No description available.

Human multidrug and toxin extrusion 1 (hMATE1) is a major candidate for being the molecular identity of organic cation/proton (OC/H+) exchange activity in the luminal membrane of renal proximal tubules (RPT). Although physiological function of hMATE1 supports luminal OC efflux, the kinetics of hMATE1-mediated OC transport have typically been characterized through measurement of uptake i.e., the interaction between outward-facing hMATE1 and OCs. To examine kinetics of hMATE1-mediated transport in a more physiologically relevant direction i.e., an interaction between inward-facing hMATE1 and cytoplasmic substrates, I measured the time course of hMATE1-mediated efflux of the prototypic MATE1-substrate, [3H]1-methyl-4-phenylpyridinium ([3H]MPP), under a variety of conditions, including different values for intra- and extracellular pH, from CHO cells that stably expressed hMATE1. I showed that an IC50/Ki for interaction between extracellular H+ and outward-facing hMATE1 determined from conventional uptake experiments [12.9 ± 1.23 nM (pH 7.89); n = 9] and from the efflux protocol [14.7 ± 3.45 nM (pH 7.83); n = 3] were not significantly different (P = 0.6). To test a hypothesis that H+ interacts symmetrically with each face of hMATE1, kinetics of interaction between intracellular H+ and inward-facing hMATE1 were determined using the efflux protocol. The IC50 for interaction with H+ was 11.5 nM (pH 7.91), consistent with symmetrical interactions of H+ with the inward-facing and outward-facing aspects of hMATE1. The efflux protocols demonstrated in this study are a potential means to examine kinetics at cytoplasmic face of hMATE1 and also a practical tool to screen uptake of substrates at extracellular face of hMATE1.

No description available.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Kidney disease has a high incidence across the globe and can be caused by acute and chronic injury. Current methods of treatment range from prevention and management with diet and extend to hemodialysis at End Stage Renal Disease (ESRD). Transmembrane Immunoglobulin Domain Containing-1 (TMIGD1) is mainly expressed in kidney and the intestines and is involved in cell-cell interaction of epithelial cells. This thesis investigated the potential role of TMIGD1 in the development of chronic kidney disease and tubular epithelial cell injury in CRISPR/Cas9-TMIGD1 transgenic mouse. Treatment of wild-type mice with adenine showed that TMIGD1 is downregulated in response to adenine-induced renal cell injury. CRISPR/Cas9-TMIGD1 -/+ mice treated with adenine displayed significantly increased tubular damage compared to wild-type mice. Additionally, expression of TMIGD1 was directly correlated with localization of C/EBPβ to nucleus, a transcription factor that is known to regulate expression of TMIGD1. In conclusion, the loss of TMIGD1 negatively impacts response to renal stress. / 2020-06-17T00:00:00Z

No description available.

Le Grapevine fanleaf virus (GFLV) est le principal agent de la maladie du court-noué de la vigne. Sa protéine de capside (CP) permet la formation des virions indispensables à la protection du génome viral, au mouvement de cellule à cellule au sein de tubules formés par la protéine de mouvement (MP) du virus, et à la transmission du GFLV par son nématode vecteur Xiphinema index. Principaux résultats : 1. un motif exposé à la surface de la CP dont la nature est critique pour transmission du GFLV par X. index a été identifié et pourrait constituer un déterminant de la spécificité de transmission. 2. Des tubules fluorescents ont été produits de façon constitutive in planta. Ils permettent de complémenter en trans un GFLV dépourvu de MP. 3. L’expression transitoire de la CP conduit à la production de pseudo-particules. Celles-ci sont modifiables à façon et font de la capside du GFLV une plateforme biotechnologique unique. De plus, c’est un puissant outil pour étudier la biologie du virus. / Grapevine fanleaf virus (GFLV) is the main agent of grapevine fanleaf degeneration disease. Its coat protein (CP) self-assembles in virions necessary for viral genome protection, for cell-to-cell movement using tubules formed by the movement protein (MP) of the virus, and for the transmission of GFLV by its nematode vector Xiphinema index.Main results: 1. An outer surface-exposed CP motif has been identified as critical for GFLV transmission by X. index and could be a determinant of transmission specificity. 2. Fluorescent tubules have been produced by constitutive expression in planta. They allow the complementation in trans of a GFLV deleted of its MP coding sequence. 3. Transient expression of the GFLV CP leads to the production of virus-like particles. They can be easily modified and show that GFLV capsid is a unique biotechnology platform. In addition, they are a powerful tool to study the biology of the virus.

Chez le xénope, le pronéphros constitue le rein fonctionnel du têtard. Le sang est filtré par le glomus et l'urine est modifiée lors de son transit dans le tubule. Au cours de ma thèse, nous avons analysé l'expression des quatre membres de la famille pou3f au cours du développement embryonnaire chez le xénope. Nous avons montré que leur expression neurale, otique ou encore épidermique est conservée entre le xénope et la souris, de même que l'expression rénale de pou3f3. Nous avons également mis en évidence une expression pronéphrique de pou3f4. Nous avons ensuite analysé le rôle de Pou3f4 au cours du développement du pronéphros. La perte de fonction de Pou3f4 entraine des défauts de différenciation terminale du tubule intermédiaire et distal. Sa surexpression aboutit au contraire à une augmentation de l'expression des marqueurs de différenciation slc12a1 et clcnkb1. Les morphants Pou3f4 présentent aussi des défauts de morphogenèse du tubule intermédiaire caractérisés par des circonvolutions réduites. Nos résultats montrent que Pou3f4 contrôle l'expression de l'ephrine efnA3 dans le tubule pronéphrique. La perte de fonction d'EfnA3 conduit à des défauts de morphogenèse du tubule, suggérant que Pou3f4, par la régulation de l'expression d'efnA3, est impliqué dans les mouvements cellulaires nécessaires à l'élongation antérieure du tubule. Enfin, nous avons observé que pou3f3 et pou3f4 ne se régulent pas l'un de l'autre. Nous avons en revanche mis en évidence une redondance fonctionnelle de ces gènes dans la morphogenèse et la différenciation du tubule puisque la perte de fonction combinée de Pou3f3 et Pou3f4 entraine un phénotype plus sévère que chaque simple perte de fonction. / In Xenopus, pronephros is the functional kidney at tadpole stage. The blood is filtrated by the glomus and the urine is modifed all along the tubule. Pou3f3 is required for intermediate and distal tubule formation in mouse. During my PhD, we have analyzed the expression of the four pou3f genes during Xenopus embryonic development. We found that neural, otic, or epidermic expression of the various pou3f genes is conserved between Xenopus and mouse. Pou3f3 expression in the pronephros is similar to that observed in mouse. We futher showed for the first time pou3f4 expression in the developping kidney. Then, we analyzed the role of Pou3f4 during pronephros development. Pou3f4 depletion inhibits the expression of terminal differentiation marker genes in the intermediate and distal tubule. Pou3f4 upregulates the expression of slc12a1 and clcnkb1 in the tubule. Moreoer, we found that Pou3f4 loss of function leads to intermediate tubule morphogenesis defects. While ephrin signaling pathway is largely described as playing crucial role in cellular movement, Pou3f4 controls efnA3 expression in the intermediate and distal tubule. EfnA3 depletion phenocopies Pou3f4 loss of function, suggesting that Pou3f4, by regulationg efnA3 expression, is implicated in cell movements necessary for anterior elongation of the tubule. Finally, we find that pou3f3 and pou3f4 do not regulate each other expression. However, they act redundantly in pronephros development since the combined Pou3f3 and Pou3f4 loss of function results in a more severe phenotype than each loss of function alone.

The intracellular Ca2+ handling system in a cardiac myocyte is of crucial importance. It regulates the contraction and relaxation of the myocyte during the excitation-contraction (EC) coupling. A normal intracellular Ca2+ handling system keeps the contraction of the heart orderly, which represents a powerful force to pump blood to the whole body. However, disarrayed or remodelled cellular structure associated with the intracellular Ca2+ handling system at the subcellular level, such as loss of T-tubule network in diseased conditions, may promote abnormal cardiac EC coupling, leading to genesis of cardiac arrhythmias impairing cardiac mechanical functions. Up to date, it is still incompletely understood how the intracellular Ca2+ handling system is altered by changes in subcellular structures of Ca2+ handling systems. In this thesis, biophysically detailed computational models for the intracellular Ca2+ handling system of a cardiac cell were developed, providing a powerful platform to investigate the spatio-temporal complexity associated with the intracellular Ca2+ handling, and its role in generating abnormal cardiac EC coupling. First, a well-validated single cell model was used to investigate how the diastolic and systolic Ca2+ concentration responded to alterations in the model parameters related to the Ca2+ handling system, from which the mechanisms underlying the rate-dependence of EC coupling were analysed. Then, a novel single cell model, with a 2D presentation of the spatial structures of subcellular Ca2+ handling and membrane action potential, of a sheep atrial myocyte was developed for simulating the abnormal intracellular Ca2+ regulation system due to the loss of T-tubules during atrial fibrillation. Variant scenarios of T-tubule loss were considered to investigate the role of the T-tubule in affecting the intracellular Ca2+ regulation. Furthermore, membrane currents' alterations due to the electrical remodelling arising from atrial fibrillation were considered together with the loss of T-tubule. Three typical types of abnormal Ca2+ cycling phenomenon, namely intracellular Ca2+ alternans, spontaneous Ca2+ sparks and intracellular Ca2+ waves were observed in AF conditions. The relationship between T-tubule loss, AF-remodelling and the genesis of delayed afterdepolarizations (DADs) was also investigated. It was shown that the loss of T-tubule in AF condition played an important role in disturbing the Ca2+ regulation system, which increases the risk for a cell to generate impaired contraction.

Lack of the enzyme 11β-hydroxysteroid dehydrogenase type 2 (11βHSD2) causes the syndrome of apparent mineralocorticoid excess (AME): low-renin hypertension, renal sodium (Na +) retention, hypokalaemic alkalosis and polyuria. This rare autosomal recessive disorder is observed in human kindreds carrying mutations in the HSD11B2 gene. Genetically modified mice, in which the homologue Hsd11b2 is rendered non-functional, have been used to study the pathogenesis of AME. Hitherto, data obtained from humans and mice have suggested that the physiological phenotype is a consequence of enhanced reabsorption of Na + through the epithelial sodium channel (ENaC) in the renal connecting tubule (CNT) and collecting duct. However, Hsd11b2 null mice exhibit epithelial hypertrophy in a different nephron segment, namely the distal convoluted tubule (DCT). The studies described herein aimed to characterise this structural phenotype and to examine the consequences for renal Na + reabsorption in AME. Hsd11b2 null mice exhibited hypertrophy and hyperplasia in the DCT, with an elevated rate of epithelial cell proliferation in this nephron segment at 60 days of age. Hsd11b2 null kidneys contained greater quantities of the thiazide-sensitive NaCl co-transporter (NCC), the dominant Na + transporter protein in the DCT. They also contained greater quantities of the phosphorylated forms of NCC that are associated with NaCl transport activity. Despite this, there was no increase in the proportion of filtered Na + that was reabsorbed in the DCT. This was assessed in anaesthetised mice, using clearance methodology to measure the thiazide-induced increment in the fractional excretion of Na + (FENa) during continuous ENaC blockade. Wild-type DCTs did not express 11βHSD2; therefore the structural and molecular changes were not a direct consequence of the loss of 11βHSD2 in affected cells. The discussion examines the likely mechanisms causing structural remodelling in the distal renal tubule of Hsd11b2 null kidneys and potential explanations for the dissociation between structural and functional phenotypes in the DCT. There are implications for our understanding of the cellular and molecular mechanisms underlying various renal phenomena including structural remodelling in the distal tubule, resolution of the ‘aldosterone paradox’ and escape from chronic aldosterone excess.