No description available.

Ewens sampling formula is the sampling distribution for a population assumed to follow a one parameter Poisson-Dirichlet distribution, where the parameter is fixed. In this project this assumption will be loosened and we will look at the parameter as a function of the sample size. This will result in sampling from a family of Poisson-Dirichlet distributions. Estimators for this new construction will be tested using two different simulation methods. / Thesis / Master of Science (MSc)

No description available.

No description available.

No description available.

No description available.

The role of highly conserved amino acids within the carboxy-terminal hydrophobic domain of herpes simplex virus I (HSV-I) glycoprotein gB was studied by introducing point mutations using the method of site directed mutagenesis. A segment of this hydrophobic domain of glycoprotein gB contains a nuclear envelope (NE) targeting signal and the effect of these point mutations on targeting to the nuclear envelope was determined. A complementation assay was employed to determine the effect these mutations have on HSV-I infectivity .The point mutations created within the transmembrane domain of glycoprotein gB had no effect on nuclear envelope targeting and localization. However, single point mutations introduced into the first and second hydrophobic domains of glycoprotein gB, G₇₄₃R and F₇₇₀S, affected the targeting and localization of full-length glycoprotein gB at the nuclear envelope. When the transmembrane domain ofHSV-I glycoprotein gB containing the following point mutations A₇₉₀Q, A₇₉₁S, A₇₈₆S, A₇₈₆Y and A₇₉₀S, was introduced into a chimeric protein consisting of the cytoplasmic domain and ectodomain of a plasma membrane protein, vesicular stomatitis virus glycoprotein G, NE targeting and localization were affected. These point mutations may affect the targeting of glycoprotein gB by altering the structure of the targeting signal within the protein. It can be hypothesized that the presence of the cytoplasmic domain. ectodomain domain, and the first and second transmembrane domains within full-length glycoprotein gB can compensate for the effect these point mutations have on nuclear envelope targeting. since the same point mutations had no effect on the targeting · and localization of full-length glycoprotein gB. Complementation assays showed that the glycoprotein gB mutants, A₇₈₆S, A₇₈₆Y, A₇₈₆N, A₇₉₀Q, A₇₉₁S, F₇₇₀S, or G₇₄₃R, were unable to complement a gB-null virus even though these mutant proteins are localized at the nuclear envelope. These proteins may not have been incorporated into the viral capsid due to misfolding or due to the fact that sequences required for interaction with other viral proteins were lost. Another possibility is that the mutant proteins were incorporated into the HSV virion but were not biologically active. / Thesis / Master of Science (MS)

Tesis por compendio / [EN] Self-incompatibility (SI) comprises a compendium of molecular intraspecific barriers, controlled by the S-locus, which enhances outcrossing and prevents inbreeding. Solanaceae, Plantaginaceae and Rosaceae exhibit the Gametophytic SI (GSI) type where specific recognition is controlled by S-RNases and S-locus F-box (SFB) proteins as the female and male S-determinants, respectively. On the other hand, unlinked S-locus genes known as modifier factors are also completely necessary for the mechanism to function. The GSI system seems to be basically preserved in Prunus but striking differences with Solanaceae and other Rosaceae have also been observed. On the basis of this background, this thesis is focused on the identification and characterization of modifiers involved in Prunus GSI to improve our understanding of the underlying mechanism. Previous works in apricot showed that an S-locus unlinked mutation expressed in pollen and located at the distal end of chr. 3 (M-locus) confers self-compatibility in the cv. 'Canino'. In this work, another self-compatible apricot cultivar, named 'Katy', was molecular and genetically analyzed. Similarly, an S-locus unlinked pollen-part mutation was found to cause the loss of self-incompatible response. A mapping strategy based on segregation distorted loci mapped 'Katy' mutation (referred as m-mutation) at the distal end of chr. 3, in a region overlapping with that identified for 'Canino' M-locus. A new screening was carried out to identify additional self-compatible mutants in apricot cultivar/accessions from germplasm banks. Through S-genotyping, three uncategorized S-alleles were recovered and two new mutations putatively conferring self-compatibility (SC) by affecting the male S-determinant SFB were detected. Additionally, M-genotyping showed that the same mutated m-haplotype was shared by 'Canino' and 'Katy', but also by 17 cultivars more from North-America and Western-Europe. A widely distributed haplotype M1-0 was proposed as the putative m-haplotype ancestor suggesting that it arose much later in time than SC-allele, a mutation in the S-locus also conferring SC in apricot. In order to identify this mutation, an integrative genetic, genomic and transcriptomic approach based on NGS data from 'Canino', 'Katy' and the self-incompatible apricot cultivar 'Goldrich' was carried out. This approach led to identify a unique polymorphism able to explain the self-compatible phenotype, a FaSt insertion type of 358-bp in coupling with the m-haplotype within a gene encoding a disulfide bond A-like oxidoreductase (named PaMDOr). PaMDOr was found to be differentially over expressed in mature anthers and the FaSt insertion is predicted to produce a truncated protein. These two findings also support PaMDOr as the pollen-part mutated modifier conferring SC in apricot. Furthermore, phylogenetic analyses suggest PaMDOr as a putative paralog of its contiguous gene (PaM-8), that emerged after the split of the Rosaceae and Solanaceae and which function became essential for the proper functioning of the GSI system in Prunus. Aimed to shed light on the differences and similarities between the S-RNase-based GSI systems in Rosaceae and Solanaceae, orthology relationships were analyzed for modifiers. Putative orthologs were found for NaTrxh, SBP1 and MdABCF in Prunus but a more complex evolutionary pattern was detected for 120K, NaStEP and NaPCCP. Thus, in spite of the differences, it can be hypothesized that part of the GSI modifier factors are shared by both families. As a whole, the multidisciplinary strategy developed in this thesis has allowed us to identify a novel modifier factor (PaMDOr) essential for the self-incompatible response in Prunus as the most significant contribution. In addition, new sources of SC have been detected in apricot and the orthology analysis helped to deepen our understanding on evolutionary aspects of the S-RNase-based GSI system exhibited by Prunus. / [ES] La autocompatibilidad (AI) comprende un conjunto de barreras moleculares intraespecíficas, controladas por el locus S, que favorecen la polinización cruzada y previenen de la endogamia. Solanáceas, Plantagináceas y Rosáceas presentan la llamada AI gametofítica (AIG) donde el reconocimiento específico está controlado por ARNasas-S y proteínas F-box del locus S (SFB) como los determinantes femenino y masculino, respectivamente. Por otra parte, genes no ligados al locus S, conocidos como factores modificadores, son también totalmente necesarios para la correcta regulación del mecanismo. El sistema AIG parece estar básicamente conservado en Prunus pero se han observado notables diferencias con Solanáceas y otras Rosáceas. Con estos antecedentes, el trabajo realizado en esta tesis se ha centrado en la identificación y caracterización de factores modificadores de la AIG en Prunus con el fin de mejorar nuestro conocimiento del mecanismo subyacente. Trabajos previos en albaricoquero mostraron la existencia de una mutación expresada en el polen y no ligada al locus S, que se localiza en el extremo distal del cr.3 (locus M) y que es capaz de conferir autocompatibilidad (AC) en el cultivar 'Canino'. En esta tesis, otro cultivar de albaricoquero autocompatible llamado 'Katy' fue genética y molecularmente analizado. De manera parecida a 'Canino', una mutación que afectaba a un factor no ligado al locus S expresado en el polen era el causante de la pérdida de la respuesta autoincompatible. La mutación en 'Katy' se consiguió mapear en el extremo distal del cr.3 (mutación m), una región que solapa con la identificada para 'Canino'. Una búsqueda para la identificación de nuevo mutantes autocompatibles en cultivares/accesiones de albaricoquero procedentes de bancos de germoplasma fue realizado. Por medio del genotipado del locus S, 3 alelos S no clasificados con anterioridad fueron hallados, mientras que 2 nuevas mutaciones autocompatibles que parecen haber afectado al determinante S masculino SFB fueron detectadas. Adicionalmente, el genotipado para el locus M mostró que el mismo haplotipo m mutado está compartido por 'Canino' y 'Katy' y 17 cultivares más del norte de América y el oeste de Europa. El haplotipo M1-0 ha sido propuesto como posible ancestro del haplotipo m, sugiriendo que éste surgió mucho más tarde que el alelo Sc, mutación del locus S que también confiere AC en albaricoquero. Con el objetivo de identificar esta mutación, un abordaje integral tanto a nivel genético como genómico y transcriptómico mediante datos NGS procedentes de 'Canino', 'Katy' y del cultivar de albaricoquero autoincompatible 'Goldrich' fue llevado a cabo. Esta aproximación sirvió para identificar un único polimorfismo capaz de explicar el fenotipo de AC, una inserción tipo FaSt de 358 pb en acoplamiento con el haplotipo m en un gen que codifica para una disulfide bond A-like oxidoreductase (PaMDOr). PaMDOr mostró estar diferencialmente sobre-expresado en anteras maduras, mientras que la inserción FaSt predice la formación de una proteína truncada. Estos dos hechos apoyan a PaMDOr como el factor modificador de la parte del polen que confiere AC en albaricoquero. Adicionalmente, análisis filogenéticos sugieren que PaMDOr podría ser un parálogo del gen contiguo (PaM-8) que surgió después de la división de Rosáceas y Solanáceas, cuya función ha llegado a ser esencial para el correcto funcionamiento del sistema en Prunus. A fin de arrojar cierta luz en las diferencias y similitudes entre los sistemas de AIG basado en ARNasas-S de Rosáceas y Solanáceas, las relaciones de ortología para factores modificadores fueron estudiadas. Ortólogos candidatos fueron encontrados para NaTrxh, SBP1 y MdABCF, sin embargo, un patrón evolutivo más complejo fue observado para NaStEP, 120K y NaPCCP. De modo que, a pesar de las diferencias, se puede hipotetizar que una parte de los modificadores de la AIG están comparti / [CA] L'autocompatibilitat (AI) comprèn un conjunt de barreres moleculars intraespecífiques, controlades pel locus S, que afavorixen la pol·linització creuada i prevé de l'endogàmia. Solanàcies, Plantaginàcies i Rosàcies presenten l'anomenada AI gametofítica (AIG) on el reconeixement específic està controlat per ARNases-S i proteïnes F-box del locus S (SFB) com a determinants femení i masculí, respectivament. Per un altra banda, gens no lligats al locus S, coneguts com factors modificadors, són també totalment necessaris per a la correcta regulació del mecanisme. El sistema AIG pareix estar bàsicament conservat en Prunus, però s'han observat notables diferències amb Solanàcies i altres Rosàcies. Amb estos antecedents, el treball realitzat durant aquesta tesi se ha focalitzat en la identificació i caracterització de factors modificadors de l'AIG en Prunus a fi d millorar el nostre enteniment del mecanisme subjacent. Treballs previs a l'albercoquer mostraren l'existència d'una mutació expressada al pol·len no lligada al locus S, la qual està localitzada a l'extrem distal del cr.3 (locus M) i es capaç de conferir autocompatibilitat (AC) al cultivar 'Canino'. En aquest treball, un altre cultivar d'albercoquer autocompatible anomenat 'Katy' va ser genètica i molecularment analitzat. De manera pareguda a 'Canino', una mutació que afecta a un factor no lligat al locus S expressat al pol·len era la causa de la perduda de la resposta autoincompatible. La mutació a 'Katy' es va mapetjar a l'extrem distal del cr.3 (mutació m) en una regió solapant amb la identificada per a 'Canino'. Una recerca per a la identificació de nous mutants autocompatibles en cultivars i/o accessions d'albercoquer procedents de bancs de germoplasma va ser portada a terme. Mitjançant el genotipatge del locus S, 3 al·lels S no classificats amb anterioritat van ser trobats, mestres que dos noves mutacions AC que pareixen haver afectat al determinant S masculí SFB varen ser detectades. Amés, el genotipatge del locus M va mostrar que el mateix haplotip m mutat està compartit per 'Canino' i 'Katy', però també per 17 cultivars més del nord d'Amèrica i l'oest d'Europa. El haplotip M1-0, ampliamente distribuït, ha sigut proposat com a possible ancestre del haplotip m, sugerint que aquest va sorgir més tard que el al·lel Sc, una mutació al locus S que també conferix AC a l'albercoquer. Amb l'objectiu d'identificar aquesta mutació, un abordatge integral tant a nivell genètic com genòmic i transcriptòmic mitjançant diversos tipus de dades NGS provinents de 'Canino', 'Katy' i del cultivar d'albercoquer autoincompatible 'Goldrich' va ser portat terme. Aquesta aproximació va permetre identificar un únic polimorfisme capaç d'explicar el fenotip AC, es tracta d'una inserció de 358 pb en adaptament amb el haplotip m en un gen que codifica per a disulfide bond A-like oxidoreductase (PaMDOr). PaMDOr va mostrar estar diferencialment sobre-expressat en anteres madures, mentres que la inserció FaSt prediu la formació d'una proteïna truncada. Estos dos fets recolzen a PaMDOr com al factor modificador de la part del pol·len que conferix AC en albercoquer. A més a més, anàlisis filogenètics suggerixen que PaMDOr podria ser un paràlog del seu gen contigu (anomenat PaM-8) que va sorgir després de la divisió de Rosàcies i Solanàcies, en la qual la funció ha arribat a ser fonamental per al correcte funcionament del sistema d'AIG a Prunus. A fi de tirar certa llum en quant a les diferències i similituds entre els sistemes d'AIG basats en ARNases-S de Rosàcies i Solanàcies, les relacions d'ortologia per als factors modificadors va ser estudiat. Ortòlogs candidat van ser trobats per a NaTrxh, SBP1 i MdABCF, no obstant, un patró evolutiu més complex va ser observat per a NaSTeP, 120K i NaPCCP. De tal manera que, a pesar de les diferències, es pot plantejar la hipòtesi de que una part dels modificadors de l'AIG estan / Muñoz Sanz, JV. (2016). Crossability barriers in Prunus: the role of modifiers in the regulation of the gametophytic self-incompatibility system [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/68489 / Compendio

Yes / Peptide-mediated synthesis and assembly of nanostructures opens new routes to functional inorganic/organic hybrid materials. However, understanding of the many factors that influence the interaction of biomolecules, specifically peptides, with metal surfaces remains limited. Understanding of the relationship between peptide sequence and resulting binding affinity and configurations would allow predictive design of peptides to achieve desired peptide/metal interface characteristics. Here, we measured the kinetics and thermodynamics of binding on a Au surface for a series of peptide sequences designed to probe specific sequence and context effects. For example, context effects were explored by making the same mutation at different positions in the peptide and by rearranging the peptide sequence without changing the amino acid content. The degree of peptide-surface contact, predicted from advanced molecular simulations of the surface-adsorbed structures, was consistent with the measured binding constants. In simulations, the ensemble of peptide backbone conformations showed little change with point mutations of the anchor residues that dominate interaction with the surface. Peptide-capped Au nanoparticles were produced using each sequence. Comparison of simulations with nanoparticle synthesis results revealed a correlation between the colloidal stability of the Au nanoparticles and the degree of structural disorder in the surface-adsorbed peptide structures for this family of sequences. These findings suggest new directions in the optimization and design of biomolecules for in situ peptide-based nanoparticle growth, binding, and dispersion in aqueous media.

Titre de l'écran-titre (visionné le 9 avril 2024) / Les myopathies liées à *RYR1*, les myopathies congénitales les plus fréquemment diagnostiquées, se caractérisent par une hypotonie musculaire squelettique et une faiblesse musculaire squelettique non progressive ou lentement progressive. Les myopathies liées à *RYR1* sont causées par des mutations dans le gène *RYR1*. L'édition Prime a le potentiel d'atteindre une efficacité élevée dans le traitement des myopathies liées à *RYR1* en ciblant les mécanismes physiopathologiques en amont. L'édition Prime utilise le même mécanisme que les systèmes CRISPR/Cas conventionnels, permettant toutes les conversions possibles de base à base et leurs combinaisons, mais n'exploite pas de modèle d'ADN double brin (dADN) ni ne confère de cassures double-brin (DSB) dans la séquence cible. La correction de la mutation T4709M est un exemple important pour le traitement potentiel des maladies liées à *RYR1*. Le maintien d'une homéostasie calcique adéquate pourrait réduire l'incidence de la faiblesse musculaire, améliorer la contraction musculaire et renforcer la fonction motrice globale. La correction des mutations du gène *RYR1* en utilisant l'édition Prime offre également la possibilité de prévenir l'apparition de complications associées aux maladies liées à *RYR1*. La correction précoce de la mutation T4709M pourrait améliorer le pronostic à long terme des personnes atteintes en fonction de l'évolution observée de la faiblesse musculaire et de l'incapacité. Cette approche offre la possibilité d'établir des schémas thérapeutiques plus efficaces et individualisés afin d'optimiser les avantages thérapeutiques et de minimiser simultanément les effets indésirables. Ainsi, cette étude vise à explorer l'application de l'édition Prime pour le traitement des myopathies liées à *RYR1*. Les myopathies liées à *RYR1* sont causées par des mutations dans le gène *RYR1*. L'efficacité de l'édition Prime dans la correction de T4709M, une mutation faux-sens de *RYR1*, a été évaluée à la fois *in vitro* et *in vivo*. Pour les expériences *in vitro*, les plasmides d'édition Prime ont été transfecté dans des cellules HEK293T, des cellules primaires de fibroblastes humains, une lignée de cellules de myoblastes humains, des cellules primaires de fibroblastes *RYR1ᵀᴹ/ᵀᴹ* de souris et des cellules C2C12 par électroporation. Pour les expériences *in vivo*, les plasmides d'édition Prime ont été injectés dans le modèle de souris *RYR1ᵀᴹ/ᵀᴹ*, suivi d'une électroporation. Plusieurs pegARN ont été testés. Les résultats des expériences *in vitro* montrent que l'efficacité de correction varie selon les pegARN. PE3 est l'un des dispositifs incrémentiels de l'édition Prime. PE3b est une version ajustée de PE3 avec une efficacité de correction améliorée et une gamme étendue de modifications génétiques ciblées. La stratégie PE3 s'est révélée plus efficace que la stratégie PE3b pour corriger la mutation ponctuelle. L'efficacité de correction de l'édition Prime peut être améliorée en effectuant plusieurs transfections ou en ajoutant une séquence ARN formant des doubles brins (appelée TevopreQ1) au pegARN. L'efficacité de correction de la mutation *RYR1* T4706M par l'édition Prime *in vivo* ne peut être confirmée. / *RYR1*-related myopathies, the most frequently diagnosed congenital myopathies, are characterized by skeletal muscle hypotonia and non-progressive or slowly progressive skeletal muscle weakness. *RYR1*-related myopathies are caused by mutations in the *RYR1* gene. Prime editing has the potential to achieve a high efficiency in the treatment of *RYR1*-related myopathies by targeting the upstream of the pathophysiological mechanisms. Prime editing employs the same mechanism as conventional CRISPR/Cas systems mediating all 12 possible base-to-base conversions and combinations but does not exploit a dDNA template or confer DSBs in the target sequence. Correction of the T4709M mutation is important example for the potential treatment of *RYR1*-related diseases. Maintenance of proper calcium homeostasis could ameliorate muscle weakness, improve muscle contractility, and enhance overall motor function. Correcting mutations of the *RYR1* gene using Prime editing also offers the potential to prevent the occurrence of complications associated with *RYR1*-related diseases. Early correction of the T4709M mutation may improve the long-term prognosis for affected individuals based on observed progression of muscle weakness and disability. This approach offers the potential for establish more effective and individualized treatment regimens to optimize therapeutic benefits and to simultaneously minimize adverse effects. Thus, this study aims to explore the application of Prime editing for treating *RYR1*-related myopathies. *RYR1*-related myopathies are caused by mutations in the *RYR1* gene. The efficiency of Prime editing in correcting T4709M, a missense mutation of *RYR1*, was evaluated both *in vitro* and *in vivo*. For *in vitro* experiments, the Prime editing plasmids were transfected into HEK293T cells, human fibroblast primary cells, human myoblast cell line, mouse *RYR1ᵀᴹ/ᵀᴹ* fibroblast primary cells, and C2C12 cells through electroporation. For *in vivo* experiments, the Prime editing plasmids were injected into *RYR1ᵀᴹ/ᵀᴹ* mouse model followed by electroporation. Multiple pegRNAs were tested. The results of *in vitro* experiments show that correction efficiency varies across pegRNAs. PE3 is one of the incremental devices in Prime editing. PE3b is an adjusted version of PE3 with improved editing efficiency and expanded range of targeted genetic modifications. The PE3 strategy was found to be a more effective than PE3b strategy for correcting the point mutation. The correction efficiency of Prime editing can be improved by conducting multiple transfections or by adding a RNA sequence forming double strands (called TevopreQ1) to pegRNA. The efficiency of correcting *RYR1* T4706M mutation by Prime editing *in vivo* cannot be confirmed.