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Bruce, David. "Antithrombin : structural variants and thrombosis." Thesis, Open University, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.386084.
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Seabra, Catarina Morais. "Rare structural variants in severe spermatogenic impairment." Master's thesis, Universidade de Aveiro, 2012. http://hdl.handle.net/10773/9537.
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Mestrado em Biomedicina MolecularA azoospermia afeta aproximadamente 15% de todos os homens inférteis e é frequentemente causada por anomalias cromossómicas e microdeleções do cromossoma Y. No entanto, em aproximadamente 70% dos casos de azoospermia não-obstrutiva (NOA) as causas permanecem por identificar. Nos últimos anos, a descoberta de variantes genómicas de número de cópia (CNVs), como as causadas por deleções, revelou uma fonte de variação genómica que afecta a dosagem génica e que poderá resultar em haploinsuficiência. De facto, observa-se uma sobre-representação de CNVs raros (<1% na população), sobretudo de grandes deleções de novo, em pacientes com diferentes distúrbios do desenvolvimento, comparados com controlos saudáveis. Porém, uma possível contribuição, para a infertilidade masculina, de variantes estruturais ligados ao cromossoma X e aos autossomas foi ainda pouco explorada. Este estudo foca-se na validação de deleções encontradas apenas em pacientes inférteis, no cromossoma X e em 11p13, que contêm genes candidatos a participar na espermatogénese. Estas deleções, previamente identificadas por arrays de oligonucleótidos, de elevada densidade (Affymetrix 6.0 SNP Array), numa coorte de 171 pacientes Portugueses com disfunção severa da espermatogénese (NOA e oligozoospermia severa), foram agora confirmadas por técnicas convencionais de genética molecular. Adicionalmente, a caraterização dos locais de quebra nestas deleções foi realizada por aCGH. Ainda que não se tenham validado as deleções menos extensas (em Xq21.1, Xq25, Xp11.4, Xq22.1 e Xq26.3), confirmou-se a nulizigotia em Xq28 nestes indivíduos, que abrange genes candidatos com uma função sugestiva na espermatogénese: MAGE-A8, expresso em testículo e em alguns cancros e o microRNA hsa-miR-4330, envolvido na regulação pós-transcricional de vários genes com expressão na linha germinal. Foi ainda validada, por MLPA, uma deleção extensa num paciente infértil não-sindrómico da nossa coorte. Estes resultados apontam a haploinsuficiência de WT1 como a causa mais provável de azoospermia neste paciente, já que não foram detetadas mutações germinais no alelo restante. Mutações no gene WT1, que codifica um factor de transcrição muito conservado, crucial para o desenvolvimento e manutenção gonadal em mamíferos, geralmente interferem com a ligação desta proteína ao DNA e estão principalmente associadas a síndromes que envolvem anomalias reprodutivas. Motivados pela nossa descoberta de uma deleção de WT1 num homem infértil embora saudável, decidimos abordar a contribuição de mutações exónicas no gene WT1 para a azoospermia isolada. Testámos a hipótese de que mutações localizadas em domínios que não aqueles essenciais à ligação ao DNA pudessem resultar na disfunção não-sindrómica da espermatogénese. Assim, analisámos a sequência codificante de WT1 num subgrupo de 40 pacientes azoospérmicos. Como resultado, descrevemos uma nova variação missense c.185C>T (P130L; ENST00000332351) no primeiro exão de WT1, inserida no domínio proteico de auto-associação. A nova variante descrita deverá ter um impacto menos drástico na função da proteína WT1, comparativamente com as mutações descritas no mesmo exão até à data, as quais resultam em proteínas truncadas e fenótipos severos de disfunção gonadal, incluindo a formação de tumores renais. Estes resultados revelam novos genes candidatos a um papel na espermatogénese e sugerem que a haploinsuficiência de proteínas importantes para o desenvolvimento do sistema reprodutor masculino podem resultar em azoospermia. Estudos futuros poderão clarificar a utilidade dos nossos genes candidatos como biomarcadores da infertilidade masculina. A implementação de novos biomarcadores beneficiaria os doentes azoospérmicos através da melhoria do diagnóstico, aconselhamento genético e acompanhamento destes pacientes, podendo vir a limitar a necessidade de procedimentos invasivos.
Azoospermia affects approximately 15% of all infertile males and it is frequently caused by chromosomal abnormalities and Yq microdeletions. However, despite considerable research efforts in the last decades, in approximately 70% of the cases of non-obstructive azoospermia (NOA) the causes are yet to be identified. In the last years, the discovery of genomic copy number variants, such as those caused by deletions, revealed a source of genomic variation which impacts gene dosage and may result in haploinsufficiency. In fact, rare CNVs (<1% population), mainly large de novo deletions, are over-represented in patients with different developmental disorders, compared to healthy controls. However, a possible contribution of X-linked and autosomal structural variants to male infertility is still largely unexplored. This study focused on the validation of rare patient-specific deletions found on the X chromosome and at 11p13 of infertile patients, which harbor candidate spermatogenesis genes. These deletions had been previously identified by high density oligonucleotide arrays (Affymetrix 6.0 SNP Array), in a cohort of 171 Portuguese patients with severe spermatogenic impairment (non-obstructive azoospermia and severe oligozoospermia) and were now confirmed by conventional molecular genetics techniques. Additionally, breakpoint characterization was carried out by aCGH. In fact, even though the smaller deletions (at Xq21.1, Xq25, Xp11.4, Xq22.1 and Xq26.3) were not validated, we confirmed nullizygosity at Xq28 in two patients, spanning either MAGE-A8, a known cancer-testis antigen, or hsa-miR-4330, a microRNA involved in post-transcription regulation, both with a suggestive role in spermatogenesis pathways. We have also validated by MLPA a large deletion at 11p13, in a non-syndromic infertile patient from our cohort. These results support WT1 haploinsufficiency as the likely cause of azoospermia in this patient, as no other germline mutations were detected in the remaining WT1 copy. Mutations in WT1, an evolutionarily conserved transcription factor crucial for gonadal development and maintenance in mammals, typically interfere with the DNA-binding properties of the protein and are mainly associated with syndromes involving reproductive abnormalities. Motivated by our finding of a WT1 deletion in an infertile but otherwise healthy man we addressed the contribution of WT1 exonic mutations to isolated azoospermia. We reasoned that mutations located in domains not essential for DNA binding could result in non-syndromic spermatogenic impairment. Thus, we analyzed the WT1 coding sequence in a subgroup of 40 azoospermic patients. As a result of the exon screening, we report a novel c.185C>T (P130L; ENST00000332351) WT1 missense variant on exon 1, within the protein self-association domain. While all exon 1 mutations as yet reported result in truncated proteins and severe phenotypes, including the formation of renal tumors, this novel variant is expected to have a milder impact on WT1 function. These results reveal new candidate genes for a role in spermatogenesis and suggest that haploinsufficiency of proteins important for the development of the male reproductive system can lead to azoospermia. Further studies will clarify the utility of our candidate genes as biomarkers of male infertility. The implementation of new biomarkers would benefit azoospermic men by improving diagnosis, genetic counseling and patient care, eventually limiting the need for invasive procedures.
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Toyama, Brandon Hiroyuki. "The structural basis of yeast prion strain variants." Diss., Search in ProQuest Dissertations & Theses. UC Only, 2009. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3378511.
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Lecompte, Lolita. "Structural variant genotyping with long read data." Thesis, Rennes 1, 2020. http://www.theses.fr/2020REN1S054.
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Les variants de structure (SVs) sont des réarrangements génomiques de plus de 50 paires de base et restent encore aujourd'hui peu étudiés malgré les impacts importants qu'ils peuvent avoir sur le fonctionnement des génomes. Récemment, les technologies de séquençage de troisième génération ont été développées et produisent des données de longues lectures qui s'avèrent très utiles car elles peuvent chevaucher les réarrangements. À l'heure actuelle, les méthodes bioinformatiques se sont concentrées sur le problème de la découverte de SVs avec des données de longues lectures. Aucune méthode n'a cependant été proposée pour répondre spécifiquement à la question du génotypage de SVs avec ce même type de données. L'objectif du génotypage de SVs vise pour un ensemble de SVs donné à évaluer les allèles présents dans un nouvel échantillon séquencé. Cette thèse propose une nouvelle méthode pour génotyper des SVs avec des longues lectures et repose sur la représentation des séquences des allèles. Notre méthode a été implémentée dans l'outil SVJedi. Nous avons testé notre outil à la fois sur des données simulées et réelles afin de valider notre méthode. SVJedi obtient une précision élevée qui dépasse les performances des autres outils de génotypage de SVs, notamment des outils de détection de SVs et des outils de génotypage de SVs de lectures courtesStructural Variants (SVs) are genomic rearrangements of more than 50 base pairs. Since SVs can reach several thousand base pairs, they can have huge impacts on genome functions, studying SVs is, therefore, of great interest. Recently, a new generation of sequencing technologies has been developed and produce long read data of tens of thousand of base pairs which are particularly useful for spanning over SV breakpoints. So far, bioinformatics methods have focused on the SV discovery problem with long read data. However, no method has been proposed to specifically address the issue of genotyping SVs with long read data. The purpose of SV genotyping is to assess for each variant of a given input set which alleles are present in a newly sequenced sample. This thesis proposes a new method for genotyping SVs with long read data, based on the representation of each allele sequences. We also defined a set of conditions to consider a read as supporting an allele. Our method has been implemented in a tool called SVJedi. Our tool has been validated on both simulated and real human data and achieves high genotyping accuracy. We show that SVJedi obtains better performances than other existing long read genotyping tools and we also demonstrate that SV genotyping is considerably improved with SVJedi compared to other approaches, namely SV discovery and short read SV genotyping approaches
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Masciangioli, Tina Marie. "Structural and dynamic studies of bacteriorhodopsin and its variants." Diss., Georgia Institute of Technology, 1999. http://hdl.handle.net/1853/30551.
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NASCIMENTO, JÚNIOR Francisco do. "ScreenVar - a biclustering-based methodology for evaluating structural variants." Universidade Federal de Pernambuco, 2017. https://repositorio.ufpe.br/handle/123456789/25375.
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The importance of structural variants as a source of phenotypic variation has grown in recent years. At the same time, the number of tools that detect structural variations using Next- Generation Sequencing (NGS) has increased considerably with the dramatic drop in the cost of sequencing in last ten years. Then evaluating properly the detected structural variants has been featured prominently due to the uncertainty of such alterations, bringing important implications for researchers and clinicians on scrutinizing thoroughly the human genome. These trends have raised interest about careful procedures for assessing the outcomes from variant calling tools. Here, we characterize the relevant technical details of the detection of structural variants, which can affect the accuracy of detection methods and also we discuss the most important caveats related to the tool evaluation process. This study emphasizes common assumptions, a variety of possible limitations, and valuable insights extracted from the state-of-the-art in CNV (Copy Number Variation) detection tools. Among such points, a frequently mentioned and extremely important is the lack of a gold standard of structural variants, and its impact on the evaluation of existing detection tools. Next, this document describes a biclustering-based methodology to screen a collection of structural variants and provide a set of reliable events, based on a defined equivalence criterion, that is supported by different studies. Finally, we carry out experiments with the proposed methodology using as input data the Database of Genomic Variants (DGV). We found relevant groups of equivalent variants across different studies. In summary, this thesis shows that there is an alternative approach to solving the open problem of the lack of gold standard for evaluating structural variants.
A importância das variantes estruturais como fonte de variação fenotípica tem se proliferado nos últimos anos. Ao mesmo tempo, o número de ferramentas que detectam variações estruturais usando Next-Generation Sequencing (NGS) aumentou consideravelmente com a dramática queda no custo de seqüenciamento nos últimos dez anos. Neste cenário, avaliar corretamente as variantes estruturais detectadas tem recebido destaque proeminente devido à incerteza de tais alterações, trazendo implicações importantes para os pesquisadores e clínicos no exame minucioso do genoma humano. Essas tendências têm impulsionado o interesse em procedimentos criteriosos para avaliar os variantes identificados. Inicialmente, caracterizamos os detalhes técnicos relevantes em torno da detecção de variantes estruturais, os quais podem afetar a precisão. Além disso, apresentamos advertências fundamentais relacionadas ao processo de avaliação de uma ferramenta. Desta forma, este estudo enfatiza questões como suposições comuns à maioria das ferramentas, juntamente com limitações e vantagens extraídas do estadoda- arte em ferramentas de detecção de variantes estruturais. Entre esses pontos, há uma muito questão bastante citada que é a falta de um gold standard de variantes estruturais, e como sua ausência impacta na avaliação das ferramentas de detecção existentes. Em seguida, este documento descreve uma metodologia baseada em biclustering para pesquisar uma coleção de variantes estruturais e fornecer um conjunto de eventos confiáveis, com base em um critério de equivalência definido e apoiado por diferentes estudos. Finalmente, realizamos experimentos com essa metodologia usando o Database of Genomic Variants (DGV) como dados de entrada e encontramos grupos relevantes de variantes equivalentes em diferentes estudos. Desta forma, esta tese mostra que existe uma abordagem alternativa para o problema em aberto da falta de gold standard para avaliar variantes estruturais.
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Lee, Seung-Joo. "Structural and functional consequences of disease-related protein variants." Case Western Reserve University School of Graduate Studies / OhioLINK, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=case1269545015.
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Boulding, Hannah. "Identifying causative elements within structural variants associated with developmental disorders." Thesis, University of Oxford, 2013. http://ora.ox.ac.uk/objects/uuid:d9af47cc-1c91-4a66-a6ac-86655f1ff375.
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It has been well established that copy number variation contributes substantially to genetic variation within human populations. However, the extent to which de novo and inherited copy number variants (CNVs) underlie human disease is not well known. In this thesis, I investigate the role of de novo and inherited CNVs in a wide range of developmental abnormalities. First, I compare disease associated and apparently benign CNVs for structural differences, with the aim of identifying distinguishing features of disease causing CNVs. I identified significant enrichments of protein-coding genes, protein-coding genes associated with disease in OMIM and miRNAs amongst disease associated disease. Conversely, inherited CNVs observed in healthy individuals show depletions of these features. Following this, I employ functional enrichment approaches to identify the copy number variable genes within these de novo CNVs that contribute to the patient’s developmental abnormalities. I predict candidate genes for 143 different developmental abnormalities, with 65% of the candidate genes not having been previously associated with disease in OMIM. Through examining the distribution of these candidate genes within the patient’s CNVs, I found evidence of extensive pleiotropy and epistasis as well as a small number of simple additive effects. Finally, I extend my analyses to examine the role of inherited CNVs as the underlying cause of human developmental disorders. I implicate inherited CNVs and their overlapping copy number variable genes in the underlying causes of 45 human developmental abnormalities. Additionally, I re-examine the patients possessing both de novo CNVs and inherited CNVs using functional enrichment analyses. I reveal significant enrichments for a greater number of human developmental abnormalities when combining both the de novo and inherited CNVs, suggesting it is de novo mutations in combination with the inherited genomic background that are responsible for many instances of human developmental abnormalities.
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Suliman, Muna. "Identifying Sortase A Variants With Higher Catalytic Effeciency." VCU Scholars Compass, 2012. http://scholarscompass.vcu.edu/etd/383.
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In the past two decades, the field of protein engineering has evolved rapidly to include new genetic and chemical techniques to alter protein function. Protein engineering seeks to improve enzyme properties through powerful methods that specifically incorporate novel or improved function in proteins. One such method is protein ligation, which is used to selectively link synthetic and recombinant polypeptides. Due to the limitations of current protein labeling techniques, simple site-specific modification methods remain in high demand. Use of enzyme-based labeling has been the focus of various studies because of its substrate specificity. Sortase-mediated transpeptidation is one approach that has been well documented. Staphylococcus aureus sortase A (SrtAstaph), a membrane-anchored cysteine transpeptidase present in gram-positive bacteria, covalently anchors virulence-associated surface proteins to the peptidoglycan cross bridge of the cell wall. SrtAstaph, one of the most characterized sortases, has found numerous applications in the semi-synthesis of protein and peptide conjugates. While current studies have demonstrated the growing range of applications for sortase A, the enzyme itself has seen very few improvements. In steady-state kinetic analysis, the calculated K cat value of SrtAstaph was 2.27 × 10−5 s−1 indicative of its slow in-vitro turnover rate. Due to sortase’s relative inefficiency, several studies documented the use of excessive amounts of the enzyme in vitro (>30μM) or reactions were incubated for long periods. Through the use of directed evolution, we aimed to improve the catalytic activity of sortase A. Using random mutagenesis and an in vivo bacterial-based screen we isolated a variant that showed a 13-fold increase in its catalytic efficiency when compared to wild-type. This sortase mutant will enable more efficient labeling of LPETG-tagged substrates and will provide further insight into the enzyme’s molecular mechanism of catalysis, which is currently limited.
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Boopathy, Sivakumar. "Investigating Structural and Functional Defects in ALS-causing Profilin 1 Variants." eScholarship@UMMS, 2009. http://escholarship.umassmed.edu/gsbs_diss/923.
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Mutations in profilin 1 (PFN1) cause amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease that targets motor neurons. PFN1 is a 15 kDa protein that is best known for its role in actin dynamics. However, little is known about the pathological mechanisms of PFN1 in ALS. In this dissertation, it is demonstrated that certain familial ALS-linked mutations severely destabilize the native conformation of PFN1 in vitro and cause accelerated turnover of the PFN1 protein in neuronal cells. This mutation-induced destabilization can account for the high propensity of ALS-linked variants to aggregate and also provides rationale for their reported functional defects in cell-based assays. The source of this destabilization is illuminated by the crystal structures of several PFN1 proteins, revealing an expanded cavity near the protein core of one ALS variant and predicting a non-surface exposed cavity in another. Functional biochemical experiments point to abnormalities in actin filament nucleation and elongation caused by PFN1 mutants. In HeLa cells, PFN1 is essential for the generation of actin-rich filopodia and expression of mutant PFN1 alters filopodia density further supporting a pathogenesis mechanism involving actin cytoskeleton. Taken together, this dissertation infers that the pathogenesis of ALS due to mutations in PFN1 can be mediated at least by two possibly related mechanisms, a destabilization of the native PFN1 structure and an impact on the actin assembly processes.
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Boopathy, Sivakumar. "Investigating Structural and Functional Defects in ALS-causing Profilin 1 Variants." eScholarship@UMMS, 2017. https://escholarship.umassmed.edu/gsbs_diss/923.
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Mutations in profilin 1 (PFN1) cause amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease that targets motor neurons. PFN1 is a 15 kDa protein that is best known for its role in actin dynamics. However, little is known about the pathological mechanisms of PFN1 in ALS. In this dissertation, it is demonstrated that certain familial ALS-linked mutations severely destabilize the native conformation of PFN1 in vitro and cause accelerated turnover of the PFN1 protein in neuronal cells. This mutation-induced destabilization can account for the high propensity of ALS-linked variants to aggregate and also provides rationale for their reported functional defects in cell-based assays. The source of this destabilization is illuminated by the crystal structures of several PFN1 proteins, revealing an expanded cavity near the protein core of one ALS variant and predicting a non-surface exposed cavity in another. Functional biochemical experiments point to abnormalities in actin filament nucleation and elongation caused by PFN1 mutants. In HeLa cells, PFN1 is essential for the generation of actin-rich filopodia and expression of mutant PFN1 alters filopodia density further supporting a pathogenesis mechanism involving actin cytoskeleton. Taken together, this dissertation infers that the pathogenesis of ALS due to mutations in PFN1 can be mediated at least by two possibly related mechanisms, a destabilization of the native PFN1 structure and an impact on the actin assembly processes.
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Ross, Gordon Andrew. "Biomedical applications of capillary electrophoresis : including the analysis of structural haemoglobin variants." Thesis, Queen Mary, University of London, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.364283.
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Awan, Sarah Jabeen. "Structural and mechanistic studies on E. coli porphobilinogen deaminase and mutant variants." Thesis, University College London (University of London), 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.244212.
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Nalin, Venkat Sameera. "Network Structural Equation Modeling of PV Minimodule Variants Under Indoor Accelerated Exposures." Case Western Reserve University School of Graduate Studies / OhioLINK, 2021. http://rave.ohiolink.edu/etdc/view?acc_num=case1619711989919366.
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MIGGIANO, RICCARDO. "Biochemical and structural studies of the Mycobacterium tuberculosis O6-Methylguanine Methyltransferase and mutated variants." Doctoral thesis, Università del Piemonte Orientale, 2014. http://hdl.handle.net/11579/41372.
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Romain, Sandra. "Identification, génotypage et représentation des variants de structure dans les pangénomes." Electronic Thesis or Diss., Université de Rennes (2023-....), 2024. https://ged.univ-rennes1.fr/nuxeo/site/esupversions/71b8c90f-bac9-4948-9bb1-a4b6d953f322.
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Les variants structuraux (SVs), des variations génomiques de plus de 50 pb, contribuent de manière significative à la diversité génétique et à l'évolution des espèces. La détection et le génotypage précis des SVs est crucial pour comprendre leur rôle dans la variation phénotypique et l'adaptation. Les graphes de variation (VGs) et graphes de pangénomes (PGs), qui représentent les variations génomiques comme des chemins alternatifs dans un graphe, offrent une approche prometteuse pour l'analyse des SVs. Cette thèse explore l'utilisation des VGs et PGs pour la détection et le génotypage des SVs, en se concentrant sur un complexe de quatre espèces de papillons Coenonympha alpins. Deux outils bio-informatiques ont été développés au cours de cette thèse : (1) SVJedi-graph, le premier génotypeur de SVs à partir de lectures longues utilisant un VG pour représenter les SVs, fournissant une précision de génotypage supérieure aux outils de l’état de l’art, en particulier pour les SVs proches et chevauchants, et (2) INVPG-annot, un outil d’identification des inversions dans les PGs, qui a permi de démontrer que les inversions sont représentées par différentes topologies dans les PGs selon l’outil de construction utilisé. L'analyse comparative des génomes des papillons Coenonympha a permis d'identifier douze grandes inversions (≥ 100 kbp) entre les quatre espèces, dont certaines pourraient jouer un rôle dans l'isolement reproductif et l'adaptation locale de deux de ces espèces. Bien que l'approche basée sur les PGs présente des avantages pour la comparaison de génomes, des défis restent à relever pour l'analyse des grands variants comme les inversionsStructural variants (SVs), genomic variations of more than 50 bp, contribute significantly to genetic diversity and species evolution. Accurate detection and genotyping SVs is crucial to understanding their role in phenotypic variation and adaptation. Variation graphs (VGs) and pangenome graphs (PGs), which represent genomic variations as alternative paths in a graph, offer a promising approach for the analysis of SVs. This thesis explores the use of VGs and PGs for the detection and genotyping of SVs, focusing on a complex of four species of alpine Coenonympha butterflies. Two bioinformatics tools were developed during this thesis: (1) SVJedi-graph, the first long-read SV genotyper using a VG to represent SVs, providing a genotyping accuracy superior to state-of-the-art tools, particularly for close and overlapping SVs, and (2) INVPG-annot, a tool for identifying inversions in PGs, which demonstrated that inversions are represented by different topologies in PGs depending on the construction tool used. Comparative analysis of the Coenonympha butterfly genomes identified twelve large inversions (≥ 100 kbp) between the four species, some of which could play a role in the reproductive isolation and local adaptation of two of these species. While the PG-based approach offers advantages for genome comparison, challenges remain for the analysis of large variants such as inversions
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Cumer, Tristan. "Etude des variants structuraux génomiques pour comprendre les processus démographiques et adaptatifs impliqués dans la domestication des petits ruminants." Thesis, Université Grenoble Alpes (ComUE), 2017. http://www.theses.fr/2017GREAV075/document.
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Les variants structuraux génomiques (SVs) composent une large part du polymorphisme observable entre les individus mais leurs impacts sur les processus micro-évolutifs restent mal connus et leur étude à large échelle est rare.La première partie de ce manuscrit est une étude de la bibliographie portant sur les SVs décrits chez les animaux domestiques. Cette partie met en avant l'importance des SVs dans la modification des gènes ou de leur régulation, impactant un grand nombre de traits sélectionnés lors de la domestication, en lien avec la productivité, la morphologie ou encore le comportement.Basée sur l’étude de données de reséquençage de 500 génomes complets de petits ruminants sauvages et domestiques, la seconde partie, ciblant trois SVs décrits dans la bibliographie, a permis (i) de réfuter l’hypothèse d’amplification en lien avec la domestication des copies endogènes protectrices du retrovirus JSRV situées dans la région 6q13 du mouton, (ii) d’identifier des duplications entourant et affectant le gène ASIP qui seraient impliquées dans les modifications de coloration du pelage en lien avec la domestication des petits ruminants, ainsi que (iii) de montrer un potentiel rôle adaptatif d'un haplotype du locus de la beta-globine lié au climat aride chez le mouton.La troisième partie se base sur une recherche sans a priori de l’ensemble des SVs présents dans des génomes complets. Au travers du développement d’une méthode de détection des SVs et de son application, cette partie permet de décrire environ 50k et 20k SVs dans les génomes des Ovis et des Capra. Parmi ces SVs, 135 chez Ovis et 70 chez Capra semblent liés à la domestication et affectent des gènes impliqués dans l’amélioration, l’immunité, la reproduction ou la survie. De plus, les distributions de 130 SVs pour les moutons et 35 SVs pour les chèvres covarient avec des variables environnementales au Maroc. Certains affectent des gènes impliqués dans la morphologie, l’immunité et le métabolisme.Ce travail met ainsi en avant de nombreux variants qui peuvent impacter des gènes et qui ont pu être ciblés lors la domestication initiale, des étapes d’amélioration ultérieure ou de l’adaptation locale des petits ruminants. Il démontre l'importance de prendre en compte les variants structuraux dans les études génomiques visant à décrire les bases génétiques de la domesticationGenomic structural variations (SVs) account for a large part of the polymorphism between individuals, but their impacts on micro-evolutionary processes remain poorly known and large-scale studies are scarce.The first part of this manuscript is a bibliographic study of SVs in domestic animals. This part highlights the importance of SVs in modifying genes or their regulation, impacting a large number of traits selected during domestication and linked to productivity, morphology or behaviour.Based on the study of resequenced data from 500 whole genomes of wild and domestic small ruminants, the second part, targeting three SVs described in the bibliography, allowed (i) to refute the hypothesis of a link between the domestication of sheep and the amplification of endogenous protective copies of the JSRV retrovirus located in the 6q13 region, l, (ii) to identify duplications surrounding and affecting the ASIP gene that could be involved in the coat color changes related to the domestication of small ruminants, as well as (iii) highlight a potential adaptive role to arid climate of an haplotype of beta-globin locus in sheep.In the third part, we conducted a whole genome survey of SVs . Through the development of a SVs detection method and its application, we could detect about 50k and 20k SVs in Ovis and Capra. Of these SVs, 135 and 70 in Ovis and Capra, respectively, appear to be linked with domestication and affect genes involved in improvement, immunity, reproduction or survival. In addition, in Morocco, the distributions of 130 SVs for sheep and 35 SVs for goats covariate with environmental variables. Some of them affect genes involved in morphology, immunity and metabolism.This work highlights that many variants impacting genes might have been targeted during initial domestication and subsequent improvement steps or during the local adaptation of sheep and goats. It demonstrates the importance of considering structural variants in genomic studies to describe the genetic basis of domestication
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Viñas, Jornet Marina. "Identificació de variants en nombre de còpies i correlació clínica en una població adulta amb discapacitat intel·lectual i trastorns psiquiàtrics i/o conductuals." Doctoral thesis, Universitat Autònoma de Barcelona, 2016. http://hdl.handle.net/10803/369041.
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El genoma humà està constituït per 3 bilions de parells de bases, que inclouen aproximadament 20.000-25.000 gens, i presenta una variabilitat en forma de variants en la seqüència i variants estructurals. L’aparició de noves tecnologies moleculars han revelat l’existència d’una gran quantitat de variants en nombre de còpies (CNVs) que representen canvis de dosi (guanys i pèrdues de material) descrits en el 4,8%-9,5% del genoma. Tot i identificar-se en població sana, s’ha reconegut que tenen una contribució en l’expressió gènica, l’estructura proteica i l’estabilitat cromosòmica i, en conseqüència, ha incrementat l’interès per entendre el paper que tenen les CNVs en malalties com la discapacitat intel·lectual i els trastorns psiquiàtrics. La discapacitat intel·lectual afecta entre l’1-3% dels individus i les millores en el seguiment mèdic dels pacients han contribuït en una major supervivència fins a etapes adultes, en les que es posa de manifest una gran incidència de trastorns psiquiàtrics i de la conducta associats.
Amb l’objectiu de determinar l’etiologia genètica del diagnòstic dual de discapacitat intel·lectual i trastorns psiquiàtrics i/o de la conducta en una cohort de 100 adults i identificar CNVs de susceptibilitat per aquesta patologia, s’ha aplicat una estratègia d’anàlisi genètica seqüencial. Inicialment es realitza un cariotip amb bandes G, un cribatge de la síndrome X fràgil i estudis moleculars dirigits a la confirmació de la sospita clínica d’una síndrome específica. Per aquells casos que són negatius, es realitza un cribatge de CNVs submicroscòpiques de les regions subtelomèriques mitjançant multiplex ligation dependent probe amplification i posteriorment un cribatge del genoma amb un array d’hibridació genòmica comparada(aCGH) d’alta resolució (400k).
S’ha establert una elevada freqüència diagnòstica (38%) en la cohort d’adults amb diagnòstic dual. La co-morbiditat d’un segon trastorn psiquiàtric augmenta la probabilitat de causa genètica. S’ha determinat un alt rendiment diagnòstic del cariotip molecular, pel que es proposa l’aCGH com a primera tècnica per l’estudi del diagnòstic dual. Mitjançant la caracterització de les CNVs, s’han identificat gens candidats que predisposen a discapacitat intel·lectual i trastorns psiquiàtrics, majoritàriament implicats en les primeres etapes del desenvolupament, amb expressió a sistema nerviós i de localització sinàptica. Hi ha gens que participen en les vies glutamatèrgiques i de les ubiquitines i gens implicats en mecanismes oxidatius. La valoració del grau de discapacitat intel·lectual, dels trastorns psiquiàtrics, dels trastorns de la conducta i la dismorfologia presents en els pacients ha permès establir una correlació genotip-fenotip, identificant CNVs associades al diagnòstic dual en el 19% dels casos i CNVs en regions candidates: dup3q29 (FBXO45, PAK2), del7q31.1 (IMMP2L), del8p23.1 (MSRA), del8q21.13 (STMN2), dup9p24.2p24.1 (SLC1A1), del10q21.3 (CTNNA3), dup15q14q15.1 (SPRED1), del15q26.2 (MCTP2), dup17q24.1q24.2 (PRKCA). Es determina que la deleció 2p16.3 és un factor de risc per discapacitat intel·lectual i trastorns psiquiàtrics amb una expressivitat variable. Es descriu per primer cop un fenotip dismòrfic comú entre els adults afectats i l’avaluació clínica dels familiars portadors identifica un patró cognitiu i psiquiàtric comú amb diferents nivells de severitat a tots els portadors de la deleció.
L’estudi d’una població adulta aporta nombrosos avantatges, tant als pacients com als familiars, per adequar el pronòstic, seguiment, tractament i consell genètic. A més a més, el coneixement obtingut en pacients adults amb trastorns psiquiàtric pot ser de gran utilitat pels infants amb discapacitat intel·lectual, ja que el diagnòstic precoç n’afavoreix la prevenció mitjançant un seguiment i tractaments específics.ABSTRACT The human genome contains nearly 3 billion base pairs that include around 20.000-25.000 genes. There are two sources of genetic variation among individuals: single nucleotide variants and structural variants. The improvement of molecular technologies has revealed a large amount of copy number variants (CNVs), which represents dose changes (gains and losses) in about 4,8%-9,5% of the genome. The CNVs contribute to the gene expression, protein structure and chromosome stability even if they are found in healthy people. Consequently, there has been a significant increase in the interest to understand the role of CNVs in diseases, such as intellectual disability and psychiatric disorders. Intellectual disability affects between 1¬3% of human population. With improvement in paediatric care, patients are most likely to survive into adulthood, in which is revealed a high incidence of psychiatric and behaviouraldisorders associated. In order to identify the genetic aetiology of dual diagnosis of intellectual disability and psychiatric and/or behavioural disorders in a cohort of 100 adults and to identify CNVs of disease susceptibility, a sequential genetic test workflow was performed. Firstly, G-banded karyotype, Fragile X syndrome screening and specific molecular technologies targeted to confirm a clinical suspicious of a syndrome were applied. In those negative cases, subtelomeric region screening by multiplex ligation dependent probe amplification and then a whole genome screening by high resolution (400k) comparative genomic hybridization array (CGHa) were performed. A high genetic diagnosis frequency (38%) has established in the adult cohort with dual diagnosis. The co-morbidity of a second psychiatric disorder increases the likelihood of genetic cause. The CNV characterization has identified candidate genes for intellectual disability and psychiatric disorder, mostly involved in the early stages of development, high expression in nervous system and synaptic localization. Some genes identified are involved in glutamatergic and ubiquitin pathways or in oxidative status. The assessment of the intellectual disability degree, psychiatric/behavioural disorders and dismorphology allowed us to establish a genotype-phenotype correlation. It has been identified CNVs associated with dual diagnosis in 19% of cases and CNVs in candidate regions: dup3q29 (FBXO45, PAK2) del7q31.1 (IMMP2L) del8p23.1 (MSRA) del8q21.13 (STMN2) dup9p24.2p24.1 (SLC1A1) del10q21.3 (CTNNA3) dup15q14q15.1 (SPRED1) del15q26.2 (MCTP2) dup17q24.1q24.2 (PRKCA). The 2p16.3 deletion is an intellectual disability and a psychiatric disorder risk factor with variable expressivity. For the first time, it has been described a common dysmorphic phenotype on those patients affected by a 2p16.3 deletion in addition to a common cognitive and psychiatric profile with different levels of severity among all carriers. Studies in an adult population provide numerous advantages in both patients and family members. Genetic diagnosis allows to adequate the prognosis, monitoring, treatment and genetic counselling. Moreover, the knowledge obtained in adult patients with psychiatric disorders can be useful for children affected by intellectual disability. The early diagnosis promotes prevention through monitoring and specific treatments.
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Sahlin, Kristoffer. "Algorithms and statistical models for scaffolding contig assemblies and detecting structural variants using read pair data." Doctoral thesis, KTH, Beräkningsbiologi, CB, 2015. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-173580.
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Advances in throughput from Next Generation Sequencing (NGS) methods has provided new ways to study molecular biology. The increased amount of data enables genome wide scale studies of structural variation, transcription, translation and genome composition. Not only is the scale of each experiment large; lowered cost and faster turn-around has also increased the frequency with which new experiments are conducted. With the data growth comes an increase in demand for efficient and robust algorithms — this is a great computational challenge. The design of computationally efficient algorithms are crucial to cope with the amount of data and it is relatively easy to verify an efficient algorithm by runtime and memory consumption. However, as NGS data comes with several artifacts together with the size the difficulty lies in verifying that the algorithm gives accurate results and are robust to different data sets. This thesis focuses on modeling assumptions of mate-pair and paired-end reads when scaffolding contig assemblies or detecting variants. Both genome assembly and structural variation are difficult problems, partly because of a computationally complex nature of the problems, but also due to various noise and artifacts in input data. Constructing methods that addresses all artifacts and parameters in data is difficult, if not impossible, and end-to-end pipelines often come with several simplifications. Instead of tackling these difficult problems all at once, a large part of this thesis concentrates on smaller problems around scaffolding and structural variation detection. By identifying and modeling parts of the problem where simplifications has been made in other algorithms, we obtain an improved solution to the corresponding full problem. The first paper shows an improved model to estimate gap sizes, hence contig placement, in the scaffolding problem. The second paper introduces a new scaffolder to scaffold large complex genomes and the third paper extends the scaffolding method to account for paired-end-contamination in mate-pair libraries. The fourth paper investigates detection of structural variants using fragment length information and corrects a commonly assumed null-hypothesis distribution used to detect structural variants.QC 20150915
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Planas, Fèlix Mercè. "Detection and classification of somatic structural variants, and its application in the study of neuronal development." Doctoral thesis, Universitat de Barcelona, 2020. http://hdl.handle.net/10803/672163.
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The identification and analysis of genomic variation across individuals has been central in biology, first through comparative genomics to answer evolutionary questions, and then in the context of biomedicine, where it is actually becoming central to the study of most diseases. Next generation sequence technologies are allowing the systematic analysis of thousands of different types of genetic variation, enhancing the identification of disease markers and the understanding of the molecular basis of disease. For the past years, there has been a burst of new methodology for genome analysis around diseases coming from hundreds of groups around the world. Specific computational methods and strategies are being designed and improved around the identification and interpretation of genomic variation. The identification and classification of different types of genomic variants in the context of biomedicine is a key and foundational step for the development of a personalized medicine.
This has been particularly central in the field of cancer genomics, which has based the research of the past ten to fifteen years in the sequencing of genomic DNA, and the identification and interpretation of (mostly) somatic and germline variation. Throughout these years, a large number of methods for variant detection have been developed with different action ranges. Despite all these developments, the identification of genomic variants has still room for improvement, not only at the level of sensitivity and specificity, but also at the computational level. Given the emergence of many initiatives for personalized medicine around the world, and the expected number of genomes that will have to be analyzed within health care systems, we require robust algorithms, designed together with a matching implementation that will minimize the computational costs of the analysis. With this aim, during this thesis, I have pushed and designed and implemented an algorithm for the efficient processing of genomic data, in close collaboration with computer scientists of our center that defined the implementation, focusing on lowering the energy and the time of the analysis. This methodology, which relies on a reference free approach of read classification, has been protected with a patent, and is being used as the foundation for the development of SMuFin2, a more accurate and computationally efficient version of the initial SMuFin from 2014. We here show that our method is able to process whole genome sequences very fast and with a minimal energy consumption, compared with existing methods, and that has great potential for the identification of all ranges of variants, including insertions of non-human DNA. Further developments on SMuFin2 are needed to finally assess its full variant calling capabilities.
Despite their great importance and their clear role in the biology of the cell, somatic variation that occurs in healthy tissues has remained diffuse in their roles. In the case of development, some hypotheses have been proposed to explain the observed somatic DNA damage that occurs during brain development (e.g., replication stress). But the real impact and the underlying mechanisms of this somatic variation are not yet understood. In order to seed light on the type and potential functional impact of somatic variation in brain development, we established a new collaboration to identify, and describe somatic DNA rearrangements induced by Pgbd5 during brain development and adult state in 36 mice neural tissue samples. The detection of somatic variants in healthy tissues presents more challenges than in the cancer scenario, where a variant is present in a significant number of cells and is easier to detect. We have identified, classified and interpreted the landscape of somatic variation in neural development and identified interesting differences between adult and embryonic variation load, and specific types of variants, as the potential result of the activity of these transposase-like genes.
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Baugh, Evan H. "Predicting the Effects of Protein Variants using Structural Modeling, Large-Scale Data Integration, and Machine Learning." Thesis, New York University, 2017. http://pqdtopen.proquest.com/#viewpdf?dispub=10247644.
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High-throughput sequencing technologies and new computational techniques for analyzing population genetics data are rapidly improving our understanding of disease susceptibility in humans and adaptation in a wide variety of organisms. These studies often discover nonsynonymous variation with large effects as even a single amino acid change can disrupt the folding, catalytic activity, and physical interactions of proteins. Current estimates predict that every human genome contains 10,000-11,000 nonsynonymous variations and, while we cannot currently characterize all this diversity experimentally, many variants that alter protein function can be identified computationally from destabilization of structural models or amino acid conservation. Methods for annotating variant effects in genome-wide association studies and exome sequencing studies use conservation and other sequence-based features to identify damaging variants but cannot predict the effect these variants have on protein function. Recent studies of de novo variants have demonstrated the power of these methods but also the need for additional information, such as physical models from the Protein Data Bank, to identify causal variants in disease association studies.
I present VIPUR, a computational framework that integrates sequence analysis and structural modeling using the Rosetta protein modeling suite to identify and interpret deleterious protein variants. To train VIPUR, I collected 9,477 protein variants with known effects on protein function from multiple organisms and curated structural models for each variant from crystal structures and homology models. VIPUR can be applied to variants in any organism’s proteome with improved generalized accuracy (AUROC .83) and interpretability (AUPR .87) compared to other methods. I show that VIPUR’s predictions of deleteriousness match the biological phenotypes for pathogenicity in ClinVar despite being trained on a different label. I use VIPUR to interpret mutations associated with inflammation and diabetes, demonstrating the structural diversity of disrupted functional sites and improved interpretation functional effects.
Generalizable tools for interpreting genetic variants are especially needed with individualized exome sequencing, where clear indications of confident predictions are necessary to identify causal variation. I demonstrate VIPUR’s ability to select candidate variants associated with human diseases by predicting the effects of de novo variants associated with Autism Spectrum Disorders (ASD) in the Simons Simplex Collection. Compared to existing methods, VIPUR deleterious predictions have the greatest enrichment for mutations found in children with ASD. VIPUR’s predictions of deleterious effects are easily combined with other protein functional data to produce a small set of candidate genes and variants with specific mechanistic predictions.
Although designed to aid in the discovery of causal variants, VIPUR can also simulate mutations to better understand specific protein functions. The distribution of VIPUR scores across all positions in a protein can be used to highlight conserved residues and provides an overall measure of protein conservation. When applied to levoglucosan kinase, a bacterial enzyme of interest for biofuel processing, VIPUR neutral predictions have a five fold enrichment for beneficial growth mutations. While VIPUR is not designed to detect gain-of-function mutations, this enrichment suggests VIPUR scores can identify potentially beneficial mutations by removing clearly deleterious ones. When applied to TP53, a human protein that is mutated in nearly half of all cancers, VIPUR score trends highlight the most common mutations in the COSMIC database, suggesting other variants that may have similar effects on tumor growth. VIPUR and the large-scale data analysis empowering it will aid in the interpretation of protein variation by providing a detailed feature space to characterize protein functional effects and confident predictions of deleterious variation in Genome-Wide Association Studies, exome sequencing initiatives, and protein engineering.
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Vicars, Caitlyn. "Investigating short structural variants within FUS, RAB27B and TARDBP for associations with sporadic amyotrophic lateral sclerosis." Thesis, Vicars, Caitlyn (2022) Investigating short structural variants within FUS, RAB27B and TARDBP for associations with sporadic amyotrophic lateral sclerosis. Honours thesis, Murdoch University, 2022. https://researchrepository.murdoch.edu.au/id/eprint/66200/.
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Over the past decade, 90% of amyotrophic lateral sclerosis (ALS) clinical trials have failed. Furthermore, 90% of sporadic cases have unknown genetic cause. Short structural variants (sSVs) are repetitive genomic regions implicated in complex diseases, with the potential to be utilised for clinical trial enrichment. The purpose of this study was to investigate sporadic ALS (sALS) missing heritability via interrogating novel sSVs, and to determine their potential as genetic biomarkers. This project aimed to develop assays for characterisation and high-throughput genotyping of sSVs within ALS-linked genes FUS and TARDBP, and the novel gene, RAB27B, to perform case-control analyses. Three loci; FUS rs34242298, TARDBP rs34839760 and RAB27B rs3060044, were selected using an sSV evaluation algorithm. Polymorphic nature was characterised via polymerase chain reaction-based assays and Sanger sequencing. High-throughput genotyping of sALS patients and healthy controls was undertaken via capillary electrophoresis, followed by case-control analyses in RStudio to identify statistically significant (p<0.05) associations with disease risk and/or site-of-onset. Reduced sALS risk was associated with Short rs3060044 alleles with ≤17GT repeats (p<0.05), driven by 16GT allele carriage. Increased disease risk correlated with carriage of a Long allele (>17GT repeats) (p<0.05), driven by the 21GT allele (p=0.00625). Analysis of rs34839760 revealed the 20A allele was associated with bulbar-onset phenotype (p<0.05), while rs34242298 was not associated with sALS. This exploratory study reinforces the requirement for sSV investigation in complex diseases. Two novel sALS genetic loci were identified; rs3060044 and rs34839760, which enhance our understanding of the aetiology and pathomechanisms underlying sALS. This data is the first to implicate RAB27B as an sALS gene, presenting a tantalising direction for future sALS exploration. Replication studies and functional investigations are warranted to explore the role of each sSV in sALS pathology. Ultimately, these genomic markers could be useful as patient stratification tools, which may enable the identification of responders to precision therapeutics.
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Josephides, Joseph Mark. "Genome-wide link between DNA replication and genome instability at the single cell level." Electronic Thesis or Diss., Université Paris sciences et lettres, 2023. http://www.theses.fr/2023UPSLS059.
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La réplication de l'ADN est essentielle pour les cellules, car elle permet de créer les quelque 30 000 milliards de cellules qui composent le corps humain à partir d'un seul zygote lors de l'embryogenèse. De plus, tout au long de la vie humaine, la réplication continue de l'ADN et la division cellulaire sont nécessaires pour remplacer les cellules âgées, mortes ou endommagées. Par conséquent, il est crucial que le programme de réplication de l'ADN fonctionne correctement à chaque division cellulaire. Cependant, de nombreux facteurs de stress, à la fois exogènes et endogènes, remettent régulièrement en question l'intégrité de l'ADN, ce qui entraîne une instabilité du génome. Cette instabilité est une cause majeure de cancers et d'autres maladies humaines.Malgré l'importance du stress de réplication et de l'instabilité génomique dans les cancers, nous ne comprenons pas complètement les mécanismes sous-jacents ni leurs impacts sur le génome. Au cours de la dernière décennie, d'énormes progrès ont été réalisés dans l'analyse des cellules individuelles. L'étude des variants structuraux (VS) au niveau cellulaire est devenue cruciale pour comprendre l'instabilité génomique, en particulier dans des populations cellulaires hétérogènes telles que les échantillons de tumeurs, qui ne peuvent pas être facilement obtenus par des analyses de masse. Des études récentes ont révélé une corrélation importante entre le timing de réplication et l'apparition de VS dans les cancers, montrant que de nombreux VS résultent de mécanismes liés à la réplication. Cependant, il existe un manque d'études détaillées sur les mécanismes précis, en particulier sur les liens entre réplication, transcription et VS au niveau de la cellule unique. Comprendre ces mécanismes est crucial pour lutter contre les principales maladies humaines.Pour répondre à cette question, ce projet développe et utilise de nouvelles méthodes informatiques basées sur l'intelligence artificielle. Il vise à (i) étudier directement le timing de réplication dans les cancers en analysant le nombre de copies au niveau de la cellule unique et (ii) examiner les interactions entre la réplication et les VS au niveau de la cellule unique. Les signatures des VS découvertes dans ce projet pourraient contribuer à améliorer le diagnostic et à définir de meilleures stratégies thérapeutiques. Dans l'ensemble, ce projet permet de mieux comprendre les mécanismes de la cancérogenèse et contribue à améliorer le diagnostic, le pronostic, le traitement et le suivi personnalisé des patientsDNA replication is a vital process of cells. Besides creating the ~30 trillion cells that comprise the human body from a single zygote during embryogenesis, continuous DNA replication and cell division is necessary during the entire human lifespan to replace the old, dead or damaged cells. It is therefore essential that the DNA replication program is correctly executed at each cell division. However, large numbers of exogenous and endogenous replication stresses routinely challenge DNA integrity and lead to genome instability, which is an important cause of cancers and many other human diseases.Although replication stress and genomic instability are two important hallmarks of cancer, we lack full comprehension of the mechanisms that lead to these deregulations and the impacts they have on the genome. During the last decade, great progress has been made in analyses of individual cells. Determination of structure variations (SVs) in single cells has become an important approach to study genomic instability in heterogeneous cell populations, such as tumour samples, that cannot easily be obtained from bulk analyses. Recent studies have revealed that replication timing shows a strong association with the occurrence of SVs in cancers, and large amounts of SVs generated during tumorigenesis result from replication-associated mechanisms. However, studies addressing the direct mechanisms and, in particular, the links between replication, transcription and SVs at the single-cell level are missing. Investigating such mechanisms is critically important to address major human diseases.To address this question, this project develops and uses novel computational methods, based on artificial intelligence, to: (i) directly investigate single cell replication timing (scRT) in cancers by single-cell copy number analysis, and (ii) examine the interactions of replication and SVs at the single cell level. The SV signatures in cancers revealed in this project might help to improve the diagnosis and better define therapeutic strategies. Altogether, this project provides further understanding of the mechanisms of carcinogenesis and contributes to improving the diagnosis, prognosis, treatment and/or personalised monitoring of patients
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Beaven, Gordon. "Structural studies of 2,4'-dihydroxyacetophenone dioxygenase, calexcitin and two plant-like variants of 5-aminolaevulinic acid dehydratase." Thesis, University of Southampton, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.418895.
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MICOZZI, DANIELA. "Exploiting Structural Analysis, in Silico Screening and functional variants characterization to identify novel inhibitors of cytidine deaminase." Doctoral thesis, Università degli Studi di Camerino, 2012. http://hdl.handle.net/11581/401792.
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This thesis work, combining a virtual screening study for cytidine deaminase ligands with a study on the effect that genetic polymorphisms have on the enzyme functionality provide a useful base to: a) understand the mechanism of nucleoside recognition by CDA and identify novel effective inhibitors; and b) study the different properties of said inhibitors toward three distinct naturally occurring CDA variants (K27, Q27 and T70). Ultimately, this may assist the future design of novel CDA inhibitors or antitumor drugs not susceptible to deamination, with the aim to get more effective personalized drug therapies.
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Romagnoli, Simone. "Identification of Structural Variants in Acute Myeloid Leukemia with normal karyotype patients by using long-reads sequencing technology." Doctoral thesis, Università di Siena, 2021. http://hdl.handle.net/11365/1157520.
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Acute Myeloid Leukemia (AML) accounts for approximately 25% of all leukemias in adults in the Western world, and therefore is the most frequent form of blood neoplasia. Leukemic stem cells show abnormal proliferation, activation of antiapoptotic pathways and the impairment normal cell differentiation resulting in the dysregulated production of not functional blood cells, known as blast. AML is an aggressive disease, with a relative survival rate for all ages 5 years after diagnosis of 29.5%, the clinical manifestations of AML reflect the accumulation of malignant, poorly differentiated myeloid cells within the bone marrow, peripheral blood and in other organs. Diagnostic tests are mainly constituted by blood cells count and morphology, AML diagnosis is established by the presence of >=20% myeloid blasts in the bone marrow or peripheral blood. The prognostic assessment of AML patients is of capital importance for the management of the disease and to set up risk adapted therapies. Although clinical factors play an important role in disease development, karyotype is the most independent prognostic factor to forecast patients’ survival and it is adopted to provide the framework for risk-adapted treatment approach (Deschler and Lübbert, 2006; De Kouchkovsky and Abdul-Hay, 2016). The European Leukemia Net (ELN) guidelines aims to standardize risk stratification in adult AML patients by incorporating cytogenetic and known molecular abnormalities in hot spot genes. Accordingly, AML patients could be stratified into distinct prognostic risk groups (favorable, intermediate or adverse) based on their cytogenetic and molecular profile. Although this classification is the gold standard for the stratification of patients, it is fulfilled for only the 75% of AML whereas it is poorly satisfying for those patients resulted with normal karyotype (nk) at the conventional cytogenetic analysis. Normal karyotype AML (nkAML) patients mostly belong to the intermediate risk category but they experience an extremely heterogeneous outcome that represents an unmet needs in the clinical context of AML (De Kouchkovsky and Abdul-Hay, 2016; Döhner et al., 2017). In the last few years, large-scale tumour-sequencing studies have demonstrated that the majority of cancers, including hematologic neoplasia, are driven by Structural Variants (SVs) that are, for instance, genomic rearrange- ments larger than 50 bp. SVs include insertions, translocations, inversions and Copy Number Alterations (CNAs) (deletions and duplications). The recent development of high-throughput sequencing platforms provided impressive insights into leukemia pathogenesis and contributed to consider SVs as the hallmark of the genome instability leading to the establishment of the neoplasia. Beside karyotype, SVs detection is currently addressed by Next Generation Sequencing (NGS) technologies that allow the simultaneous and accurate detection of recurrent SVs breakpoints (Schütte et al., 2019), nothwithstanding, NGS faces inaccuracy and limitations when applied to resolve wide and structurally complex SVs due to the short length (100-500 bp) of the sequencing read employed (Norris et al., 2016).
In this study, we exploited the long-reads Oxford Nanopore Sequencing technology to explore the genome of a cohort of 152 AML patient with normal cytogenetics, aiming to address the genomic analysis challenges and to identify new potential genomic biomarkers able to refine the prognostic forecasting for nkAML patients. Of 152 bone marrow samples collected at diagnosis, 85 referred to the hematology unit of the A.O.U.Careggi and 67 were prospectively collected for the AML #1310 study by the Italian Hematologic Network GIMEMA (Venditti et al., 2019).
The DNA purified from nkAML samples was used to sequence the whole genome by the nanopore long-reads approach and further analysed by the bioinformatic pipeline specifically developed for SVs calling. Two SVs caller, Sniffles (Sedlazeck et al., 2018) and cuteSV (Jiang et al., 2020), were employed for the identification of an high-confidency callset of SVs that were further clustered and filtered before correlating them with patients’ outcome data. We employed an univariate Cox proportional-hazards analysis to weight the correlation between patients’ survival and each predictor variables. Further, to better estimate the cumulative impact of multiple genome and clinical variables, we developed a multi- variate Cox regression model including those SVs selected by Cox univariate model (pvalue <.05) and other predictors such as age, white blood cells count and the known molecular abnormalities in specific hotspot genes included in the ELN guidelines (Fms related Receptor Tyrosine Kinase 3 (FLT3)-ITD, Nucleophosmin 1 (NPM1), CCAAT Enhancer Binding Protein alpha (CEBPa)). Multivariate analysis allowed to select 12 SVs, represented by genomic deletions or insertions, with high impact on patients’ leukemia free and Overall Survival (OS). Of those, 8 resulted with an HR >1 (also referred as High Risk SVs (hrSVs)), thus associated with an increased risk of death, the other with an Hazard Ratio (HR) <1 (also referred as Low-risk SVs (lrSVs)) were associated to a reduced risk of death. The following stratification of the study cohort based on the presence of hrSVs enabled the identification of a high risk group of patients (accounting for the 17% of the cohort) with an extremely poor survival (median OS time 8.27 months for the group harbouring the hrSVs compared to 62.7 month fo the other, LogRank pvalue <.0001) and a low rate of response to therapy (46% for the patients with hrSVs compared to the 80%, pvalue <.0001). Taking together, these data suggest that the employ of an emerging long-reads sequencing technology capable to detect wide SVs together with a dedicated analysis pipeline could represent a powerful tool to accurately screen the whole genome of AML patients and identify new genomic biomark- ers for the prognostic assessment of nkAML patients capable to refine the actual ELN prognostic assessment in our cohort.
inversions and Copy Number Alterations (CNAs) (deletions and duplications). The recent development of high-throughput se- quencing platforms provided impressive insights into leukemia pathogenesis and contributed to consider SVs as the hallmark of the genome instability leading to the establishment of the neo- plasia. Beside karyotype, SVs detection is currently addressed by Next Generation Sequencing (NGS) technologies that allow the simultaneous and accurate detection of recurrent SVs breakpoints (Schütte et al., 2019), nothwithstanding, NGS faces inaccuracy and limitations when applied to resolve wide and structurally com- plex SVs due to the short length (100-500 bp) of the sequencing read employed (Norris et al., 2016).
In this study, we exploited the long-reads Oxford Nanopore Se- quencing technology to explore the genome of a cohort of 152 AML patient with normal cytogenetics, aiming to address the genomic analysis challenges and to identify new potential genomic biomarkers able to refine the prognostic forecasting for nkAML patients. Of 152 bone marrow samples collected at diagnosis, 85 referred to the hematology unit of the A.O.U.Careggi and 67 were prospectively collected for the AML #1310 study by the Italian Hematologic Network GIMEMA (Venditti et al., 2019).
The DNA purified from nkAML samples was used to sequence the whole genome by the nanopore long-reads approach and further analysed by the bioinformatic pipeline specifically developed for SVs calling. Two SVs caller, Sniffles (Sedlazeck et al., 2018) and cuteSV (Jiang et al., 2020), were employed for the identification of an high-confidency call-set of SVs that were further clustered and filtered before correlating them with patients’ outcome data. We employed an univariate Cox proportional-hazards analysis to weight the correlation between patients’ survival and each predic- tor variables. Further, to better estimate the cumulative impact of multiple genome and clinical variables, we developed a multi- variate Cox regression model including those SVs selected by Cox univariate model (pvalue <.05) and other predictors such as age, white blood cells count and the known molecular abnormalities in specific hotspot genes included in the ELN guidelines (Fms related Receptor Tyrosine Kinase 3 (FLT3)-ITD, Nucleophosmin 1 (NPM1), CCAAT Enhancer Binding Protein alpha (CEBPa)). Multivariate analysis allowed to select 12 SVs, represented by genomic deletions or insertions, with high impact on patients’
leukemia free and Overall Survival (OS). Of those, 8 resulted with an HR >1 (also referred as High Risk SVs (hrSVs)), thus associ- ated with an increased risk of death, the other with an Hazard Ratio (HR) <1 (also referred as Low-risk SVs (lrSVs)) were as- sociated to a reduced risk of death. The following stratification of the study cohort based on the presence of hrSVs enabled the identification of a high risk group of patients (accounting for the 17% of the cohort) with an extremely poor survival (median OS time 8.27 months for the group harbouring the hrSVs compared to
62.7 month fo the other, LogRank pvalue <.0001) and a low rate of response to therapy (46% for the patients with hrSVs compared to the 80%, pvalue <.0001). Taking together, these data suggest that the employ of an emerging long-reads sequencing technology capable to detect wide SVs together with a dedicated analysis pipeline could represent a powerful tool to accurately screen the whole genome of AML patients and identify new genomic biomark- ers for the prognostic assessment of nkAML patients capable to refine the actual ELN prognostic assessment in our cohort.
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Loegler, Victor. "The genotype-phenotype relationship through the pangenome perspective." Electronic Thesis or Diss., Strasbourg, 2024. http://www.theses.fr/2024STRAJ071.
Full textAbstract:
La variation génomique au sein d'une espèce constitue la base de la variation phénotypique héréditaire sur laquelle agit la sélection naturelle. Cependant, explorer le rôle des variants structurels (SV, plus de 50 paires de bases) sur la variation des traits reste un défi en raison de la difficulté à les détecter. Cette thèse vise à étudier l'impact phénotypique de ces variants en utilisant une population de plus de 1000 isolats de la levure Saccharomyces cerevisiae. La construction du pangénome à l'aide d'assemblages quasi-télomères-à-télomères a permis la création d'un catalogue complet de variants génomiques. Des études d'association avec plus de 8 000 caractères ont révélé l'impact relativement plus important des SV sur la variation des caractères, ainsi que des divergences entre l’architecture génétique de différents types de caractères. L'ensemble de ces travaux met en évidence le large impact phénotypique des grands variants génomiques au niveau de l'espèceGenomic variation within a species provides the basis for the heritable phenotypic variation upon which natural selection acts. However, exploring the role of structural variants (SVs, more than 50 base pairs) on trait variation remains a challenge due to the difficulty of detecting them. This thesis research aims to address the phenotypic impact of such variants by leveraging a natural population of over a thousand isolates of the budding yeast Saccharomyces cerevisiae. Pangenome construction using near telomere-to-telomere assemblies enabled the creation of a comprehensive catalog of genomic variants. Association studies with more than 8,000 molecular and organismal traits revealed the relatively higher impact of SVs on traits variation, and discrepancies in the genomic basis of different types of traits. Together, this work highlights the strong phenotypic effect of large genomic variants at the species level
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Gordon, Lynsey. "Biosynthesis of the natural and novel structural variants of calcium-dependent antibiotic produced by streptomyces coelicolor A3(2)." Thesis, University of Manchester, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.506564.
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The aims of this project are to better understand the biosynthesis of CDA and then use that knowledge to genetically manipulate Streptomyces to produce novel compounds. The natural structural variants of CDA which have been characterised contain several unusual amino acids residues. CDA contains both D-figured and non-proteinogenic residues within its structure. In all of the CDA structures isolated so far, position six is occupied by a D-figured hydroxyphenylglycine residue (D-4-HPG). The precursor biosynthetic pathway for HPG has been elucidated in A. orientalis and it has been shown previously that the cda cluster contains homologues to these genes. A mutant strain of S. coelicolor in which the hmaS (4-hydroxymandelic acid synthase) gene had been disrupted had previously been created and shown to be deficient in CDA production. During this study the proposed intermediates of the HPG biosynthetic pathway were fed into the mutant and CDA production re-established thus proving the pathway for HPG biosynthesis in S. coelicolor mirrors that seen in other organisms. Feeding analogues of the pathway intermediates to the mutants resulted in the mutasynthesis of novel lipopeptides with modified arylglycine residues. This study also identified a gene from the cda cluster, hasP, which in silico analysis predicted to encode a 3-hydroxyasparaginyl phosphotransferase responsible for the biosynthesis of 3-phosphohydroxyasparagine sometimes found at position nine in the CDA structure. Deletion of the majority of the gene resulted in only non-phosphorylated CDA variants being produced.
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Tica, Jelena [Verfasser], and Jan [Akademischer Betreuer] Korbel. "Investigating origin and functional impact of genomic structural variants with next-generation sequencing / Jelena Tica ; Betreuer: Jan Korbel." Heidelberg : Universitätsbibliothek Heidelberg, 2015. http://d-nb.info/1180499433/34.
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Imsland, Freyja. "Monogenic Traits Associated with Structural Variants in Chicken and Horse : Allelic and Phenotypic Diversity of Visually Appealing Traits." Doctoral thesis, Uppsala universitet, Institutionen för medicinsk biokemi och mikrobiologi, 2015. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-259621.
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Domestic animals have rich phenotypic diversity that can be explored to advance our understanding of the relationship between molecular genetics and phenotypic variation. Since the advent of second generation sequencing, it has become easier to identify structural variants and associate them with phenotypic outcomes. This thesis details studies on three such variants associated with monogenic traits. The first studies on Rose-comb in the chicken were published over a century ago, seminally describing Mendelian inheritance and epistatic interaction in animals. Homozygosity for the otherwise dominant Rose-comb allele was later associated with reduced rooster fertility. We show that a 7.38 Mb inversion is causal for Rose-comb, and that two alleles exist for Rose-comb, R1 and R2. A novel genomic context for the gene MNR2 is causative for the comb phenotype, and the bisection of the gene CCDC108 is associated with fertility issues. The recombined R2 allele has intact CCDC108, and normal fertility. The dominant phenotype Greying with Age in horses was previously associated with an intronic duplication in STX17. By utilising second generation sequencing we have examined the genomic region surrounding the duplication in detail, and excluded all other discovered variants as causative for Grey. Dun is the ancestral coat colour of equids, where the individual is mostly pale in colour, but carries intensely pigmented primitive markings, most notably a dorsal stripe. Dun is a dominant trait, and yet most domestic horses are non-dun in colour and intensely pigmented. We show that Dun colour is established by radially asymmetric expression of the transcription factor TBX3 in hair follicles. This results in a microscopic spotting phenotype on the level of the individual hair, giving the impression of pigment dilution. Non-dun colour is caused by two different alleles, non-dun1 and non-dun2, both of which disrupt the TBX3-mediated regulation of pigmentation. Non-dun1 is associated with a SNP variant 5 kb downstream of TBX3, and non-dun2 with a 1.6 kb deletion that overlaps the non-dun1 SNP. Homozygotes for non-dun2 show a more intensely pigmented appearance than horses with one or two non-dun1 alleles. We have also shown by genotyping of ancient DNA that non-dun1 predates domestication.
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Sekar, Aswin. "A natural allelic series of complex structural variants and its influence on the risk of lupus and schizophrenia." Thesis, Harvard University, 2014. http://nrs.harvard.edu/urn-3:HUL.InstRepos:13070061.
Full textAbstract:
The human genome's strongest influences on two common diseases, systemic lupus erythematosus (SLE) and schizophrenia, arise from genetic variation in the Human Leukocyte Antigen (HLA) locus. However, the genes and functional alleles driving these genetic relationships have remained unknown. We hypothesized that a complex, multi-allelic form of structural variation in the Complement component 4 (C4) gene, within the HLA locus, underlies these relationships.
Loci that exist in many structural forms and vary widely in copy number have been difficult to analyze molecularly. As a result, we know little about their population genetic properties or their influence on phenotypes. In this work, we developed molecular and statistical methods to characterize such loci and to evaluate their contribution to phenotypes.
Applying these methods to the C4 locus, we found that C4 segregates in four common and at least eleven low-frequency structural forms in human populations. Although there was only partial correlation between C4 structural variation and individual single nucleotide polymorphisms (SNPs), we developed an imputation approach to enable statistical prediction of C4 structural states from flanking SNP haplotypes.
C4 structural variation associated to gene expression in lymphoblastoid cell lines and human brain tissue. Applying our imputation strategy to SLE and schizophrenia case-control cohorts totaling > 75,000 individuals, we found that structural variation in C4 contributes to risk of both phenotypes in a manner predicted by its effect on gene expression in relevant tissues, and with largely opposite directions of effect - alleles that were protective for schizophrenia increased risk for SLE, and vice versa. Leveraging a natural allelic series of C4 structural forms, we developed a novel form of association testing and showed that the association to C4 is unlikely to be caused by correlation with HLA SNPs. C4 was expressed in human neurons, whereas other upstream complement pathway genes were expressed primarily by microglia. Mice lacking C4 showed a deficit in synaptic pruning that was rescued by human C4.
The methods developed in this thesis enable analysis of complex structural variation, and our results identify a novel form of genome variation as making a strong contribution to phenotypes.
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Hanson, Christopher Jon. "Exploration of the Gossypium raimondii Genome Using Bionano Genomics Physical Mapping Technology." BYU ScholarsArchive, 2018. https://scholarsarchive.byu.edu/etd/6854.
Full textAbstract:
Cotton is a crop with a large global economic impact as well as a large, complex genome. Most industrial cotton production is from two tetraploid species (Gossypium hirsutum L. and Gossypium barbadense L.) which contain two subgenomes, specifically the AT and DT subgenomes. The DT subgenome is nearly half the size of the AT subgenome in tetraploid cotton and is closely related to an extant D-genome Gossypium species, G. raimondii Ulbr. Characterization of the structural variants present in diploid D-genome should provide greater insight into the evolution of the DT subgenome in the tetraploid cotton. Bionano (BNG) optical mapping uses patterns of fluorescent labels inserted at specific endonuclease sites to create physical maps of the genomes which can then be examined for structural variation. To develop optical maps in G. raimondii, we first developed a de novo PacBio long read sequence assembly of G. raimondii. This sequence assembly consisted of 2,379 contigs, an average contig length of 413 Kb and a contig N50 of 4.9 Mb. Using BNG technology, we developed two optical maps of the diploid D genome of G. raimondii. One was created using the Nt.BssSI endonuclease and one with the Nt.BspQI endonuclease. Using the BNG optical maps, the PacBio assembly was hybrid scaffolded into 100 scaffolds (+ 5 unscaffolded contigs) with an average scaffold length of 7.5 Mb and a scaffold N50 of 13.1 Mb. A comparison between the Nt. BssSI BNG optical map and the two sequence assemblies identified 3,195 structural variants. These were used to validate the accuracy of the reference sequence of G. raimondii and structural variants were used to create a new phylogeny of nine major cotton species.
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Moss, Tiffanie. "CHARACTERIZATION OF STRUCTURAL VARIANTS AND ASSOCIATED MICRORNAS IN FLAX FIBER AND LINSEED GENOTYPES BY BIOINFORMATIC ANALYSIS AND HIGH-THROUGHPUT SEQUENCING." Case Western Reserve University School of Graduate Studies / OhioLINK, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=case1333648149.
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Blissing, Annica. "Thiopurine S-methyltransferase - characterization of variants and ligand binding." Licentiate thesis, Linköpings universitet, Kemi, 2017. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-136558.
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Thiopurine S-methyltransferase (TPMT) belongs to the Class I S-adenosylmethionine-dependent methyltransferase (SAM-MT) super family of structurally related proteins. Common to the members of this large protein family is the catalysis of methylation reactions using S-adenosylmethionine (SAM) as a methyl group donor, although SAM-MTs act on a wide range of different substrates and carry out numerous biologically important functions. While the natural function of TPMT is unknown, this enzyme is involved in the metabolism of thiopurines, a class of pharmaceutical substances administered in treatment of immune-related disorders. Specifically, methylation by TPMT inactivates thiopurines and their metabolic intermediates, which reduces the efficacy of clinical treatment and increases the risk of adverse side effects. To further complicate matters, TPMT is a polymorphic enzyme with over 40 naturally occurring variants known to date, most of which exhibit lowered methylation activity towards thiopurines. Consequently, there are individual variations in TPMTmediated thiopurine inactivation, and the administered dose has to be adjusted prior to clinical treatment to avoid harmful side effects. Although the clinical relevance of TPMT is well established, few studies have investigated the molecular causes of the reduced methylation activity of variant proteins. In this thesis, the results of biophysical characterization of two variant proteins, TPMT*6 (Y180F) and TPMT*8 (R215H), are presented. While the properties of TPMT*8 were indistinguishable from those of the wild-type protein, TPMT*6 was found to be somewhat destabilized. Interestingly, the TPMT*6 amino acid substitution did not affect the functionality or folding pattern of the variant protein. Therefore, the decreased in vivo functionality reported for TPMT*6 is probably caused by increased proteolytic degradation in response to the reduced stability of this protein variant, rather than loss of function. Also presented herein are novel methodological approaches for studies of TPMT and its variants. Firstly, the advantages of using 8-anilinonaphthalene-1-sulfonic acid (ANS) to probe TPMT tertiary structure and active site integrity are presented. ANS binds exclusively to the native state of TPMT with high affinity (KD ~ 0.2 μm) and a 1:1 ratio. The stability of TPMT was dramatically increased by binding of ANS, which was shown to co-localize with the structurally similar adenine moiety of the cofactor SAM. Secondly, an enzyme activity assay based on isothermal titration calorimetry (ITC) is presented. Using this approach, the kinetics of 6-MP and 6-TG methylation by TPMT has been characterized.
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VanPelt, Jamie L. "NMR Studies of Klebsiella Pneumoniae Carbapenemase-2 Inhibition and Structural Characterization of New Delhi Metallo-β-Lactamase Variants and Ligand Complexes." Miami University / OhioLINK, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=miami1542725553898546.
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Gronow, Joana Verfasser], Frank [Akademischer Betreuer] [Sönnichsen, and Ulrich [Gutachter] Lüning. "Structural Stabilization of α-Helical Antifreeze Protein Variants Using the Trp-cage Protein / Joana Gronow ; Gutachter: Ulrich Lüning ; Betreuer: Frank D. Sönnichsen." Kiel : Universitätsbibliothek Kiel, 2020. http://nbn-resolving.de/urn:nbn:de:gbv:8-mods-2020-00047-1.
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Gronow, Joana [Verfasser], Frank D. [Akademischer Betreuer] Sönnichsen, and Ulrich [Gutachter] Lüning. "Structural Stabilization of α-Helical Antifreeze Protein Variants Using the Trp-cage Protein / Joana Gronow ; Gutachter: Ulrich Lüning ; Betreuer: Frank D. Sönnichsen." Kiel : Universitätsbibliothek Kiel, 2020. http://d-nb.info/1206179678/34.
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Garimella, Kiran. "Hypothesis-free detection of genome-changing events in pedigree sequencing." Thesis, University of Oxford, 2016. http://ora.ox.ac.uk/objects/uuid:5153dbfa-bf2b-4bcc-a5fd-71492b2b2138.
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In high-diversity populations, a complete accounting of de novo mutations can be difficult to obtain. Most analyses involve identifying such mutations by sequencing pedigrees on second-generation sequencing platforms and aligning the short reads to a reference assembly, the genomic sequence of a canonical member (or members) of a species. Often, large regions of the genomes under study may be greatly diverged from the reference sequence, or not represented at all (e.g. the HLA, antigenic genes, or other regions under balancing selective pressure). If the haplotypic background upon which a mutation occurs is absent, events can easily be missed (as reads have nowhere to align) and false-positives may abound (as the software forces the reads to align elsewhere). This thesis presents a novel method for de novo mutation discovery and allele identification. Rather than relying on alignment, our method is based on the de novo assembly of short-read sequence data using a multi-color de Bruijn graph. In this data structure, each sample is assigned a unique index (or "color"), reads from each sample are decomposed into smaller subsequences of length k (or "kmers"), and color-specific adjacency information between kmers is recorded. Mutations can be discovered in the graph itself by searching for characteristic motifs (e.g. a "bubble motifs", indicative of a SNP or indel, and "linear motifs" indicative of allelic and non-allelic recombination). De novo mutations differ from inherited mutations in that the kmers spanning the variant allele are absent in the parents; in a sense, they facilitate their own discovery by generating "novel" sequence. We exploit this fact to limit processing of the graph to only those regions containing these novel kmers. We verified our approach using simulations, validation, and visualization. On the simulations, we developed genome and read generation software driven by empirical distributions computed from real data to emit genomes with realistic features: recombinations, de novo variants, read fragment sizes, sequencing errors, and coverage profiles. In 20 artifical samples, we determined our sensitivity and specificity for novel kmer recovery to be approximately 98% and 100% at worst, respectively. Not every novel stretch can be reconstituted as a variant, owing to errors and homology in the graph. In simulations, our false discovery rate was 10% for "bubble" events and 12% for "linear" events. On validation, we obtained a high-quality draft assembly for a single P. falciparum child using a third-generation sequencing platform. We discovered three de novo events in the draft assembly, all three of which are recapitulated in our calls on the second-generation sequencing data for the same sample; no false-positives are present. On visualization, we developed an interactive web application capable of rendering a multi-color subgraph that assists in visually distinguishing between true variation and sequencing artifacts. We applied our caller to real datasets: 115 progeny across four previously analyzed experimental crosses of Plasmodium falciparum. We demonstrate our ability to access subtelomeric compartments of the genome, regions harboring antigenic genes under tremendous selective pressure, thus highly divergent between geographically distinct isolates and routinely masked and ignored in reference-based analyses. We also show our caller's ability to recover an important form of structural de novo variation: non-allelic homologous recombination (NAHR) events, an important mechanism for the pathogen to diversify its own antigenic repertoire. We demonstrate our ability to recover the few events in these samples known to exist, and overturn some previous findings indicating exchanges between "core" (non-subtelomeric) genes. We compute the SNP mutation rate to be approximately 2.91 per sample, insertion and deletion mutation rates to be 0.55 and 1.04 per sample, respectively, multi-nucleotide polymorphisms to be 0.72 per sample, and NAHR events to be 0.33 per sample. These findings are consistent across crosses. Finally, we investigated our method's scaling capabilities by processing a quintet of previously analyzed Pan troglodytes verus (western chimpanzee) samples. The genome of the chimpanzee is two orders of magnitude larger than the malaria parasite's (3, 300 Mbp versus 23 Mbp), diploid rather than haploid, poorly assembled, and the read dataset is lower coverage (20x versus 120x). Comparing to Sequenom validation data as well as visual validation, our sensitivity is expectedly low. However, this can be attributed to overaggressiveness in data cleaning applied by the de novo assembler atop which our software is built. We discuss the precise changes that would likely need to be made in future work to adapt our method to low-coverage samples.
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Iraola, Guzmán Susana. "AnáIisis de la herencia epigenética en trastornos neurológicos." Doctoral thesis, Universitat de Barcelona, 2012. http://hdl.handle.net/10803/101096.
Full textAbstract:
Las enfermedades neurodegenerativas, como la enfermedad de Alzheimer (EA) y la enfermedad de Parkinson (EP), representan un grave problema de salud pública, sobre todo en los países occidentales, donde el envejecimiento creciente de la población augura un incremento sustancial de la prevalencia de estas patologías. A pesar de que ciertos tratamientos proporcionan una disminución de las manifestaciones clínicas, el avance del proceso neurodegenerativo es irreversible. La identificación de los mecanismos, como la interacción entre factores genéticos y medio-ambientales, implicados en la etiología y evolución de estas patologías es de importancia capital. En el presente trabajo de tesis se explora el papel de la metilación del ADN genómico y el mosaicismo genético en enfermedades neurodegenerativas. El análisis del perfil de metilación del ADN se realizó empleando dos arrays de metilación: “HumanMethylation” (27K y 450K, IlIumina), cuyas sondas distribuidas estratégicamente por todo el genoma, permiten detectar cuantitativamente el estado de mutilación de unos 27.000 y 450.000 dinucleótidos CpG, respectivamente. La comparación de un total de 60 individuos (28 con enfermedad de Alzheimer, 3 con enfermedad de Parkinson y 29 controles) ha permitido identificar el perfil de metilación del genoma de distintas áreas del sistema nervioso central (SNC) (corteza, amígdala, hipocampo, hipotálamo, protuberancia, sustancia negra y cerebelo), mostrando la existencia de un patrón diferencial entre hombres y mujeres, asociado a la inactivación del cromosoma X, un patrón independiente para cerebelo, y un patrón de metilación de un conjunto de dianas característico de los estadíos 3 y 4 de Braak de la EA. Asimismo, se observaron diferencias significativas de metilación (1.112 CpGs, p<0,0l) en el cerebelo asociadas a la EA, confirmando su implicación en la enfermedad. El análisis del mosaicismo somático del cerebro se realizó empleando el "SurePrint G3 human CGH array 400K" (Agilent). Tomando como área de referencia el cerebelo se detectaron ganancias o pérdidas de material genómico entre áreas del cerebro de un mismo individuo. Dos muestras de corteza, pertenecientes a dos controles, presentaron una ganancia de material genómico en el gen WWOX, mientras que tan solo una muestra mostró una ganancia de material genómico en el gen ADAM5P3A. La elevada frecuencia de variantes en el número de copia en WWOX y su posible implicación en EA llevó a genotipar un mayor número de individuos, aunque ninguno mostró mosaicismo somático. El análisis del estado de metilación de las sondas ubicadas en WWOX permitió observar una disminución significativa de la metilación entre pacientes y controles en 14 sondas (T-student, p<0,05), sugiriendo que la regulación epigenética de WWOX puede estar alterada en la EA. En conjunto, estos resultados muestran la alteración de los perfiles de mutilación del SNC en relación con la EA tardía (estadíos 3 y 4 de Braak). Principalmente, en una de las regiones cuya afectación patológica en la EA ha sido más controvertida, cerebelo. Es especialmente interesante remarcar que la aparición de las lesiones características de cerebelo tienen lugar en estadíos más avanzados, indicando la posibilidad de que la alteraciones epigenéticas observadas podrían corresponder a un evento prematuro en la progresión de la patología.Neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), represent a major issue of public health in developing countries where the aging of the population is leading to a progressive increase of its prevalence rates. Currently, several therapeutic strategies help to palliate clinical symptoms, but the neurodegeneration is progressive and irreversible. Identification of underlying mechanisms leading to these disorders is essential to improve patient's life expectancy and quality. In this context, many efforts have been focused on identifying genetics and environment causes of these disorders with little success, highlighting the need to evaluate new mechanisms and factors involved. The present thesis project has explored the implication of new mechanisms, such as DNA methylation and somatic mosaicism in AD and PD. The analysis of DNA methylation was performed with a new methylation array technology: 'HumanMethylation' (27K and 450K, IlIumina), whose probes strategically distributed along the human genome, enables to quantify the methylation state of around 27,000 and 450,000 CpG sites, respectively. The pattern of methylation of 60 subjects (28 AD, 3 PD and 29 unaffected) with four to seven brain regions (cortex, amygdala, hippocampus, hypothalamus, pons, substantia nigra and cerebellum) has been assessed. The study has shown three ma in clusters depending on gender (female/male), brain area (cerebellum vs others) and disease stage (AD3 vs AD4). In addition, a' differential analysis performed in individual CpG sites proved the presence of significant differences associated to AD patient's cerebellum (1112 CpG sites, p<0.01). Somatic mosaicism analysis has been carried out with a 'SurePrint G3 human CGH array 400K' (Agilent) to detect intra-individual genomic gains and losses compared to cerebellum. A total of two cortex samples showed a genomic gain in the WWOX gene, whereas only one sample showed a gain on ADAM5P3A. WWOX has been considered as a potential candidate gene in previous AD studies, and was further analyzed in a larger cohort of human brain samples. Genotyping assays did not confirm the presence of new somatic mosaicism cases, but it was possible to determine the genotype distribution and compared data between samples. A significant hypomethylation of the WWOX promoter region was observed in AD patients compared to controls subjects (T-test, p<0.05) in 14 probes, suggesting a potential regulation of expression by methylation. Overall, these results highlight the implication of epigenetic mechanisms in neurodegenerative disorders, as AD. In particular, it is remarkable the specific pattern of methylation in the cerebellum in intermediate stages of AD, suggesting an overlap with early modifications, which could contribute to unraveling new mechanisms implicated in AD.
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Villa, Marcos Olaya. "Caracterización de reordenamientos cromosómicos asociados a fenotipo." Doctoral thesis, Universitat Pompeu Fabra, 2009. http://hdl.handle.net/10803/7193.
Full textAbstract:
El establecimiento de correlaciones entre fenotipo y genotipo es uno de los principales objetivos de la genética. La obtención de un diagnóstico ajustado facilita el manejo clínico del paciente, así como poder ofrecer un correcto consejo genético, con asesoramiento reproductivo a las familias de pacientes con enfermedades genéticas. La identificación de genes asociados a patología desde alteraciones citogenéticas asociadas a fenotipo es uno de los métodos de clonación posicional. En este trabajo nos hemos basado en dos tipos de modelos de anomalías citogenéticas: balanceadas y no balanceadas (translocaciones y cromosomas marcadores). Hemos caracterizado las alteraciones citogenéticas de cinco pacientes de cada modelo con fenotipos diversos, empleando una combinación de técnicas citogenéticas y moleculares, con el objetivo de proponer genes candidatos asociados a cada fenotipo.One of the main objectives of Genetics is the establishment of phenotype-genotype correlations. A correct diagnosis facilitates the clinical management of the patient and the possibility to offer a genetic counselling, with reproductive assessment to the families with a patient with a genetic disease. The identification of genes associated to pathology from cytogenetic alterations associated to phenotype is one of the methods of positional cloning. In this work we have based in two different models of cytogenetic alterations: balanced and unbalanced anomalies (translocations and marker chromosomes). We have characterized five patients of each group with different phenotypes, using a combination of cytogenetic and molecular techniques, with the objective of establish candidate genes associated to disease.
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Martínez, Fundichely Alexander 1978. "Bioinformatic characterization and analysis of polymorphic inversions in the human genome." Doctoral thesis, Universitat Pompeu Fabra, 2013. http://hdl.handle.net/10803/384837.
Full textAbstract:
Within the great interest in the characterization of genomic structural variants
(SVs) in the human genome, inversions present unique challenges and
have been little studied. This thesis has developed "GRIAL", a new algorithm
focused specifically in detect and map accurately inversions from
paired-end mapping (PEM) data, which is the most widely used method
to detect SVs. GRIAL is based on geometrical rules to cluster, merge and
refine both breakpoints of putative inversions. That way, we have been
able to predict hundreds of inversions in the human genome. In addition,
thanks to the different GRIAL quality scores, we have been able to
identify spurious PEM-patterns and their causes, and discard a big fraction
of the predicted inversions as false positives. Furthermore, we have created
â ˘ AIJInvFESTâ˘A˙I, the first database of human polymorphic inversions,
which represents the most reliable catalogue of inversions and integrates
all the associated information from multiple sources. Currently, InvFEST
combines information from 30 different studies and contains 1092 candidate
inversions, which are categorized based on internal scores and manual
curation. Finally, the analysis of all the data generated has provided information
on the genomic patterns of inversions, contributing decisively to
the understanding of the map of human polymorphic inversions.Dentro del estudio de las variantes estructurales en el genoma humano, las inversiones han sido las menos han consolidado sus resultados y constituye uno de los principales retos en la actualidad. Esta tesis aborda el tema a través de la implementación de "GRIAL" un nuevo algoritmo específicamente diseñado para la detección más precisa posible de las inversiones usando el mapeo de secuencias apareadas (del inglés PEM) que es el método más utilizado para estudiar la variación estructural. GRIAL se basa en reglas geométricas para agrupar los patrones de PEM que señalan un posible punto de rotura (del inglés breakpoint) de inversión, además une cada breakpoint correspondientes a inversiones independientes y refina lo más exacto posible su localización. Su uso nos permitió predecir cientos de inversiones. Un gran aporte de nuestro método es la creación de índices (del inglés score) de fiabilidad para las predicciones mediante los cuales identificamos patrones de inversión incorrectos y sus causas. Esto nos permitió filtrar nuestro resultado eliminando un gran número de predicciones posiblemente falsas. Además se creó "InvFEST", la primera base de datos especialmente dedicada a inversiones polimórficas en el genoma humano la cual representa el catálogo más fiable de inversiones, integrando además a cada inversión conocida la información asociada disponible. Actualmente InvFEST contiene (y mantiene la clasificación según el nivel de certeza) un catálogo de 1092 inversiones clasificadas, a partir de datos de 30 estudios diferentes. Finalmente el análisis de toda la información generada nos permitió describir algunos patrones de las inversiones polimórficas en el genoma humano contribuyendo de este modo a la comprensión de esta variante estructural y el estado de su información en los estudios del genoma humano.
Inversió genòmica
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Uddin, Md Mesbah. "Identification of causal factors for recessive lethals in dairy cattle with special focus on large chromosomal deletions." Thesis, Paris, Institut agronomique, vétérinaire et forestier de France, 2019. http://www.theses.fr/2019IAVF0018/document.
Full textAbstract:
L'objectif général de cette thèse est d'identifier les variants causaux ou, à défaut, un ensemble de marqueurs prédictifs - qui présentent un déséquilibre de liaison élevé avec les variants causaux - pour la fertilité des vaches laitières. Nous avons abordé cet objectif général dans cinq articles: (i) décrit une approche systématique de cartographie des variants létaux récessifs chez les bovins Normands français basée sur la recherche de déficit en haplotypes homozygotes (HHD). Cette étude montre l’influence de la taille de l’échantillon, de la qualité des génotypes, de la qualité du phasage des génotypes en haplotypes et de l’imputation, de l’âge de l’haplotype et enfin, de la définition des seuils de signification prenant en compte les tests multiples, sur la découverte et la reproductibilité des résultats de HHD. Elle illustre également l’importance de la cartographie fine avec les données de généalogie et de séquence de génome entier (WGS), l’annotation intégrative (entre espèces) pour hiérarchiser les mutations candidates et, enfin, le génotypage à grande échelle de la mutation candidate, pour valider ou invalider les mutations initiales. (ii) décrit une cartographie à haute résolution de grandes délétions chromosomiques de séquences du génome dans une population de 175 animaux appartenant à trois races laitières nordiques. Cette étude utilise trois approches différentes pour valider les résultats de la cartographie. Le chapitre décrit les propriétés génétiques des populations et l’importance fonctionnelle des délétions identifiées. (iii) traite de trois questions liées à l’imputation de variants structuraux, ici de délétions chromosomiques importantes: la disponibilité des génotypes de délétion, la taille du panel de référence d'haplotypes et, enfin, l’imputation elle-même. Pour aborder les deux premières questions, cette étude décrit une approche basée sur un modèle de mélange gaussien dans laquelle les données de profondeur de lecture provenant de fichiers au format VCF (variant call format) sont utilisées pour génotyper un locus de délétion connu, en l’absence d’information sur la séquence brute. Enfin, il présente un pipeline pour l'imputation conjointe de variants WGS et de grandes délétions chromosomiques. (iv) décrit des études d'association pangénomiques de la fertilité femelle dans trois races de bovins laitiers nordiques à l'aide de variants WGS imputés et de grandes délétions chromosomiques. Cette étude concerne huit caractères de fertilité et utilise des analyses d'association mono-marqueur, conditionnelles et conjointes. Cette étude montre qu’une surestimation, ou « inflation », des statistiques de test peut être observée même après correction pour la stratification de la population à l'aide de composantes principales génomiques et pour les structures familiales à l'aide de matrices de relations génomiques. Ce biais était connu pour les caractères très polygéniques. Enfin, cette étude présente plusieurs locus de traits quantitatifs (QTL) nouveaux et confirme plusieurs autres déjà connus. Elle souligne également l’importance d’inclure les grandes délétions (imputées) pour la cartographie par association des caractères de fertilité. (v) décrit la prédiction des valeurs génomiques de fertilité (ou indice de fertilité) à l'aide de génotypes à puces SNP, de QTL sélectionnés et de délétions chromosomiques importantes. En utilisant la méthode de meilleure prédiction linéaire sans biais génomique (GBLUP) avec une ou plusieurs matrices de relations génomiques dérivées d'un ensemble de marqueurs sélectionnés, cette étude rapporte une précision de prédiction améliorée. Cette étude met également en évidence l’influence de la sélection des marqueurs les plus prédictifs, en particulier pour une race ayant une population d’apprentissage réduite, sur la précision des prédictions génomiques. Enfin, les résultats démontrent que les grandes délétions ont en général un pouvoir prédictif élevéThe overall aim of this PhD thesis is to identify causal variants for recessive lethal mutations and select a set of predictive markers that are in high linkage-disequilibrium with the causal variants for female fertility in dairy cattle. We addressed this broad aim under five articles: (i) describes a systematic approach of mapping recessive lethals in French Normande cattle using homozygous haplotype deficiency (HHD). This study shows the influence of sample size, quality of genotypes, quality of (genotype) phasing and imputation, age of haplotype (of interest), and last but not the least, multiple testing corrections, on discovery and replicability of HHD results. It also illustrates the importance of fine-mapping with pedigree and whole-genome sequence (WGS) data, (cross-species) integrative annotation to prioritize candidate mutation, and finally, large-scale genotyping of the candidate mutation, to validate or invalidate initial results. (ii) describes a high-resolution population-scale mapping of large chromosomal deletions from whole-genome sequences of 175 animals from three Nordic dairy breeds. This study employs three different approaches to validate identified deletions. Next, it describes population genetic properties and functional importance of these deletions. (iii) deals with three main issues related to imputation of structural variants, in this case, large chromosomal deletions, e.g. availability of deletion genotypes, size of haplotype reference panel, and finally, imputation itself. To address the first two issues, this study describes a Gaussian mixture model-based approach where read-depth data from the variant call format (VCF) file is used to genotype a known deletion locus, without the need for raw sequence (BAM) file. Finally, it presents a pipeline for joint imputation of WGS variants along with large chromosomal deletions. (iv) describes genome-wide association studies for female fertility in three Nordic dairy cattle breeds using imputed WGS variants including large chromosomal deletions. This study is based on the analyses of eight fertility related traits using single-marker association, conditional and joint analyses. This study illustrates that inflation in association test-statistics could be seen even after correcting for population stratification using (genomic) principal components, and relatedness among the samples using genomic relationship matrices; however, this was known for traits with strong polygenic effects, among other factors. Finally, mapping of several new quantitative trait loci (QTL), along with the previously known ones, are reported in this study. This study also highlights the importance of including (imputed) large deletions for association mapping of fertility traits. (v) describes prediction of genomic breeding values for fertility using SNP array-chip genotypes, selected QTL and large chromosomal deletion. Using genomic best linear unbiased prediction (GBLUP) method with one or several genomic-relationship matrices derived from a set of selected markers, this study reports higher prediction accuracy compared with previous report. This study also highlights the influence of selecting markers with best predictability, especially for a breed with small training population, in accuracy of genomic prediction. The results demonstrate that large deletions in general have a high predictive performance
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Jorge, Susan Elisabeth Domingues Costa 1983. "Correlação estrutura-função de variantes da hemoglobina humana = Structure-function relations of human hemoglobin variants." [s.n.], 2013. http://repositorio.unicamp.br/jspui/handle/REPOSIP/310885.
Full textAbstract:
Orientadores: Maria de Fatima Sonati, Munir Salomão SkafTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas
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Resumo: O resumo poderá ser visualizado no texto completo da tese digital
Abstract: The complete abstract is available with the full electronic document
Doutorado
Ciencias Biomedicas
Doutora em Ciências Médicas
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Soundiramourtty, Abirami. "Exploring the transpositional landscape and recent transposable element activity in beech trees using long read mobilome and genome sequencing and with new computational tools." Electronic Thesis or Diss., Perpignan, 2024. http://www.theses.fr/2024PERP0043.
Full textAbstract:
L’adaptation des organismes aux changements environnementaux est devenue une question fondamentale de la recherche, en particulier face aux impacts du réchauffement climatique. Un axe clé de recherche consiste à comprendre comment les éléments génétiques sous jacent, tels que les éléments transposables (ET). Les ET sont des séquences d'ADN répétés présentes chez tous les Eucaryotes, possédant la capacité unique de se déplacer au sein du génome, un phénomène appelé transposition active. Ainsi, ils peuvent provoquer des mutations en générant des insertions polymorphiques d'ET (TIPs) entre individus, voire des insertions somatiques. En général, les ET restent inactifs grâce à des mécanismes épigénétiques qui limitent leur prolifération incontrôlée. Cependant, ils peuvent être réactivés par divers stimuli environnementaux, rendant la transposition active relativement rare. Cette mobilité des ET peut être révélée en utilisant l'ADN circulaire extrachromosomique (ADNecc) comme marqueur de transposition. Le paysage transpostionnel des TEs et leur activité récente ont été décrits chez des organismes modèles, mais restent inexploités chez les espèces pérennes comme les arbres. Cette étude vise à explorer l’activité transpositionelle récente et la mobilité en cours des ET chez des espèces pérennes non modèles en utilisant le hêtre européen (Fagus sylvatica) comme notre modèle d’étude. Nous avons cherché à étudier l'activité récente des ET et leur mobilité continue en identifiant les variants causés par les ET au sein d'une population et chez un individu (à l'échelle somatique) en utilisant le séquençage du génome complet (WGS) et le séquençage du mobilome (ou ADNecc). Nous avons réalisé le séquençage WGS et du mobilome d'arbres de la forêt de Verzy, connue pour abriter des hêtres nains et tortillards, également appelés « mutants ». Ces arbres présentent des traits morphologiques instables, avec chez certains arbres de nouvelles branches qui se développent avec une forme normale. Nous avons identifié deux ET appartenant au type des Miniature Inverted Repeats Transposable Elements (MITEs), nommés SQUIRREL1 et SQUIRREL2, qui se mobilisent activement dans ces arbres, produisant une grande quantité dADNecc et causant même des variations somatiques. SQUIRREL1 et SQUIRREL2 sont également actifs dans les hêtres de la forêt de la Massane. De plus, dans tous ces arbres, plusieurs d’autres ET, principalement des MITEs, produisent une grande quantité dADNecc, bien que leur niveau d’activité semble varier en fonction des tissus, suggérant que l'activité des ET varie selon le stade de développement et indiquant une transposition dominée par les MITEs chez le hêtre. Parallèlement, nous avons étudié les TIPs dans une population de hêtres de la forêt de la Massane, une forêt ancienne classée au patrimoine mondial de l'UNESCO. En séquençant 150 arbres, nous avons cherché à comprendre comment les ET contribuent à la diversité génétique de l'ensemble de la population en détectant les TIPs générés par les Long Terminal Repeats rétrotransposons (LTR RT) et les MITEs en utilisant le séquençage WGS. Nous avons détecté environ 30 000 TIPs de LTR-RT chez chaque individu, contre 70 000 TIPs de MITEs. La plupart de ces TIPs restent à faible fréquence mais de nombreux MITE-TIPs restent localisés près de gènes fonctionnels et conservés au sein de la population. À partir des TIPs, nous avons identifié plusieurs points chauds de variation et des régions conservées le long du génome du hêtre permettant d’abordant la structuration du génome chez cette espèce. Pour conclure, notre étude met en lumière l’importance des ET dans la structuration du paysage génomique des arbres, en particulier dans la manière dont ces éléments contribuent à l’évolution des espèces à longue durée de vie. Les recherches futures pourraient étendre ces travaux à d’autres espèces d'arbres et explorer si les schémas observés se retrouvent dans d’autres espèces d’arbresThe adaptation of organisms to environmental changes has become a fundamental research question,particularly in the context of climate change. A key area of this research is to identify underlying genetic elements, such as transposable elements (TEs), contributing to this process. TEs are repetitive DNA sequences found across all eukaryotes, possessing the unique ability to move within the genome, a phenomenon known as active transposition. They can cause mutations by generating transposable element insertion polymorphisms (TIPs) between individuals, and even somatic insertions. Generally, TEs remain inactive by epigenetic mechanisms that limit their uncontrolled proliferation. However, they can be reactivated upon various environmental stimuli, making active transposition relatively rare. TE mobility can be detected using extrachromosomal circular DNA (eccDNA) as a marker of transposition. The transpositional landscape of TEs and their recent activity have been documented in model organisms but remain underexplored in perennial species such as trees. This study aims to investigate recent transpositional activity and ongoing mobility of TEs in non-model perennial species, using European beech (Fagus sylvatica) as our model. We sought to study recent TE activity and their continuous mobility byidentifying TE-induced variants within a population and in an individual (at the somatic scale) using whole-genome sequencing (WGS) and mobilome sequencing (eccDNA). We conducted WGS and mobilome sequencing of trees from the Verzy forest, known for its dwarf and tortuous beeches, also referred as "mutants." These trees exhibit unstable phenotypical traits, with some trees developing new normal branches. We identified two TEs belonging to the Miniature Inverted Repeat Transposable Elements (MITEs) type, named SQUIRREL1 and SQUIRREL2, which are actively mobilizing in these trees, producing large amounts of eccDNA and even causing somatic variations.SQUIRREL1 and SQUIRREL2 are also active in beech trees from the Massane forest. Furthermore, in all these trees, several other TEs,mainly MITEs, produce significant amounts of eccDNA, although their activity levels appear to vary depending on the tissues, suggesting that TE activity could be tissue-specific indicating MITE-dominated transposition in beech. Simultaneously, we investigated TIPs in a population of beech trees from the Massane forest, an ancient forest classified as a UNESCO World Heritage site. By sequencing 150 trees, we aimed to understand how TEs contribute to the genetic diversity of the entire population by detecting TIPs generated by Long Terminal Repeat retrotransposons (LTR-RTs) and MITEs using WGS. We detected approximately 30,000 LTR-RT TIPs in each individual, compared to 70,000 MITE TIPs. While most of these TIPs remain at low frequency, many MITE-TIPs are located near functional genes and more conserved within the population. Using these TIPs, we identified several hotspots of variation and conserved regions along the beech genome, providing insights into genome structure in this species. In conclusion, our study highlights the importance of TEs in shaping the genomic landscape of trees, particularly in understanding how these elements contribute to the evolution of long-lived species. Future research could expand this work to other tree species and explore whether the patterns observed in beeches are common in other types of trees
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Wigmore, Eleanor May. "Regional brain volumes and antidepressant treatment resistance in major depressive disorder." Thesis, University of Edinburgh, 2018. http://hdl.handle.net/1842/31291.
Full textAbstract:
Major depressive disorder (MDD) is a heritable and highly debilitating condition with antidepressants, first-line treatment, demonstrating low to modest response rates. No current biological mechanism substantially explains MDD but both neurostructural and neurochemical pathways have been suggested. Further explication of these may aid in identifying subgroups of MDD that are better defined by their aetiology. Specifically, genetic stratification provides an array of tools to do this, including the intermediate phenotype approach which was applied in this thesis. This thesis explores genetic overlap with regional brain volume and MDD and the genetic and non-genetic components of antidepressant response. The first study utilised the most recent published data from ENIGMA (Enhancing Neuroimaging Genetics through Meta-analysis) Consortium's genome-wide association study (GWAS) of regional brain volume to examine shared genetic architecture between seven subcortical brain volumes and intracranial volume (ICV) and MDD. This was explored using linkage disequilibrium score regression (LDSC), polygenic risk scoring (PRS) techniques, Mendelian randomisation (MR) analysis and BUHMBOX (Breaking Up Heterogeneous Mixture Based On Cross-locus correlations). Results indicated that hippocampal volume was positively genetically correlated with MDD (rg= 0.46, P= 0.02), although this did not survive multiple comparison testing. Additionally, there was evidence for genetic subgrouping in Generation Scotland: Scottish Family Health Study (GS:SFHS) MDD cases (P=0.00281), however, this was not replicated in two other independent samples. This study does not support a shared architecture for regional brain volumes and MDD, however, provided some evidence that hippocampal volume and MDD may share genetic architecture in a subgroup of individuals, albeit the genetic correlation did not survive multiple testing correction and genetic subgroup heterogeneity was not replicated. To explore antidepressant treatment resistance, the second study utilised prescription data in (GS:SFHS) to define a measure of (a) treatment resistance (TR) and (b) stages of resistance (SR) by inferring antidepressant switching as non-response. GWAS were conducted separately for TR in GS:SFHS and the GENDEP (Genome-based Therapeutic Drugs for Depression) study and then meta-analysed (meta-analysis n=4,213, cases=358). For SR, a GWAS on GS:SFHS only was performed (n=3,452). Additionally, gene-set enrichment, polygenic risk scoring (PRS) and genetic correlation analysis were conducted. No significant locus, gene or gene-set was associated with TR or SR, however power analysis indicated that this analysis was underpowered. Pedigree-based correlations identified genetic overlap with psychological distress, schizotypy and mood disorder traits. Finally, the role of neuroticism, psychological resilience and coping styles in antidepressant resistance was investigated. Univariate, moderation and mediation models were applied using logistic regression and structural equation modelling techniques. In univariate models, neuroticism and emotion-orientated coping demonstrated significant negative association with antidepressant resistance, whereas resilience, task-orientated and avoidance-orientated coping demonstrated significant positive association. No moderation of the association between neuroticism and TR was detected and no mediating effect of coping styles was found. However, resilience was found to partially mediate the association between neuroticism and TR. Whilst the first study does not indicate a genetic overlap between regional brain volumes and MDD, it demonstrates the utility of the intermediate approach in complex disease. Antidepressant resistance was associated with neuroticism both genetically and phenotypically, indicating its role as an intermediate phenotype. Nonetheless, larger sample sizes are needed to adequately address the components of antidepressant resistance. Further work in antidepressant non-response may help to identify biological mechanisms responsible in MDD pathology and help stratify individuals into more tractable groups.
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Villatoro, Gómez Sergio. "Estudio de variantes estructurales del genoma humano asociadas a trastornos del neurodesarrollo." Doctoral thesis, Universitat Autònoma de Barcelona, 2016. http://hdl.handle.net/10803/400662.
Full textAbstract:
El síndrome de Angelman (SA) y el de Prader Wili (SPW) son trastornos del neurodesarrollo cuya principal etiología molecular es la deleción de la región 15q11.2-q13. Esta deleción está promovida por la Recombinación Homóloga No Alélica (NAHR) y mediada por secuencias altamente repetitivas de bajo número de copias (LCRs) que la flanquean. La orientación de estas LCRs predispone al reordenamiento final que se obtendrá por NAHR. Las LCRs en orientación directa generan deleciones y duplicaciones mientras las que presentan orientación invertida originan inversiones. Estas inversiones pueden facilitar una recombinación inadecuada entre las LCRs que flanquean la región 15q11.2-q13 dando lugar a la deleción en la descendencia. En este trabajo se presenta un nuevo análisis de la frecuencia de la inversión 15q11.2-q13 en 23 controles de población general, 21 progenitores de pacientes con SA y 32 con descendencia por SPW. Se han analizado un total de 9540 cromosomas informativos mediante FISH de sondas de BACs, encontrando la inversión en el 4,61% de los cromosomas estudiados en la población control. También se ha analizado la frecuencia de la inversión en progenitores de pacientes con SA y SPW observando un incremento significativo de inversiones en las madres de hijos con SA por deleción y en los padres de SPW por deleción frente a la población control (p=8x10-7 y p=0.007, respectivamente). Nuestros resultados indican que la inversión 15q11.2-q13 es un polimorfismo presente en población general. Así mismo, el incremento de la frecuencia de la inversión en madres de SA y padres de SPW con deleción sugiere que la inversión podría ser una estructura que promueve el mal alineamiento de los LCRs y facilita la generación de deleciones en 15q11.2-q13. El SA es diagnosticado molecularmente en el 90% de los pacientes, sin embargo, en el 10% restante, aún presentando unas características clínicas bien definidas, su etiología molecular es todavía una incógnita (SA-like). Se han analizado 20 pacientes SA-like mediante a-CGH previo cribado de variaciones en número de copias (CNVs) en regiones sindrómicas y subteloméricas. Las regiones variables en número de copia con una frecuencia poblacional inferior al 5% o que no se encuentran estudiadas según la Database of Genomic Variants se han seleccionado para ser confirmadas mediante Multiplex Ligation-depenent Probe Amplificatio (MLPA). Se han evaluado las CNVs en 20 pacientes SA-like y sus progenitores y se ha ampliado el estudio para cribar pacientes con
discapacidad intelectual (n=296), trastornos del espectro autista (n=164) y controles de población general española (n=453). Se ha identificado una deleción de novo (1q44), dos duplicaciones heredadas de la madre (Xp11.23 y Xq28) y 20 regiones heredadas con CNVs en los pacientes SA-like pero que no se observan en la población control. En tres pacientes la concomitancia de una deleción y un SNP puede estar originando una discapacidad intelectual de herencia recesiva, sugiriendo que el gen MYH13 y los ARNs no codificantes largos podrían estar involucrados en el fenotipo SA-like. En lo referente a los pacientes con discapacidad intelectual y trastornos del espectro autista se han identificado alteraciones de gran tamaño implicadas en la etiología: del(1)(p36), del(1)(q44), dup(10)(q21.1), dup(X)(q11.23q28) y dup(X)(q28) en tres casos. También se han detectado 29 regiones con CNVs heredadas que no son variables en población general, 12 de las cuales son compartidas con los pacientes SA-like. Nuestros resultados respaldan la idea de que regiones con CNVs pueden ser, probablemente, responsables de un alto porcentaje de trastornos del neurodesarrollo. Las CNVs identificadas en pacientes, pero no detectadas en población control, aún siendo heredadas, podrían estar asociadas a algunas características clínicas de la enfermedad desenmascarando, en genes específicos, mutaciones de carácter recesivo relacionados con los fenotipos.Angelman syndrome (AS) and Prader Willi syndrome (PWS) are neurodevelomental disorders in which main molecular etiology is the 15q11.2-q13 deletion. This deletion is leaded by Non Allelic Homologous Recombination (NAHR) mediated by flanking high repetitive sequences named Low Copy Repeats (LCRs). The orientation of these LCRs leads the final product of NAHR. LCRs in direct orientation are solved in deletions or duplications while LCRs in inverted orientation lead inversions. These inversions could facilitate abnormal recombination between flanking LCRs and could mediate interstitial deletion of chromosome 15q11.2-q13 in the offspring. Herein we report a new analysis of the frequency of inversion 15q11.2-q13 in 23 controls from general population, 21 AS parents and 32 PWS parents. Molecular cytogenetic analysis was performed using FISH with BACs probes by examining a total of 9540 informative chromosomes. First, the 15q11.2-q13 inversion was detected on average in 4.61% of chromosomes of Spanish control population. Then we analyzed the frequency of the 15q11.2-q13 inversion in parents of AS and PWS and a significant increase in AS mothers and PWS fathers with offspring affected by deletion was observed in front of control group (p= 8x10-7and p=0,007, respectively). Our results indicate that 15q11.2-q13 inversion is a polymorphism presents in general population. Moreover, the high inversion frequency observed in AS mothers and PWS fathers of offspring affected by deletion suggest that the inversion could be a structure that promotes misalignment between the LCRs and facilitates the occurrence of 15q11.2-q13 deletions. AS has a recognizable molecular cause in about 90% of cases, nevertheless in 10% with well-defined clinical features the molecular etiology is still unknown (AS-like). We have analysed 20 AS-like patients by a-CGH after screening the patients for syndromic and subtelomeric copy number alterations (CNVs). Regions that contained rare CNVs or not reported in the Database of Genomic Variants were selected for validation using custom Multiplex Ligation-dependent Probe Amplification (MLPA) assays. We assessed the CNV status in the 20 AS-like cases and in their parents, and also expanded the study to larger sets of samples of individuals suffering idiopathic intellectual disability (n=296), autism spectrum disorders (n=164) as well as to a control cohort of normal individuals (n=453). We have identified one de novo deletion (1q44), two maternally inherited duplications (Xp11.23 and Xq28) and 20 inherited altered regions present in AS-like cases that have not been present in control population. In three patients a concomitance of a deletion and SNPs is leading a possible recessive intellectual disability disease suggesting that MYH13 and long non-coding RNAs could be involved in AS-like. Concerning intellectual disability and autism spectrum disorders big alterations: del(1)(p36), del(1)(q44), dup(10)(q21.1), dup(X)(q11.23q28) and dup(X)(q28) in three patients, have been associated with the etiology. We also have identified 29 inherited genomic variants that were not present in the general population, 12 out of them shared with AS-like patients. Our results support the point of view that a considerable proportion of genomic regions showing variability in copy number could be responsible for neurodevelopment disorders. The inherited CNVs identified in cases, but not detected in controls, suggesting that even if they are inherited, they could be responsible for some of the clinical features perhaps unmasking, in specific genes, recessive mutations involved in the phenotypes.
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Chu, Nam Ky [Verfasser], Christian F. W. [Akademischer Betreuer] Becker, and Michael [Akademischer Betreuer] Sattler. "The effect of posttranslational modifications on biochemical and structural characteristics of Prion Protein variants and the SMN Tudor domain / Nam Ky Chu. Gutachter: Christian F. W. Becker ; Michael Sattler. Betreuer: Christian F. W. Becker." München : Universitätsbibliothek der TU München, 2013. http://d-nb.info/1038527333/34.
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Bruneau, Alix. "Régulation de l'expression membranaire du transporteur de phospholipides biliaires ABCB4 : effet de mutations Functional Defect of Variants in the Adenosine Triphosphate–Binding Sites of ABCB4 and Their Rescue by the Cystic Fibrosis Transmembrane Conductance Regulator Potentiator, Ivacaftor (VX-770) Structural analogues of roscovitine rescue the intracellular traffic and the function of ER-retained ABCB4 variants in cell models." Thesis, Sorbonne université, 2019. http://www.theses.fr/2019SORUS048.
Full textAbstract:
ABCB4 est exprimé à la membrane canaliculaire des hépatocytes où il sécrète un composant majeur de la bile : la phosphatidylcholine. Plus de 500 mutations d’ABCB4 sont associées à des maladies biliaires. La pathologie la plus sévère est la PFIC3, qui se développe tôt dans l’enfance et progresse rapidement vers l’insuffisance hépatique. La transplantation hépatique reste la seule thérapie efficace. Le développement d’alternatives représente donc un enjeu majeur. Cette thèse s’intéresse à l’effet de cinq mutations décrites chez des patients et situées dans les sites de liaison à l’ATP d’ABCB4. En combinant la modélisation 3D avec des études in vitro, nous avons montré que ces mutations sont responsables d’un défaut d’activité d’ABCB4. Nous avons mis en évidence que le VX-770, un médicament approuvé en clinique pour traiter les patients atteints de mucoviscidose, restaure l’activité des cinq mutants. Ces travaux ouvrent des perspectives de repositionnement du VX-770 pour le traitement ciblé de patients atteints de pathologies biliaires liées à ABCB4. La seconde partie de cette thèse consiste à étudier le rôle de l’interaction du domaine N-terminal d’ABCB4 avec la kinase MRCKalpha. L’inhibition ou l’extinction de cette kinase montrent que MRCKalpha joue un rôle dans la régulation de l’expression membranaire d’ABCB4 en contrôlant son internalisation depuis la membrane plasmique. En inhibant la protéine MLC2, effecteur de MRCKa, nous avons ensuite montré que l’effet de MRCKalpha sur l’expression d’ABCB4 passe par MLC2, qui est un partenaire du domaine linker d’ABCB4. Notre travail montre un rôle commun de ces deux partenaires dans la régulation de l’internalisation d’ABCB4ABCB4 is exclusively expressed at the canalicular membrane of hepatocytes where its function is to translocate phosphatidylcholine (PC) into bile. Variations in ABCB4 gene sequence are associated with several chronic and progressive liver diseases. The most severe is PFIC3 which develops early in childhood and most often requires liver transplantation. Less severe diseases are the intrahepatic cholestasis of pregnancy and the low phospholipid- associated cholelithiasis syndrome which occur in young adults. Up to now, about 500 disease-causing ABCB4 variants have been reported. A challenge is to find pharmacological treatments for the severe forms of the diseases. We have studied the effect of five disease-causing variations that reside in the highly conserved motifs of ABC transporters, involved in ATP binding. Using three-dimension structural modeling and in vitro studies, we showed that the five mutants were normally processed and targeted to the plasma membrane, whereas their PC secretion activity was dramatically decreased. PC secretion activity of the mutants was rescued by the clinically approved CFTR potentiator ivacaftor (VX-770). These results pave the way for personalized therapy in ABCB4-related diseases.The second part of my project was aimed at investigating the potential role of two ABCB4 partners, the kinase MRCKalpha and its effector the myosin light chain II (MLCII) in the expression and function of ABCB4. We found that downregulation of both partners didn’t affect the canalicular localization of ABCB4 but led to a reduction of its endocytosis. Our results open new insights into the mechanisms underlying the regulation of ABCB4 expression and function
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Obri, Arnaud. "Etude structurale et fonctionnelle de la variante d'histone H2AZ." Phd thesis, Université de Strasbourg, 2012. http://tel.archives-ouvertes.fr/tel-00912335.
Full textAbstract:
La variante d'histone H2AZ joue un rôle important dans l'activation de la transcription, la prolifération cellulaire, le développement et la différentiation. H2AZ orne les promoteurs de la majorité des gènes, mais les mécanismes de bases de cette localisation sont inconnus. La compréhension de l'assemblage et du désassemblage du nucléosome passe par la caractérisation de la dynamique du nucléosome et des chaperonnes d'histones. L'objectif de ma thèse était d'identifier des chaperonnes spécifiques impliqués dans la dynamique de H2AZ en utilisant une approche de protéomique. Pour élucider les mécanismes de déposition/éviction de H2AZ, j'ai purifié le complexe prénucléosomale de H2AZ et j'ai caractérisé toutes les protéines associées. J'ai trouvé que Anp32e fait partie du complexe p400/TIP60 qui est présumée pour être responsable de l'échange d'H2AZ sur la chromatine. Anp32e présente une spécificité pour le dimère H2AZ-H2B, car il n'interagit pas avec le dimère H2A-H2B. L'interaction est accomplie au niveau d'une petite région dans le domaine d'ancrage sur H2AZ et au niveau d'un nouveau domaine ZID sur Anp32e. Finalement, j'ai montré que la suppression d'Anp32e entraine : un défaut dans la dé-répression des gènes dont l'expression est contrôlée par une hormone et une accumulation sur les promoteurs de ces derniers. Dans l'ensemble ces résultats identifient Anp32e comme une nouvelle chaperonne de la variante d'histoneH2AZ impliquée dans l'éviction de H2AZ chez les mammifères.
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ROQUEPLO, GIMENEZ ANNE-PAULE. "Structure et fonction de l'angiotensionogene humain : etude des variants naturels et des variants obtenus par mutagenese dirigee." Paris 6, 1999. http://www.theses.fr/1999PA066440.
Full textAbstract:
L'angiotensinogene est le substrat unique et specifique de la renine. Il a ete recemment mis en evidence une relation positive entre le gene de l'angiotensinogene humain et l'hypertension arterielle essentielle. Nous avons realise l'etude des mutations naturelles du gene et caracterise la mutation y248c qui induit une glycosylation anormale de la proteine et un diminution de sa production in vitro et in vivo. Nous avons etudie certaines proprietes biochimiques de la proteine recombinante. La glycosylation de la proteine est entierement responsable de son heterogeneite. Nous avons produit un angiotensinogene mutant recombinant, homogene, completement deglycosyle, fonctionnel et utilise ce mutant pour demontrer que l'angiotensinogene presente un pont disulfure entre la cys 1 8 et la cys 1 3 8. La purification de l'angiotensinogene humain recombinant a conduit a montrer un clivage c-terminal entre la gln 4 1 2 et la leu 4 1 3, suggerant l'existence d'une fonction serpin-like pour l'angiotensinogene. La capacite de l'angiotensinogene de former un complexe covalent avec la proforme de la proteine majeure basique des granules eosinophiles (prombp) a ete recemment demontree. Nous avons produit in vitro le complexe angiotensinogene-prombp et montre que l'hydrolyse du complexe par la renine est 7 fois plus lente que l'hydrolyse de la forme monomerique. La liaison disulfire reliant les deux proteines fait intervenir la cys 2 3 2 de l'angiotensinogene. Le polymorphisme m235t de l'nagiotensinogene influe sur la formation du complexe avec une proportion plus faible du rapport complexe/monomere pour l'angiotensinogene 235t suggerant une nouvelle explication physiopathologique pour son association avec l'hta gravidique. Le clivage de l'angiotensinogene par la renine libere d'une part l'angiotensine i, fondamentale pou le fonctionnement du systeme renine-angiotensine, et d'autre part le des-angiotensine i-angiotensinogene, par l'intermediaire du clivage de la region c-terminal et la cys 2 3 2 hyperreactive dont la relevance physiologique reste a demontrer.