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Abstract Rab-family small monomeric GTP-binding proteins are well known for their role in regulating intracellular vesicular transport required for signal transduction, cell growth, cell differentiation, and development. GTP-hydrolysis activating proteins (i.e. GAPs) function as negative regulators of Rabs for controlling the lifetime of Rabs at active state. RN-tre is an evolutionarily conserved Rab GAP and has been shown to possess GAP activity towards Rab5 in vitro. Previous cell culture studies on mammalian RN-tre, however, show controversial results regarding whether RN-Tre is involved in regulating endocytosis. As genetic analysis of RN-tre has not been performed in any experimental organism, the biological function of RN-tre remains unknown. In my M. Sc project, I performed genetic experiments to determine the function of RN-Tre in Drosophila. The results from overexpression experiments show that increasing the level of RN-Tre decreased the surface level of cell surface proteins and induced the accumulation of membrane proteins in vesicle-like structures. Mutations in the RN-Tre gene, or knocking down the expression of RN-Tre with RNA interference, significantly inhibited Notch endocytosis. Consistently, reducing the dosage of the RN-Tre gene suppressed the wing phenotype caused by Notch haploinsufficiency. These results support that RN-Tre regulates the steady-state level of cell surface receptors by promoting endocytosis. The genetic interaction between RN-Tre and Rab5 suggests a model in which RN-Tre promotes endocytosis by facilitating the recycling of Rab5. My M. Sc. study thus reveals for the first time the in vivo function of RN-Tre, and provides novel and important insights into functional relationship between Rabs and GAPs during development. / RésuméLes protéines de liaison du GTP de la famille Rab sont bien connues pour le rôlequ'elles jouent dans la régulation du transport vésiculaire intracellulaire requis lors de latransduction de signaux, ainsi que la croissance, la différentiation et le développementcellulaire. Les protéines d'hydrolyse du GTP (les GAPs) agissent comme régulateursnégatifs pour contrôler la durée de vie des Rabs à l'état actif. RN-Tre est un GAP Rabconservé évolutivement et il a été démontré que cette protéine était GAP-active sur Rab5in vitro. Cependant, des cultures cellulaires précédentes sur le RN-Tre mammifère ontmontré des résultats controversés par rapport au rôle du RN-Tre dans la régulation del'endocytose. Étant donné que l'analyse génétique du RN-Tre n'a pas été effectué chezaucun organisme expérimental, la fonction biologique du RN-Tre demeure inconnue. Aucours de mon projet de maîtrise, j'ai réalisé des expériences génétiques pour déterminer lafonction du RN-Tre chez la drosophile. Les résultats des expériences de surexpressiondémontrent que l'augmentation des niveaux de RN-Tre réduisait le niveau de surface desprotéines de surface et induisait l'accumulation de protéines membranaires dans desstructures ressemblant à des vésicules. Des mutations dans le gène du RN-Tre ou unknock-down de l'expression du RN-Tre grâce à l'ARN interférent inhiba de façonsignificative l'endocytose de Notch. De la même manière, en réduisant le dosage du gèneRN-Tre suppréssait le phénotype ailes causé par une haplo-insuffisance de Notch. Cesrésultats supportent l'hypothèse que le RN-Tre régule l'équilibre dynamique desrécepteurs en surface cellulaire en promouvant l'endocytose. L'interaction génétique entreRN-Tre et Rab5 suggère un model selon lequel RN-Tre promouvoit l'endocytose enfacilitant le recyclage de Rab5. Mon étude de maîtrise révèle pour la pr

A colloid-free, reduced pressure in vitro normothermic perfusion system was developed to assess rabbit kidney function following cryobiological manipulation. Rabbit kidneys retained significant levels of function with stable or increasing GFR, sodium reabsorption of 40 to 60%, and glucose reabsorption of greater than 80% of the filtered load. / Including dextran in the perfusate as a source of colloid osmotic pressure was deleterious to kidney function. The kidney demonstrated sensitivity to exogenous energy substrate; supplementation of lactate and glucose resulted in better function over butyrate with glucose or glucose alone. The system proved sensitive to ischemia and to stimulation with an antidiuretic preparation. This system was utilized to examine the effects of treatment of rabbit kidneys with dimethylsulfoxide(Me(,2)SO). A recirculating constant pressure hypothermic (10(DEGREES)C) perfusion apparatus was assembled and kidneys were perfused with potassium-rich, sodium-poor solution with elevated glucose and mannitol. The pharmacokinetics of 3 M Me(,2)SO equilibration was studied through estimation of the inulin and Me(,2)SO spaces using radiochemical markers. Medullary tissue equilibration was achieved prior to cortical equilibration, which was complete after 35 minutes of exposure to 3 M Me(,2)SO. Me(,2)SO perfusion resulted in increased fluid retention by the kidney. / Function of rabbit kidneys was assessed at normothermia following Me(,2)SO introduction at two different rates, 35 minute equilibration at 3 M Me(,2)SO, and removal by an osmotic counterbalance system commencing with either 800 or 600 mOs/kg washout solution made hypertonic with mannitol. / Kidneys exhibited excellent in vitro function following perfusion with Me(,2)SO. Washout with solutions of hyperosmolarity commencing at 600 mOs/kg with gradual and stepwise reduction to 400 mOs/kg allowed immediate removal of Me(,2)SO without damage; initiation of washout at 800 mOs/kg resulted in reduced function at 37(DEGREES)C.