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1
Röhrs, Susanne. « A neuraminidase-negative variant of highly pathogenic avian influenza virus H5N1 ». Diss., Ludwig-Maximilians-Universität München, 2014. http://nbn-resolving.de/urn:nbn:de:bvb:19-177974.
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Hochpathogene Aviäre Influenzaviren (HPAIV) des Subtypes H5N1 zirkulieren seit den ersten Ausbrüchen vor mehr als 10 Jahren in Wirtschaftsgeflügel und Wildvögeln, vor allem in Asien und Afrika. Vakzinekampagnien mit ungeeigneten oder unwirksamen Impfstoffen sowie Transporte von infiziertem Geflügel, sowie Wildvogelzüge führten nicht nur zur Verbreitung der Viren, sondern auch zu Reassortierungen mit anderen zirkulierenden Influenzaviren und zur antigenetischen Drift. Bis heute ist kein Impfstoff verfügbar der ausreichenden und schnellen Schutz vor einer HPAIV H5N1 Infektion, sowie der Ausscheidung bietet, genauso wie eine einfache Applikation ermöglicht und sowohl im Säugetier, als auch Vogel einsetzbar ist. Im Falle eines Ausbruches wäre eine solche Vaccine das perfekte Werkzeug, um eine Pandemie zu verhindern.
Die in dieser Dissertation vorgestellten Arbeiten zeigen die in-vitro und in-vivo Charakterisierung und einer Neuraminidase-negativen H5N1 Mutante, so wie den Einsatz als Impfstoffmodel zur Untersuchung der Frühimmunisierung.
Ein hochpathogenes H5N1 Isolat des Stammes 2.1 (A/swan/Germany/R65/2006) wurde fünfzigmal im embryonierten Hühnerei passagiert. Den Passagen wurde bei jedem Durchgang polybasisches Hühnerserum mit Antikörper gegen H5 und N2 beigemischt, um einen selektiven Druck auf das Virus zu erzeugen. Die daraus resultierende Mutante zeigte überaschenderweise nicht die erwarteten Veränderungen und Anpassungen im Haemagglutinin, sondern Deletionen und „Segment-shuffling“ im Segment 6, welches für das Neuraminidaseprotein kodiert. Des Weiteren konnte keine Neuraminidaseaktivität mehr nachgewiesen werden.
Die Deletionen führten zu Veränderungen im Wachstumsverhalten des Virus und der Pathogenität im Tier. So konnte hier gezeigt werden, dass die Mutante ohne Zugabe von externer Neuraminidase oder Adaptation des Haemagglutinins in Zellkultur und Ei hohe Infektionstiter erreichen kann. Im Vergleich zum Ursprungsvirus aber Wachstumsdefizite aufweist, die sich in kleineren Plaques und langsamerem Wachstum auf Zellkultur zeigen.
Infektionsversuche im Huhn zeigten, dass das Virus weder Klinik auslöst, noch ausgeschieden oder übertragen wird, aber eine gute Immunantwort induziert. Hohe Antikörper konnten nur erreicht werden, wenn Hühner intramuskulär infiziert wurden, wohingegen eine Applikation über den oronasalen Weg nicht bei allen Tieren gelang.
Die gute Immunantwort und Apathogenität des Virus machten es im weiteren Verlauf zu einem geeigneten Kandidaten für Frühimmunisierungsversuche im Säugetier- und Vogelmodell. So wurden Balb/C Mäuse, Frettchen und Hühner ein, drei und sieben Tage vor einer H5N1 Belastungsinfektion mit der H5N1 Mutante intramuskulär oder intranasal immunisiert. Dabei konnte ein 100% Schutz vor klinischen Symptomen und der Ausscheidung des Challenge-Virus nach nur 7 Tagen gezeigt werden. Darüber hinaus waren die Tiere vor Klinik bereits drei Tage nach der Immunisierung geschützt, wobei aber virale RNA in oronasalen Proben und auch den Organen nachgewiesen werden konnte.
Die Neuraminidasedeletion der Mutante ermöglichte außerdem eine Unterscheidung von immunisierten zu infizierten Tieren, da erstere im ELISA NP- aber keine NA-Antikörper zeigten, wohingegen infizierte Tiere Antikörper gegen beide Proteine bildeten.
In Zukunft könnten NA negative Influenzaviren zusätzlich oder als Alternative zu den gängigen stamping out Strategien eingesetzt werden. Dafür aber sind weiter Untersuchungen essentiell. Die hochpathogene Spaltstelle im HA von „EscEgg50A“ impliziert das Risiko mit zirkulierenden AI Stämmen zu reassortieren und ist daher für den Einsatz zum Beispiel in Zuchtherden ungeeignet. Darüber hinaus ist das Wissen über das NA Protein und seine Funktionsweise sehr lückenhaft und NA-negative Mutanten könnten für zukünftige Untersuchungen genutzt werden.
2
CICCARESE, GIULIA. « GENOTYPE – PHENOTYPE CORRELATIONS IN CUTANEOUS MELANOMA PATIENTS CARRIER OF THE MITF p.E318K PATHOGENIC VARIANT ». Doctoral thesis, Università degli studi di Genova, 2019. http://hdl.handle.net/11567/945094.
Texte intégralRésumé :
Background: The p.E318K variant of the microphthalmia-associated transcription factor (MITF) has been implicated in genetic predisposition to melanoma as an intermediate penetrance allele. However, the impact of this variant on clinico-phenotypic, as well as on dermoscopic patterns features of affected patients is not entirely defined. The purpose of our study was to assess the association between the p.E318K germline variant and clinic-phenotypical features of MITF+ compared to non-carriers (MITF-), including dermoscopic findings of melanomas and dysplastic nevi.
Methods: we retrospectively analyzed a consecutive series of 1386 patients recruited between 2000 and 2017 who underwent genetic testing for CDKN2A, CDK4, MC1R and MITF germline variants in our laboratory for diagnostic/research purposes. The patients were probands of melanoma-prone families and apparently sporadic single or multiple primary melanoma patients. For all, we collected clinical, pathological information and dermoscopic images of the histopathologically diagnosed melanomas and dysplastic nevi, when available.
Results: After excluding patients positive for CDKN2A/CDK4 pathogenic variants and those affected by non-cutaneous melanomas, our study cohort comprised 984 cutaneous melanoma patients, 22 MITF+ and 962 MITF-. MITF+ were more likely to develop dysplastic nevi and multiple primary melanomas. Nodular melanoma was more common in MITF+ patients (32% compared to 19% in MITF-). MITF+ patients showed more frequently dysplastic nevi and melanomas with uncommon dermoscopic patterns (unspecific), as opposed to MITF- patients, whose most prevalent pattern was the multicomponent.
Conclusions: MITF+ patients tend to develop melanomas and dysplastic nevi with histopathological features, frequency and dermoscopic patterns often different from those prevalent in MITF- patients. Our results emphasize the importance of melanoma prevention programs for MITF+ patients, including dermatologic surveillance with digital follow-up.
3
Almanza, Deanna J. « Medical Decision Making among Individuals with a Variant of Uncertain Significance in a Hereditary Cancer Gene and those with a CHEK2 Pathogenic Variant ». Scholar Commons, 2019. https://scholarcommons.usf.edu/etd/7726.
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Despite national guidelines, women with a BRCA VUS or CHEK2 pathogenic variant are choosing to have risk-reducing surgeries such as bilateral mastectomies which are not aligned with their level of cancer risk based on genetic test results alone. Semi-structured telephone interviews were conducted with 6 women with a BRCA VUS and 12 with a CHEK2 pathogenic variant exploring the factors influencing their decision-making process when considering medical management options. Patients from a cancer registry agreed to a recorded telephone interview. Coding was performed using the main constructs from the Ottawa Patient Decision Guide including: knowledge, uncertainty, values, and support. Iterative analysis was used to identify emerging themes.
Analysis of the interviews revealed overlapping of the four constructs in the decision-making process. The knowledge sought to make medical management decisions was driven by the uncertainty associated with the genetic test results. Participants often contextualized their risk by building on the risk associated with genetic test results with family history, variant re-interpretation, and the knowledge that the risks associated with other genes may be higher. Patients generally made the decision they thought was best for them, even though it was more difficult if that decision was not supported by healthcare providers, friends, or family. When faced with uncertain cancer risks and presented with options for medical management, values were weighed against the negatives of each option. Often mental health was prioritized over the negatives associated with ‘removing body parts’.
These findings offer a look into the decisional needs of patients such as accurate knowledge, certainty, decisional support, and attention to personal values. Better understanding of the unmet needs of these patients and working to rectify them through provider education, outreach, counseling strategies to mitigate uncertainty, and research on how to best address and identify each patient’s specific decisional needs can contribute to the goal of risk-appropriate and values-based decision-making. With a better understanding of patients’ decisional needs, healthcare providers can better advocate for tailored counseling sessions which explore and address specific patient needs to help them make informed, risk-appropriate, and value-based medical management decisions.
4
Mondal, Shankar Prosad. « Molecular characterization of the features of antigenic and pathogenic variant strains of infectious bronchitis virus / ». For electronic version search Digital dissertations database. Restricted to UC campuses. Access is free to UC campus dissertations, 2005. http://uclibs.org/PID/11984.
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Darmawan, Hariyanto. « Transport of a pathogenic bacterium and its non-pathogenic variant strain through a granular porous medium : from a simple system to a real system ». Thesis, McGill University, 2011. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=104768.
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The attachment efficiency of two strains of Escherichia coli O157:H7 – one pathogenic strain and another non-pathogenic strain – was measured over a range of solution ionic strengths and in two different granular systems: a simple system made of clean quartz sand and a real system made of natural subsurface soil. In this study, the relevance of the non-pathogenic strain of E. coli O157:H7 as a potential surrogate for its pathogenic counterpart was investigated. The results suggest that it is not straightforward to find an appropriate surrogate for the pathogenic strain. Different porous media begets different attachment efficiency of the potential surrogate strain relative to the attachment efficiency of the toxigenic strain. A modest attempt was also made to build an artificial system that mimics the natural soil, by coating the clean granular sand with humic acids and adding clay component.Pour étudier la contamination d'eaux souterraines, l'efficacité d'adhésion de deux variétés d'E. coli O157:H7 – une pathogène et une autre non-pathogène – a été mesurée sur une gamme de force ionique dans deux systèmes granulaires : un système simple fait de sable de quartz propre et un système naturel de sol souterrain. Dans cette étude, la pertinence de la variété non-pathogène (E. coli O157:H7) comme substitut potentiel pour sa contrepartie fut étudiée. Les résultats suggèrent qu'il est très difficile de trouver un substitut approprié de la variété pathogène pour ce type d'études, car différents médias porreaux engendrent différentes efficacités d'adhésion de la variété substitut potentielle. Une tentative a aussi été faite de construire un système artificiel dans le labo qui imite le sol naturel, en enrobant le sable de quartz avec des acides humiques et par l'addition d'un composé d'argile.
6
Al-Saadi, Abdulwahid. « Phenotypic characterization and sequence analysis of pthA homologs from five pathogenic variant groups of Xanthomonas citri ». [Gainesville, Fla.] : University of Florida, 2005. http://purl.fcla.edu/fcla/etd/UFE0011580.
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Kvist, Alexander. « Identifying Pathogenic Amino Acid Substitutions in Human Proteins Using Deep Learning ». Thesis, KTH, Skolan för kemi, bioteknologi och hälsa (CBH), 2018. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-233513.
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Many diseases of genetic origin originate from non-synonymous single nucleotide polymorphisms (nsSNPs). These cause changes in the final protein product encoded by a gene. Through large scale sequencing and population studies, there is growing availability of information of which variations are tolerated and which are not. Variant effect predictors use a wide range of information about such variations to predict their effect, often focusing on evolutionary information. Here, a novel amino acid substitution variant effect predictor is developed. The predictor is a deep convolutional neural network incorporating evolutionary information, sequence information, as well as structural information, to predict both the pathogenicity as well as the severity of amino acid substitutions. The model achieves state-of-the-art performance on benchmark datasets.
8
HASSAN, AMAL. « FROM PROTEIN STRUCTURE TO DRUG DESIGN (DISCOVERY) : TARGETING THE ION CHANNEL ASIC1 AND A PATHOGENIC VARIANT OF HUMAN GELSOLIN ». Doctoral thesis, Università degli Studi di Milano, 2019. http://hdl.handle.net/2434/629877.
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La conoscenza della struttura tridimensionale di un potenziale target farmacologico apre la via a nuove strategie terapeutiche (ad esempio tramite structure-based drug design (SBDD)) ed è requisito fondamentale per la bioinformatica strutturale.
In questo contesto, durante la mia tesi di dottorato, sono state studiate due proteine di interesse biomedico. La prima è una proteina di membrana, l’isoforma 1 dell’Acid Sensing Ion Channel (ASIC), implicata in diverse malattie neurodegenerative. In studi precedenti il diminazene aceturato (DA) si era dimostrato un potente inibitore del canale. Diversi analoghi di DA sono stati progettati su base strutturale e la loro affinità per ASIC analizzata tramite docking molecolare. Le molecole migliori sono state testate in saggi cellulari per valutarne l’efficacia. Per caratterizzare la capacità inibitoria degli analoghi sintetizzati in vitro, è stato messo a punto un protocollo per la produzione della proteina ASIC1, utilizzando diversi sistemi di espressione eterologa. La purificazione della proteina è stata effettuata usando un approccio high-throughput per supportare successivamente la cristallizzazione della proteina, al fine di ottenere informazioni più dettagliate sul meccanismo d’azione degli analoghi del DA e, di conseguenza, disegnare nuovi farmaci, isoforma-selettivi e in grado di attraversare la barriera emato-encefalica.
In secondo luogo, ho studiato la proteina Gelsolin (GSN), responsabile di una malattia familiare degenerativa (detta amiloidosi AGel). Lo scopo di questo progetto era quello di investigare l'effetto della mutazione D187N sulla struttura di GSN e la sua propensione ad aggregare e/o degradarsi in maniera anomala. Il D187N GSN è stato il primo mutante ad essere identificato, ma, ad oggi, non si avevano informazioni sulla sua struttura. In uno studio precedente, era stato identificato un nanobody (Nb11) in grado di proteggere la proteina dalla degradazione, ma il meccanismo di protezione non era stato chiarito. Nel mio lavoro ho risolto la struttura del complesso Nb11:D187N a 1.9 Å, permettendo la caratterizzazione molecolare del meccanismo di azione del Nb11. I dati strutturali ottenuti sono stati completati con una caratterizzazione biofisica e biochimica, estesa anche ad altre due varianti patologiche della GSN, recentemente identificate (G167R e N184K).Knowledge of the three-dimensional structure of therapeutically relevant proteins paves the way for novel strategies in pharmacological research (such as the structure-based drug design (SBDD) method) and establishes the foundations for structural bioinformatics. In this context, during my PhD Thesis, two therapeutically relevant proteins have been studied. First, a membrane protein, Acid Sensing Ion Channel (ASIC) isoform 1, a validated target in neurodegenerative disorders, was selected. Previous studies showed that diminazene aceturate (DA) is a potent small-molecule inhibitor of ASIC channels. Here, several DA analogues were screened by molecular docking and the best binders were tested in cell-based assays to further assess their efficacy. In order to determine the inhibitory capability of the synthesized analogues in vitro on the purified protein, the expression of ASIC1 was undertaken, using different organisms of expression. The protein purification was performed in a high-throughput approach in order to recover enough protein for crystallization, with the final aim of studying the mechanism of action of DA analogs, and support the design of new, isoform-selective and brain-penetrant drugs. Secondly, the soluble protein Gelsolin (GSN), responsible for a familial degenerative disease (AGel amyloidosis) was studied. Aim of this project was to understand the impact of the D187N mutation on GSN structure and its propensity to aberrant aggregation and/or degradation. D187N GSN mutant was the first identified in man, but its crystal structure had until now eluded any characterization. Conversely, a nanobody (Nb11) was shown to protect GSN from aberrant proteolysis, but its mechanism of protection remained unclear. Here, the structure of the Nb11:D187N complex was solved at 1.9 Å resolution, enabling the characterization of the Nb11action mechanism. The structural data were complemented with biophysical and biochemical characterisations. These studies were then extended to two recently identified pathological variants of GSN (G167R and N184K).
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Röhrs, Susanne [Verfasser], et Gerd [Akademischer Betreuer] Sutter. « A neuraminidase-negative variant of highly pathogenic avian influenza virus H5N1 : generation, characterization and use as a model for early onset of immunity / Susanne Röhrs. Betreuer : Gerd Sutter ». München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2014. http://d-nb.info/1065610890/34.
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Bryen, Samantha Jane. « Identification and molecular mechanisms of pathogenic splicing variants in neuromuscular disorders ». Thesis, The University of Sydney, 2021. https://hdl.handle.net/2123/26955.
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Background
For families with rare Mendelian disorders, obtaining a precise genetic diagnosis is essential to enable personalised and preventative medicine. However, only ∼50% of families receive a genetic diagnosis following genetic testing, as there are still many challenges in the clinical interpretation of genetic variation. One such challenge is the identification and classification of variants that disrupt precursor messenger RNA (pre-mRNA) splicing, hereafter termed splicing variants. Even though splicing variants are well recognised as a common cause of Mendelian disorders, they are regularly classified as Variants of Uncertain Significance (VUS) rendering them clinically unactionable. It is our inability to accurately predict if and how a variant disrupts pre-mRNA splicing that prevents definitive classification of putative splicing variants.
Aims
To explore the molecular mechanisms by which variants disrupt pre-mRNA splicing, thereby improving our ability to detect and accurately assign pathogenicity to splicing variants in the context of genetically diagnosing individuals with neuromuscular disorders.
Methods
We have a cohort of 214 families with neuromuscular disorders for whom we have iteratively applied exomic, genomic and transcriptomic diagnostic sequencing approaches. Putative splicing variants were identified within this cohort or referred to us as VUSs from collaborators. Functional studies were performed to assess variant effect on pre-mRNA splicing. Analysis of datasets derived from variant databases and the human reference genome provided additional insights.
Results
Numerous complex splicing variants were investigated and established as pathogenic for a variety of neuromuscular conditions. Splicing variants analysed included intronic deletions, deep intronic variants, coding variants, structural rearrangements, and extended splice site variants. The mechanistic basis of aberrant pre-mRNA splicing arising from these variants were explored, revealing novel or under-recognised disease mechanisms.
Conclusions
We reveal important mechanistic insights behind pre-mRNA splicing that can be incorporated into diagnostic pipelines and used to inform new splicing prediction algorithms. The discoveries and lessons learned within this thesis are applicable to all rare Mendelian disorders and cancer genomics.
11
Riera, Ribas Casandra. « Novel approaches in the identification of pathogenic variants in the clinical diagnosis ». Doctoral thesis, Universitat Autònoma de Barcelona, 2016. http://hdl.handle.net/10803/399559.
Texte intégralRésumé :
El rápido crecimiento experimentado por las técnicas de secuenciación de última generación ha impulsado a su vez el desarrollo de aplicaciones bioinformáticas destinadas a la anotación funcional e interpretación de las variantes identificadas. De hecho, el uso de estas herramientas es cada vez más popular, habiéndose extendido al ámbito del diagnóstico clínico. Sin embargo, la tasa de éxito promedio de estos métodos se sitúa en torno al 80 %, bastante por debajo todavía de los niveles requeridos para su uso independiente en casos de diagnóstico. En la presente tesis se aborda este problema con la finalidad de extender la precisión de estos métodos y así mejorar su aplicabilidad. Para ello abordamos este desafío desde cuatro perspectivas distintas. En primer lugar, identificamos la existencia de un límite en la tasa de acierto de estas herramientas, y determinamos que la aproximación denominada “protein-specific” (específica de proteína) es realmente prometedora. En segundo lugar, aplicamos dicha aproximación al caso de la enfermedad de Fabry, desarrollando un predictor que identifica sus variantes causales con una tasa de acierto del 90-95 %, compitiendo holgadamente con la de los métodos habitualmente utilizados (ej. SIFT, PolyPhen-2, etc.). En tercer lugar, extendemos esta aproximación a un conjunto de 82 proteínas, contrastando la calidad de los predictores específicos con la de un amplio conjunto de herramientas estándar. Finalmente, proponemos una nueva forma de comparar los métodos de predicción basada en el coste. Este planteamiento considera de forma implícita tanto la enfermedad como los tratamientos asociados disponibles. Como resultado se presenta un criterio de selección de predictores más adaptado al contexto clínico.The rapid growth experienced by next-generation sequencing techniques has fuelled the development of bioinformatic applications for the functional annotation and interpretation of the variants identified. In fact, the use of these tools is becoming increasingly popular, having been extended to the field of clinical diagnosis. However, the average success rate of these methods is around 80%, still well below the levels required for their independent use in diagnosis. In this thesis we address this problem with the goal of extending the accuracy of pathogenicity predictors and thus improve their applicability. We have approached this challenge from four different directions. First, we have identified the existence of an upper limit in the success rate of these tools and determined that the approach known as "protein-specific" is a good option to surpass this threshold. Second, we have applied this approximation to Fabry disease, developing a predictor that identifies causal variants with a success rate of 90-95%, comfortably competing with common methods (e.g. SIFT, PolyPhen-2, etc.). Thirdly, we have extended this approach to a set of 82 proteins, benchmarking the quality of the resulting protein-specific predictors against that of standard tools. Finally, we have proposed a new way to compare prediction methods, based on the cost. This approach implicitly considers both the disease and the associated treatments available. As a result, it constitutes a criterion for selecting predictors adapted to the clinical context.
12
Harvey, John Steven. « Metachromatic leukodystrophy : the role of non-pathogenic sequence variants in the causation of disease / ». Title page, contents and abstract only, 1996. http://web4.library.adelaide.edu.au/theses/09PH/09phh341.pdf.
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VITALE, Alessandra Maria. « GENETIC NEUROCHAPERONOPATHIES ASSOCIATED WITH CCT5 AND HSP60 VARIANTS : ANALYSIS OF THEIR MOLECULAR ANATOMY AND POSSIBLE PATHOGENIC IMPLICATIONS ». Doctoral thesis, Università degli Studi di Palermo, 2022. https://hdl.handle.net/10447/563680.
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Padilla, Sirera Natàlia. « Novel approaches for in silico identification of pathogenic variants in BRCA1 and BRCA2 hereditary breast and ovarian cancer predisposition genes ». Doctoral thesis, Universitat Autònoma de Barcelona, 2020. http://hdl.handle.net/10803/670705.
Texte intégralRésumé :
Variants germinals a les proteïnes BRCA1 i BRCA2 poden alterar la funció protectora d’aquestes a l’ADN, incrementant el risc de desenvolupar càncer de mama i ovari hereditari (HBOC). Identificació d’aquells individus portadors de variants patogèniques permet canalitzar-los en programes específics de prevenció i vigilància, augmentant les seves taxes de supervivència. Per això, en primer lloc, cal identificar quines de les variants són patogèniques. Malauradament, no sempre hi ha prou informació per arribar a una conclusió. En aquesta situació, els predictors de patogenicitat dissenyats per estimar computacionalment el dany causat per les variants poden proporcionar una valuosa informació.
En aquest treball presentem una nova família de predictors de patogenicitat per BRCA1 i BRCA2. Aquests predictors difereixen en el seu objectiu: un està entrenat per estimar l’impacte molecular de les variants sobre la funció HDR de BRCA1 i BRCA2, i l’altre està entrenat per estimar la significació clínica d’una variant, és a dir, si la seva classificació és patogènica o neutra. Els seus rendiments han estat provats i són comparables als d’altres mètodes àmpliament utilitzats en el camp. Addicionalment, vam presentar els predictors al repte ENIGMA de la 5a Avaluació Crítica de la Interpretació del Genoma (CAGI), trobant que els nostres mètodes, especialment aquells que estimen l’impacte funcional de les variants, es classifiquen en les primeres posicions en comparació amb les altres eines.
Per tal de difondre aquesta família de predictors a la comunitat científica, hem construït el lloc web BRASS (https://www.biotoclin.org/BRASS), on els usuaris poden analitzar les seves variants de BRCA1 i BRCA2 amb canvi de sentit. Els usuaris més avançats també poden interpretar les prediccions mitjançant una mètrica de fiabilitat i diversos gràfics contextualitzant la seva puntuació amb la d’un conjunt de variants curades manualment.
Independentment, hem aplicat els nostres coneixements sobre predictors de patogenicitat en un gran projecte internacional per caracteritzar un nou trastorn neurològic pediàtric causat per variants patogèniques a la histona H3.3. Vam combinar l’ús de predictors de patogenicitat estàndard amb evidències d’anàlisis estructurals i càlculs biofísics per proporcionar una visió mecanicista de l’impacte de les variants causals.Variantes germinales en las proteínas BRCA1 y BRCA2 pueden alterar la función protectora de estas en el ADN, incrementando el riesgo de desarrollar cáncer de mama y ovario hereditario (HBOC). Identificación de aquellos individuos portadores de variantes patogénicas permite canalizarlos hacia programas específicos de prevención y vigilancia, aumentando sus tasas de supervivencia. Para ello, en primer lugar, es necesario identificar cuáles de las variantes son patogénicas. Desafortunadamente, no siempre hay suficiente información para llegar a una conclusión. En esta situación, los predictores de patogenicidad diseñados para estimar computacionalmente el daño causado por las variantes pueden proporcionar una valiosa información. En este trabajo presentamos una nueva familia de predictores de patogenicidad para BRCA1 y BRCA2. Estos predictores difieren en su objetivo: uno está entrenado para estimar el impacto molecular de las variantes en la función HDR de BRCA1 y BRCA2, y el otro está entrenado para estimar la significancia clínica de una variante, es decir, si su clasificación es patogénica o neutra. Sus rendimientos han sido probados y son comparables a los de los métodos ampliamente utilizados en el campo. Además, presentamos los predictores al desafío ENIGMA de la 5ª Evaluación Crítica de la Interpretación del Genoma (CAGI), encontrando que nuestros métodos, especialmente aquellos que estiman el impacto funcional de las variantes, se clasifican en las primeras posiciones en comparación con las otras herramientas. Para difundir esta familia de predictores a la comunidad científica, hemos construido el sitio web BRASS (https://www.biotoclin.org/BRASS), donde los usuarios pueden analizar sus variantes de BRCA1 y BRCA2 con cambio de sentido. Los usuarios más avanzados también pueden interpretar las predicciones utilizando una métrica de confiabilidad y varios gráficos que contextualizan su puntuación a la de un conjunto de variantes seleccionadas manualmente. De forma independiente, aplicamos nuestro conocimiento sobre los predictores de patogenicidad en un gran proyecto internacional para caracterizar un nuevo trastorno neurológico pediátrico causado por variantes patogénicas en la histona H3.3. Combinamos el uso de predictores patogénicos estándar con evidencia de análisis estructurales y cálculos biofísicos para proporcionar una visión mecanicista del impacto de las variantes causales.
Germline variants in BRCA1 and BRCA2 can disrupt the DNA protective role of these proteins resulting in an increased risk of developing hereditary breast and ovarian cancer (HBOC). Identification of those individuals carrying pathogenic variants will allow channeling them into specific programs of prevention and surveillance, incrementing their survival rates. For this purpose, first, it is necessary to identify which of the variants are pathogenic. Unfortunately, there is not always enough information to reach a conclusion. In this situation, pathogenicity predictors designed to computationally estimate the damage caused by variants, can provide valuable information. Here, we present a novel family of pathogenicity predictors for BRCA1 and BRCA2. These predictors differ in their objective: one is trained to estimate the molecular impact of variants on the HDR function of BRCA1 and BRCA2, and the other is trained to estimate the clinical significance of a variant, that is, whether it should be classified as pathogenic or neutral. Their performances have been tested and are comparable to those of widely used predictors in the field. Additionally, we presented them to the ENIGMA challenge from the 5th Critical Assessment of Genome Interpretation (CAGI), finding that our predictors, especially those estimating the functional impact of variants, ranked in the top positions compared to other tools. In order to disseminate this family of predictors to the scientific community, we have built the BRASS website (https://www.biotoclin.org/BRASS), where users can analyze their missense BRCA1 and BRCA2 variants. More advanced users can also interpret the predictions using a reliability metric and several plots contextualizing the score to that of a set of manually curated variants. Independently, we applied our knowledge about pathogenicity predictors in a large international effort to characterize a novel pediatric neurologic disorder caused by pathogenic variants in histone H3.3. We combined the use of standard pathogenic predictors with evidence from structural analyses and biophysical computations to provide a mechanistic view of the impact of the causative variants.
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Baird, Denis Andrew. « Statistical and bioinformatics approaches for discovering pathogenic single nucleotide variants in idiopathic early on-set nephrotic syndrome using exome sequencing ». Thesis, University of Bristol, 2017. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.723503.
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Matar, Suzan. « Characterization of staphylococcal small colony variants and their pathogenic role in biomaterial-related infections with special reference to Staphylococcus epidermidis ». Thesis, University of Nottingham, 2004. http://eprints.nottingham.ac.uk/12135/.
Texte intégralRésumé :
There are many surgical implanted devices in current use and all are prone to biomaterial-related infections (BRI) associated with staphylococcal biofilm formation. BRI are usually associated with S. epidermidis or S. Aureus and are characterized by treatment failure and chronicity resulting in reoperation, removal of the implant, and loss of function or death. Staphylococcal small colony variants (SCVs) may be generated by exposure to sublethal concentrations of antibiotics or nutrient limitation which may occur in biofilms. Although the characteristics of S. aureus SCVs have been well studied, little information on SCVs of S. epidermidis and their potential role in BRI is currently available. This study was designed to investigate the biochemical and phenotypic characteristics of S. epidermidis SCVs to further identify characteristics which may contribute to their ability to cause these increasingly important infections. Exposure to two to four times the gentamicin MIC led to the emergence of stable S. epidermidis SCVs, and the ability to produce SCVs was strain dependent. These variants were isogenic by PFGE and less immunogenic by western blotting, and SDS-PAGE analysis of whole cell preparations and cell wall fractions showed altered protein profiles when compared to wild type strains. S epidermidis SCVs were resistant to aminoglycosides such as amikacin and/or netilmicin and they were thiamine and/or menadione auxotrophs. Chemiluminescence assays showed a decreased ATP content reflecting the deficiency in electron transport systems which results in a growth rate – all characteristics similar to those of S. aureus SCVs. Analysis of virulence factor production indicated that S. epidermidis SCVs showed increased lipolytic and proteolytic activity when compared to those of S. aureus. Some S. epidermidis SCVs showed phase variation in exopolysaccharide production which enabled them to be more adherent to uncoated plastic -a property that may also be important for the later stages of development of biofilms. Invasion assays demonstrated that some S. epidermidis and S. aureus SCVs were internalised by HUVECs by a receptor-mediated mechanism which differed from that of the wild type strains. Interaction of staphylococci with HUVECs induced cytokine production but SCVs stimulated production of IL1, IL-6 and IL-8 at lower concentrations than their related wild type parents in the first 6 hours of co-incubation. SCVs were also less damaging to the HUVEC cell line after 24 hours when compared to wild type strains. This study supports the suggestion that a switch to the S. epidermidis SCV phenotype could be a mechanism exploited by the wild type strains to facilitate their survival inside the host. The chronicity and increased antibiotic resistance associated with BRI could in part, be explained by the characteristics of SCVs identified in this study. In particular the ability to survive intracellularly combined with reduced immunogenicity and resulting decreased cytokine production, may contribute to persistence of infection. Although SCVs are resistant to some antibiotics, surviving intracellularly may further protect staphylococci from other drugs which are unable to enter mammalian cells. Resistance may be further enhanced for some strains in biofilms where enhanced polysaccharide production may also limit antibiotic access.
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Vanderwal, April. « Factors that Influence the Management Recommendations Breast Surgeons Provide to Women with Pathogenic Variants in Moderate Penetrance Breast Cancer Susceptibility Genes ». University of Cincinnati / OhioLINK, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1592133660069865.
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Fanelli, Antonella. « Screening of the SHOX gene in a large cohort of short stature italian patients. Identification of novel likely pathogenic variants through functional characterization ». Doctoral thesis, Università del Piemonte Orientale, 2019. http://hdl.handle.net/11579/102444.
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Henderson, Melissa. « Patient-physician Dialogue Matters : Factors that Impact Medical Management Decisions among Women with Pathogenic Variants in Moderate-penetrance Genes Associated with Hereditary Breast Cancer ». University of Cincinnati / OhioLINK, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1554213725302437.
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Supinger, Rachel Christine. « Process Review of GJB6 Reflex Testing in Individuals with 0 or 1 GJB2 Pathogenic Variants and Non-Syndromic Hearing Loss ». The Ohio State University, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=osu1492470064058105.
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Egbers, Anna [Verfasser], et Stefan [Akademischer Betreuer] Kindler. « Das Fragile X Syndrom : Untersuchung des zellulären Interaktionsmusters der physiologischen und einer pathogenen FMRP-Variante / Anna Egbers ; Betreuer : Stefan Kindler ». Hamburg : Staats- und Universitätsbibliothek Hamburg, 2017. http://d-nb.info/1128820382/34.
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Kremer, Laura Sophie Verfasser], Thomas [Akademischer Betreuer] [Floß, Bernhard [Gutachter] Küster, Thomas [Gutachter] Floss et Johannes [Gutachter] Mayr. « Discovery and validation of coding and non-coding pathogenic variants in mitochondrial disorders / Laura Sophie Kremer ; Gutachter : Bernhard Küster, Thomas Floss, Johannes Mayr ; Betreuer : Thomas Floss ». München : Universitätsbibliothek der TU München, 2017. http://nbn-resolving.de/urn:nbn:de:bvb:91-diss-20171219-1364275-1-5.
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Kremer, Laura Sophie [Verfasser], Thomas [Akademischer Betreuer] Floss, Bernhard [Gutachter] Küster, Thomas [Gutachter] Floss et Johannes [Gutachter] Mayr. « Discovery and validation of coding and non-coding pathogenic variants in mitochondrial disorders / Laura Sophie Kremer ; Gutachter : Bernhard Küster, Thomas Floss, Johannes Mayr ; Betreuer : Thomas Floss ». München : Universitätsbibliothek der TU München, 2017. http://d-nb.info/1152006525/34.
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LE, ROMANCER MARC. « La maladie des necroses annulaires superficielles des tubercules de la pomme de terre : identification et caracterisation de l'agent pathogene : le variant yntn du virus y de la pomme de terre ». Rennes 1, 1993. http://www.theses.fr/1993REN10089.
Texte intégralRésumé :
La maladie des necroses annulaires superficielles des tubercules est apparue recemment en europe. L'agent causal est un nouveau variant du pvy (pvy-ntn). Son aptitude a infecter capsicum annuum et chenopodium species, sa capacite a contourner ou a abaisser la resistance gouvernee par les genes ny-dms et ry-sto le differencient des autres biotypes de pvy. Le variant y-ntn induit une reaction d'hypersensibilite foliaire et tuberculaire en l'absence de gene specifique de resistance. L'absence de symptomes necrotiques tuberculaires est controlee chez l'hote par un gene dominant. Les sequences nucleotidiques et en acides amines de la region 3 terminale du genome viral presentent une variabilite qui permet de distinguer deux principaux groupes de souches de pvy. Ces groupes correspondent assez bien aux deux serotypes majeurs du pvy. Les methodes d'analyses genomique et serologique devraient permettre de mieux ordonner les differentes populations de pvy infeodees au tabac, au poivron, a la tomate et a la pomme de terre
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Pestrak, Matthew James Pestrak. « Investigation of the Pseudomonas aeruginosa biofilm exopolysaccharide Psl and its role during infection ». The Ohio State University, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=osu1543240479329587.
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Schüller, Anna Katharina [Verfasser]. « The glmS ribozyme is an antibacterial target : Mode of action analysis, investigation of potential metabolite analogs and characterization of glmS ribozyme variants of human pathogens / Anna Katharina Schüller ». Bonn : Universitäts- und Landesbibliothek Bonn, 2018. http://d-nb.info/1161462201/34.
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Graf, Elisabeth [Verfasser], Hans-Rudolf [Akademischer Betreuer] Fries, Thomas [Gutachter] Meitinger et Hans-Rudolf [Gutachter] Fries. « Etablierung und Anwendung der Exom-Sequenzierung und eines NGS-LIMS zur Identifikation seltener pathogener genetischer Varianten bei monogenen Erkrankungen / Elisabeth Graf ; Gutachter : Thomas Meitinger, Hans-Rudolf Fries ; Betreuer : Hans-Rudolf Fries ». München : Universitätsbibliothek der TU München, 2019. http://d-nb.info/1190818701/34.
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Jaber, Dana. « Identification et caractérisation d’un nouveau gène dont les mutations sont responsables d’Arthrogrypose Multiple Congénitale De Novo Mutations of SCN1Aare Responsible for Arthrogryposis Broadening the SCN1A– Related Phenotypes ». Thesis, université Paris-Saclay, 2020. http://www.theses.fr/2020UPASL047.
Texte intégralRésumé :
L’arthrogrypose multiple congénitale (AMC) est une maladie rare caractérisée par des rétractions d’au moins deux articulations distinctes présentes à la naissance. L’AMC est génétiquement hétérogène et des variants dans de très nombreux gènes ont été identifiés. Notre projet a eu pour but d’identifier de nouvelles causes génétiques de l’AMC en utilisant le séquençage de l’exome entier (WES).Dans le gène SCN1A, nous avons identifié des variants pathogéniques dominants survenus de novo chez trois patients non apparentés. SCN1A code Nav1.1, un composant des canaux sodiques voltage-dépendants exprimés au niveau de l’AIS (Axon Initial Segment) et des nœuds de Ranvier et contribue à l’initiation et la propagation des potentiels d’action. Nous avons montré que SCN1A est exprimé dans le cerveau et la moelle épinière mais pas dans le muscle à des stades du développement similaires à l’âge de l’apparition de l’AMC et que l’AMC est due à un défaut de SCN1A dans le cortex moteur.Nous montrons pour la première fois que des variants de SCN1A sont responsables d’un déficit moteur sévère provoquant l’AMC, suggérant un rôle critique de SCN1A dans le développement moteur. Ces résultats élargissent le spectre phénotypique de SCN1A allant d’une encéphalopathie épileptique sévère isolée ou associée à un défaut moteur causant l’arthrogrypose (Jaber et al. sous presse). Nos données élargissent ainsi le groupe des AMC liées à une atteinte du système nerveux central et précisément celles causées par des anomalies des canaux ioniques tels que NALCN ou CACNA1EArthrogryposis multiplex congenita (AMC) is a rare disease characterized by the presence of multiple joint contractures at birth. AMC includes a large spectrum of diseases which result from variants in genes encoding components required for neuromuscular junctions, skeletal muscle, motor neurons, peripheral nerves or connective tissues. AMC may also result from central nervous involvement. Despite important progress in this field, many AMC patients remain genetically undiagnosed. Our project aimed at identifying novel genes in which variants can cause AMC. We identified pathogenic de novo dominant variants in SCN1A gene in three unrelated patients. SCN1A encodes Nav1.1 voltage-dependent sodium channel, a critical component of axon initial segment and nodes of Ranvier. We showed that SCN1A is expressed in both brain and spinal cord but not in skeletal muscle at a developmental stage similar to that of AMC observation and showed that AMC is caused by brain involvement.We showed that SCN1A variants are responsible for early onset motor defect leading to AMC indicating a critical role of SCN1A in prenatal motor development and broadening the phenotypic spectrum of variants in SCN1A (Jaber et al. in press). Altogether, our data enlarge the group of AMC linked to central nervous system involvement and caused by variants of genes encoding channels such as NALCN or CACNA1E
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Hovhannisyan, Yeranuhi. « Modélisation cardiaque des myopathies myofibrillaires à l'aide de cellules souches pluripotentes induites pour explorer la pathogenèse cardiaque Polyacrylamide Hydrogels with Rigidity-Independent Surface Chemistry Show Limited Long-Term Maintenance of Pluripotency of Human Induced Pluripotent Stem Cells on Soft Substrates Modéliser la myopathie myofibrillaire pour élucider la pathogenèse cardiaque Synemin-related skeletal and cardiac myopathies : an overview of pathogenic variants Desmin prevents muscle wasting, exaggerated weakness and fragility, and fatigue in dystrophic mdx mouse Effects of the selective inhibition of proteasome caspase-like activity by CLi a derivative of nor-cerpegin in dystrophic mdx mice ». Thesis, Sorbonne université, 2020. http://www.theses.fr/2020SORUS095.
Texte intégralRésumé :
La myopathie myofibrillaire est une maladie neuromusculaire à évolution lente caractérisée par de graves troubles musculaires causés par des mutations dans le gène codant pour des protéines du cytosquelette. L'un des gènes affectés en relation avec le développement de la MFM est DES. Des mutations dans le gène de la desmine entraînent des myopathies des muscles squelettiques et cardiaques. Cependant, les évènements qu'elles entraînent et qui sont à l’origine des phénotypes pathologiques cardiaques restent mal connus. Mon objectif est de créer un modèle in vitro de MFM basé sur des cellules souches pluripotentes humaines afin d'étudier le rôle des mutations spécifiques dans la desmine sur le développement et la fonction des cellules cardiaques. Pour atteindre cet objectif, en collaboration avec les docteurs A. Behin, K. Wahbi et la société Phenocell, nous avons généré des iPSC à partir des cellules sanguines périphériques de patients souffrant d'une forme de cardiomyopathie induite par une mutation de la desmine. Les lignées iPSC générées contenant les mutations du gène codant la desmine ont permis d’étudier le rôle d’une mutation dans la spécification et la fonction des cardiomyocytes. La bioénergétique mitochondriale, la structure cellulaire et la fonction contractiles ont été évaluées au niveau cellulaire. En conclusion, il convient de noter que les mutations de la desmine conduisent à une désorganisation des structures des sarcomères dans les cardiomyocytes et à une perturbation de l'expression des protéines mitochondriales. Ce qui conduit à une altération des fonctions de la mitochondrie. Ces données permettent d’améliorer notre compréhension des mécanismes moléculaire qui sous-tendent le développement de la MFMMyofibrillar Myopathy is a slowly progressive neuromuscular disease characterized by severe muscular disorders caused by mutations in the gene encoded cytoskeletal proteins. One of the genes described in connection with the development of MFM is DES. Mutations in the desmin gene lead to skeletal and cardiac muscles myopathies. However, the cardiac pathological consequences caused by them remain poorly understood. My objective is to create an in vitro human stem cell model of MFM to specifically investigate the role of patient-specific mutations in desmin on cardiac lineage development and function. To achieve that objective, in collaboration with Drs. Behin and K. Wahbi and Phenocell, we generate patient-specific iPSC from peripheral blood cells of the patient suffering severel form of desmin-deficient cardiomyopathy. The generated iPSC lines carrying DES gene mutations enable a powerful examination of the role of desmin mutation on cardiomyocyte specification and function. Bioenergetic, structural, and contractile function will be assessed in a single cell. In conclusion, it should be noted that desmin mutations lead to a disorganization of sarcomere structures in cardiomyocytes and to a perturbation of mitochondrial protein expression. This leads to a distortion of functions in the mitochondria. These data facilitate the understanding of the molecular pathway underlying the development of desmin-related myopathy. And the system we have created could also allow us to better evaluate the correlation between the desmin genotype and phenotype in terms of effect on the heart
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Bacelar, Clara Gonçalves. « Renal manifestations in adults with mitochondrial disease from the mtDNA m.3243A>G pathogenic variant ». Master's thesis, 2021. https://hdl.handle.net/10216/134352.
Texte intégralRésumé :
As doenças mitocondriais são um grupo de doenças heterogéneo, tanto do ponto de vista clínico como genético, de atingimento tipicamente multissistémico. A variante patogénica m.3243A>G constitui o defeito no DNA mitocondrial mais frequente, e pode resultar em diferentes síndromes clínicas, incluindo a diabetes e surdez de hereditariedade materna (MIDD) e a encefalopatia mitocondrial, acidose láctica e episódios tipo AVC (MELAS). O atingimento renal nestas doenças não é muito comum, mas pode ser subestimado e constituir um fator agravante na morbilidade destes doentes. Manifesta-se geralmente por proteinúria subnefrótica e agravamento progressivo da função renal. A apresentação das doenças mitocondriais no adulto pode ser de difícil reconhecimento, especialmente em casos oligossintomáticos ou apenas com manifestações renais. No entanto, a suspeita deve surgir sempre que existe história familiar, especialmente no lado materno, e envolvimento multissistémico, mais frequentemente do sistema nervoso central e músculo-esquelético. As opções terapêuticas atuais para as mitocondriopatias são predominantemente de suporte. O aconselhamento genético é importante, para que os pacientes disponham de informação precisa sobre o prognóstico, risco de recorrência e opções de planeamento familiar.Mitochondrial diseases are a clinically and genetically heterogeneous group of disorders, that typically have a multisystemic involvement. The m.3243A>G pathogenic variant is the most frequent mitochondrial DNA defect, and it causes several different clinical syndromes, such as the mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS), and the maternally inherited diabetes and deafness (MIDD) syndromes. Not frequently reported, renal involvement in these diseases is probably underestimated, yet it represents an important aggravating factor in the morbidity of these patients. It generally manifests as subnephrotic proteinuria and progressive deterioration of kidney function. Adult presentation of mitochondrial diseases is hard to recognize, especially in oligosymptomatic patients or those with exclusive kidney involvement. However, suspicion should always arise when family history, particularly on the maternal side, and multisystemic symptoms, most often of the central nervous system and skeletal muscles, are present. Current treatment options for mitochondrial cytopathies are largely supportive. Genetic counselling is important in mitochondrial disorders, to provide patients with accurate information about prognosis, recurrence risk, and reproductive choices.
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Bacelar, Clara Gonçalves. « Renal manifestations in adults with mitochondrial disease from the mtDNA m.3243A>G pathogenic variant ». Dissertação, 2021. https://hdl.handle.net/10216/134352.
Texte intégralRésumé :
As doenças mitocondriais são um grupo de doenças heterogéneo, tanto do ponto de vista clínico como genético, de atingimento tipicamente multissistémico. A variante patogénica m.3243A>G constitui o defeito no DNA mitocondrial mais frequente, e pode resultar em diferentes síndromes clínicas, incluindo a diabetes e surdez de hereditariedade materna (MIDD) e a encefalopatia mitocondrial, acidose láctica e episódios tipo AVC (MELAS). O atingimento renal nestas doenças não é muito comum, mas pode ser subestimado e constituir um fator agravante na morbilidade destes doentes. Manifesta-se geralmente por proteinúria subnefrótica e agravamento progressivo da função renal. A apresentação das doenças mitocondriais no adulto pode ser de difícil reconhecimento, especialmente em casos oligossintomáticos ou apenas com manifestações renais. No entanto, a suspeita deve surgir sempre que existe história familiar, especialmente no lado materno, e envolvimento multissistémico, mais frequentemente do sistema nervoso central e músculo-esquelético. As opções terapêuticas atuais para as mitocondriopatias são predominantemente de suporte. O aconselhamento genético é importante, para que os pacientes disponham de informação precisa sobre o prognóstico, risco de recorrência e opções de planeamento familiar.Mitochondrial diseases are a clinically and genetically heterogeneous group of disorders, that typically have a multisystemic involvement. The m.3243A>G pathogenic variant is the most frequent mitochondrial DNA defect, and it causes several different clinical syndromes, such as the mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS), and the maternally inherited diabetes and deafness (MIDD) syndromes. Not frequently reported, renal involvement in these diseases is probably underestimated, yet it represents an important aggravating factor in the morbidity of these patients. It generally manifests as subnephrotic proteinuria and progressive deterioration of kidney function. Adult presentation of mitochondrial diseases is hard to recognize, especially in oligosymptomatic patients or those with exclusive kidney involvement. However, suspicion should always arise when family history, particularly on the maternal side, and multisystemic symptoms, most often of the central nervous system and skeletal muscles, are present. Current treatment options for mitochondrial cytopathies are largely supportive. Genetic counselling is important in mitochondrial disorders, to provide patients with accurate information about prognosis, recurrence risk, and reproductive choices.
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Perera, Needra Sheron. « Assessment of Missense Alterations in MLH1 and their Pathogenic Significance ». Thesis, 2010. http://hdl.handle.net/1807/32048.
Texte intégralRésumé :
Germline mutations in mismatch repair genes predispose individuals to Lynch Syndrome, the most common colorectal cancer predisposition syndrome. MLH1 is a key mismatch repair gene that is mutated in Lynch syndrome and about a third of the genetic alterations identified in MLH1 are missense variants of unclassified clinical significance. We hypothesize that missense alterations in distinct domains of MLH1 likely affect its expression and function(s) to varying degrees. To address this we utilized several approaches to investigate the molecular basis of the pathogenicity of a panel of unclassified variants. Our results demonstrate that the MLH1 variants p.R265C and p.K618A significantly decrease the stability of the MLH1 protein, while the variant p.L749Q compromises heterodimerization of the MLH1-PMS2 complex. Given the limitations and complexity of in vitro assessment strategies, we conducted a proof-of-principle study to investigate whether missense variants in MLH1 lead to allelic imbalances at the transcriptional level. Our analysis using the PeakPicker software indicated that the missense variants c.350C>T, c.793C>T, c.1852_1853AA>GC, as well as the truncating variant c.1528C>T were associated with significantly unbalanced allelic expression. This illustrates a novel method of investigating the pathogenicity of unclassified genetic variants, which has the potential to be applied in the diagnostic setting. Previous genetic epidemiology studies from our laboratory have demonstrated that the MLH1 c.-93G>A promoter variant is strongly associated with the microsatellite instability phenotype in colorectal tumours. Additionally, this promoter variant was associated with an elevated risk of endometrial cancer in case-control studies. Results from our functional studies indicate that the c.-93G>A variant significantly alters the promoter activity of MLH1. The MLH1 promoter is bi-directional with the EPM2AIP1 gene located on the antisense strand. Interestingly, we observed that this variant significantly affected EPM2AIP1 transcription as well. Furthermore, our experiments suggest that c.-93G>A variant affects transcription by altering the affinity of nuclear factors that bind this region. Combined, these findings shed light on the possible mechanisms by which missense variants affect MLH1 expression and function, which in conjunction with results from other functional assays will help increase the accuracy and efficiency of genetic testing of inherited cancers.
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DINDO, MIRCO. « Molecular analysis of the dimerization and aggregation processes of human alanine:glyoxylate aminotransferase and effect of mutations leading to Primary Hyperoxaluria Type I ». Doctoral thesis, 2017. http://hdl.handle.net/11562/960999.
Texte intégralRésumé :
Primary Hyperoxaluria Type 1 (PH1) is a rare autosomal recessive disorder characterized by the deposition of insoluble calcium oxalate crystals at first in the kidneys and urinary tract and then in the whole body. PH1 is caused by the deficiency of human liver peroxisomal alanine:glyoxylate aminotransferase (AGT). AGT is a pyridoxal 5'-phosphate (PLP)-dependent enzyme, which converts glyoxylate to glycine, thus preventing glyoxylate oxidation to oxalate and calcium oxalate formation. Only two curative therapeutic approaches are currently available for PH1: the administration of pyridoxine (PN), a precursor of PLP, which is only effective in a minority of patients (25- 35%), and liver transplantation, a very invasive procedure. AGT is encoded by the AGXT gene, which is present in humans as two polymorphic forms: the major allele (encoding AGT-Ma) and the minor allele (encoding AGT-Mi). PH1 is a very heterogeneous disease with respect to the clinical manifestations, the response to treatment and the pathogenic mechanisms. In fact, more than 200 pathogenic mutations have been identified so far and the molecular mechanisms by which missense mutations cause AGT deficiency span from functional, to structural and to subcellular localization defects or to a combination of them. Several lines of evidence at both molecular and cellular level, indicate that many disease-causing missense mutations interfere with AGT dimer stability and/or aggregation propensity. However, neither the dimerization nor the aggregation process of AGT have been analyzed in detail. Therefore, we engineered a mutant form of AGT stable in solution in the monomeric form and studied its biochemical properties and dimerization kinetics. We found that monomeric AGT is able to bind PLP and that the coenzyme stabilizes the dimeric structure. Moreover, the identification of key dimerization hot-spots at the monomer-monomer interface allowed us to unravel the mechanisms at the basis of the aberrant mitochondrial mistargeting of two of the most common PH1-causing variants. We also elucidated the molecular and cellular consequences of the pathogenic mutations R36H, G42E, I56N, G63R and G216R, involving residues located at the dimer interface, and tested their in-vitro responsiveness to the treatment with PN. The latter results allowed us to suggest a possible correlation between the structural defect of a variant and its degree of responsiveness to PN. Finally, by combining bioinformatic and biochemical approaches, we analyzed in detail the tendency of AGT to undergo an electrostatically-driven aggregation. We found that the polymorphic changes typical of the minor allele have opposite effect on the aggregation propensity of the protein, and we predicted the possible effect/s of pathogenic mutations of residues located on the AGT surface. Overall, the results obtained allow not only to better understand PH1 pathogenesis, but also to predict the response of the patients to the available therapies as well as to pave the way for the development of new therapeutic strategies.
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Pereira, Maria da Conceição Moutinho Pedroso. « Pathogenic variants in movement disorders : modifiers, interactors and disease models ». Doctoral thesis, 2019. https://hdl.handle.net/10216/123074.
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Pereira, Maria da Conceição Moutinho Pedroso. « Pathogenic variants in movement disorders : modifiers, interactors and disease models ». Tese, 2019. https://hdl.handle.net/10216/123074.
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Chuang, Kai-Wen, et 莊凱文. « Clustering Ensemble Based Imbalanced Learning for Predicting Pathogenic Non-coding Variants ». Thesis, 2019. http://ndltd.ncl.edu.tw/handle/n9y34b.
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碩士國立臺灣大學
生物產業機電工程學研究所
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With the help of Next Generation Sequencing (NGS) and whole-genome sequencing (WGS), many variants in the non-coding regions were found in the human genome, but the ensured pathogenic variants were only a minority. It is a challenge to find pathogenic variants from such a large number of non-coding variants. Recently, a method, HyperSMURF, was previously proposed to tackle this problem by using both sampling and over-sampling techniques to balance the data. Through reproducing the analytic results of HyperSMURF, we observed that this approach might generate samples that did not help with training in minority or reduced the samples that might benefit training in majority. In this regard, this study aims at presenting a machine learning framework, CE-SMURF. The CE-based (Clustering Ensemble-based) method is used to find the samples of the center in majority and the samples of the boundary in minority, and then use the resampling technique to balance the ratio of data. Moreover, in order to improve the learning performance, we used the ensemble method to build multiple models, and computed the final scores by averaging the probability of variants in each model. It is found that CE-SMURF can significantly improve the performance of the predicting non-coding pathogenic variants.
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Cheng-YuLiao et 廖正宇. « Exome sequencing identifies pathogenic variants in the patients with severe combined immunodeficiency ». Thesis, 2015. http://ndltd.ncl.edu.tw/handle/44uakj.
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Bastos, Hélder Novais. « Identification of human and pathogen molecular variants associated to tuberculosis heterogeneity ». Doctoral thesis, 2017. http://hdl.handle.net/1822/50226.
Texte intégralRésumé :
Tese de Doutoramento em MedicinaTuberculosis (TB) imposes high human and economic tolls. One of the most striking features of TB is the variability of infection outcomes, which has been classically attributed to environmental and host determinants. More recently, studies uncovering Mycobacterium tuberculosis complex (MTBC) genomic diversity have shown the potential importance of pathogen-related factors to the disease pathogenesis. We approached this question from different angles, by combining the study of the pathogen properties, the host immune response and the clinical features of TB, within a cohort of 681 culture-confirmed pulmonary TB (PTB) cases diagnosed at the Hospital de São João, a major healthcare center in Porto, Portugal, between 2007 and 2013. We started by developing a severity assessment tool for stratifying mortality risk in PTB patients. Five risk features were selected for the prediction model: hypoxemic respiratory failure (OR 4.7, 95% CI 2.8-7.9), age ≥50 years (OR 2.9, 95% CI 1.7-4.8), bilateral lung involvement (OR 2.5, 95% CI 1.4-4.4), ≥1 significant comorbidity – HIV infection, diabetes mellitus, liver failure or cirrhosis, congestive heart failure and chronic respiratory disease – (OR 2.3, 95% CI 1.3- 3.8), and hemoglobin <12 g/dL (OR 1.8, 95% CI 1.1-3.1). A TB risk assessment tool (TReAT) was developed, stratifying patients with low (score ≤2), moderate (score 3-5) and high (score ≥6) mortality risk. The mortality associated with each group was 2.9%, 22.9% and 53.9%, respectively. The model performed equally well in the validation cohort. After focusing on the host clinical prognostic predictors, in the second part of the project we assessed the impact of M. tuberculosis diversity on the disease clinical severity. We started by developing a clinical decision tree to classify the severity of the disease and by applying it to a selected group of 133 individuals that in our cohort did not present known predictor or precipitator TB factors. We found that, for this group of patients, no association existed between the severity of disease and the phylogeny of the infecting bacteria. We also found that M. tuberculosis isolates from patients with mild disease grew significantly slower, while strains associated to moderate outcome had a longer lag phase and reached the highest plateau, after a steep exponential phase. To gain in-depth knowledge of the genetic basis for differential mycobacterial growth, we performed whole genome sequencing analysis. We detected several single nucleotide polymorphisms (SNPs) in genes that were previously associated with growth suppression and identified novel gene candidates involved in membrane transport and biosynthetic pathways. Finally, in the third part of this work, we studied the architecture of the immune response triggered by the different isolates of M. tuberculosis. Sixteen clinical isolates associated with different clinical severity of TB were selected and used to infect peripheral blood mononuclear cells (PBMCs) from nontreated/ non-recent latent TB infected (LTBI) donors or past/cured TB patients. Independently of the host genetics, we identified two distinct groups of M. tuberculosis isolates: high versus low inflammatory triggers. Furthermore, we report that PBMCs from past TB patients produced less IL-1β than those from LTBI participants in response to a variety of isolates, whereas the opposite was observed for IL-1RA. LTBI subjects elicited responses with significantly higher IL- 1β/IL-1RA ratios than those from TB patients, thus suggesting this ratio as a discriminator of risk for latent to active TB progression. Overall, we provide a new clinical prediction rule for the risk of death in TB patients and propose a new classification tree for TB severity. On the pathogen side, we unveiled the differential growth of clinical isolates associated with moderate outcomes of TB as a distinctive feature. On the host side, we suggest the ratio IL-1β/IL-1RA as a possible biomarker of disease resistance versus susceptibility to TB. Our findings present new platforms for active and latent TB management and open new avenues for basic research, to unveil host and pathogen determinants of TB outcomes.
A tuberculose (TB) continua a impor elevados custos económicos e humanos. Uma das características mais notáveis da TB é a variabilidade de resultados da infeção, que tem sido classicamente atribuída a determinantes ambientais e do hospedeiro. Trabalhos mais recentes estudaram a diversidade genómica do Mycobacterium tuberculosis complex (MTBC) e revelaram o potencial impacto dos fatores da bactéria na patogénese da TB. Esta questão foi abordada por diferentes ângulos, combinando o estudo das propriedades do patogénio, a resposta imune do hospedeiro e as características clínicas de TB, a partir de uma coorte de 681 casos de TB pulmonar confirmados por cultura, diagnosticados no Hospital de São João, um centro clínico de excelência do Porto, Portugal, entre 2007 e 2013. Começámos por desenvolver uma ferramenta de avaliação de gravidade clínica para estratificar o risco de mortalidade de doentes com TB pulmonar. Cinco fatores de risco foram selecionados para o modelo de predição: insuficiência respiratória hipoxémica (OR 4.7, 95% CI 2.8-7.9), idade ≥50 anos (OR 2.9, 95% CI 1.7-4.8), envolvimento pulmonar bilateral (OR 2.5, 95% CI 1.4-4.4), ≥1 comorbilidade significativa – infeção HIV, diabetes mellitus, insuficiência hepática ou cirrose, insuficiência cardíaca congestiva e doença respiratória crónica – (OR 2.3, 95% CI 1.3-3.8), e hemoglobina <12 g/dL (OR 1.8, 95% CI 1.1-3.1). Desenvolveu-se a tuberculosis risk assessment tool (TReAT), estratificando doentes com baixo (pontuação ≤2), moderado (pontuação 3-5) e alto (pontuação ≥6) risco de mortalidade. A mortalidade em cada grupo foi de 2.9%, 22.9% e 53.9%, respetivamente. O modelo manteve um bom desempenho na coorte de validação. Após nos termos focado nos preditores clínicos de prognóstico do hospedeiro, na segunda parte do projeto quisemos analisar o impacto da diversidade de M. tuberculosis na gravidade da doença. Desenvolvemos uma árvore de decisão clínica para classificar a gravidade da doença e aplicámo-la a um grupo selecionado de 133 doentes que na nossa coorte não apresentavam fatores preditores ou precipitantes conhecidos de TB. Neste grupo não se verificou uma associação entre a gravidade clínica e a filogenia da bactéria infetante. Porém, mostrámos que os isolados de M. tuberculosis de indivíduos com doença ligeira crescem de forma significativamente mais lenta, enquanto que as estirpes associadas com TB moderada apresentam uma fase lag mais longa e atingem um patamar mais elevado, após uma fase uma íngreme fase exponencial. Para conhecer em detalhe as bases genéticas do crescimento micobacteriano, realizámos uma análise de sequenciação genómica. Detetaram-se diversos single nucleotide polymorphisms (SNPs) em genes que previamente tinham sido associados com a supressão de crescimento e identificámos novos genes candidatos envolvidos no transporte de membrana e em vias biossintéticas. Finalmente, na terceira parte do trabalho, estudámos a arquitetura da resposta imune desencadeada por diferentes estirpes de M. tuberculosis. Dezasseis isolados clínicos associados a diferentes gravidades clínicas de TB foram selecionados e usados para infetar células mononucleares do sangue periférico (PBMCs) de dadores com infeção latente não recente e não tratada ou de doentes com TB passada/curada. Independentemente da genética do hospedeiro, identificámos dois grupos distintos de isolados de M. tuberculosis muito e pouco inflamatórias. Além disso, mostrámos que PBMCs de doentes com TB passada produzem menos IL-1β em resposta a uma variedade de isolados, enquanto o oposto se verificou para IL-1RA. Dadores com infeção latente apresentaram respostas com razão IL-1β/IL-1RA significativamente mais elevada. Em suma, propomos uma nova regra de predição clínica para o risco de mortalidade por TB e uma nova árvore de classificação de gravidade da doença. Na vertente do patogénio, desvendámos um perfil de crescimento distinto dos isolados clínicos associados com TB moderada. Na vertente do hospedeiro, os nossos resultados sugerem que a razão IL-1β/IL-1RA poderá ser um biomarcador de resistência versus suscetibilidade para TB. Estes dados fornecem novas plataformas para a investigação básica dos determinantes do hospedeiro e patogénio na heterogeneidade da TB.
The work presented in this dissertation was performed in the Microbiology and Infection Research Domain of the Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, Braga, Portugal (ICVS/3B’s – PT Government Associate Laboratory, Braga/Guimarães, Portugal); in Centro Hospitalar São João, Porto, Portugal; and at the Institute for Molecular and Cell Biology (IBMC), University of Porto, Portugal. The financial support was provided by Fundação Amélia de Mello/José de Mello Saúde, Sociedade Portuguesa de Pneumologia (SPP) and by a Research Grant from the European Society for Clinical and Infectious Diseases (ESCMID). The study was cofunded by Programa Operacional Regional do Norte (ON.2 – O Novo Norte), Quadro de Referência Estratégico Nacional (QREN), through the Fundo Europeu de Desenvolvimento Regional (FEDER).
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Davison, Kenneth Mark. « Pathogenic mutations and novel variants in MLH1 and MSH2 in a South African colon cancer cohort ». Thesis, 2013. http://hdl.handle.net/10539/12571.
Texte intégralRésumé :
Identification of mutations in the mismatch repair genes of hereditary non-polyposis colorectal cancer (HNPCC) families can lead to improved management and screening of affected family members. This study aimed to characterise the mutation profile of MLH1 and MSH2 in a South African colorectal cancer cohort. Twenty patient samples were screened for mutations in MLH1 and MSH2 using Sanger sequencing and Multiplex Ligation-dependent Probe Amplification (MLPA). Three previously reported pathogenic mutations were found using Sanger sequencing, two in MLH1 (c.454-13A>G and c.731_734delGTTA) and one in MSH2 (c.2006-6T>C). Six novel variants were detected using Sanger sequencing, two in MLH1 and four in MSH2. Further investigation of the novel variants strongly suggests that one variant in MLH1 (c.885-1G>A) is pathogenic and two have an unknown contribution towards disease. Molecular diagnostic screening of mutations in MLH1 and MSH2 has the potential to improve surveillance and management of HNPCC in South Africa.
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Cardoso, Ana Rita Pereira. « Influence of repetitive elements on pathogenic copy number variants (CNVs) associated with X-Linked Intellectual Disability (XLID) ». Master's thesis, 2016. https://repositorio-aberto.up.pt/handle/10216/95132.
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Cardoso, Ana Rita Pereira. « Influence of repetitive elements on pathogenic copy number variants (CNVs) associated with X-Linked Intellectual Disability (XLID) ». Dissertação, 2016. https://repositorio-aberto.up.pt/handle/10216/95132.
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