neuromuscular junction (NMJ). Congenital myasthenic syndrome (CMS) due to RAPSN mutations compromises neuromuscular transmission through a deficiency of AChR at the NMJ. RAPSN-CMS is autosomal recessive, and most patients harbour a common mutation p.Asn88Lys. A definitive genetic diagnosis for patients who do not carry the p.Asn88Lys allele can be challenging. In this thesis 10 novel variants in RAPSN are shown to impair AChR clustering in vitro, and are thus defined as pathogenic. The properties of RAPSN mutations p.Val45M, p.Asn88Lys, p.Arg91Leu and p.Ala153Thr were studied and were found to be diverse, though common mechanisms were found to underlie the AChR deficiency. The mutations reduce the stability of rapsyn and the stability of AChR-rapsyn clusters formed on cultured myotubes. A unique missense mutation in the AChR d-subunit encoding gene, p.Glu381Lys, phenotypically mimics RAPSN-CMS, and investigations revealed that this mutation also causes instability of the AChR clusters. Salbutamol, a medication that is believed to stabilise NMJ, has recently been found to benefit AChR deficiency patients when used in combination with pyridostigmine. However, the mode of action of salbutamol at the NMJ is undefined. A pilot study was performed to evaluate the efficacy and mechanism of salbutamol therapy in a mouse model of AChR deficiency. Methodologies were established for the analysis of the effects of salbutamol and will provide the basis for a more detailed study of its beneficial action in disorders of neuromuscular transmission.
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Alotibi, Raniah Saleem. "Investigating the mechanisms of telomeric mutation in human cells." Thesis, Cardiff University, 2015. http://orca.cf.ac.uk/84320/.
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Telomeres are nucleoprotein structures that contain non-coding (TTAGGG) tandem repeats and associated telomere binding proteins at the end of chromosomes. As a consequence of end-replication losses, telomeres undergo gradual erosion with ongoing cell division. It is hypothesised that in addition to the end-replication problem, mutational mechanisms may contribute to telomere erosion by generating large-scale telomeric deletion events. As short dysfunctional telomeres are capable of fusion to other chromosome ends, large-scale telomeric deletions can lead to genomic instability which in turn may drive tumour progression. The primary aim of this thesis was to investigate putative mutational mechanisms that could lead to large-scale telomeric deletion. The role of oxidative stress and it potential contribution to telomere dynamics was assessed. The induction of fragility and replication inhibition at telomeres was also examined. Furthermore, the role that G-quadruplex structure within telomere repeat sequences and the possible induction of replication fork stalling and resolution as single or double stranded breaks was also considered as a mutational mechanism that could lead to telomere deletion. High-resolution analysis of telomere dynamics using Single Telomere Length Analysis (STELA), following the induction of oxidative stress in IMR90 fibroblasts, revealed that oxidative damage does not appear to affect the rate of telomere erosion, or the frequency of large-scale telomeric deletion. The data are more consistent with the view that premature senescence does not arise as a consequence of accelerated telomere erosion, but instead more likely results from stochastic DNA damage across the rest of the genome. The analysis of telomere dynamics following the induction of chromosome fragility, showed that telomere length in Seckel cell (SCK) fibroblasts were significantly different from those of untreated cells following treatments with aphidicolin with an increase in stochastic telomeric deletion. Whilst in MRC5 fibroblasts, the induction of the telomere fragility impacted on the upper to lower allele ratio, with a loss of the longer telomere length distributions. The stabilisation of G-quadruplex structures using the G-quadruplex ligand (RHPS4), together with ATRX knockdown, showed that an absence of ATRX sensitised cells to the ligand, but that the stabilisation of G-quadruplexes, did not significantly affect the telomere dynamics as determined using STELA. Taken together, the data presented in this thesis are not consistent with a role for oxidative stress, or the formation of G-quadruplex structures, in generating large-scale telomeric deletion; however telomeric mutational events may occur following the induction of chromosome fragile sites, specifically in the context of an ATR deficiency.
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Motley, William Washburn. "Glycyl-tRNA synthetase mutations in neurological disease : mechanisms and models." Thesis, University of Oxford, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.558205.
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Charcot-Marie- Tooth disease type 2D (CMT2D) is a dominantly inherited axonal neuropathy caused by missense mutations in the glycyl-tRN A synthetase gene (CARS). Dominant mutations in tyrosyl-tRNA synthetase and alanyl-tRNA synthetase also cause CMT, suggesting a shared mechanism for all three diseases. The goal of this thesis was to investigate possible mechanisms and narrow the potential ways that mutations in CARS could lead to axon loss. GARS mutations are distributed throughout the protein in multiple functional domains. The localization, dimerization, and degradation of GARS were examined as in vitro measures of protein function. Dimer function was preserved in most mutants. Similarly, no differences in wild-type and mutant localization or degradation were seen. In vitro experiments did not show evidence of a loss of function in most GARS mutants. Progress has also been made towards the development of a Drosophila model of CMT2D. Overexpression of mutant Aats-gly, the Drosophila ortholog of CARS, was found to cause lethality in fruit flies, while overexpression of the wild-type did not reduce viability. Results suggest that CMT2D can be modeled in fly, that the toxicity of the mutant protein is tissue autonomous, and that mutations in CARS and YARS have similar effects in Drosophila, hinting at a shared mechanism. There are two murine models of CMT2D with different mutations in the Cars gene. While they have very different levels of severity, they are both reliable models of the human disease. Each disease model was crossed to mice ubiquitously overexpressing wild-type GARS in order to determine whether the disease phenotype is caused by a loss of function in GARS. Using behavioral, histological, and electrophysiological measures of nerve function, it was determined that overexpression of wild-type CARS cDNA mitigates, but does not dramatically improve neuromuscular function in either model. The wild-type transgenes were able to rescue compound heterozygous CarsC201RIXM256 mice from embryonic lethality proving that the protein product of the two transgenes is functional. In summary, my work in cells, fruit flies, and mice provides evidence that CARS mutations cause CMT2D primarily by a pathogenic gain of function.
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Murray, Lydia Soraya. "Dissecting the mechanisms of disease of COL4A1 and COL4A2 mutations." Thesis, University of Glasgow, 2014. http://theses.gla.ac.uk/5143/.
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The best characterised collagen IV disease is Alport syndrome, caused by α3.α4.α5(IV) mutations. Mutations affecting α1(IV)2α2(IV) can cause diseases, such as haemorrhagic stroke, aneurysm, haematuria, proteinuria, cataracts, vascular tortuosity and iris hypoplasia. Recent data has shown that α and COL4A2 mutations may be more common in the population than previously anticipated. However, the mechanisms of disease of all collagen IV mutations are poorly understood. The aim of this thesis was to investigate the disease mechanism in a selection of COL4A1, COL4A2 and COL4A5 mutations. Collagen IV is folded in the endoplasmic reticulum (ER) then secreted to provide the major structural component of the basement membrane (BM). As such, a combination of ex situ biopsies, in vitro cell cultures and an in vivo mouse model provided an excellent platform for investigating the intracellular and extracellular effects of mutant trimers. Primary dermal fibroblasts containing COL4A2 and COL4A5 mutations, and a Col4a1 mouse model were treated with the FDA approved chemical chaperone phenyl-4-butyric acid (PBA) to determine its effects on intracellular ER retention of trimers (which can activate ER stress), extracellular trimer incorporation into the BM, and disease phenotypes. In vitro and ex situ investigation of a COL4A2 mutation suggested that intracellular α1(IV)2α2(IV) accumulation and ER stress associated with severe BM defects and the disease. Intracellular accumulation and ER stress were reduced by PBA treatment, and treatment of a Col4a1 mouse model displayed elevated α1(IV)2α2(IV) incorporation onto the BM. In vivo PBA treatment did not exacerbate any defects, rescued some disease phenotypes, and had no effect on others. In addition, one of five COL4A5 mutations analysed in vitro displayed intracellular α3.α4.α5(IV) accumulation and ER stress that were ameliorated by PBA. Collectively these data suggest that ER stress and/or reduced α1(IV)2α2(IV) BM incorporation underlie some collagen IV pathologies, and provide novel evidence that they may be amenable to chemical chaperone therapy. More investigation is now needed to further dissect the relative contributions of ER stress and BM defects to collagen IV diseases.
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Koskiniemi, Sanna. "Dynamics of the bacterial genome rates and mechanisms of mutation /." Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis : Univ.-bibl. [distributör], 2010. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-111428.
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Kubica, Barbara. "Fitness Effects and Resistance Mechanisms of Beneficial Mutations in 'Pseudomonas aeruginosa'." Thèse, Université d'Ottawa / University of Ottawa, 2012. http://hdl.handle.net/10393/23123.
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Beneficial mutations are the fuel for adaptation but remain poorly studied. Extreme value theory has been used extensively as a model for understanding the distribution of fitness effects (DFE) among beneficial mutations prior to selection, however there is little empirical data available to test the most compelling predictions of the theory. This thesis aims to test the prediction that the DFE among beneficial mutations is a negative exponential, with many mutations of small effect and few of large effect. To do this, I collected a wide range of Pseudomonas aeruginosa mutants resistant to the antibiotic ciprofloxacin and measured their fitness in the absence of the drug. I reject the exponential as an adequate descriptor of the data and show that the DFE among beneficial mutations is better described as being located in the Weibull domain of attraction, which is characterised by having a right-hand bound on the magnitude of the largest fitness effects. I also sequenced a number of genes known to be targets of fluoroquinolone resistance to shed light on the range and number of genetic targets involved in resistance. I found a number of mutations, and often multiple mutations, in known genetic targets. Evidence suggests that mutations in regions of the genome not sequenced are also important in determining fitness under permissive conditions. Taken together, these results provide valuable insight into one of the most fundamental problems in evolution, the nature and fitness effects of beneficial mutations.
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Gomes, Cláudia, Puchol Sandra Martínez, Noemí Palma, Gertrudis Horna, Lidia Ruiz-Roldán, Maria J. Pons, and Joaquim Ruiz. "Macrolide resistance mechanisms in Enterobacteriaceae: Focus on azithromycin." Taylor & Francis, 2016. http://hdl.handle.net/10757/620710.
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From its introduction in 1952 onwards, the clinical use of macrolides has been steadily increasing,
both in human and veterinary medicine. Although initially designed to the treatment of Grampositive
microorganisms, this antimicrobial family has also been used to treat specific Gram-negative
bacteria. Some of them, as azithromycin, are considered in the armamentarium against
Enterobacteriaceae infections. However, the facility that this bacterial genus has to gain or
develop mechanisms of antibiotic resistance may compromise the future usefulness of these antibiotics
to fight against Enterobacteriaceae infections. The present review is focused on the mechanisms
of macrolide resistance, currently described in Enterobacteriaceae.
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Wiberg, Jörgen. "Mechanisms controlling DNA damage survival and mutation rates in budding yeast." Doctoral thesis, Umeå universitet, Institutionen för medicinsk kemi och biofysik, 2012. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-54203.
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All living organisms are made of cells, within which genetic information is stored on long strands of deoxyribonucleic acid (DNA). The DNA encodes thousands of different genes and provides the blueprint for all of the structures and activities occurring within the cell. The building blocks of DNA are the four deoxyribonucleotides, dATP, dGTP, dTTP, and dCTP, which are collectively referred to as dNTPs. The key enzyme in the production of dNTPs is ribonucleotide reductase (RNR). In the budding yeast Saccharomyces cerevisiae, the concentrations of the individual dNTPs are not equal and it is primarily RNR that maintains this balance. Maintenance of the dNTP pool balance is critical for accurate DNA replication and DNA repair since elevated and/or imbalanced dNTP concentrations increase the mutation rate and can ultimately lead to genomic instability and cancer. In response to DNA damage, the overall dNTP concentration in S. cerevisiae increases. Cell survival rates increase as a result of the elevated concentration of dNTPs, but the cells also suffer from a concomitant increase in mutation rates. When the replication machinery encounters DNA damage that it cannot bypass, the replication fork stalls and recruits specialized translesion synthesis (TLS) polymerases that bypass the damage so that replication can continue. We hypothesized that elevated dNTP levels in response to DNA damage may allow the TLS polymerases to more efficiently bypass DNA damage. To explore this possibility, we deleted all known TLS polymerases in a yeast strain in which we could artificially increase the dNTP concentrations. Surprisingly, even though all TLS polymerases had been deleted, elevated dNTP concentrations led to increased cell survival after DNA damage. These results suggest that replicative DNA polymerases may be involved in the bypass of certain DNA lesions under conditions of elevated dNTPs. We confirmed this hypothesis in vitro by demonstrating that high dNTP concentrations result in an increased efficiency in the bypass of certain DNA lesions by DNA polymerase epsilon, a replicative DNA polymerase not normally associated with TLS activity. We asked ourselves if it would be possible to create yeast strains with imbalanced dNTP concentrations in vivo, and, if so, would these imbalances be recognized by the checkpoint control mechanisms in the cell. To address these questions, we focused on the highly conserved loop2 of the allosteric specificity site of yeast Rnr1p. We introduced several mutations into RNR1-loop2 that resulted in changes in the amino acid sequence of the protein. Each of the rnr1-loop2 mutation strains obtained had different levels of individual dNTPs relative to the others. Interestingly, all of the imbalanced dNTP concentrations led to increased mutation rates, but these mutagenic imbalances did not activate the S-phase checkpoint unless one or several dNTPs were present at concentrations that were too low to sustain DNA replication. We were able to use these mutant yeast strains to successfully correlate amino acid substitutions within loop2 of Rnr1p to specific ratios of dNTP concentrations in the cells. We also demonstrated that specific imbalances between the individual dNTP levels result in unique mutation spectra. These mutation spectra suggest that the mutagenesis that results from imbalanced dNTP pools is due to a decrease in fidelity of the replicative DNA polymerases at specific DNA sequences where they are more likely to make a mistake. The mutant rnr1-loop2 strains that we have created with defined dNTP pool imbalances will be of great value for in vivo studies of polymerase fidelity, translesion synthesis by specialized DNA polymerases, and lesion recognition by the DNA repair machinery.
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Hardwick, Robert John. "Mechanisms of mutation at mouse expanded simple tandem repeat (ESTR) loci." Thesis, University of Leicester, 2007. http://hdl.handle.net/2381/30375.
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Mouse expanded simple tandem repeat (ESTR) loci are non-coding, unstable arrays of short repeat units with high spontaneous mutation rates (up to 15% per gamete). Exposure to ionising radiating and some chemicals can elevate the mutation rate further, which makes them useful tools for the assessment of genomic mutation rates.;ESTRs mutate by either gaining or losing repeat units to result in the generation of differentially-sized mutant alleles. However, the mechanism by which this occurs remains unclear, and this hinders the reliable extrapolation of ESTR data to other loci. To address this issue, this thesis aimed to investigate the mechanism of mutation at the ESTR locus Ms6-hm.;Recent evidence has suggested that events at DNA replication are responsible for generating ESTR mutants. It this is the case, then it would be expected that ESTR mutation rate would increase in line with the number of cell division events in a tissue system. In this thesis, the speculated link between cell division rate and ESTR mutation rate was tested in three different tissues of varying mitotic proficiencies: brain (low mitotic index), bone marrow (intermediate mitotic index), and sperm (high mitotic index). In the first instance, spontaneous mutation rates in each of these tissues were assessed in mice of four different age groups: 12, 26, 48 and 96 weeks old; and in the second part of this thesis, in utero-irradiated mice were analysed to evaluate the mitosis-proficient process of embryogenesis.;I report increases in ESTR spontaneous mutation rate in sperm with age, but not in brain, and I also demonstrate that ESTR mutation rates are elevated in each tissue --- including brain --- following in utero -irradiation. I conclude that DNA replication is an important process in the generation of ESTR mutants.
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Kingswell, Nicola Jo. "Pathological mechanisms underpinning amelogenesis imperfecta in mice carrying an amelogenin mutation." Thesis, University of Leeds, 2015. http://etheses.whiterose.ac.uk/10619/.
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Mice carrying a Y64H amelogenin mutation phenotypically mimic human amelogenesis imperfecta. Affected ameloblasts are characterised by the presence of abnormal cytoplasmic vesicles of retained amelogenin. Protein-protein binding studies using recombinant wild type and Y64H amelogenin revealed that the mutation increased amelogenin-amelogenin binding. This may drive intracellular aggregation of Y64H amelogenin, explaining the abnormal retention. Intracellular protein aggregation causes ER stress which triggers the UPR. The UPR attempts to restore proteostasis but as a last resort triggers apoptosis. SEM of affected enamel showed initially secreted enamel is normal; coincident with UPR in pro-survival mode. The final outer enamel is abnormal; indicative of UPR induced ameloblast apoptosis. Q-RT-PCR was used to measure ER stress related gene expression in affected ameloblasts. Expression levels of ER stress genes increased but not significantly (significance was reached in later studies by others in the research consortium). Amelogenin expression was shown to be significantly reduced in affected ameloblasts; reduced protein expression being a known pro-survival tactic employed during ER stress. A steady-state in vitro mineralisation system was used to examine the effect of the Y64H mutation on mineral nucleation by recombinant amelogenins in isolation or in conjunction with recombinant 32 kDa enamelin. Data showed that the Y64H mutation did not affect the nucleating potential suggesting that the pathological mechanism driving AI in affected mice is linked to ER stress rather than dysfunction of secreted amelogenin. An unexpected finding was that the 32 kDa enamelin (much lauded in the literature as a functional species) may be unique to pig amelogenesis and its functional significance is therefore debateable In summary, the mechanism driving AI in these mice is associated with intracellular ER stress. Extracellular dysfunction of mutated enamel proteins has been the focus of AI research but the involvement of ER stress provides additional therapeutic options for treating AI.
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Liquori, Alessandro. "Deciphering molecular mechanisms of unusual variants in Usher Syndrome." Thesis, Montpellier, 2015. http://www.theses.fr/2015MONTT016.
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Le syndrome de Usher (USH) est une maladie transmise selon le mode autosomique récessif caractérisée par l’association d’une surdité congénitale (HL) et d’une rétinite pigmentaire (RP), et dans certains cas, d’une aréflexie vestibulaire. Une hétérogénéité clinique et génétique est reconnue. Environ 10 % des cas USH restent non résolus après analyse moléculaire exhaustive des différents gènes. Ces cas incluent les patients qui ne portent aucune mutation dans un des gènes USH connus ainsi que les patients porteurs d’une seule mutation dans un gène USH. Au cours de cette thèse, nous nous sommes intéressés à l’étude des patients porteurs d’une seule mutation dans les gènes USH2A et PCDH15.Dans la première partie de la thèse, nous avons analysé une cohorte de patients avec un phénotype USH2A bien défini : 5 patients pour lesquels une seule mutation à l’état hétérozygote avait été identifiée dans le gène USH2A et un patient porteur d’un variant silencieux en trans d’une mutation non-sens.Pour les 5 patients, nous avons émis l’hypothèse que la seconde mutation, restant à être identifiée, pourrait se trouver dans des régions introniques profondes. Pour cela, nous avons développé une approche de séquençage à haut débit (NGS) de l’ADN pour identifier les variants introniques profonds dans le gène USH2A et évaluer leurs conséquences sur l’épissage. Comme preuve de concept et pour valider l’approche, y compris le pipeline bio-informatique et l’évaluation des outils de prédiction de l’épissage, nous avons analysé un patient porteur d’un pseudoexon (PE) connu dans le gène USH2A. Ensuite, les 5 patients ont été étudiés en utilisant le pipeline défini, ce qui a conduit à l’identification de 3 nouveaux variants introniques profonds chez 4 d’entre eux. Tous les variants ont été prédits comme pouvant avoir un impact sur l’épissage et aboutir à l’insertion de PE. Ces prédictions ont été validées par les essais minigènes. Grâce à cette étude, nous présentons une stratégie innovante pour identifier les mutations introniques profondes, lorsque l’analyse des transcrits n’est pas possible. Par ailleurs, le pipeline bio-informatique développé fonctionne indépendamment de la taille du gène analysé, ce qui permet l’application possible de cette approche à n’importe quel gène. Par ailleurs, un oligonucléotide antisens de type morpholino (AMO) a été évalué in vitro afin de rétablir l’altération d’épissage induite par une des mutations identifiées. Les résultats ont montré un taux d’exclusion élevé du transcrit aberrant et suggèrent une application possible en thérapie moléculaire. Nous avons ensuite effectué des études sur le variant USH2A c.1377T>A, un variant silencieux afin d’évaluer son effet sur l’épissage. L’analyse de l’ARN issu de cellules nasales du patient a montré que ce variant conduit au saut de l’exon 8 dans les transcrits USH2A. Ceci a été confirmé par un essai minigène. En outre, des études préliminaires ont été réalisées en utilisant des outils de prédictions et des essais minigènes pour évaluer l’implication des éléments cis-régulateurs dans le défaut d’épissage observé chez le patient. Dans la deuxième partie de la thèse, nous avons analysé une patiente USH1, pour laquelle une seule mutation avait été identifiée dans le gène PCDH15. Dans ce cas, nous avons combiné la culture des cellules épithéliales nasales avec l’analyse des transcrits PCDH15. Celle-ci a été réalisée par séquençage de cinq RT-PCR chevauchantes. Grâce à cette analyse, nous avons réussi à délimiter une région d’intérêt dans le transcrit, dont l’amplification a échoué exclusivement pour l’allèle porteur de la mutation non identifiée. D’autres analyses ont été effectuées dans la région génomique correspondante par capture ciblée couplée au séquençage NGS et LongRange PCR suivi de séquençage Sanger. Cependant, aucun variant candidat n’a été identifié à ce jour. Nous suggérons l’implication de mécanismes moléculaires complexes qui restent à être caractérisésUsher syndrome (USH) is an autosomal recessive disorder characterized by the association of sensorineural hearing loss (HL) and retinitis pigmentosa (RP), and in some cases, vestibular areflexia. Clinical and genetic heterogeneity are recognised. Indeed, three clinical types can be caused by mutations in one of the 10 known genes and USH2A represents the most frequently involved gene.Approximately 10 % of the USH cases remain genetically unsolved after extensive molecular analysis of the different genes, which includes sequencing of the exons and their intronic boundaries, combined to large rearrangements screening by array CGH. These unsolved cases include patients who do not carry any mutation in any of the known USH genes and patients who carry a single USH mutation. During this thesis we focalised on the study of patients carrying a single mutation in USH2A and PCDH15 gene.First, we have analysed a cohort of well-defined USH2A patients: five patients, for whom a single USH2A heterozygous mutation had been identified and one patient carrying a silent variant in trans to a nonsense mutation. For the 5 patients, we supposed that the second mutation remaining to be found could be localised deep in the introns. Indeed, a deep intronic mutation resulting in the inclusion of a pseudoexon (PE 40) in USH2A transcripts had been identified, following RNA analysis from nasal cells. Unfortunately, analysing USH2A transcripts still represent a challenging approach in a diagnostic settings and it is not always possible. To circumvent this issue, we have developed a DNA-Next Generation Sequencing (NGS) approach to identify deep intronic variants in USH2A and evaluate their consequences on splicing. As a proof of concept and to validate this approach, including the bioinformatics pipeline and the assessment of splicing predictor tools, the patient carrying the PE 40 was analysed at first. Then, the 5 patients were studied using the defined pipeline, which led to the identification of 3 distinct novel deep intronic variants in 4 of them. All were predicted to affect splicing and resulted in the insertion of PEs, as shown by minigene assays. Through this study, we present a new and attractive strategy to identify deep intronic mutations, when RNA analyses are not possible. In addition, the bioinformatics pipeline developed is independent of the gene size, implying the possible application of this approach to any disease-linked gene. Moreover, an antisense morpholino oligonucleotide (AMO) tested in vitro for its ability to restore the splicing alterations caused by one of the identified mutation provided high inhibition rates. These results are indicative of a potential application for molecular therapy.In the second case, we have performed studies on the USH2A c.1377T>A silent variant to investigate its effect on splicing. Analysis of RNA from nasal cells of patients showed that this variant led to the skipping of exon 8 in USH2A transcripts. This was confirmed by minigene assay. Moreover, preliminary studies have been performed using prediction tools and minigene assays to assess the involvement of cis-acting elements in causing the aberrant splicing.In the second part of the thesis, we have analysed an USH1 patient, for whom only one mutation had been identified in the PCDH15 gene. In this case, we combined nasal epithelial cells culture with the analysis of the PCDH15 transcripts. This was performed by sequencing five overlapping RT-PCRs. Through this analysis, we were able to delimit a region within the transcript, which failed to be amplified exclusively in the allele carrying the unidentified mutation. Further analyses have been performed in the corresponding genomic region by NGS-target capture and LongRange PCR associated with Sanger sequencing. However, no evident mutation has been identified so far. Therefore, we suggest the involvement of complex molecular mechanisms that remain to be characterised
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Morais, Sara Peres de. "Juvenile Parkinson disease caused by parkin mutations: large deletions and pathogenic mechanisms." Master's thesis, Faculdade de Ciências e Tecnologia, 2011. http://hdl.handle.net/10362/6723.
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Dissertação para obtenção do Grau de Mestre em
Genética Molecular e BiomedicinaAutosomal recessive juvenile Parkinson disease (AR-JP) is mainly caused by mutations in PARK2. AR-JP presents with rigidity, bradykinesia and resting tremor, usually before age 40 years. Large PARK2 deletions account for 50% of the mutations identified in patients with AR-JP of Portuguese origin. The PARK2 gene encodes parkin, an E3 ubiquitin ligase, an important part of the cellular machinery that covalently tags target proteins with ubiquitin for degradation by the ubiquitinproteasome system (UPS), the main cellular protein degradation system responsible for targeted degradation of damaged and misfolded proteins. This project aims were: determine the breakpoints of the deletion found in Portuguese patients in order to identify the genomic mechanisms underlying these gene rearrangements and to explore the pathogenic mechanisms of parkin mutations by assessing the dynamics of formation and degradation of aggregates by UPS and also by determining its effects in the UPS degradation capacity and its relation with neuronal death. A successful approach was developed to narrow the deletion breakpoint intronic position. Cellular models expressing wild-type and mutant parkin were developed and characterized regarding mRNA and protein expression, as well as, aggregate formation, cell viability and proteasome activity. Our data show that the different studied mutations do not have an impact on cell viability, although resulted in differences in the number of cell with aggregates for the cells expressing N52MfsX29, L358RfsX77 and R275W mutants as well as in the number of aggregates present in each cell. We were also able to show that proteasome inhibition has as impact both in cell viability and in aggregate formation, resulting in decreased viability and increased aggregate formation. The study of the cellular mechanisms resulting in neuronal dysfunction is crucial for the identification of potential therapeutic targets for Parkinson disease.
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Bohman, Lova. "Pathological Mechanisms of Sarcomere Mutations in the Disease Hypertrophic Cardiomyopathy : A Review." Thesis, Linköpings universitet, Institutionen för fysik, kemi och biologi, 2021. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-176045.
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Hypertrophic cardiomyopathy is a heart disease that is characterized by an enlarged heart muscle. Mutations to sarcomere proteins in the muscle fibers give rise to the disease, and this review aims to compile the mechanisms by which the mutations cause the disease phenotype. β-myosin heavy chain mutants affect the thick filament structure and contraction velocity of the muscle. Mutations to the myosin-binding protein C produces truncated proteins with decreased expression in the cells. Troponin T mutants cause myofibrillar disarray, alters affinity to α-tropomyosin, and are linked to a higher risk of sudden death. Troponin I is an unpredictable mutant that needs to be further researched but is thought to cause regulatory problems. Mutations to α-tropomyosin and the regulatory myosin light chain both affect the Ca2+-affinity of the proteins and leads to contractile problems. Hypercontractility as a result of the mutations seems to be the primary cause of the disease. Hypertrophic cardiomyopathy is linked to sudden death, and factors such as a family history of sudden death, multiple simultaneous mutations, unexplained syncope, non-sustained ventricular tachycardia, abnormal blood pressure response and extreme hypertrophy (>30 mm) heightens the risk of a sudden death. An increased knowledge about the disease will aid in the mission to better the treatments for the affected, but further investigation of pathological pathways needs to be performed.
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Oonthonpan, Lalita. "Two human Mitochondrial Pyruvate Carrier mutations reveal distinct mechanisms of molecular pathogenesis." Diss., University of Iowa, 2019. https://ir.uiowa.edu/etd/7006.
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The Mitochondrial Pyruvate Carrier (MPC) occupies a central metabolic node by transporting cytosolic pyruvate into the mitochondrial matrix, thereby linking glycolysis with mitochondrial metabolism. Two reported human MPC1 mutations cause developmental abnormalities, neurological problems, metabolic deficits, and for one patient, early death. We aimed to understand biochemical mechanisms by which the human patient c.C289T and c.T236A MPC1 alleles disrupt MPC function. MPC1 c.C289T encodes two protein variants, a mis-spliced, truncation mutant (A58G) and full-length point mutant (R97W). MPC1 c.T236A encodes a full-length point mutant (L79H). Using human patient fibroblasts and complementation of CRISPR-deleted, MPC1 null mouse C2C12 cells, we investigated how MPC1 mutations cause MPC deficiency. Truncated MPC1 A58G protein was intrinsically unstable and failed to form MPC complexes. The MPC1 R97W protein was less stable but when overexpressed formed complexes with MPC2 that retained pyruvate transport activity. Conversely, MPC1 L79H protein formed stable complexes with MPC2, but these complexes failed to transport pyruvate. These findings inform MPC structure-function relationships and delineate three distinct biochemical pathologies resulting from human patient MPC1 mutations and inform fundamental MPC structure-function relationships. These results also demonstrate an efficient molecular genetic system using the mouse C2C12 cell line to mechanistically investigate human inborn errors in pyruvate metabolism.
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Starker, Lee. "New Insights in Genetic and Epigenetic Mechanisms Involved in Parathyroid Tumorigenesis." Doctoral thesis, Uppsala universitet, Institutionen för kirurgiska vetenskaper, 2013. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-205587.
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Primary hyperparathyroidism (pHPT) is a pathology associated with one or multiple hyperfunctioning parathyroid glands. The disease prevalence occurs in roughly 1-2% of the population primarily post-menopausal women. The molecular pathology of the disease is poorly understood. Elevated serum calcium levels in the setting of an inappropriately elevated parathyroid hormone level are indicative of the disease process. The ultimate treatment of the disease is to remove the hyperfunctioning gland. The aim of this thesis was to examine potential genetic and epigenetic aberrations that are potentially disease causing. The methylation signature of normal and pathological parathyroid tissue has yet to be investigated. DNA was bisulphite modified and analyzed using the Infinium HumanMethylation27 BeadChip. Distinct hierarchical clustering of genes with altered DNA methylation profiles in normal and pathologic parathyroid tissue was evident. DNA hypermethylation of CDKN2B, CDKN2A, WT1, SFRP1, SFRP2, and SFRP4 known to be important in the development of parathyroid tumors were associated with reduced gene expression in both benign and malignant parathyroid tumors. Familial primary hyperparathyroidism (FPHPT) may occur due to an underlying germ-line mutation in the MEN1, CASR, or HRPT2/CDC73 genes. Eighty-six young (≤45 years of age) patients with clinically non-syndromic PHPT underwent genetic analysis. Eight of 86 (9.3%) young patients with clinically non-familial PHPT displayed deleterious germ-line mutations in the susceptibility genes (4 MEN1, 3 CASR, and 1 HRPT2/ CDC73). Accumulation of non-phosphorylated active β -catenin has been reported to commonly occur in parathyroid adenomas from patients with primary hyperparathyroidism (pHPT). We assessed possible β-catenin stabilizing mutations in a large series of parathyroid adenomas. A total of one hundred and eighty sporadic parathyroid adenomas were examined for mutations in exon 3 of the CTNNB1gene. The mutation S33C (TCT >TGT) was detected by direct-DNA sequencing of PCR fragments in 1 out of 180 sporadic parathyroid adenomas (0.68 %). Eight matched tumor-constitutional DNA pairs from patients with sporadic parathyroid adenomas underwent whole-exome capture and high-throughput sequencing. Four of eight tumors displayed a frame shift deletion or nonsense mutations within the MEN1 gene, which was accompanied by loss of heterozygosity of the remaining wild-type allele. One tumor harbored a Y641N mutation of the histone methyltransferase EZH2 gene, previously linked to myeloid and lymphoid malignancy formation. Targeted sequencing in the additional 185 parathyroid adenomas revealed a high rate of MEN1 mutations (35%).
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Ball, Nathan B. "Design and characterization of a gel loading mechanism for an ultra-high throughput mutational spectrometer." Thesis, Massachusetts Institute of Technology, 2005. http://hdl.handle.net/1721.1/32881.
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Thesis (S.B.)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering, 2005.Includes bibliographical references (leaf 51).
A process known as Constant Denaturant Capillary Electrophoresis is used to separate mutant from wild-type DNA at fractions down to 10-7. A device known as an Ultra-high Throughput Mutational Spectrometer is being created to run 10,000 parallel channels of CDCE in order to correlate multiple point mutations in DNA with the diseases that they can cause, such as cancer. By separating the DNA in large populations, the underlying causes of such diseases can be identified. To successfully run CDCE, a high viscosity polymer gel must be loaded into each of the 10,000 channels, each of which are composed of an individual glass capillary with a 75 m inner diameter. A mechanism was designed and tested which loaded gel into 8 channels simultaneously. The mechanism was used to test the relationship between gel loading time in relation to varied pressure and capillary length, through 45 total runs, with 8 channels per run. The relationships were characterized, resulting in two equations that enable an accurate prediction of the fill time necessary to load 10,000 parallel channels simultaneously under varied conditions.
by Nathan B. Ball.
S.B.
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Harris, Reuben Stewart. "On a molecular mechanism of adaptive mutation in Escherichia coli." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp05/nq21574.pdf.
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Frida, Jonsson. "Underlying genetic mechanisms of hereditary dystrophies in retina and cornea." Doctoral thesis, Umeå universitet, Institutionen för medicinsk biovetenskap, 2017. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-130538.
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Inherited retinal and corneal dystrophies represent a group of disorders with great genetic heterogeneity. Over 250 genes are associated with retinal diseases and 16 genes are causative of corneal dystrophies. This thesis is focused on finding the genetic causes of corneal dystrophy, Leber congenital amaurosis (LCA), Stargardt disease and retinitis pigmentosa in families from northern Sweden. By whole exome sequencing a novel mutation, c.2816C>T, p.Thr939Ile, in Collagen Type XVII, Alpha 1 chain, COL17A1, gene was identified in several families with epithelial recurrent erosion dystrophy (ERED). We showed that the COL17A1 protein is expressed in the basement membrane of the cornea, explaining the mutation involvement in the corneal symptoms. We could link all the families in this study to a couple born in the late 1700s confirming a founder mutation in northern Sweden. Our finding highlights role of COL17A1 in ERED and suggests screening of this gene in patients with similar phenotype worldwide. Furthermore the genetic causes in several retinal degenerations were identified. In one family with two recessive disorders, LCA and Stargardt disease, a novel stop mutation, c.2557C>T, p.Gln853Stop, was detected in all LCA patients. In the Stargardt patients two intronic variants, the novel c.4773+3A>G and c.5461-10T>C, were detected in the ABCA4 gene. One individual was homozygous for the known variant c.5461-10T>C and the other one was compound heterozygote with both variants present. Both variants, c.4773+3A>G and c.5461-10T>C caused exon skipping in HEK293T cells demonstrated by in vitro splice assay, proving their pathogenicity in Stargardt disease. Finally, in recessive retinitis pigmentosa, Bothnia Dystrophy (BD), we identified a second mutation in the RLBP1 gene, c.677T>A, p.Met226Lys. Thus, BD is caused not only by common c.700C>T variant but also by homozygosity of c.677T>A or compound heterozygosity. Notably, known variant, c.40C>T, p.R14W in the CAIV gene associated with a dominant retinal dystrophy RP17 was detected in one of the compound BD heterozygote and his unaffected mother. This variant appears to be a benign variant in the population of northern Sweden. In conclusion, novel genetic causes of retinal dystrophies in northern Sweden were found demonstrating the heterogeneity and complexity of retinal diseases. Identification of the genetic defect in COL17A1 in the corneal dystrophy contributes to understanding ERED pathogenesis and encourages refinement of IC3D classification. Our results provide valuable information for future molecular testing and genetic counselling of the families.
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Liu, Ping. "Structural, Kinetic and Mutational Analysis of Two Bacterial Carboxylesterases." Digital Archive @ GSU, 2007. http://digitalarchive.gsu.edu/biology_diss/26.
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The crystal structures of two thermostable carboxylesterase Est30 and Est55 from Geobacillus stearothermophilus were determined to help understand their functions and applications in industry or medicine. The crystal structure of Est30 was determined at 1.63 Å resolution by the multiple anomalous dispersion method. The two-domain Est30 structure showed a large domain with a modified alpha/beta hydrolase core including a seven, rather than an eight-stranded beta sheet, and a smaller cap domain comprising three alpha helices. A 100 Da tetrahedral ligand, propyl acetate, was observed to be covalently bound to the side chain of Ser94 in the catalytic triad. This ligand complex represents the first tetrahedral intermediate in the reaction mechanism. Therefore, this Est30 crystal structure will help understand the mode of action of all enzymes in the serine hydrolase superfamily. Est55 is a bacterial homologue of the mammalian carboxylesterases involved in hydrolysis and detoxification of numerous peptides and drugs and in prodrug activation. Est55 crystals were grown at pH 6.2 and pH 6.8 and the structures were determined at resolutions of 2.0 and 1.58 Å respectively. Est55 folds into three domains, a catalytic domain, an α/β domain and a regulatory domain. This structure is in an inactive form; the side chain of His409, one of the catalytic triad residues, is pointing away from the active site. Moreover, the adjacent Cys408 is triply oxidized and lies in the oxyanion hole, which would block the entry of substrate to its binding site. This structure suggested a self-inactivation mechanism, however, Cys408 is not essential for enzyme activity. Mutation of Cys408 showed that hydrophobic side chains at this position were favorable, while polar serine was unfavorable for enzyme activity. Both Est30 and Est55 were shown to hydrolyze the prodrug CPT-11 into the active form SN-38. Therefore, Est30 and Est55 are potential candidates for use with irinotecan in cancer therapy. The catalytic efficiency (kcat/Km) of Est30 is about 10-fold lower than that of Est55. The effects of the Cys408 substitutions on Est55 activity differed for the two substrates, p-NP butyrate and CPT-11. Mutant C408V may provide a more stable form of Est55.
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Donnelly, Steven. "Molecular mechanisms underlying mutations in Connexin 26 associated with genetically inherited skins disorders." Thesis, Glasgow Caledonian University, 2013. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.688293.
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Kawanishi, Masanobu. "Molecular mechanisms of mutations induced by environmental pollutants, 3-nitrobenzanthrone, crotonaldehyde and acrolein." Kyoto University, 1998. http://hdl.handle.net/2433/157051.
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本文データは平成22年度国立国会図書館の学位論文(博士)のデジタル化実施により作成された画像ファイルを基にpdf変換したものであるKyoto University (京都大学)
0048
新制・課程博士
博士(工学)
甲第7521号
工博第1762号
新制||工||1124(附属図書館)
UT51-98-W265
京都大学大学院工学研究科環境地球工学専攻
(主査)教授 松井 三郎, 教授 森澤 眞輔, 教授 齋藤 烈
学位規則第4条第1項該当
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Lorenzi, Roberto. "Studies on gene conversion as a mutational mechanism in the evolution of major histocompatibility complex genes." Thesis, University of Cambridge, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.336766.
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Moens, Thomas Grover. "Molecular mechanisms of pathogenesis in Drosophila models of C9orf72 mutation associated ALS/FTD." Thesis, University College London (University of London), 2018. http://discovery.ucl.ac.uk/10046295/.
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A GGGGCC hexanucleotide repeat expansion within the C9orf72 gene is the most common genetic cause of both amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD). Toxicity has been proposed to be due to loss of function of the gene, or by a toxic gain of function, mediated either by the transcription of repetitive sense and antisense RNA molecules, or by translation of RNA into five repetitive dipeptide proteins (DPRs) via repeat associated non-ATG initiated translation. In order to fully assess the role of sense and antisense RNA in vivo, Drosophila models were created where expression of sense or antisense RNA was induced whilst suppressing the formation of DPRs. Despite the formation of cardinal pathological features (RNA binding protein sequestering intranuclear RNA foci) toxicity was not observed in these models suggesting that repeat RNA plays a limited role in disease pathogenesis. When individual DPRs are expressed in Drosophila neurons a strong toxicity is induced by the arginine containing DPRs (poly-GR and poly-PR). To gain insight into the mechanism(s) by which this toxicity occurs, the protein-interactome of these DPRs was investigated in vivo using novel transgenic Drosophila that inducibly express affinity tagged DPR constructs, with identification of interacting proteins using mass spectrometry. In parallel, inclusions of dipeptide proteins were laser-capture microdissected from patient brain tissue and enriched proteins identified by mass spectrometry. The overlap of these datasets suggested that translation may be impaired by the arginine-containing DPRs and methods were adapted to assess the rate of translation in adult Drosophila brains. In parallel, enzymelinked immunosorbent assays (ELISAs) were developed against poly-GR and an abundant non-toxic DPR poly-GP. Measurement of these proteins was performed in various model systems (transfected immortalised cell lines, induced pluripotent stem cell derived neurons, Drosophila models) to confirm the validity of the assays and the potential therapeutic value of interventions.
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Wang, Zhibin. "Molecular mechanism of Arabidopsis CBF mediated plant cold-regulated gene transcriptional activation." Columbus, Ohio : Ohio State University, 2006. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1158600906.
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Liu, Fengling. "Kinetic and Crystallographic Studies of Drug-Resistant Mutants of HIV-1 Protease: Insights into the Drug Resistance Mechanisms." Digital Archive @ GSU, 2007. http://digitalarchive.gsu.edu/biology_diss/19.
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HIV-1 protease (PR) inhibitors (PIs) are important anti-HIV drugs for the treatment of AIDS and have shown great success in reducing mortality and prolonging the life of HIV-infected individuals. However, the rapid development of drug resistance is one of the major factors causing the reduced effectiveness of PIs. Consequently, various drug resistant mutants of HIV-1 PR have been extensively studied to gain insight into the mechanisms of drug resistance. In this study, the crystal structures, dimer stabilities, and kinetics data have been analyzed for wild type PR and over 10 resistant mutants including PRL24I, PRI32V, PRM46L, PRG48V, PRI50V, PRF53L, PRI54V, PRI54M, PRG73S and PRL90M. These mutations lie in varied structural regions of PR: adjacent to the active site, in the inhibitor binding site, the flap or at protein surface. The enzymatic activity and inhibition were altered in mutant PR to various degrees. Crystal structures of the mutants complexed with a substrate analog inhibitor or drugs indinavir, saquinavir and darunavir were determined at resolutions of 0.84 – 1.50 Å. Each mutant revealed distinct structural changes, which are usually located at the mutated residue, the flap and inhibitor binding sites. Moreover, darunavir was shown to bind to PR at a new site on the flap surface in PRI32V and PRM46L. The existence of this additional inhibitor binding site may explain the high effectiveness of darunavir on drug resistant mutants. Moreover, the unliganded structure PRF53L had a wider separation at the tips of the flaps than in unliganded wild type PR. The absence of flap interactions in PRF53L suggests a novel mechanism for drug resistance. Therefore, this study enhanced our understanding of the role of individual residues in the development of drug resistance and the structural basis of drug resistance mechanisms. Atomic resolution crystal structures are valuable for the design of more potent protease inhibitors to overcome the drug resistance problem.
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Fearon, Abbie Elizabeth. "Dissection of drug resistance mechanisms in FGFR2 mutant endometrial cancer." Thesis, Queen Mary, University of London, 2015. http://qmro.qmul.ac.uk/xmlui/handle/123456789/9027.
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Mutations in FGFR2 are common in a subset of endometrial carcinomas. Given the emergence of small molecule inhibitors specific to this receptor tyrosine kinase, FGFR2 is an attractive therapeutic target. However, compensatory and adaptation mechanisms limit the clinical utility of compounds that target nodes in the receptor tyrosine kinase network. Here, we analysed the impact of FGFR inhibition in endometrial cancer cells and observed the emergence of a resistant population in an FGFR2-mutant cell line. To understand the mechanisms underlying this adaptation response, we used a phosphoproteomics approach to measure the kinase network in an unbiased manner. These experiments led to the identification of an AKT-related compensatory mechanism underpinning this resistance. Further dissection of this resistance mechanism utilising gene expression analysis showed PHLDA1, a negative regulator of AKT, was significantly down-regulated in resistant cells. This was further confirmed at the protein level. siRNA knockdown of PHLDA1 conferred immediate drug resistance in the FGFR2-mutant endometrial cancer cell line. Therefore, we identified PHLDA1 down-regulation as a mediator of drug resistance in FGFR2 mutant cancer cells, the first demonstration of the role of PHLDA1 in the acquisition and maintenance of drug resistance. Using a 3D physiomimetic model, we demonstrated that AKT inhibition alone also led to generation of a drug-resistant population. Most importantly, dual-drug therapy inhibited proliferation and induced cell death. Our data highlight how mass spectrometry and microarray gene expression analysis can complement each other in the identification of novel resistance mechanisms in cancer cells. These data suggest that combination treatment of FGFR2-mutant endometrial cancers, targeting both FGFR2 and AKT, represents a promising therapeutic approach.
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Verkerk, Johanna Maria Henriëtta. "The molecular basis of the fragile X syndrome expansion of a trinucleotide repeat, a new mutational mechanism /." [S.l.] : Rotterdam : [The Author] ; Erasmus University [Host], 1994. http://hdl.handle.net/1765/13739.
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Thornburg, Adrienne. "Resolving the molecular mechanisms of inherited deafness caused by missense mutations in cadherin 23." The Ohio State University, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=osu1461284758.
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Sanjuan, Ruiz Inmaculada. "Pathophysiological mechanisms involved in amyotrophic lateral sclerosis caused by mutations in the FUS gene." Thesis, Strasbourg, 2019. http://www.theses.fr/2019STRAJ056.
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La sclérose latérale amyotrophique (SLA) et la démence frontotemporale (DFT) sont deux maladies neurodégénératives incurables. Même si ces deux maladies sont distinctes, elles sont liées par une série de facteurs cliniques, génétiques et histologiques donnant lieu à ce qu’on connaît comme le continuum SLA-DFT. Des mutations du gène FUS sont liées à la SLA, et des défauts de la protéine FUS sont retrouvés dans la DFT, les deux caractérisées par la formation d’agrégats de FUS.Nous avons étudié les mécanismes d’autorégulation de FUS dans un modèle murin, par l’activation d’une voie alternative d’épissage en insérant un transgène FUS humain sauvage, ce qui nous a permis de potentiellement élucider de nouvelles pistes envisageant des thérapies géniques. De plus, nos souris développent des symptômes liés à la DFT. Nos résultats indiquent une altération des synapses corticales qui pourraient être à l’origine des défauts comportementaux que nous avons observés, ainsi que des défauts du système cholinergiqueAmyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are two untreatable neurodegenerative diseases. Even thought they are two distinctive diseases, they share a series of clinical, genetic and histological hallmarks, thus defining the ALS-FTD continuum. Mutations in the FUS gene have been linked with ALS, whereas alterations in FUS proteins have been detected in FTD patients. Both diseases are characterized by the presence of cytosolic FUS aggregates.We have studied the autoregulation mechanisms of FUS in a mouse model via de activation of an alternative splicing pathway by the insertion of a human wild-type FUS transgene, which has allowed us to potentially elucidate new therapeutic approaches by gene therapy. Furthermore, our mice develop FTD-like symptoms. Our results suggest an alteration in cortical synapses which could originate the observed cognitive and behavioural deficits, accompanied by alterations in the cholinergic system
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Segovia, Ugarte Romulo M. "Synthetic hypermutation : gene-drug mutation rate synergy reveals a translesion synthesis mechanism." Thesis, University of British Columbia, 2017. http://hdl.handle.net/2429/61269.
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Gene-gene or gene-drug interactions are typically quantified using fitness as a readout because the data is continuous and easily measured in high-throughput. However, to what extent fitness captures the range of other phenotypes that show synergistic effects is usually unknown. Using Saccharomyces cerevisiae, and focusing on a matrix of DNA repair mutants and genotoxic drugs, I quantified 76 gene-drug interactions based on both mutation rate and fitness and find that these parameters are not connected. Independent of fitness defects I identified seven cases of synthetic hypermutation, where the combined effect of the drug and mutant on mutation rate was greater than predicted. One example occurred when yeast lacking RAD1 were exposed to cisplatin and I characterized this interaction using whole-genome sequencing. Our sequencing results indicate mutagenesis by cisplatin in rad1∆ cells depended almost entirely on interstrand crosslinks at GpCpN motifs. Interestingly, our data suggest that the 3’ base in this motif templates the addition of the mutated base. This result differs from cisplatin mutation signatures in XPF-deficient C. elegans and supports a model in which translesion synthesis polymerases perform a slippage and realignment extension across from the damaged base. Accordingly, DNA polymerase zeta activity was essential for mutagenesis in cisplatin-treated rad1∆ cells. Together these data reveal the potential to gain new mechanistic insights from non-fitness measures of gene-drug interactions and extend the use of mutation accumulation and whole-genome sequencing analysis to define DNA repair mechanisms.Medicine, Faculty of
Medical Genetics, Department of
Graduate
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Moloney, Dominique Martine. "What is the mechanism of the high mutation rate in Apert syndrome." Thesis, University of Oxford, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.284421.
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Sun, Song. "Dynamics and Mechanisms of Adaptive Evolution in Bacteria." Doctoral thesis, Uppsala universitet, Institutionen för medicinsk biokemi och mikrobiologi, 2012. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-172786.
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Determining the properties of mutations is fundamental to understanding the mechanisms of adaptive evolution. The major goal of this thesis is to investigate the mechanisms of bacterial adaptation to new environments using experimental evolution. Different types of mutations were under investigations with a particular focus on genome rearrangements. Adaptive evolution experiments were focused on the development of bacterial resistance to antibiotics. In paper I, we performed stochastic simulations to examine the role of gene amplification in promoting the establishment of new gene functions. The results show that gene amplification can contribute to creation of new gene functions in nature. In paper II, the evolution of β-lactam resistance was studied by evolving S. typhimurium carrying a β-lactamase gene towards increased resistance against cephalosporins. Our results suggest that gene amplification is likely to provide an immediate solution at the early stage of adaptive evolution and subsequently facilitate further stable adaptation. In paper III, we isolated spontaneous deletion mutants with increased competitive fitness, which indicated that genome reduction could be driven by selection. To test this hypothesis, independent lineages of wild type S. typhimurium were serially passaged for 1000 generations and we observed fixation of deletions that significantly increased bacterial fitness when reconstructed in wild type genetic background. In paper IV, we developed a new strategy combining 454 pyrosequencing technology and a ‘split mapping’ computational method to identify unique junction sequences formed by spontaneous genome rearrangements. A high steady-state frequency of rearrangements in unselected bacterial populations was suggested from our results. In paper V, the rates, mechanisms and fitness effects of colistin resistance in S. typhimurium were determined. The high mutation rate and low fitness costs suggest that colistin resistance could develop in clinical settings. In paper VI, a novel Metallo-β-lactamase (MBL) with low resistance against β-lactam antibiotics was employed as the ancestral protein in a directed evolution experiment to examine how an enzyme evolves towards increased resistance. For most isolated mutants, in spite of their significantly increased resistance, both mRNA and protein levels were decreased as compared with the parental protein, suggesting that the catalytic activity had increased.
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Zorzet, Anna. "Mechanisms of Adaptation to Deformylase Inhibitors." Doctoral thesis, Uppsala universitet, Institutionen för medicinsk biokemi och mikrobiologi, 2010. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-123242.
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Antibiotic resistance is a growing problem on a global scale. Increasing numbers of bacteria resistant toward one or multiple antibiotics could return us to the high mortality rates for infectious diseases of the pre-antibiotic era. The need for development of new classes of antibiotics is great as is increased understanding of the mechanisms underlying the development of antibiotic resistance. We have investigated the emergence of resistance to peptide deformylase inhibitors, a new class of antibiotics that target bacterial protein synthesis. The fitness of resistant mutants as well as their propensity to acquire secondary compensatory mutations was assessed in order to gain some insight into the potential clinical risk of resistance development. Most of this work was done in the bacterium Salmonella typhimurium, due to the availability of excellent genetic tools to study these phenomena. In addition, we have studied the bacterium Staphylococcus aureus as peptide deformylase inhibitors have been shown to have the greatest effect on Gram-positive organisms. In the course of this work we also examined the mechanistic aspects of translation initiation. Using a cell-free in vitro translation system we studied the effects of various components on translation initiation. These results have been combined with results obtained from resistant and compensated bacterial strains in vivo to gain new insights into the mechanisms of translation initiation.
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Jamieson, Quentin. "The Inactivation Mechanisms of Shaker IR and Kv2.1 Potassium Channels: Lessons from Pore Mutation." Case Western Reserve University School of Graduate Studies / OhioLINK, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=case1396357775.
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Hassan, A. Quamrul. "Molecular complementation of mutant thyroid hormone receptors that disrupt transactivation mechanism." Access to citation, abstract and download form provided by ProQuest Information and Learning Company; downloadable PDF file 9.11 Mb., 175 p, 2006. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&res_dat=xri:pqdiss&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&rft_dat=xri:pqdiss:3205433.
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Chatron, Nolan. "VKORC1 et résistance aux antivitamines K : étude par modélisation moléculaire." Thesis, Université Paris-Saclay (ComUE), 2017. http://www.theses.fr/2017SACLN008/document.
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VKORC1 est une enzyme membranaire du réticulum endoplasmique, responsable de la réduction de la vitamine K époxyde en vitamine K quinone, activant la synthèse des facteurs de coagulation. VKORC1 constitue ainsi une cible thérapeutique privilégiée des anticoagulants de type anti-vitamine K (AVKs). Cependant, certaines mutations de VKORC1 induisent une dérégulation physiologique et/ou une résistance aux AVKs. En l’absence de données structurales, plusieurs modèles topologiques de VKORC1 ont été proposés à partir des données biochimiques et biophysiques, fréquemment contradictoires. La topologie de VKORC1 ainsi que l'implication des résidus de cystéine (strictement conservés chez toutes les VKORs) dans le mécanisme enzymatique de la protéine restent incertaines. Nous avons construit, par des méthodes in silico, un modèle 3D de la VKORC1 humaine sauvage (hVKORC1WT) à l'échelle atomique. Des simulations de dynamique moléculaire de la protéine, en considérant tous les résidus de cystéine sous leur forme oxydée (SH), nous ont permis d'identifier les résidus de cystéine les plus susceptibles de former des ponts disulfures. Nous avons par conséquent décrit les conformations métastables de hVKORC1WT mimant les états fonctionnels de la protéine. L’étude de la reconnaissance des formes époxyde et réduites de la vitamine K par les conformations prédites de hVKORC1WT a mis en évidence leur sélectivité réciproque. Les conformations de hVKORC1WT ciblées par chaque forme de la vitamine K et leur rôle dans le mécanisme de réduction de la molécule ont ainsi été caractérisés. Nous avons postulé à partir de ces résultats le mécanisme enzymatique détaillé de hVKORC1WT et proposé la structure-cible des AVKs. Les interactions entre la cible prédite - hVKORC1WT à l'état actif - et trois AVKs différents ont été décrites en termes d’énergie libre de liaison. Ces résultats ont été corrélés avec les constantes d'inhibition mesurées in vivo, validant nos prédictions théoriques. Notre protocole, développé pour hVKORC1WT, est applicable aux formes mutées de l’enzyme. Il permettra la description des mécanismes modifiant l'activité réductase de hVKORC1 et/ou provoquant une résistance aux AVKs. Cette description ouvre la voie à la conception d'une nouvelle génération d'inhibiteurs surmontant les résistances aux AVKs. Notre concept et le protocole établi pour l’étude de hVKORC1 peuvent être étendus à l'analyse des VKORs mammaliennes et aux autres enzymes de la famille des oxydoréductasesVKORC1 is an endoplasmic reticulum membrane-resident enzyme, responsible for vitamin K epoxide reduction to vitamin K quinone that activates coagulation factors synthesis. VKORC1 is thus a prominent target of vitamin K agonists (VKAs) in anticoagulant therapies. However, some VKORC1 mutations lead to physiological dysregulation and/or VKAs resistance. No VKORC1 structural data is available, and postulated topological models are based on biochemical and biophysical experimental observations frequently contradicting. Topology of VKORC1 and involvement of cysteines residues (highly conserved in VKORs) in the protein enzymatic mechanism remain unclear. We built an in silico 3D model of the wild-type human VKORC1 (hVKORC1WT) at the atomistic scale. Molecular dynamics simulations of the protein model, carrying all the cysteines residues in their oxidized form (SH), were used for identification of cysteines residues which may plausibly form disulfide bridges. We thus described hVKORC1WT metastable conformations depicting the functionally relevant states of protein. Study of the vitamin K (in epoxide and in reduced forms) recognition by the predicted conformations of hVKORC1WT revealed their reciprocal selectivity. The hVKORC1WT conformations targeted by each vitamin K form were established and their role in the reduction mechanism of this molecule was explained. Using our results, we postulated the comprehensive enzymatic mechanism of hVKORC1WT and we proposed the 3D structure as the VKAs target. Interactions between the predicted target - hVKORC1WT active state - and three different VKAs were characterized. The obtained affinities (free binding energy) were in good correlation with in vivo measured inhibition constants (Ki), thus validating our theoretical predictions. Our protocol developed for hVKORC1WT is suitable for a study of its mutants. Description of the enzymatic mechanisms of mutated hVKORC1 will lead to understanding of the modified reductase activity or/and to explaining of its resistance to VKAs. Such data are crucial for the development of novel strategies in the design of a new generation of inhibitors overcoming VKAs resistance. Our concept and the established in silico protocol can be extended to analysis of mammalian VKORs and other oxidoreductases family proteins
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Sander, Max [Verfasser]. "Die molekularen Mechanismen des angeborenen Wachstumshormonmangels verursacht durch Cystein-Mutationen / Max Sander." Berlin : Medizinische Fakultät Charité - Universitätsmedizin Berlin, 2021. http://d-nb.info/1228860483/34.
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Thibodeau, Isabelle. "Mutations in Connexin43 and Connexin40 Associated with Human Atrial Fibrillation; Physiology and Mechanism." Thesis, University of Ottawa (Canada), 2010. http://hdl.handle.net/10393/28834.
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Atrial Fibrillation (AF), the most common sustained cardiac arrhythmia, is a disease that affects the electrical conductance in the atria. Gap junction channels, composed of connexin (Cx) proteins, allow the electrical signal to propagate between cardiac myocytes. Mutations in two highly expressed cardiac connexin genes, GJA1 (Cx43) and GJA5 (Cx40), have been identified by our group in a cohort of patients with lone AF. To characterize trafficking of a novel somatic frameshift Cx43 mutant and three novel Cx40 mutants, fluorescently tagged connexins were visualized in HeLa cells using confocal microscopy. Electrophysiological studies were performed by a member of our lab to test the function of mutant gap junction channels. The Cx43 frameshift mutant (Cx43-932delC) caused intracellular retention of Cx43, resulting in impaired electrical coupling between paired cells. All three Cx40 mutants (F30L, G311S and R113S) demonstrated normal trafficking and electrical coupling between paired N2A cells. Enhanced hemichannel function was observed for the Cx40-F30L mutant, leading to cell death in Xenopus oocytes. Increased sensitivity to intracellular pH caused closure of gap junction channels composed of Cx40-G311S or Cx40-R113S. Intracellular localization of a previously published Cx40 mutation (P88S) was determined to be retained within the ER and Golgi compartments. Trafficking rescue was observed when P88S was co-expressed with wild-type Cx40 or Cx43. My results and those of our collaborators demonstrate a novel finding that a Cx43 mutation is associated with AF and also further support reported findings on the association of Cx40 mutations with AF.
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Lasser, Micaela Cari. "Using Xenopus laevis to investigate developmental mechanisms underlying human neurodevelopmental disorders and intellectual disabilities:." Thesis, Boston College, 2020. http://hdl.handle.net/2345/bc-ir:108957.
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Thesis advisor: Laura Anne LoweryThesis advisor: Sarah McMenamin
Development of the central nervous system (CNS) is a complex process that requires the proper function of many genes in order for neurons to proliferate and divide, differentiate, and subsequently migrate long distances to form connections with one another. Abnormalities in any one of these cellular processes can lead to detrimental developmental defects. Growing evidence suggests that genetic mutations caused by rare copy number variants (CNVs) are associated with neurodevelopmental disorders including intellectual disabilities (ID), Autism spectrum disorder (ASD), and schizophrenia. Additionally, these pathogenic CNVs are characterized by extensive phenotypic heterogeneity, as affected individuals often present with microcephaly, craniofacial and heart defects, growth retardation, and seizures. Despite their strong association as risk factors towards neurodevelopmental disorders, the developmental role of individual CNV-affected genes and disrupted cellular mechanisms underlying these mutations remains poorly understood. Moreover, it is unclear as to how the affected genes both individually and combinatorially contribute to the phenotypes associated with pathogenic CNVs. Thus, in this thesis, we explore the functional basis of phenotypic variability of pathogenic CNVs linked to neurodevelopmental disorders. In particular, we focus on the 3q29 deletion and 16p12.1 deletion, to provide insight towards the convergent cellular, molecular, and developmental mechanisms associated with decreased dosage of the affected gene homologs using two complementary model systems, Xenopus laevis and Drosophila melanogaster. First, we examine the role of individual homologs of several CNV-affected genes at chromosome 3q29 and their interactions towards cellular processes underlying the deletion. We find that multiple 3q29-affected genes, including NCBP2, DLG1, FBXO45, PIGZ, and BDH1, contribute to disruptions in apoptosis and cell cycle pathways, leading to neuronal and developmental defects. We then expand further upon this work by discerning the individual contribution of four CNV-affected genes at chromosome 16p12.1, POLR3E, MOSMO, UQCRC2, and CDR2, towards neurodevelopment and craniofacial morphogenesis. We demonstrate that several of these genes affect multiple phenotypic domains during neurodevelopment leading to brain size alterations, abnormal neuronal morphology, and cellular proliferation defects. We then explore their functions during vertebrate craniofacial morphogenesis and demonstrate that some 16p12.1-affected genes are enriched in migratory neural crest, and contribute to early craniofacial patterning and formation of cartilaginous tissue structures. Together, these data are the first to suggest that signature neurodevelopmental phenotypes demonstrated in the 3q29 deletion and 16p12.1 deletion may stem from convergent cellular mechanisms including aberrations in neuronal proliferation, apoptosis and cell cycle regulation, and neural crest cell development
Thesis (PhD) — Boston College, 2020
Submitted to: Boston College. Graduate School of Arts and Sciences
Discipline: Biology