<p>  </p> <p>Zebrafish (<em>Danio rerio</em>) is a well-established model to study neutrophil biology. However, a lack of standard tissue-specific knockdown or knockout technique in the zebrafish field has limited the power of this model organism when studying developmental essential genes, such as those related to mitochondrial function. We have developed a robust and flexible neutrophil-restricted knockout in zebrafish based on CRISPR/Cas9 system, with which we gained insights into the role of Rac2 in regulating the actin cytoskeleton and the subcellular location of Rac activation in zebrafish neutrophils.</p> <p>Previous study in our lab using another neutrophil-specific knockout system addressed multiple mitochondrial proteins regulate neutrophil motility in zebrafish. Interestingly, we observed <em>Mfn2</em>-deficient neutrophils trapped in the vasculature in zebrafish embryos. Here we further characterized the function of MFN2 in regulating cell migration with neutrophil-like HL-60 cells and mice embryonic fibroblasts (MEFs). We found significant changes in actin organization in both <em>MFN2</em>-deficient neutrophil-like cells and MEFs and mechanistically, disrupted mitochondria-ER interaction, increased intracellular Ca2+ levels. We also investigated the cytoskeleton proteins and observed hyperactivation of RhoA and Myosin light chain kinase, along with accumulation of phosphorylated myosin light chain at the cell boundary in <em>MFN2</em>-deficient MEFs. These altered MFN2-Ca2+-RhoA/MLCK-myosin signaling finally affects the peripheral actin bundle architecture and forms the “Peripheral Actin Myosin Belt (PAMB)” structure. The formation of PAMB hampered cell adhesive migration in <em>Mfn2</em>-null MEFs. </p> <p>Altogether, our research gained new insights into the essential role of MFN2 in cytoskeleton regulation and the underlying molecular mechanisms, which may provide a new direction to understand the relevance of this gene in immune cell dysfunction and other MFN2-associated diseases.</p>

Pour répondre aux défis environnementaux actuels, des solutions en rupture par rapport aux turbomachines existantes sont actuellement encours de développement. Elles s’appuient sur des cycles thermodynamiques plus efficients.L’objectif de ces travaux de thèse est d’étudier expérimentalement les mécanismes de transition de régime de combustion pour ce type d'applications en utilisant un surrogate de kérosène, le n-décane. Pour cela, une déflagration est initiée dans une enceinte fermée et comprime les gaz frais. La pression et la température de ces derniers augmentent jusqu’à atteindre les conditions propices à l’apparition de l’autoinflammation.3 régimes de combustion successifs sont caractérisés dans la chambre de combustion au moyen de diagnostics optiques rapides. Un premier dégagement de chaleur associé à la flamme froide pré-oxyde les gaz frais, il est suivi du dégagement de chaleur principal (Main Heat Release,MHR). Pour les températures initiales de mélange les plus élevées, une détonation est observée à la fin du processus. Deux chemins de transition différents sont mis en évidence : la transition Déflagration-Auto-inflammation (DAIT) et la transition Déflagration-Auto-inflammation-Détonation (DAIDT). La sensibilité des transitions de régime aux conditions initiales de pression, de température et de richesse a été caractérisée au moyen de plusieurs études paramétriques. Dans ce but, les conditions de température, de pression et de composition du mélange sont calculées aux instants d’apparition des différents fronts réactifs (flamme froide, MHR et détonation). Il a notamment été observé que les dégagements de chaleur successifs de l’auto-inflammation se déroulaient aux mêmes températures (740 K pour la flamme froide et 1050 K pour le MHR)quelles que soient les conditions initiales. L’étude s’est concentrée ensuite sur l’analyse d’un point de fonctionnement particulier. L’étude de ce point de fonctionnement, différents vitesses de front d’auto-inflammation ont été observées, mettant en évidence le mécanisme de SWACER lors de la transition.Un critère de transition de régime depuis l’auto-inflammation proposé de Zander et al., dans le cadre d’études numériques, a été testé dans notre configuration expérimentale. Un critère modifié a été développé en lui adjoignant la notion d’effets de compressibilité dans l’écoulement réactif. L’application de ce critère à l’ensemble des essais permet de prédire l’apparition de la détonation dans les conditions où 0 et 100 % de DAIDT sont observés. Les différents domaines de transition de régime ont également été positionnés sur le diagramme de Bradley (ξ, ϵ). Les modes de combustion prédits par le diagramme sont consistants avec ceux qui sont atteints dans la chambre.L’influence de la distribution initiale de température sur les modes de combustion atteignables dans la chambre a été étudiée. Trois topologies d’auto-inflammation ont été mises en évidence pour trois distributions de température dans la chambre. Ces topologies sont séparées en deux catégories, celles privilégiant une direction particulière lors de l’auto-inflammation séquentielle et celle présentant un comportement tridimensionnel.Les essais ayant un comportement tridimensionnel présentent une très forte propension à la DAIDT mais une propagation lente des fronts d’auto-inflammation. Dans ce cas, un autre mécanisme de transition vers la détonation est mis en évidence : l’auto-inflammation d’une poche homogène de gaz génère des ondes de choc et déclenchent des auto-inflammations successives pendant leur propagation. Le couplage choc/front réactif entraine la formation de la détonation.Différents mécanismes de transition vers la détonation ont été observés et étudiés sur une large plage de conditions de pression, température,richesse et gradient thermique. Les résultats obtenus permettront d’appuyer les études numériques réalisées sur le sujet, manquant jusque-là de données expérimentales en conditions académiques. / To meet the current environmental challenges, breakthrough solutions compared to existing turbomachines are currently under development.They rely on the use of more efficient thermodynamic cycles.The objective of this thesis is to study experimentally the mechanisms of transition of combustion regime using a kerosene surrogate, n-decane.For this purpose, a deflagration is initiated in a closed chamber and compresses the fresh gases. The pressure and the temperature of the endgas increase until reaching the conditions favorable to the appearance of the autoignition in the chamber.3 successive combustion regimes are characterized in the combustion chamber by means of fast optical diagnostics. A first heat release,associated with the cool flame phenomenon, pre-oxidizes the fresh gases, it is followed by the Main Heat Release (MHR). For the highest initial temperatures, a detonation is observed at the end of the process. Two different transition paths are highlighted: the Deflagration-Autoignition Transition (DAIT) and the Deflagration-Autoignition-Detonation Transition (DAIDT).The sensitivity of regime transitions to the initial conditions of pressure, temperature and mixture composition was characterized by means of several parametric studies. For this purpose, the conditions of temperature, pressure and composition of the mixture are calculated at the onset of the different reactive fronts (cool flame, MHR and detonation). In particular, it has been observed that the successive heat releases of theauto-ignition start at the same temperatures (740 K for the cool flame and 1050 K for the MHR) whatever the initial conditions. The study, then, focused on the analysis of a particular operating point. During the study of this operating point different self-ignition front velocities were observed, highlighting the mechanism of SWACER during the transition.A regime transition criterion proposed by Zander et al. based on numerical studies has been tested in our experimental setup. A modified criterion has been developed to take into account compressibility effects in the reactive flow. The application of this criterion to all the dataset makes possible to predict the appearance of the detonation under the conditions where 0 and 100% of DAIDT are observed. The different regime transition domains have also been positioned on the Bradley diagram (ξ, ε). The modes of combustion predicted by the diagram are consistent with those reached in the chamber.The influence of the initial temperature distribution on the combustion modes achievable in the chamber has been studied. Three topologies of autoignition have been demonstrated for three initial temperature distributions in the chamber. These topologies are separated into two categories, those favoring a particular direction during sequential self-ignition and that exhibiting a three-dimensional behavior.Three-dimensional tests show a very high propensity for DAIDT but a slow spread of autoignition fronts. In this case, another mechanism of transition to detonation is evidenced: the self-ignition of an homogeneous gas pocket generates shock waves and triggers successive autoinflammations during their propagation. The shock coupling / reactive front causes the formation of the detonation. Different transition mechanisms to detonation have been observed and studied over a wide range of pressure, temperature, equivalence ratio and thermal gradient conditions. The obtained results will be useful to support the numerical studies carried out on the subject, which lacks experimental data in academic conditions.

Cities are rapidly expanding, and global warming is intensified in urban environments due to the urban heat island effect. Therefore, urban animals may be particularly susceptible to warming associated with ongoing climate change. Thus, I used a comparative and manipulative approach to test three related hypotheses about the determinants of heat tolerance or critical thermal maximum (CTmax) in urban ants—specifically, that (1) body size, (2) hydration status, and (3) preferred micro-environments influence CTmax. I further tested a fourth hypothesis that native species are particularly physiologically vulnerable in urban environments. I manipulated water access and determined CTmax for 11 species common to cities in California's Central Valley that exhibit nearly 300-fold variation in body mass. Inter- (but not intra-) specific variation in body size influenced CTmax where larger species had higher CTmax. The sensitivity of ants’ CTmax to water availability exhibited species-specific thresholds where short-term water limitation (8 h) reduced CTmax in some species while longer-term water limitation (32 h) was required to reduce CTmax in other species. However, CTmax was not influenced by the preferred foraging temperatures of ants. Further, I did not find support for my fourth hypothesis because native species did not exhibit reduced thermal safety margins, or exhibit CTmax values that were more sensitive to water limitation relative to non-native species. In sum, understanding the links between heat tolerance and water availability will become critically important in an increasingly warm, dry, and urbanized world that may be selecting for smaller (not larger) body size.

No description available.

This licentiate thesis concerns one dimensional flow simulation of turbocharged spark ignited engines. The objective has been to contribute to the improvement of turbocharged SI engines’ performance as well as 1 D simulation capabilities. Turbocharged engines suffer from poor gas exchange due to the high exhaust pressure created by the turbine. This results in power loss as well as high levels of residual gas, which makes the engine more prone to knock. This thesis presents an alternative gas exchange concept, with the aim of removing the high exhaust pressure during the critical periods. This is done by splitting the two exhaust ports into two separate exhaust manifolds. The alternative gas exchange study was performed by measurements as well as 1-D simulations. The link between measurements and simulations is very strong, and will be discussed in this thesis. As mentioned, turbocharged engines are prone to knock. Hence, finding a method to model knock in 1-D engine simulations would improve the simulation capabilities. In this thesis a 0-D knock model, coupled to the 1-D engine model, is presented / QC 20101112

Hepatitis E Virus (HEV), the causative agent of hepatitis E, is a zoonotic pathogen of worldwide significance. The genus Orthohepevirus A of the family Hepeviridae includes all mammalian strains of HEV and consists of 8 recognized genotypes. Genotypes 1 and 2 HEVs only infect humans and genotypes 3 and 4 infect humans and several other animal species including pigs and rabbits. An ever-expanding host range of genetically-diversified strains of HEV now include bat, fish, rat, ferret, moose, wild boar, mongoose, deer, and camel. Additionally, the ruminant species goats, sheep, and cattle have been implicated as potential reservoirs as well. My dissertation research investigates a novel animal model for HEV, examines the immune dynamics during acute infection, and evaluates the possibility of additional animal reservoirs of HEV. The first project established an immunoglobulin (Ig) heavy chain knock-out JH (-/-) gnotobiotic piglet model that mimics the course of acute HEV infection observed in humans and evaluated the pathogenesis of HEV infection in this novel animal model. The dynamics of acute HEV infection in gnotobiotic pigs were systematically determined with a genotype 3 human strain of HEV. We also investigated the potential role of immunoglobulin heavy-chain JH in HEV pathogenesis and immune dynamics during the acute stage of virus infection. This novel gnotobiotic pig model will aid in future studies into HEV pathogenicity, an aspect which has thus far been difficult to reproduce in the available animal model systems. The objective of the second project for my PhD dissertation was to determine if cattle in the United States are infected with a bovine strain of HEV. We demonstrated serological evidence of an HEV-related agent in cattle populations with a high level of IgG anti-HEV prevalence. We demonstrated that calves from a seropositive cattle herd seroconverted to IgG binding HEV during a prospective study. We also showed that the IgG anti-HEV present in cattle has an ability to neutralize genotype 3 human HEV in vitro. However, our exhaustive attempts to detect HEVrelated sequence from cattle in the United States failed, suggesting that one should be cautious in interpreting the IgG anti-HEV serological results in bovine and other species. Collectively, the work from my PhD dissertation delineated important mechanisms in HEV pathogenesis and established a novel animal model for future HEV research. / Ph. D. / Hepatitis E Virus (HEV), the causative agent of hepatitis E, is a zoonotic pathogen of worldwide significance. According to the World Health Organization, there are approximately 20 million HEV infections annually, which result in 3.3 million cases of acute hepatitis E and >44,000 HEV-related deaths. Hepatitis E is a self-limiting acute disease in general, but carries the ability to cause high mortality in pregnant women and chronic hepatitis in immunocompromised individuals. The underlying mechanisms of HEV host tropism and progression of disease to chronicity are unknown. My dissertation work investigates a novel animal model for HEV, evaluates the possibility of additional animal reservoirs of HEV, and examines the immune dynamics during acute infection. The first project established an immunoglobulin (Ig) heavy chain knock-out JH (-/-) gnotobiotic piglet model that mimics the course of acute HEV infection observed in humans. The dynamics of acute HEV infection were determined in both the knock-out and wild-type piglets with a genotype 3 strain of human HEV. We also investigated the potential role of immunoglobulin heavy-chain JH in HEV pathogenesis and virus infection. In the second project, we determined if cattle in the United States are infected with a bovine strain of HEV. We showed serological evidence of an HEV-related agent in cattle as well as calves born in a seropositive herd. Despite the detection of specific antibodies recognizing HEV in cattle, definitive evidence of virus infection could not be demonstrated. Our exhaustive attempts to detect HEV-related sequence from cattle in the United States failed, suggesting that one should be cautious in interpreting the IgG anti-HEV serological results in bovine and other species. Collectively, the work from my PhD dissertation research delineated important mechanisms in HEV pathogenesis and established a novel animal model for future HEV research.

No description available.

Thesis (MScMedSc (Biomedical Sciences. Medical Biochemistry))--University of Stellenbosch, 2010. / Bibliography / ENGLISH ABSTRACT: The genome of the pathogen Mycobacterium tuberculosis contains five copies of the ESAT-6 (ESX) gene cluster region, which encodes for a novel type VII secretion system. These gene cluster regions, which are directly involved in pathogenicity and phagosomal escape, contain genes encoding exported T-cell antigens ESAT-6 and CFP-10. The mechanism of action of the ESX secretion system however, remains largely unknown. This study focused on ESX gene cluster region 4 (ESX-4), which has been shown to be the most ancestral region and is also present in other species of Mycobacteria and even in other high G+C Gram-positive bacteria, such as Corynebacterium diptheriae and Streptomyces coelicolor. This project aimed to investigate the protein-protein interactions of ESX-4 of M. tuberculosis in the model organism Mycobacterium smegmatis by means of Mycobacterial Protein Fragment Complementation (M-PFC). M-PFC is a two-hybrid technique which employs two cloning vectors, pUAB300 (conferring resistance to hygromycin B) and pUAB400 (conferring resistance to kanamycin). Genes of interest are cloned into these vectors and co-transformed into the model organism M. smegmatis after which it is expressed as fusion proteins. Interaction of the proteins allows selective growth on a medium containing the antibiotic trimethoprim. Various interactions were identified throughout this region, including selfinteractions as well as the expected interaction between the ESAT-6 and CFP-10 protein family members esxT and esxU. Since this region is ancestral, ESX-4 provides the basic model of the mechanism of secretion of the type VII secretion system. Many similarities were apparent when the interactions identified for ESX-4 were compared to the interactions previously identified in ESX-3. Interactions identified by means of M-PFC provide a basis for the further study of the structure of this secretion system, and should be confirmed by means of other techniques, such as co-immunoprecipitation. Despite the ability of M-PFC to identify protein-protein interactions in a mycobacterial system, and thus overcoming some of the limitations of the classical yeast two-hybrid model, it must still be regarded as a fishing experiment for potential interactions. A further aim of the project was to construct a knock-out of ESX-4 in the model organism M. smegmatis, which contains three ESX regions, namely ESX-1, -3 and -4. Homologous recombination proved to be an effective technique for the construction of the knock-out, also indicating that ESX-4 is not essential for in vitro growth of M. smegmatis. The knock-out strain showed no morphological differences to the wild type strain of M. smegmatis. The knock-out strain will in future be compared to the wild type strain in various functional studies in order to determine the function of the ancestral ESX region. / AFRIKAANSE OPSOMMING: Die genoom van die patogeen Mycobacterium tuberculosis bavat vyf kopieë van die ESAT-6 geen groep gebiede wat kodeer vir ‘n unieke tipe VII sekresie sisteem. Die geen groep gebiede, wat direk betrokke is by patogenisiteit en fagosomale ontsnapping, bevat gene wat kodeer vir die gesekreteerde T-sel antigene ESAT-6 en CFP-10. Die meganisme van die ESX sekresie sisteem is egter steeds tot ‘n groot mate onbekend. Hierdie studie het gefokus op die ESX geen groep gebied 4 (ESX-4), wat voorheen bepaal is om die vroegste kopie van die gebied te wees en wat ook in ander species van Mikobakterieë en hoë G+C Gram-positiewe bakterieë, soos Corynebacterium diptheriae en Streptomyces coelicolor, voorkom. Hierdie projek was daarop gemik om die proteïen-proteïen interaksies van ESX-4 van M. tuberculosis in die model organisme Mycobacterium smegmatis te ondersoek deur middel van Mikobakteriële Proteïen Fragment Komplementasie (M-PFK). M-PFK is ‘n twee-hibried tegniek wat van twee kloningsvektore, naamlik pUAB300 (wat weerstand teen hygromycin B bied) en pUAB400 (wat weerstand teen kanamycin bied) gebruik maak. Gene van belang word in die vektore ingekloneer en in die model organisme, M. smegmatis geko-transformeer, waarna dit as fusieproteïene uitgedruk word. Indien ‘n interaksie tussen die proteïene plaasvind, sal selektiewe groei op ‘n medium wat die antibiotikum trimethoprim bevat, waargeneem word. Verskeie interaksies is in hierdie gebied geïdentifiseer, insluitende self-interaksies, sowel as die verwagte interaksie tussen die ESAT-6 en CFP-10 proteïen familielede esxT en esxU. Aangesien hierdie gebied die vroegste kopie is, bied ESX-4 die basiese model vir die meganisme van sekresie van die tipe VII sekresie sisteem. Wanneer interaksies wat vir ESX-4 geïdentifiseer is met die wat voorheen vir ESX-3 geïdentifiseer is vergelyk word is daar heelwat ooreenkomste. Interaksies wat deur middel van M-PFK geïdentifiseer is, verskaf ‘n basis vir die vêrdere studie van interaksies van hierdie gebied, en sal bevestig moet word deur gebruik te maak van aanvullende tegnieke, soos ko-immunopresipitasie. Ten spyte van die vermoë van M-PFK om proteïen-proteïen interaksies in ‘n mikobakteriële sisteem, wat dus sommige van die beperkings van die klassieke gis twee-hibriedmodel oorkom, te bestudeer, behoort dit steeds as ‘n voorlopige metode van identifikasie beskou te word. ‘n Vêrdere doel van die projek was om ‘n uitslaanmutant van ESX-4 in die model organisme M. smegmatis, wat drie van die ESX gebiede, naamlik ESX-1, -3 en -4 bevat, te skep. Homoloë rekombinasie is bewys om ‘n effektiewe tegniek te wees vir die skep van ‘n uitslaanmuntant en het daarop gedui dat ESX-4 nie essensieel is vir die in vitro groei van M. smegmatis nie. Die uitslaanstam het ook geen morfologiese verskille getoon teenoor die oorspronklike stam nie. Die uitslaanmutant sal in die toekoms gebruik word in ‘n verskeidenheid funksionele studies waar dit vergelyk sal word met die oorspronklike stam, ten einde die funksie van die vroegste ESX-gebied te bepaal. / Medical Research Council of South Africa / National Research Foundation of South Africa / Ernst and Ethel Eriksen Trust

L’Ins(1,4,5)P3 joue un rôle évident dans la signalisation cellulaire : il permet la libération du Ca 2+ des stocks intracellulaires par son action au niveau de récepteurs spécifiques. Pour mettre fin à son action, l’Ins(1,4,5)P3 peut être dégradé par une Ins(1,4,5)P3 5-phosphatase en Ins(1,4)P2, un métabolite inactif. L’Ins(1,4,5)P3 peut aussi être transformé en Ins(1,3,4,5)P4 par une Ins(1,4,5)P3 3-kinase. L’Ins(1,3,4,5)P4 semble posséder des capacités de signalisation propres ou au contraire liées à celles de l’Ins(1,4,5)P3. L’Ins(1,3,4,5)P4 est aussi le point de départ de toute une série d’inositol hautement phosphorylés, dont les rôles ne sont pas clairs. Trois isoformes de l’Ins(1,4,5)P3 3-kinase existent (A, B et C). Ces isoformes possèdent un domaine catalytique carboxy-terminal bien conservé. Par contre, les domaines amino-terminaux sont spécifiques et leur permettraient d’établir des interactions ou de subir des régulations propres. Pour tenter d’élucider le rôle fonctionnel de l’Ins(1,3,4,5)P4, nous avons généré et analysé des souris déficientes pour les isoformes B et C de cette enzyme. Les souris déficientes pour l’Ins(1,4,5)P3 3-kinase C ne présentent pas de phénotype évident, ce qui suggère que son rôle n’est pas crucial ou que son absence peut être compensée par une autre enzyme. Les souris déficientes pour l’Ins(1,4,5)P3 3-kinase B, par contre, présentent une immunodéficience caractérisée par une absence spécifique des lymphocytes T αβ périphériques. Cette absence fait suite à un blocage dans la différenciation du précurseur du lymphocyte, le thymocyte. Les caractéristiques de la signalisation induite par le récepteur de surface (TCR) permettent la sélection des thymocytes, de manière à constituer un pool de lymphocytes T restreints pour le MHC et tolérants pour le soi. Nous avons montré que ces phénomènes de sélection étaient défectueux dans les thymocytes mutants, du fait de leur hyporéactivité à la stimulation par le TCR. Le mécanisme responsable de cette hyporéactivité n’est pas encore élucidé. A première vue, la mobilisation de Ca 2+ ne semble pas altérée dans ces thymocytes mutants en réponse à des stimulations classiques. Cependant, d’autres types de stimulation, se rapprochant plus de celles réellement rencontrées par le thymocyte in vivo, doivent encore être investigués. L’intégrité d’autres voies de signalisation cruciales du lymphocyte T doit aussi être vérifiée. En conclusion, l’isoforme B de l’Ins(1,4,5)P3 3-kinase et l’Ins(1,3,4,5)P4 qu’il produit jouent un rôle crucial dans la différenciation du thymocyte, par un mécanisme qui reste encore à déterminer.

No description available.