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Implication de l'unfolded protein response et de l'autophagie dans la morphogénèse du rotavirus. / Implication of the unfolded protein response and autophagy in the morphogenesis of rotavirusVu, Lan Trang 18 December 2014 (has links)
Le rotavirus est le principal agent étiologique des gastro-entérites infantiles. Après l'assemblage dans le réticulum endoplasmique (RE), les virions sont relargués au niveau du pôle apical des entérocytes selon une voie de trafic atypique ne passant pas par l'appareil de Golgi. Ce travail a pour but d'étudier les mécanismes de sortie du RE et le trafic atypique du rotavirus. Des acteurs de la machinerie réticulaire de repliement et de contrôle qualité des protéines ont été retrouvés associés aux intermédiaires d'assemblage du rotavirus. En corrélation avec cela, l'infection provoque un stress du RE et active une réponse cellulaire spécifique, l'Unfolded Protein Response (UPR). Le rotavirus module de manière différentielle les trois voies de signalisation de l'UPR et seules les voies IRE1 et PERK sont requises pour la morphogénèse virale. L'autophagie, en dehors de son rôle dans la dégradation du matériel cellulaire, a été récemment impliquée dans des voies de sécrétion non conventionnelles. Dans les cellules épithéliales intestinales Caco-2, la différenciation cellulaire se manifeste par l'augmentation de l'expression des marqueurs autophagiques et la diminution du flux autophagique. Quelque soit l'état de différenciation des cellules, l'infection à rotavirus bloque à la fois la formation et la maturation des autophagosomes. Uniquement dans les cellules Caco-2 non différenciées, l'infection induit une lipidation de LC3 qui n'est pas associée à l'autophagie, mais qui corrèle avec un clivage de la protéine ATG3 impliquée dans le processus de lipidation. Ni l'autophagie, ni la lipidation de LC3 ne sont requises pour la morphogénèse du rotavirus dans les cellules Caco-2. / Rotavirus is the major causative agent of severe gastroenteritis in young children worldwide. After assembly steps in the Endoplasmic Reticulum (ER), virions are released without any cellular lysis at the apical side of enterocytes, following an atypical trafficking pathway that bypasses the Golgi apparatus. This work is aimed at understanding the mechanisms of ER exit of rotavirus particles as well as their unconventional trafficking. Components of the protein folding and quality control machinery in the ER were found associated with viral assembly complexes. Consistent with this observation, viral infection induced ER stress, which activates a specific cellular response named the Unfold Protein Response (UPR). Rotavirus infection modulated differently the three UPR signaling pathways and only the IRE1 and PERK pathways were required for viral morphogenesis. Autophagy, besides being a degradative process, has recently been shown to be potentially involved in unconventional secretion pathways. We showed that the differentiation process of human intestinal epithelial Caco-2 cells into enterocyte-like phenotype was marked by the increase in expression of autophagic markers and the reduction of autophagic flux. Rotavirus infection blocked both the initiation and late steps of autophagy, in both undifferentiated and differentiated Caco-2 cells. Surprisingly, only in undifferentiated Caco-2 cells, rotavirus infection induced a lipidation of LC3 that was not associated with autophagy but correlated with a cleavage of ATG3, a protein directly involved in the lipidation process. Neither autophagy nor the lipidation of LC3 were required for rotavirus morphogenesis in Caco-2 cells.
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Applying optical tweezers in vivo : A biophysical study of mechanical forces in Drosophila Melanogaster at the onset of gastrulationBambardekar, Kapil 20 January 2015 (has links)
Nous avons développé un dispositif combinant pinces optiques et imagerie par feuillet de lumière. Nous montrons que les interfaces cellulaires de l'épithélium précoce de l'embryon de Drosophile peuvent être piégées et manipulées directement avec des pinces optiques. La manipulation optique est réalisée à la fin de la cellularisation, processus par lequel des membranes cellulaires séparent les noyaux pour donner naissance à un épithélium ; à ce stade, les mouvements cellulaires sont minimes et les cellules ont des formes hexagonales similaires. En imposant un mouvement sinusoïdal au piège perpendiculairement à une interface, nous étudions la déflection de l'interface en fonction de la puissance laser, de l'amplitude du mouvement du piège et de la fréquence d'oscillation. En outre, des expériences de déflection-relaxation par déplacement instantané puis arrêt du piégeage, ont été réalisées, fournissant une alternative à l'analyse fréquentielle pour étudier les propriétés viscoélastiques de l'interface. Un modèle de type solide linéaire standard rend compte des observations et permet d'extraire les paramètres viscoélastiques de l'interface. Nous mettons également en évidence que la déflection imposée à une interface se propage aux interfaces voisines en s'affaiblissant exponentiellement sur une distance d'une à deux cellules. Cette technique étant établie, nous l'utilisons pour mesurer les tensions durant l'extension de la bandelette germinale. Les tensions sont anisotropes, les jonctions parallèles à la direction dorsoventrale ayant une tension trois fois plus élevée que celles perpendiculaires. Ce travail fournit des mesures absolues des tensions intercellulaire. / Here, an optical tweezers setup was developed on a pre-existing single-plane illumination (SPIM) setup. The cell-cell interface in embryonic epithelia could be trapped and manipulated directly with optical tweezers. The interaction of the interface with the trap was initially characterized at the end of cellularization where the tissue has minimal movements and actomyosin turnover. With a sinusoidal trap excursion, the interface amplitude was found to increase linearly with applied laser power as well as trap amplitude and time period. Furthermore, push and pull experiments on the interface responding to a stationary trap, provided another way to address the viscoelastic properties of the interface. The interface kinetics in stationary experiments could fit adequately to a passive viscoelastic model. This model also explained well the linear response to trap amplitude and time period, and formed the basis of estimating interface tension from its amplitude. Moreover, the propagation of the sinusoidal movement to neighbouring interfaces decayed rapidly with minimal phase lag in both experiments and the model. Having established a suitable regime of trapping conditions, where interface deflection is small and linear, the mechanical anisotropy of the epithelium was at the onset of gastrulation. The interface tension increased by 2-3 fold, exhibiting both apico-basal and dorso-ventral polarization of tension, concomitant with polarized accumulation of myosin. The role of myosin was established further through ROCK-inhibition. Perturbation of actin also decreased the interface tension. My work provides a crucial insight into the mechanical behaviour of dynamic epithelia.
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Controle do metabolismo e desenvolvimento da orquídea epífita Catasetum fimbriatum em resposta à incidência de luz no sistema radicular / Control of metabolism and development of the epiphyte orchid Catasetum fimbriatum in response to light incidence on the root systemPaulo Marcelo Rayner Oliveira 09 May 2017 (has links)
O ambiente epifítico é considerado um dos habitats mais desafiadores para as plantas, pois a disponibilidade hídrica e nutricional pode ser bastante escassa. Além disso, as plantas que colonizam este ambiente estão mais expostas aos fatores ambientais, dentre eles a luminosidade se mostra bastante importante, uma vez que esta atua tanto como fonte de energia na fotossíntese quanto como sinal ambiental em repostas fotomorgênicas. Sabe-se, por exemplo, que a luz exerce forte influência sobre a morfogênese radicular de plantas em geral, porém impacta ainda mais o desenvolvimento de orquídeas epífitas, dada a frequente exposição de suas raízes aéreas à incidência luminosa. Tendo em vista que a auxina ácido indolil-3-acético (AIA), o ácido abscísico (ABA) e o etileno são moduladores cruciais no controle da arquitetura radicular na maioria das plantas, sendo também mediadores chave em várias respostas fotomorfogênicas, este estudo propôs-se a investigar o possível envolvimento destes hormônios durante diferentes respostas morfo-fisiológicas desencadeadas pela exposição à luz do sistema radicular de plantas de C. fimbriatum. A ausência de incidência luminosa sobre às raízes resultou em maiores taxas de crescimento e volume radicular, porém, com menor acúmulo de biomassa em relação às raízes expostas à luz. O incremento na biomassa em raízes expostas à luz esteve correlacionado ao espessamento da parede celular na região cortical, o qual ocorreu em resposta especificamente à luz azul. Em termos gerais, a exposição das raízes à luz induziu o aumento nos níveis de AIA e ABA, enquanto que os teores de ACC foram superiores em raízes protegidas da incidência luminosa. Estes resultados sugerem que a luz pode modular o desenvolvimento radicular de C. fimbriatum através de um fino controle hormonal que depende de ajustes coordenados dos níveis de AIA ABA e ACC. Também foi investigado o potencial envolvimento das auxinas e do ABA durante a remobilização de carboidratos entre pseudobulbos e folhas de plantas que tiveram seus sistemas radiculares expostos à (ou protegidos da) luz. Os resultados revelaram que a manutenção das raízes sob condições de escuro levou ao aumento dos teores de AIA e de todas as fontes de carbono estudadas (especialmente de glicose e frutose) nos pseudobulbos, enquanto que as raízes cobertas apresentaram apenas um leve aumento no conteúdo de AIA. O tratamento concomitante das raízes com a condição de escuro e a aplicação de um inibidor do transporte polar de auxina causou uma diminuição abrupta nos teores de AIA em todos os órgãos analisados e a elevação do conteúdo de ABA no sistema radicular. De maneira interessante, essa última condição experimental induziu um conspícuo acúmulo de carboidratos nos pseudobulbos, principalmente de sacarose. Assim, os dados deste trabalho reforçam a importante participação do AIA e ABA como possíveis mediadores da sinalização desencadeada pela luz incidente no sistema radicular de C. fimbriatum, cujas respostas induzidas regulam não somente a morfogênese de tecidos radiculares, mas também influenciam na regulação da partição de carbono no sistema caulinar por meio de um provável mecanismo de sinalização à longa distância / The epiphytic environment is considered one of the most challenging for plants, due to frequent scarcity of water and nutrients. Furthermore, the plants that colonized this biotope are usually more exposed to the environmental cues. Light is considered one of the most important signals controlling plant development because it can act as both an energy source for photosynthesis and an environmental signal for photomorphogenic responses. Besides, light can influence the root morphology of most plants, with even stronger impacts expected in aerial roots of epiphytic orchids due their frequent exposition to direct light. Since indole-3-acetic acid (IAA), abscisic acid (ABA) and ethylene are crucial hormonal signals modulating the root architecture in most plant species, and key mediators during numerous photomorphogenic responses, this study investigated the potential involvement of these hormones in different morpho-physiological responses regulated by either the darkness treatment or the light exposure of Catasetum fimbriatum root system. The absence of light incidence on the roots resulted in higher root volume and growth rate, but lower dry mass accumulation than the light-exposed ones. The higher accumulation of biomass in the light-exposed roots was closed correlated with a more intense cell wall thickening in the root cortex, which appeared to be specifically induced by the blue light. In general, root exposure to light induced increasing levels of ABA and AIA, while the ACC content was higher in roots protected from light. This suggests that light might modulate C. fimbriatum root development through a fine-tuned hormonal mediation, which depends on coordinated adjustments of IAA, ABA and ACC levels. This study also investigated the potential involvement of auxin and ABA during the (re)mobilization of carbohydrates in pseudobulbs and leaves of plants that had their root systems either exposed to (or protected from) light. The results revealed that covering the roots increased in pseudobulbs the levels of AIA and all carbon sources studied (specially glucose and fructose), while the covered roots showed slightly higher levels of AIA. The concomitant treatment with NPA and root covering caused a sharp decrease of AIA levels in all organs and an ABA increase in the root system. Interestingly, this last condition induced a conspicuous carbohydrate accumulation in pseudobulbs, with sucrose as the predominant form. The data obtained in this study reinforce the remarkable participation of IAA and ABA as possible mediators of the signaling cascades triggered by the light incidence on C. fimbriatum root system, which was capable of inducing photomorphogenic responses not only in root tissues, but was also able to influence the carbon portioning in the shoot system by a potential long-distance signaling mechanism
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Compreendendo os fluxos de biomassa de Andropogon lateralis e Paspalum notatum em um campo nativo heterogêneo / Understanding the biomass flows of Andropogon lateralis and Paspalum notatum in a heterogeneous native grasslandSilva, Jonatas Cezar da January 2018 (has links)
Em um ecossistema pastoril natural, entender as relações entre a produção primária, o processo de pastejo e o ambiente é de inegável importância, pois a compreensão destes fatores influenciará no correto gerenciamento dos ambientes pastoris, garantindo a produtividade e contribuindo para a segurança alimentar. O experimento foi conduzido na Estação Experimental Agronômica da Universidade Federal do Rio Grande do Sul (EEA-UFRGS), município de Eldorado do Sul, Rio Grande do Sul, Brasil, em uma pastagem natural heterogênea, característica do Bioma Pampa. O objetivo do trabalho foi compreender os fluxos de biomassa, bem como, determinar as características morfogênicas e entender o processo de desfolhação. Duas espécies de gramíneas mais representantes do Bioma, pertencentes a dois grupos funcionais distintos: paspalum notatum, representando as espécies utilizadoras de recursos, e andropogon lateralis, representando as conservadoras de recursos, foram avaliadas. Os tratamentos consistiram de quatro ofertas de forragem: 4, 8, 12 e 16% (kg de matéria seca para cada 100 kg de peso vivo animal), manejados sob pastoreio contínuo, dispostos em um delineamento em blocos casualizado, em arranjo fatorial 4X2, com duas repetições Adotou-se a técnica de perfilhos marcados, com 30 perfilhos marcados por espécie, distribuídos em três transectas por unidade experimental. Houve interação entre oferta de forragem e espécie forrageira para taxa de elongação foliar, comprimento final de folhas e densidade de perfilhos (P<0,05). A maioria das características morfogênicas apresentou diferença entre espécies, sendo maiores para a espécie utilizadora de recursos (P. notatum). As ofertas de forragem influenciaram as taxas de senescência, número de folhas senescentes e de folhas novas surgidas por perfilho. O filocrono encontrado foi de 208,3 ºC para P. notatum e, 366,2 ºC para A. lateralis. Houve diferença entre espécies para o intervalo entre desfolhas sucessivas, sendo de 25 dias para P. notatum e 27 dias para A. lateralis (P=0,0140), bem como para intensidade de desfolha, de 52% para P. notatum e 61% para A. lateralis. Os fluxos de biomassa apresentaram interação oferta de forragem e espécie forrageira para os fluxos de senescência e consumo. O balanço final do fluxo de biomassa (crescimento menos senescência e consumo) apresentaram balanço negativo tanto para ofertas como entre os grupos funcionais estudados. / Understanding the relationship between pasture primary production, the grazing process and the environment in natural pastoral ecosystems is of undeniable importance, since the interactions between these factors will influence the correct ecosystems management, ensuring productivity and contributing to food security. The study was carried out at the Experimental Station of the Federal University of Rio Grande do Sul (EEA-UFRGS), in the municipality of Eldorado do Sul, Rio Grande do Sul state, Brazil, in a natural heterogeneous grassland typical of the Pampa Biome. The aim of this study was to understand the biomass fluxes, as well as to determine morphogenetic characteristics and to understand the defoliation process. Two species of grasses with different growth strategies: paspalum notatum, representing higher growth species (or acquisitive resource plant strategy) and andropogon lateralis, representing lower growth species (or conservative resource plant strategy), were evaluated. Treatments consisted of four forage allowances: 4, 8, 12 and 16% (kg of dry matter per 100 kg of animal live weight), managed under continuous grazing. We used the marked tillers technique, with 30 tillers per species and per treatment, divided into three transects The experimental design was a randomized complete block with factorial arrangement 4x2, in two replicates. There was interaction between forage allowance and species for leaf elongation rate, leaf final length and tiller density (P<0.05). Most of the morphogenetic characteristics presented differences between the species, being higher for the acquisitive resource plant strategy (P. notatum). The forage allowances influenced senescence rates, number of senescent leaves and new leaves emerged per tiller. The phyllochron was 208.3 ºC for P. notatum and 366.2 ºC for A. lateralis. There were differences in the interval between two successive defoliation, being 25 days for P. notatum and 27 days for A. lateralis (P=0,0140), and defoliation intensity, of 52% for P. notatum and 61% for A. lateralis. The biomass fluxes showed interaction forage allowance specie for the variables senescence flow and consumption flow. The final balance of the biomass flow (growth minus senescence and consumption) presented a negative balance both for forage allowances and between the functional groups studied.
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Étude du mécanisme de génération de forces mécaniques par les cellules apoptotiques et leur transmission au reste du tissu / Study of the mecanical force generator mecanism within apoptotic cells and their transmission to surrounding tissueAmbrosini, Arnaud 24 September 2018 (has links)
La morphogénèse épithéliale est une caractéristique clé du développement des organismes multicellulaires. Parmi les différents types d'évènement morphogénétique, la capacité à créer des invaginations est cruciale pour la mise en forme des organismes. Un aspect fondamental de la morphogénèse repose sur la capacité des cellules à exercer, échanger et résister aux stress mécaniques pour permettre la mise en forme des tissues. Au cours des décennies passées, l'importance des forces mécaniques générées au niveau des jonctions adhérentes et notamment sur le plan parallèle au plan apical a été largement démontrée. Cependant, le rôle des forces mécaniques générées sur un plan perpendiculaire au plan apical (dans l'axe apico/basal) est loin d'être aussi bien compris. Récemment, l'équipe a montré que les cellules apoptotique au sein de l'épithélium de patte de drosophile sont capables de générer une force apico/basale qui est requise pour la formation des plis distaux, préfigurant les articulations de la future patte. Même si le rôle d'une structure verticale d'acto-myosine (nommée câble) dans la génération de cette force, a été clairement démontré, rien n'est connu à propos de sa régulation ou du point d'ancrage que ce câble d'acto-myosine pourrait utiliser pour générer cette force. De plus, seuls les effets apicaux de cette force ont été étudiés. Mes travaux de thèses se sont articulés autour de deux objectifs principaux : (1) Déchiffrer les mécanismes intracellulaires requis pour le processus de génération de force apico-basal via le câble, en étudiant en parallèle les effets de la génération de force sur le processus apoptotique lui-même. (2) Etudier les conséquences de l'application de cette force mécanique sur le pôle basal de l'épithélium. Au cours de ma thèse, j'ai montré que pour exercer une force apico-basale, les cellules apoptotiques créent une structure apico-basale comprenant, de l'apical vers le basal : les jonctions adhérentes, un câble d'acto-myosine, le noyau et les jonctions basales.[...] / Epithelium morphogenesis is a key feature during the development of multicellular organism. Within morphogenetic events, the ability to create a fold is crucial to shape multicellular organism. A fundamental aspect of morphogenesis lies on the ability of cells to exert, exchange and resist mechanical stress in order to shape the tissue. During the past decades, the importance of mechanical force generated at the level of adherent junctions, parallel to the apical plan has been greatly elucidated. However, the role of mechanical forces generated perpendicular to the apical plan (in the apico/basal axe) is far from being understood. Recently, the team demonstrated that apoptotic cells in the leg disc epithelium of the drosophila, are able to generate an apico/basal force that is required for the fold formation that foreshadows the future articulation of the adult leg. Even if the role of acto-myosin structure in the generation of this force has been demonstrated, nothing is known about other regulators or even anchoring points that could help this structure in generating this force. Moreover, the effects of this force have only been observed for the apical side of the epithelium. My Phd aims at two goals: (1) Deciphering the intracellular structure that are required for this force generation process and the possible effect of force generation for the apoptotic process per se.(2) Analysing the consequences of those forces on the basal side of the epithelium. During my Phd, I have shown that in order to exert an apico/basal force, the apoptotic cell needs to generate an apico/basal structure comprising from the apical to the basal: adherent junctions, acto-myosin cable, nucleus and basal adhesions. More precisely, I observed that acto-myosin structures called "cables" that have been implicated in the force generation process, spawn from the adherent junctions and grows progressively until reaching the nucleus of the cell. I observed that apoptotic cells have a basally localised nucleus. Following that, I observed that nucleus is anchored by a basal actin meshwork, that restraints apoptotic nucleus movements. What is more, I observed that apoptotic cells maintain basal cell/matrix adhesions. [...]
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Identification des modules de la signalisation auxinique impliqués dans la morphogenèse foliaire / Identifying auxin signaling modules involved in leaf serrationBoudin, Manon 28 November 2017 (has links)
L’auxine est une hormone essentielle au développement des plantes, tant pour la division quel’expansion cellulaire. La transcription des gènes de réponses à l’auxine est permise par un mécanisme designalisation faisant intervenir des complexes ubiquitine ligase E3 impliquant des protéines à F-boxTIR1/AFBs, qui en présence d’auxine peuvent interagir avec les répresseurs transcriptionnels AUX/IAA pourinduire leur dégradation et activer la transcription de gènes de réponses à l’auxine. Cette transcriptionimplique des facteurs de transcription ARFs (Auxin Response Factors). Dans la feuille, un maximum d’auxinerésultant de l’activité des transporteurs d’efflux d’auxine PIN1 participe à l’initiation des dents à la marge. Lefacteur de transcription CUC2 qui permet la formation des domaines frontières intervient dans le contrôle del’orientation des transporteurs PIN1. L’auxine réprime également CUC2 limitant ainsi son expression au sinus,ce qui semble nécessaire pour la formation des dents. Les acteurs de la signalisation auxinique impliquésdans la formation des dents ne sont néanmoins pas connus chez Arabidopsis.Dans cette thèse, les profils d’expression des ARFs ont été mis en évidence dans les jeunes feuillesd’Arabidopsis au moment de la formation des dents et une cartographie fine a été établie. Trois ARFspotentiellement répresseurs ARF1, ARF3, ARF18 et trois activateurs ARF5, ARF6, ARF8 ont été identifiéscomme étant exprimés dans la zone dent/sinus. La modification de leurs profils d’expression dans des formesde feuilles modifiées par des variations d’expression de CUC2 a également été étudiée. Afin de déterminer sices ARFs sont impliqués dans la morphogénèse foliaire et plus particulièrement dans l’initiation des dents, lesimple mutant arf5-2/mpS319, et les doubles mutants arf6-2 arf8-3, arf1-5 arf3-1, arf1-5 arf18-2 et arf3-1arf18-2 ont été générés et analysés. Les feuilles matures du mutant nul arf5-2/ mpS319 présentent une tailleplus importante que celles de Col-0, suggérant que ARF5 serait impliqué dans l’expansion cellulaire. / Auxin is essential for plant development, more particularly by participating in cellular division andexpansion. Transcription of auxin response genes is allowed by the auxin signaling pathway involving the F-boxTIR1/AFBs proteins associated to ubiquitin ligase E3, which can interact with the AUX/IAA repressors in thepresence of auxin to trigger their degradation and activate transcription of early auxin response genes involvingthe Auxin Response Factors (ARF). At the leaf margin, an auxin maximum resulting from the transport of auxinmediated by the auxin efflux carrier PIN1 is required for teeth formation. The transcription factor CUC2 thatdefines boundary domains, somehow redirects PIN1 to create a convergent flux to the apex of the tooth. Auxinrepresses CUC2 thus limiting its expression to the sinus. In Arabidopsis, the actors of the auxin signalingpathway involved in leaf serration are unknown.In this thesis, the expression profiles of ARFs genes have been investigated in young leaves of Arabidopsis, morespecifically during teeth formation and a detailed map was built. Three ARFs acting as putative repressors ARF1,ARF3 and ARF18 and three activators ARF5, ARF6 and ARF8 were identified as been expressed in thesinus/teeth area. Their expression profiles were also studied in leaves exhibiting modified shapes as a result ofvariations in CUC2 expression. To determine if these ARFs are involved in leaf morphogenesis and moreparticularly in tooth initiation, the arf5-2 null mutant and the double null mutants arf6-2 arf8-3, arf1-5 arf3-1,arf1-5 arf18-2 and arf3-1 arf18-2 were generated and serration was analyzed. Length of arf5-2 mature leaves islonger than the wild type, suggesting that ARF5 could be involved in cell expansion.
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Engineering Reporter Tags in Flaviviruses to Probe Viral Structure and MorphogenesisMatthew T Lerdahl (8726223) 24 April 2020 (has links)
<div>The family Flaviviridae includes important genera such as flavivirus and hepacivirus which comprise significant human pathogens that affect hundreds of millions annually. The understanding of these viruses, the viral life cycle, and pathogenicity is vital when it comes to developing therapeutics. Flavivirus virions undergo major conformational rearrangements during the life cycle, including the assembly and maturation steps. In order to create a reagent to investigate these processes, luminescent reporter viruses have been constructed. Luminescent reporter tags have yet to be incorporated into the structural proteins of dengue virus (DENV) without significantly affecting replication or infectivity and successful tagging would allow for targeted studies examining access to specific structural epitopes. Engineering tags in DENV structural proteins is particularly difficult because most reporter tags involve large insertions which may create steric hindrance and inhibit proper protein folding. However, the reporter system described here, developed by Promega, is much smaller than a full-size luciferase protein. It involves an eleven amino acid subunit (HiBiT) tagged to a viral protein that creates measurable luminescence when incubated with the larger subunit (LgBiT). Using the structure of the virion as a guide, the HiBiT reporter tag was incorporated into the structural region of the DENV genome including sites in capsid (C) as well as the glycoproteins membrane (M) and envelope (E). Resulting recombinant viruses were characterized and tag sites within the C protein membrane anchor as well as the transmembrane domain of M protein were found to tolerate HiBiT insertion and produce infectious particles. The recombinant virus possessing HiBiT in C protein was found to be stable over three rounds of serial passaging while virus containing the M protein tag site was found to be unstable. HiBiT activity of the capsid tagged virus was also found to directly correlate with purified infectious particles, suggesting the capsid membrane anchor may remain associated with the virus even after polyprotein processing. Additionally, insert composition was found to be a key determinant for the production of infectious virus. The lessons learned from engineering HiBiT in the DENV system were then applied to hepatitis C virus (HCV). </div><div>The highly lipophilic and pleiomorphic nature of HCV has made structural studies particularly difficult. However, by constructing multi-tagged reporter viruses containing both HiBiT and various purification tags, researchers will save time and resources in preparation for structural studies which are vital for vaccine development. In this study, HiBiT was incorporated into sites within HCV previously shown to tolerate tags of various sizes. Different insert compositions were engineered within the genome and the construct containing both FLAG and HiBiT tags within the N-terminus of E2 yielded highly infectious and quantifiable, luminescent virus. The recombinant HCV containing FLAG and HiBiT displayed similar peak titer as compared to WT while also demonstrating HiBiT activity. Furthermore, the FLAG peptide was found to be partially surface exposed and capable of being used for virus purification purposes. The multi-tagged reporter virus characterized in this study provides a robust platform for quantification and purification of HCV, two facets of research that are critical for the determination of viral structure via cryo-EM and other imaging techniques. The findings from both the DENV and HCV studies provide a robust foundation for future tagging of viruses within the family Flaviviridae and offer insight on the structural proteins that compose the virion.</div>
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Relationship Between Physiological Umbilical Herniation and Liver Morphogenesis During the Human Embryonic Period: A Morphological and Morphometric Study / ヒト胚子期にみられる生理的臍帯ヘルニアの発生要因について‐肝形成との関連に基づいた形態及び形態計測学的検討Kanahashi, Toru 23 March 2020 (has links)
京都大学 / 0048 / 新制・論文博士 / 博士(人間健康科学) / 乙第13333号 / 論人健博第7号 / 新制||人健||5(附属図書館) / 京都大学大学院医学研究科人間健康科学系専攻 / (主査)教授 藤井 康友, 教授 岡 昌吾, 教授 長尾 美紀 / 学位規則第4条第2項該当 / Doctor of Human Health Sciences / Kyoto University / DGAM
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Sonic hedgehog expands neural stem cells in the neocortical region leading to an expanded and wrinkled neocortical surface / Sonic hedgehogは大脳新皮質領域の神経幹細胞数を増大させ、大脳新皮質表面積の拡大と皺形成をもたらすMOHAMMED, J.M. SHQIRAT 24 September 2021 (has links)
京都大学 / 新制・課程博士 / 博士(医学) / 甲第23464号 / 医博第4771号 / 新制||医||1053(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 林 康紀, 教授 伊佐 正, 教授 高橋 淳 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM
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Phenotypic Variations In Animal Morphogenesis : Sea Urchin Twins And Cloned Rabbits / Variations phénotypiques de la morphogénèse animale : jumeaux d'oursins et lapins clonésFabrèges, Dimitri 11 January 2016 (has links)
La variabilité est une propriété intrinsèque aux systèmes biologiques, essentielle pour l'évolution et l'embryogénèse. Souvent considérée comme du bruit, ce n'est que récemment que l'aléatoire des processus biologiques a commencé à être systématiquement étudié. Cette thèse pose les questions suivantes : qu'est-ce qu'un développement normal ? Quel est l'étendue et le rôle de la variabilité dans la robustesse et la résilience du développement embryonnaire ?Ces questions sont posées pour le lapin (Oryctolagus cuniculus) et l'oursin (Paracentrotus lividus et Sphaerechinus granularis).Nous nous sommes aussi intéressé à la quantification du déterminisme de la variabilité embryonnaire à l'aide d'oursins jumeaux et de lapins clonés.La mesure des comportements cellulaires est effectuée sur des lignages cellulaires obtenus à partir d'imagerie 3D+temps. Nous montrons que les oursins jumeaux peuvent se développer selon trois phénotypes différents, jamais observés chez le normal, avant de converger vers une blastula d'apparence normale. De plus, les comparaisons entre et au sein des pairs de jumeaux montrent que le phénotype et la survie ne dépend que de l'histoire individuelle des embryos.Nos mesures quantitatives des pairs de jumeaux amènent des questions ouvrant de nouveaux horizons de recherche : les jumeaux sont-ils robustes ou résilient ?Le développement pré-implantatoire des lapins a été étudié sur cinq embryons numériques (trois sauvages et deux clones), du stade 32-cellules à l'éclosion.Nous montrons que les divisions asymétriques internes et externes régulent la variation du nombre de cellules internes ainsi que la taille de la masse cellulaire.De plus, la variation du nombre de cellules internes est plus grande que pour les cellules externes, ce qui semble directement lié au taux de morts cellulaires.Notre hypothèse est que le potentiel de bon développement des clones est assuré par une grande plasticité épigénétique des cellules donneuses.Ce travail espère définir des méthodes et des concepts fondateurs pour une exploration quantitative et une modélisation multi-échelle de la morphogénèse animale. / Variability is an intrinsic characteristic of biological systems, essential for evolution and embryogenesis.Considered as noise for centuries, it is only recently that the stochasticity of biological processes has began to be systematically explored.The present thesis addresses the following questions: What is a normal development?What is the extent and role of variability in developmental robustness and resilience?We tackle these issues in rabbit (Oryctolagus cuniculus) and sea urchin (Paracentrotus lividus and Sphaerechinus granularis).We also aimed to quantify determinism and stochasticity of developmental variability by means of sea urchin twins and cloned rabbits.Variations in cell behaviors were investigated through reconstruction of cell lineage from 3D+time imaging.We showed that sea urchin twins can follow three different developmental paths never observed in normal embryo, before converging to normal looking blastula.Moreover, comparisons between and within pairs of twins revealed that phenotype and survival depend on individual history alone.Our quantitative observation of twin pairs raises question opening a future line of research: are twins robust or resilient?Rabbit preimplantation development was explored with five digital specimens (three wild-types and two clones) from the 32-cell stage to hatching.We showed that inner and outer asymmetric divisions regulate the variation of inner cell number and may control inner cell mass size.In addition, the variation of inner cell number in clones is higher than outer cells which seems to be directly correlated to their cell death ratio.Our current hypothesis is that the potential to lead to viable clones requires plasticity of donor's epigenetic state.This work is expected to ground concepts and methods for a quantitative exploration and further multilevel modeling of morphogenetic processes.
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