No description available.

Argonaute proteins play substantial roles in post-transcriptional regulation of gene expression within RNA interference (RNAi) pathways, making them crucial subjects for research, aimed at understanding their interactions with small non-coding RNAs (ncRNAs) and other RNAi components. This study focuses on investigating these properties of Argonaute proteins, particularly Argonaute protein A (AgnA), in the social amoeba Dictyostelium discoideum that is renowned for its broad genetic toolbox and unique life cycle. While previous studies have examined the disruption of three Argonaute genes (agnB, agnC, agnE) and their effect on mRNA levels and small ncRNA expression, this study extends to agnA gene, which remains less studied. Key questions surrounding the influence of AgnA on the cellular processes such as the cell growth rate, development, gene expression, as well as potential targets and small ncRNA binding, remain unanswered. A well-established approach that could provide the necessary answers is the disruption of the gene through traditional homologous recombination, by insertion of a drug-resistance cassette flanked by homology arms complementary to the target locus. However, the emerging CRISPR/Cas9 gene editing tool on contrary offers straightforward protocols for disruption of gene expression through efficient induction of genomic knockouts, point mutations and deletions. In this study, both approaches were applied in parallel to knockout the agnA gene, enabling comparison of knockout efficiency and further study of the growth rate, development and gene expression in the knockout strains. Moreover, important information regarding the growth patterns of both wild-type and agnE knockout strains were also elucidated, complementing the previous growth rate analyses. The obtained data from this research could provide valuable insights for future studies ofthe RNAi machinery components and particularly the function of Argonaute proteins in D. discoideum.

Parkinson's disease (PD) is the second most common neurodegenerative disease, caused by the loss of dopaminergic neurons (DN) in the substantia nigra. It is characterized by significant inflammation occurring in both the central nervous system and peripheral areas. Specifically, PINK1 variants have been detected to cause an autosomal-recessive form of the disease. PINK1 plays a crucial role in developing PD and the inflammation process. Therefore, we developed a PINK1 knock-out (KO) model in Thp1 cells using the CRISPR/CAS9 technique to assess the modulation of macrophage activities in the peripheral immune system. Our PINK1 KO models exhibited a decreased Mitochondrial Complex I activity, confirming the disruption of the PINK1 function. Moreover, PINK1 KO clones displayed changes in the expression levels of Tau protein. Furthermore, PINK1-KO macrophages showed increased expression levels of the cytokines IL-6, IL-1β, and IL-23, confirming their pro-inflammatory predisposition. Finally, immunostaining results revealed a reduction in the average of lipids droplets per cell, similar to the reduced expression levels observed in SCD5 in our PINK1-KO. Altogether, these data support the role of PINK1 in mitochondrial dysfunction, activation of the peripheral immune system, and lipid dysfunction. Our study suggests that SCD5 could be a potential target for novel therapies. These findings could aid in the diagnosis and open the path to unveiling key pathogenic mechanisms in PBMCs/macrophages that could potentially be used as a disease biomarker in the future. / La maladie de Parkinson (MP) est la deuxième maladie neurodégénérative la plus courante et est causée par la perte de neurones dopaminergiques (ND) dans la substance noire. Elle se caractérise par une inflammation importante, survenant à la fois dans le système nerveux central et dans les zones périphériques. Plusieurs éléments soutiennent l'implication de mécanismes immunitaires dans l'étiologie de la maladie. Plus précisément, des variants de PINK1 ont été détectés comme provoquant une forme autosomique récessive de la maladie. PINK1 joue un rôle crucial dans le développement de la maladie de Parkinson et dans le processus d'inflammation. Par conséquent, nous avons développé un modèle PINK1 knock-out (KO) dans les cellules Thp1 en utilisant la technique CRISPR/CAS9 pour évaluer fonctionnellement la modulation des activités des macrophages dans le système immunitaire périphérique. Nos modèles PINK1 KO présentent une diminution de l'activité du complexe mitochondrial I, confirmant la perturbation de la fonction PINK1. De plus, les clones PINK1 KO présentent des changements dans les niveaux d’expression de la protéine Tau. Les macrophages PINK1 KO ont montré une augmentation des niveaux d’expression des cytokines IL-6, IL-1β et IL-23 confirmant leur prédisposition pro-inflammatoire. Enfin, nos résultats d'immunomarquage ont révélé une réduction de la moyenne des gouttelettes lipidiques par cellule, similaire aux niveaux d’expression réduits observés dans SCD5 dans notre PINK1 KO. Ensemble, ces données soutiennent le rôle de PINK1 dans le dysfonctionnement mitochondrial, l'activation du système immunitaire périphérique et le dysfonctionnement lipidique. Notre étude a montré que SCD5 pourrait être une cible potentielle pour de nouvelles thérapies. Les résultats de nos recherches pourraient faciliter le diagnostic et ouvrir la voie à la révélation de mécanismes pathogéniques clés dans les PBMC/macrophages pouvant être potentiellement utilisés comme biomarqueurs de la maladie à l’avenir.

Les myopathies héréditaires sont des maladies affectant les fibres musculaires, résultant d’un variant génétique pathogénique transmis par un ou les deux parents. Le diagnostic de ces maladies est complexe en raison de la similarité de certains symptômes avec d’autres maladies neuromusculaires et de leur hétérogénéité. Malgré les progrès des connaissances sur les maladies musculaires héréditaires, de nombreux patients ne reçoivent toujours pas de diagnostic moléculaire malgré les divers tests et le dépistage génétique. Actuellement, 13 variants dans le gène MLIP ont été rapportés chez 15 patients atteints d’une myopathie récessive. Les symptômes, les atteintes et l’âge au début de la maladie varient grandement entre ces patients. Ces variant sont suspectés d’entraîner une perte de fonction ou une altération majeure de la fonction protéique de MLIP et/ou de provoquer des dysfonctionnements dans les voies biologiques associées avec MLIP. De plus, les variants sont localisés autour de l’exon 4 contenant la séquence de localisation nucléaire et autour de l’exon 9 contenant, avec l’exon 10, un crochet AT permettant une interaction avec la chromatine. Pour comprendre la fonction de MLIP dans le muscle et expliquer la variabilité phénotypique, trois objectifs ont été définis : 1) définir le patron d’expression des transcrits chez trois patients; 2) modéliser le variant du patient adulte Z46 par CRISPR-Cas9 dans des myoblastes humains immortalisés; 3) analyser l’impact fonctionnel et pathologique des variants des trois patients. Le séquençage à longue lecture a révélé une architecture transcriptomique différente selon le groupe d’âge et a permis de répertorier 11 nouveaux transcrits non connus chez MLIP. De plus, les techniques de qRT-PCR et de Western Blot ont montré qu’uniquement le patient adulte Z46 présente un mécanisme compensatoire des niveaux de LMNA lors d’une diminution ou une perte de MLIP dans le noyau. Ce mécanisme serait spécifique au tissu ou au type cellulaire, car il n’est retrouvé dans les myoblastes porteurs du variant du patient. En analysant tous les résultats, la variabilité phénotypique semble être liée à la perte de la séquence de localisation nucléaire, mais aussi à l’intensité et l’efficacité de l’interaction entre MLIP et la chromatine. / Hereditary myopathies are diseases affecting muscle fibers, resulting from a pathogenic genetic variant transmitted by one or both parents. The diagnosis of these diseases is complex due to the similarity of some symptoms with other neuromuscular diseases and their heterogeneity. Despite advances in knowledge of hereditary muscle diseases, many patients still do not receive molecular diagnosis despite various tests and genetic screening. Currently, 13 variants in the MLIP gene have been reported in 15 patients with recessive myopathy. Symptoms, conditions, and age at the beginning of the disease vary greatly between these patients. These variants are suspected to cause loss of function or major impairment of MLIP protein function and/or to cause dysfunctions in the biological pathways associated with MLIP. Moreover, the variants are located around exon 4 containing the nuclear localization sequence, and around exon 9 containing, with exon 10, an AT-hook allowing an interaction with chromatin. To understand the function of MLIP in muscle and explain phenotypic variability, three objectives were defined: 1) to define the pattern of expression of transcripts in three patients; 2) to model the variant of adult patient Z46 by CRISPR-Cas9 in immortalized human myoblasts; 3) analyze the functional and pathological impact of the variants of the three patients. Long-Read Sequencing revealed a different transcriptomic architecture according to the age group and identified 11 new transcripts not known in MLIP. In addition, qRT-PCR and Western Blot techniques have shown that only the adult Z46 patient has a compensatory mechanism for LMNA levels during a decrease or loss of MLIP in the nucleus. This mechanism would be specific to tissue or cell type because it is not found in myoblasts carrying the variant of the same patient. By analyzing all the results, phenotypic variability seems to be related to the loss of the nuclear localization sequence, but also to the intensity and efficiency of the interaction between MLIP and chromatin.

La régulation de l’expression génique chez les mammifères dépend de l’organisation tridimensionnelle des chromosomes, en particulier à l’échelle des communications entre les séquences régulatrices et leurs promoteurs cibles. Ainsi, les chromosomes sont organisés en une nouvelle architecture consistant en domaines d’interactions topologiques (TADs, acronyme anglais). Mon projet de thèse avait pour but de caractériser les mécanismes moléculaires impliqués dans cette architecture et leurs importances au cours du développement embryonnaire, pour un locus bien particulier, le Xic (acronyme anglais pour X-inactivation centre). Le Xic contient les éléments régulateurs nécessaires pour initier l’inactivation du chromosome X (ICX), un phénomène épigénétique spécifique du développement des mammifères femelles, rendant l’un des deux chromosomes X inactif du point de vue transcriptionnelle. L’ICX permet d’égaliser l’expression des gènes liés au X entre les sexes chez les mammifères. Le Xic est organisé au moins en deux TADs mais une partie du locus reste encore non identifiée. Je présente ici une analyse fonctionnelle approfondie des différents éléments régulateurs au sein du Xic, comprenant des enhancers, des gènes d’ARNs non codants et des éléments structurels. Après avoir créé une série d’allèles mutés chez la souris et les cellules souches embryonnaires murines, j’ai caractérisé l’impact de ces réarrangements génomiques sur le paysage structurel et transcriptionnel du Xic. J’ai identifié des nouveaux acteurs dans la régulation de ce locus, en particulier des séquences régulatrices conservées chez les mammifères placentaires et des éléments structurels importants pour la formation d’une frontière entre les deux TADs du Xic, importante pour leur séparation et régulation. Je décris aussi la découverte de communication entre ces TADs, ce qui constitue un mécanisme inédit de régulation génique pendant le développement. Ce travail contribue à un nouveau niveau de compréhension des lois qui régissent l’organisation des TADs dans le contexte de la régulation génique chez les mammifères. / Mammalian gene regulatory landscapes rely on the folding of chromosomes in the recently discovered topologically associating domains (TADs), which ensure appropriate communication between cis-regulatory elements and their target promoters. The aim of my PhD project was to characterise the molecular mechanisms that govern this novel architecture and its functional importance in the context of a critical and developmentally regulated locus, the X-inactivation centre (Xic). The Xic contains the necessary elements to trigger X-chromosome inactivation, an epigenetic phenomenon that occurs during the development of female mammals to transcriptionally silence one of the X-chromosomes and equalise X-linked gene expression between sexes. The Xic is partitioned into at least two TADs, but its full extent is unknown. Here, I present a comprehensive functional analysis of different cis-regulatory elements within the Xic, including enhancer-like regions, long noncoding RNA loci and structural elements. Upon generating a series of mutant alleles in mice and murine embryonic stem cells, I characterised the impact of these genomic rearrangements in the structural and transcriptional landscape of the Xic and identified novel players in the regulation of this locus, including cis-acting elements conserved across placental mammals and structural elements critical for the insulation between the Xic TADs. I also found evidence for communication across TADs at this locus, which provides new insights into how regulatory landscapes can work during development. This study also extends our understanding of the rules governing the organisation of TADs and their chromatin loops in the context of mammalian gene regulation. / Nos mamíferos, a regulação da expressão genética depende da organização tridimensional dos cromosomas, em particular ao nível da comunicação regulatória entre promotores e enhancers. A esta escala, descobriu-se recentemente que os cromossomas estão organizados em domínios de interações topológicas (conhecidos como TADs, no acrónimo inglês) que se pensa providenciarem uma base estrutural para as paisagens de regulação transcricional dos genes. O meu projecto de tese teve como objectivo caracterizar os mecanismos moleculares responsáveis por esta arquitectura e a sua importância funcional no contexto de um locus crítico para o desenvolvimento embrionário, o centro de inactivação do cromossoma X (Xic, acrónimo inglês). O Xic contém os elementos genéticos necessários e suficientes para iniciar a inactivação do cromossoma X, um fenómeno epigenético que ocorre durante o desenvolvimento das fêmeas de mamíferos para silenciar um dos cromosomas X e igualar a expressão dos genes do X entre indivíduos XX e XY. O Xic está organizado em pelo menos dois TADs, mas o seu intervalo genético completo permanece desconhecido. Apresento nesta tese uma análise funcional e detalhada de diferentes sequências reguladoras presentes no Xic, incluindo regiões do tipo enhancer, genes de ARNs não codificantes e elementos estruturais. Após a criação de diversos alelos mutantes (deleções, inserções, inversões) em ratinho e em células estaminais embrionárias, através das recentes técnicas de engenharia genética, TALENs e CRISPR/Cas9, caracterizei o impacto destes rearranjos genéticos na paisagem topológica e transcricional do Xic, o que permitiu a identificação de novos actores moleculares na regulação deste locus. Em particular, descobrimos sequências de regulação transcricional altamente conservadas em mamíferos placentários e elementos estruturais importantes para a formação da fronteira entre os dois TADs do Xic. Descrevo também evidência de que há comunicação entre os dois TADs neste locus, o que compromete os modelos actuais do modus operandis dos TADs, e por isso contribui para um novo nível de compreensão dos mecanismos que regulam a expressão genética durante o desenvolvimento.

No description available.

Rare diseases, when considered as a whole, affect up to 7% of the population, which would represent 3.5 million individuals in the United Kingdom alone. However, while 'personalised medicine' is now yielding remarkable results using recent sequencing technologies in terms of diagnosing genetic conditions, we have made much less headway in translating this patient information into therapies and effective treatments. Even with recent calls for greater research into personalised treatments for those affected by a rare disease, progress in this area is still severely lacking, in part due to the astronomical cost and time involved in bringing treatments to the clinic. Gene correction using the recently-described genome editing technology CRISPR/Cas9, which allows precise editing of DNA, offers an exciting new avenue of treatment, if not cure, for rare diseases; up to 80% of which have a genetic component. This system allows the researcher to target any locus in the genome for cleavage with a short guide-RNA, as long as it precedes a highly ubiquitous NGG sequence motif. If a repair sequence is then also provided, such as a wild-type copy of the mutated gene, it can be incorporated by homology-directed repair (HDR), leading to gene correction. As both guide-RNA and repair template are easily generated, whilst the machinery for editing and delivery remain the same, this system could usher in the era of 'personalised medicine' and offer hope to those with rare genetic diseases. However, currently it is difficult to test the efficacy of CRISPR/Cas9 for gene correction, especially in vivo. Therefore, in my PhD I have developed a novel fluorescent reporter system which provides a rapid, visual read-out of both non-homologous end joining (NHEJ) and homology-directed repair (HDR) driven by CRISPR/Cas9. This system is built upon a cassette which is stably and heterozygously integrated into a ubiquitously expressed locus in the mouse genome. This cassette contains a strong hybrid promoter driving expression of membrane-tagged tdTomato, followed by a strong stop sequence, and then membrane-tagged EGFP. Unedited, this system drives strong expression of membrane-tdTomato in all cell types in the embryo and adult mouse. However, following the addition of CRISPR/Cas9 components, and upon cleavage, the tdTomato is rapidly excised, resulting via NHEJ either in cells without fluorescence (due to imperfect deletions) or with membrane-EGFP. If a repair template containing nuclear tagged-EGFP is also supplied, the editing machinery may then use the precise HDR pathway, which results in a rapid transition from membrane-tdTomato to nuclear- EGFP. Thereby this system allows the kinetics of editing to be visualised in real time and allows simple scoring of the proportion of cells which have been edited by NHEJ or corrected by HDR. It therefore provides a simple, fast and scalable manner to optimise reagents and protocols for gene correction by CRISPR/Cas9, especially compared to sequencing approaches, and will prove broadly useful to many researchers in the field. Further to this, I have shown that methods which lead to gene correction in our reporter system are also able to partially repair mutations found in the disease-causing gene, Zmynd10; which is implicated in the respiratory disorder primary ciliary dyskinesia (PCD), for which there is no effective treatment. PCD is an autosomal-recessive rare disorder affecting motile cilia (MIM:244400), which results in impaired mucociliary clearance leading to neonatal respiratory distress and recurrent airway infections, often progressing to lung failure. Clinically, PCD is a chronic airway disease, similar to CF, with progressive deterioration of lung function and lower airway bacterial colonization. However, unlike CF which is monogenic, over 40 genes are known to cause PCD. The high genetic heterogeneity of this rare disease makes it well suited to such a genome editing strategy, which can be tailored for the correction of any mutated locus.

No description available.

Mutations in the gene encoding the bone morphogenetic protein type 2 receptor (BMPR2) are the most common genetic cause of heritable pulmonary arterial hypertension (PAH). However, given the reduced penetrance of BMPR2 mutations in affected families, a major outstanding question is the identity of additional factors or pathways that are responsible for the manifestation of clinical disease. Furthermore, limited human tissue is available for study and usually only from patients with end-stage disease, making it difficult to understand how PAH is established and progresses. Alternative human models of PAH are therefore required. This thesis describes the characterisation of the first human iPSC-derived smooth muscle cell (iPSC-SMC) model of PAH and elucidates the role of BMPR2 deficiency in establishing PAH-associated phenotypes in iPSC-derived SMCs. To achieve this, I used CRISPR-Cas9 gene editing to generate wild-type and BMPR2+/- iPSC lines with isogenic backgrounds which were subsequently differentiated into lineage-specific iPSC-SMCs that displayed a gene expression profile and responses to BMP signalling akin to those present in distal pulmonary artery smooth muscle cells (PASMCs). Using these cells, I found that the introduction of a single BMPR2 mutation in iPSC-SMCs was sufficient to recapitulate the pro-proliferative and anti-apoptotic phenotype of patient-derived BMPR2+/- PASMCs. However, acquisition of the mitochondrial hyperpolarisation phenotype was enhanced by inflammatory signalling and required an interaction between BMPR2 mutations and environmental stimuli provided by exposure to serum over time. Furthermore, I showed that BMPR2+/- iPSC-SMCs had an altered differentiation state and were less contractile compared to wild-type iPSC-SMCs, phenotypes which have not been observed previously in PAH-derived PASMCs. Finally, RNA sequencing analysis identified genes that were differentially expressed between wild-type and BMPR2+/- iPSC-SMCs and may hence provide further insights into PAH pathobiology. The iPSC-SMC model described in this study will be useful for identifying additional factors involved in disease penetrance and for validating therapeutic approaches that target BMPR2.

Fundamentally altering cellular function at a genetic level is a major area of interest in the biologic sciences and the medical community. By engineering transfectable constructs that can be inserted to dysfunctional cellular systems, scientists can mitigate aberrant genetic behavior to produce proper molecular function. While viral vectors have been a mainstay in the past, there are many limitations, particularly related to safety, that have changed the focus of genome editing to incorporate alternative methods for gene delivery. Lance Array Nanoinjection (LAN), a second-generation microfabricated transfection biotechnology, is one of these alternative technologies. LAN works by utilizing both simultaneous electrostatic interaction with molecular loads and physical lancing of hundreds of thousands of target cell membranes. The purpose of this work is to demonstrate LAN in the context of in vitro transfection of immortalized culture cells and primary cells. As part of that exploration, three distinct areas of investigation are considered, which include: characterizing environmental factors that impact LAN transfection, demonstrating LAN genetic modification of immortalized HeLa 229 culture cells using an indicator marker, and lastly, investigating the effects of LAN on human primary, neonatal fibroblasts.