No description available.

Background: The focus of this study was to investigate how producing human bone marrow (hBM) derived mesenchymal stromal cell (MSC) extracellular vehicles (EVs) in a high purity isolation system would affect their established characterization criteria and address the validity of these methods of EV production. Additionally, we set out to functionally characterize the hBM-MSC-EVs for their identified immunomodulatory ability while also assessing the presence of novel MSC-EV marker NRP1 identified by our group to further affirm its validity as a functional MSC-EV identity marker. Methods: Each hBM-MSC-EV donor was cultured in a hollow-fiber bioreactor system in non-stimulating serum/xeno-free conditions for 25 days to produce EVs persistently under quiescent conditions to characterize the hBM-MSC-EVs in their native form. EVs were isolated by traditional PEG-based precipitation for preliminary characterization to monitor bioreactor production wherein they were characterized using multimodal tangential flow filtration coupled with fast protein liquid chromatograph (FPLC) size exclusion/high-affinity purifications to obtain the final highly purified EV sample. Additionally, functional analysis of their immunomodulatory ability, EVs and MSCs were incubated with activated peripheral blood mononuclear cells (PBMCs) as an in-vitro model to evaluate their potency. Results: The hBM-MSC-EVs produced from the bioreactor system showed consistent characterization in accordance with the MISEV2018 establish criteria. We were also able to demonstrate their functional ability by observing statistically significantly immunomodulatory ability of activated PBMCs equivalent to native MSC ability. We were also able to validate the present of NRP1 on all hBM-MSC-EV samples produced confirming its validity as a MSC-EV marker. Conclusion: The significance of the results obtained from this study confirms the production of MSC-EV using a bioreactor and high purity isolation for obtaining consistent MSC-EVs for downstream investigation. Additionally, we were able to demonstrate the significance of MSC-EVs on MSC signaling for immunomodulation by showing equivalent functional potency when tested in-vitro. These results contribute to further understanding the biological attributes of MSC-EVs and contribute to the validation of currently established characterization guidelines.

OBJECTIF: La mauvaise clairance des lipoprotéines riches en triglycérides par le tissu adipeux blanc (TAB) entraîne l’hypertriglycéridémie, la résistance à l’insuline et la sécrétion hépatique d’apolipoprotéine B (apoB). Ce mémoire tente de déterminer si le LDL entraîne une clairance réduite des lipoprotéines riches en triglycérides par le TAB. MÉTHODES/RÉSULTATS: Suivant l’ingestion d’un repas riche en gras marqué à la trioléine-13C, des femmes obèses postménopausées avec apoB plasmatique élevé (> médiane 0.93 g/L, N=22, 98% sous forme de IDL/LDL) avaient une clairance réduite de triglycérides-13C et acides gras non-estérifiés-13C (AGNE), comparées à celles avec un apoB plus bas. L'aire sous la courbe à 6 heures des triglycérides-13C et AGNE-13C plasmatiques corrélait avec l'apoB, suggérant une moindre captation dans les tissus périphériques chez les femmes avec apoB élevé. Ex vivo, suivant une incubation de 4 heures de biopsies de TAB avec de la trioléine-3H, l’apoB des patientes corrélait négativement avec les lipides-3H intracellulaires. Le traitement des biopsies de TAB des participantes avec leur propre LDL menait à une réduction de l’hydrolyse et de la captation de la trioléine-3H et à l’accumulation d’AGNE-3H dans le médium. In vitro, le LDL inhibait l’activité de la LPL. De plus, les adipocytes 3T3-L1 différenciés en présence de LDL avaient une hydrolyse et une captation réduite des lipoprotéines riches en trioléine-3H. CONCLUSION: Ce mémoire suggère que le LDL diminue la clairance des lipoprotéines riches en triglycérides par le TAB humain, ce qui pourrait expliquer la résistance à l’insuline observée chez des sujets avec apoB élevé. / OBJECTIVE: Delayed plasma clearance of postprandial triglyceride-rich lipoproteins (TRL) by white adipose tissue (WAT) promotes hypertriglyceridemia, insulin resistance and hepatic secretion of apoB-lipoproteins. The aim of this memoir was to examine whether low-density lipoproteins (LDL) induced delayed clearance of TRL by WAT. METHOD/RESULTS: Six hours following the ingestion of a high-fat meal, 22 postmenopausal obese women were separated based on plasma apoB levels (above/below median of 0.93 g/L). The high apoB group had delayed plasma clearance of postprandial triglyceride and non-esterified fatty acids (NEFA) compared to women with low apoB. There was no group difference in triolein oxidation rate, suggesting a lower NEFA uptake and storage in peripheral tissue in women with high apoB. Ex vivo, following a 4 hour incubation of participant’s WAT with synthetic 3H-triolein-TRL, plasma apoB correlated negatively with incorporated 3H-lipids. Incubation of women’s WAT with their own LDL (90% of apoB-lipoproteins in plasma) decreased 3H-TRL hydrolysis and increased medium 3H-NEFA accumulation. In vitro, LDL directly inhibited LPL activity. Finally, LDL-differentiated 3T3-L1 adipocytes had lower 3H-TRL hydrolysis and 3H-NEFA storage. CONCLUSION: This thesis suggests that LDL delay clearance and storage of TRL in human WAT, possibly explaining the increased insulin resistance observed in subjects with high apoB.

Human α-N-Acetylglucosaminidase (Naglu) is a lysosomal acid hydrolase implicated in tthe rare metabolic storage disorder known as mucopolysaccharidosis type IIIB (MPS IIIB; also Sanfilippo syndrome B). Absence of this enzyme results in cytotoxic accumulation of heparan sulphate in the central nervous system, causing mental retardation and a shortened lifespan. Enzyme replacement therapy is not currently effective to treat neurological symptoms due to the inability of exogenous Naglu to access the brain. This laboratory uses a Spodoptera frugiperda (Sf9) insect cell system to express Naglu fused to a synthetic protein transduction domain with the intent to facilitate delivery of Naglu across the blood-brain barrier. The project described herein may be broken down into three main sections. Firstly, the impact of two cryptic splice sites on Naglu expression levels was analyzed in both transiently expressing Sf9 cultures and stably selected cell lines. Secondly, the effectiveness of the native Naglu secretion-signaling peptide in the Sf9 system was examined. Finally, purification of a Naglu fusion protein from suspension culture medium was performed using hydrophobic interaction chromatographic techniques. The ultimate goal of this research is to develop an efficient system for economical, large-scale production of a human recombinant Naglu fusion protein that has the potential to be successfully used for enzyme replacement therapy to treat MPS IIIB. / Graduate

OBJECTIF: La mauvaise clairance des lipoprotéines riches en triglycérides par le tissu adipeux blanc (TAB) entraîne l’hypertriglycéridémie, la résistance à l’insuline et la sécrétion hépatique d’apolipoprotéine B (apoB). Ce mémoire tente de déterminer si le LDL entraîne une clairance réduite des lipoprotéines riches en triglycérides par le TAB. MÉTHODES/RÉSULTATS: Suivant l’ingestion d’un repas riche en gras marqué à la trioléine-13C, des femmes obèses postménopausées avec apoB plasmatique élevé (> médiane 0.93 g/L, N=22, 98% sous forme de IDL/LDL) avaient une clairance réduite de triglycérides-13C et acides gras non-estérifiés-13C (AGNE), comparées à celles avec un apoB plus bas. L'aire sous la courbe à 6 heures des triglycérides-13C et AGNE-13C plasmatiques corrélait avec l'apoB, suggérant une moindre captation dans les tissus périphériques chez les femmes avec apoB élevé. Ex vivo, suivant une incubation de 4 heures de biopsies de TAB avec de la trioléine-3H, l’apoB des patientes corrélait négativement avec les lipides-3H intracellulaires. Le traitement des biopsies de TAB des participantes avec leur propre LDL menait à une réduction de l’hydrolyse et de la captation de la trioléine-3H et à l’accumulation d’AGNE-3H dans le médium. In vitro, le LDL inhibait l’activité de la LPL. De plus, les adipocytes 3T3-L1 différenciés en présence de LDL avaient une hydrolyse et une captation réduite des lipoprotéines riches en trioléine-3H. CONCLUSION: Ce mémoire suggère que le LDL diminue la clairance des lipoprotéines riches en triglycérides par le TAB humain, ce qui pourrait expliquer la résistance à l’insuline observée chez des sujets avec apoB élevé. / OBJECTIVE: Delayed plasma clearance of postprandial triglyceride-rich lipoproteins (TRL) by white adipose tissue (WAT) promotes hypertriglyceridemia, insulin resistance and hepatic secretion of apoB-lipoproteins. The aim of this memoir was to examine whether low-density lipoproteins (LDL) induced delayed clearance of TRL by WAT. METHOD/RESULTS: Six hours following the ingestion of a high-fat meal, 22 postmenopausal obese women were separated based on plasma apoB levels (above/below median of 0.93 g/L). The high apoB group had delayed plasma clearance of postprandial triglyceride and non-esterified fatty acids (NEFA) compared to women with low apoB. There was no group difference in triolein oxidation rate, suggesting a lower NEFA uptake and storage in peripheral tissue in women with high apoB. Ex vivo, following a 4 hour incubation of participant’s WAT with synthetic 3H-triolein-TRL, plasma apoB correlated negatively with incorporated 3H-lipids. Incubation of women’s WAT with their own LDL (90% of apoB-lipoproteins in plasma) decreased 3H-TRL hydrolysis and increased medium 3H-NEFA accumulation. In vitro, LDL directly inhibited LPL activity. Finally, LDL-differentiated 3T3-L1 adipocytes had lower 3H-TRL hydrolysis and 3H-NEFA storage. CONCLUSION: This thesis suggests that LDL delay clearance and storage of TRL in human WAT, possibly explaining the increased insulin resistance observed in subjects with high apoB.