The lymphatic system has fundamental physiological roles in maintaining fluid homeostasis, immune cell trafficking and lipid transport from the small intestine to the venous circulation. Lymphatic vessels are the main functional organ responsible for the diverse transport roles the system plays. Unlike the blood vasculature, the lymphatic system does not have a central pump, such as the heart, and relies on a variety of factors to move lymph through. It was long thought that only external factors, such as skeletal muscle contraction and lymph formation, played a role in the functional transport capacity of these vessels. With the advancement of imaging capabilities (both hardware and software), it has become clear in the past two decades or so that the main factor in driving lymph transport is the ability of these vessels to intrinsically contract whereby each vessel is comprised of a chain of ‘mini pumps’ in series. The functional capacity of these vessels is thus now understood to be primarily determined by this pumping activity that has been shown to be regulated by various mechanical and biochemical cues. Lymphatic vessel dysfunction has been implicated in a variety of diseases including many lipid related pathologies and a neglected tropical disease known as lymphatic filariasis. While it has been possible to study the vessel function in the context of fluid drainage and immune cell trafficking, the capability to understand the role of lymphatic vessels in lipid transport has not been available due to the lack of experimental animal models and acquisition systems. As part of this thesis, we sought to develop an experimental animal model along with hardware and software tools to investigate the interplay between lymphatics and their lipid content. We report the first functional measurements of how vessels respond to elevated lipid loads. We further utilized our engineering expertise to develop an experimental platform allowing us to further understand the parasite known as B. malayi that migrates to and resides in lymphatic vessels.

The small intestine is the main organ for food digestion and nutrient absorption. It is constantly exposed to antigen and immunomodulatory agents from diet and commensal microbiota. Thus, the intestine is the largest compartment of the immune system in the body. Peanuts and many other allergen resources contain triglycerides, which may affect the antigen absorption through the intestine, but their effects on sensitization and anaphylaxis are unknown. We found that medium chain triglycerides (MCT) promoted antigen absorption into Peyer’s Patches, rather than into the blood directly. Both gavage and feeding of MCT plus peanut protein induced spontaneous allergic sensitization. MCT-sensitized mice experienced the IgG-dependent anaphylaxis from systemic challenges and the IgE-dependent anaphylaxis from oral challenges. Furthermore, MCT alone had direct pharmaceutical effect on enterocytes, like stimulating Jejunal-epithelial Th2 cytokine responses compared with what was seen in the long chain triglycerides (LCT) treated group. Moreover, the oral challenges conducted with peanut protein in MCT significantly exacerbated anaphylaxis compared with the LCT challenges. The intestine also plays an important role in whole body cholesterol homeostasis due to its exclusive function in cholesterol absorption. The researchers found that the intestine function in cholesterol secretion and elimination, but it has not been proven directly until recently. This pathway that facilitate the cholesterol secretions through intestine was named the Transintestinal Cholesterol Efflux (TICE) and has not been well studied yet. To find the possible transporter candidates involved in TICE, we compared both biliary and intestinal cholesterol excretion rates in wild-type (WT) and G5G8 deficient (KO) mice of both sexes. All mice were maintained on a plant-sterol free diet beginning at weaning to prevent the development of secondary phenotypes associated with Sitosterolemia. We found that WT mice had higher biliary cholesterol excretion rates compared to their G5G8 KO littermates as previously reported. However, this difference is significantly greater in females compared to males. Interestingly, intestinal cholesterol excretions increased in female KO mice compared to their WT littermates, a difference not observed in males. This data suggests a sexually dimorphic adaptive mechanism to maintain cholesterol elimination in the absence of G5G8. Whereas male mice maintain a greater level of biliary output in the absence of G5G8, female mice upregulate an alternate intestinal elimination route. To determine the origin of intestinally secreted cholesterol, we compared both hepatobiliary and intestinal cholesterol secretion rates in male wild-type (WT) and CETP transgenic (CETP TG) mice at the age of 12 weeks. Cholesteryl ester transfer protein (CETP) facilitates the transport of cholesteryl esters and triglycerides between lipoproteins in plasma and alters the lipoprotein distribution of plasma cholesterol. We found that WT and CETP TG mice did not differ in either biliary or intestinal cholesterol secretion rates when maintained on a standard chow diet. However, CETP TG mice showed increased biliary cholesterol secretion rates and decreased intestinal cholesterol secretion rates compared to the WT group in response to a Western diet. We next determined the effect of CETP on the delivery of radiolabeled HDL-cholesterol ester to bile and intestinal lumen. Unlike bulk cholesterol secretions, HDL-derived cholesterol esters were preferentially delivered to the intestine in CETP TG mice. This data suggests that CETP alter the routes of total and HDL cholesterol elimination from the body in mice.

[Truncated abstract] Hereditary Motor and Sensory Neuropathy Lom (HMSNL) is a severe autosomal recessive peripheral neuropathy, the most common form of demyelinating Charcot-Marie-Tooth (CMT) disease in the Roma (Gypsy) population. The mutated gene, N-myc downstream-regulated gene 1 (NDRG1) on chromosome 8q24, is widely expressed and has been implicated in a wide range of processes and pathways. In this study we have aimed to assess the overall contribution of this gene to the pathogenesis of peripheral neuropathies, in cases where the most common causes of CMT disease havebeen excluded, as well as to gain clues about its function through the identification of its interactions with other proteins. Sequence analysis of NDRG1 in 104 patients with CMT disease and of diverse ethnicity identified one novel disease-causing mutation, IVS8-1G>A (g.2290787G>A), which affects the splice-acceptor site of IVS8 and results in the skipping of exon 9 . . . The results suggest a defect in Schwann cell lipid trafficking as a major pathogenetic mechanism in CMT4D. At the same time, database searches showed that the chromosomal location of NDRG1 coincides with a reported High-Density Lipoprotein-Cholesterol Quantitive Trait Locus (HDL-CQTL) in humans and in mice. A putative role of NDRG1 in the general mechanisms of HDL-mediated cholesterol transport was supported by biochemical studies of blood lipids, which revealed an association between the Gypsy founder mutation, R148X, and decreased HDL-C levels. These findings suggest that while peripheral neuropathy is the drastic result of NDRG1 deficiency, the primary role of the protein may be related to general mechanisms of lipid transport⁄metabolism.

No description available.

Les membranes plasmiques et les membranes du trans-Golgi des cellules eucaryotes présentent une asymétrie des lipides qui les composent, avec les aminophospholipides (APLs : phosphatidylsérine et phosphatidyléthanolamine) enrichis dans le feuillet cytosolique. La dissipation de cette asymétrie est impliquée dans de nombreux processus (patho)physiologiques. Plusieurs études suggèrent que les ATPases P4 sont les candidats les plus probables pour le transport des APLs et le maintien de leur distribution asymétrique ; leur délétion dans la levure inhibe le trafic membranaire. En outre, des études ont montré que les ATPases P4 interagissaient avec les protéines de la famille CDC50 ; cette interaction est essentielle pour l’adressage et peut-être aussi la fonction des ATPases P4. Afin de contribuer à la compréhension du mécanisme de transport des lipides par les ATPases P4, l’objectif de ce travail a été de mettre au point la co-expression fonctionnelle, dans la levure, de l’ATPase P4 Drs2p et de sa protéine partenaire Cdc50p. Nous avons obtenu une fraction membranaire enrichie à 3% avec la protéine Drs2p, majoritairement en complexe avec Cdc50p. L’étude fonctionnelle du complexe nous a permis de mettre en évidence un rôle crucial du phosphatidylinositol-4-phosphate (PI(4)P), un important régulateur du trafic membranaire, au cours d’une étape particulière du cycle catalytique. Nous avons également développé un protocole de purification sur résine streptavidine du complexe Drs2p/Cdc50p. Enfin, comme un site potentiel d’interaction avec le PI(4)P est présent sur l’extrémité C-terminale de Drs2p, nous avons engendré différentes constructions de Drs2p, dans lesquelles une partie de l’extrémité C-terminale a été délétée ; dans une autre construction, l’extrémité N-terminale a également été délétée. Notre travail ouvre la voie à la caractérisation fonctionnelle et structurale détaillée du complexe Drs2p/Cdc50p, et à l’étude du rôle du transport de lipides dans le trafic membranaire. / Trans-Golgi membranes and plasma membranes of eukaryotic cells are asymmetric, with their cytosolic leaflet enriched in aminophospholipids (APLs: phosphatidylserine and phosphatidylethanolamine). Dissipation of this asymmetry is involved in many (patho)physiological processes. P4 ATPases are prime candidates for APL transport and for maintaining asymmetry across membranes. In addition, yeast deleted for P4 ATPases display membrane trafficking defects. Besides, CDC50 proteins have been shown to interact physically with P4 type ATPases, and this interaction is important for addressing the complex to the right destination, and possibly also for its function. To gain insight into the molecular mechanism of lipid transport by P4 ATPases, the goal of my thesis was to develop the co-expression, in yeast, of a functional P4 ATPase, Drs2p, together with its partner, Cdc50p. The strategy we developed allowed us to obtain a membrane fraction enriched in Drs2p (~3%), mainly in complex with Cdc50p. Functional characterization of the complex identified phosphatidylinositol-4-phosphate (PI4P), a major regulator of membrane trafficking, as a crucial component for rapid completion of the Drs2p/Cdc50p catalytic cycle. We also purified the complex in one step on streptavidin beads. Finally, we started investigating the potential auto-inhibitory roles of the C-terminus (as the C-terminus of Drs2p contains a PI4P binding site) and the N-terminus of Drs2p, by expressing various truncated versions of Drs2p. Our work sets the stage for detailed functional and structural characterization of the Drs2p/Cdc50p complex and its role in membrane traffic.

Background: Ceramide synthesis and metabolism is a promising target in cancer drug development. γ-tocotrienol (GT3), a member of the vitamin E family, orchestrates multiple effects that ensure the induction of apoptosis in both, wild-type and RAS-mutated pancreatic cancer cells. Here, we investigated whether these effects involve changes in ceramide synthesis and transport. Methods: The effects of GT3 on the synthesis of ceramide via the de novo pathway, and the hydrolysis of sphingomyelin were analyzed by the expression levels of the enzymes serine palmitoyl transferase, ceramide synthase-6, and dihydroceramide desaturase, and acid sphingomyelinase in wild-type RAS BxPC3, and RAS-mutated MIA PaCa-2 and Panc 1 pancreatic cancer cells. Quantitative changes in ceramides, dihydroceramides, and sphingomyelin at the cell membrane were detected by LCMS. Modulation of ceramide transport by GT3 was studied by immunochemistry of CERT and ARV-1, and the subsequent effects at the cell membrane was analyzed via immunofluorescence of ceramide, caveolin, and DR5. Results: GT3 favors the upregulation of ceramide by stimulating synthesis at the ER and the plasma membrane. Additionally, the conversion of newly synthesized ceramide to sphingomyelin and glucosylceramide at the Golgi is prevented by the inhibition of CERT. Modulation ARV1 and previously observed inhibition of the HMG-CoA pathway, contribute to changes in membrane structure and signaling functions, allows the clustering of DR5, effectively initiating apoptosis. Conclusions: Our results suggest that GT3 targets ceramide synthesis and transport, and that the upregulation of ceramide and modulation of transporters CERT and ARV1 are important contributors to the apoptotic properties demonstrated by GT3 in pancreatic cancer cells.

<p> The liver X receptors (LXRs) are members of the nuclear hormone receptor (NHR) superfamily of transcription factors and are activated by oxysterols. As such, LXRs act as "cholesterol sensors" and play an integral role in cholesterol homeostasis by modulating the expression of genes involved in lipid transport and metabolism as well as inflammation. </p> <p> This thesis begins by describing the modulation of LXR transactivation by PKC. Specifically, transactivation by LXRα is decreased upon activation of PKC signalling pathways as assessed by LXR reporter gene analysis and endogenous target gene expression. These findings reveal a mode of regulation of LXRα that may be relevant to disease conditions where aberrant PKC signalling is observed. </p> <p> The second and third part of the thesis turns the attention to the role of LXR in human airway smooth muscle (hASM), a crucial effector cell in asthma progression. For the first time, research described here indicates that primary human ASM cells express functional LXRs. Moreover, LXR target genes ABCA 1 and ABCG I were highly induced upon the addition of LXR agonists leading to enhanced cholesterol efflux to apoAI and HDL, a process dependant entirely on ABCA I. Furthermore, activation of LXR inhibited the expression of multiple cytokines in response to inflammatory mediators and inhibited the proliferation and migration of hASM cells, two important processes that contribute to the airway remodelling observed in the asthmatic lung. </p> <p> This body of work suggests that modulation of LXR offers prospects for new therapeutic approaches in the treatment of asthma. Furthermore, it establishes a critical role for ABCA 1 in lipid transport in ASM cells and suggests that dysregulation of cholesterol homeostasis in these cells may be important. These findings have broad implications in the association of hypercholesterolemia and AHR and places LXR at the forefront of novel therapeutic avenues to treat inflammatory lung disease. </p> / Thesis / Doctor of Philosophy (PhD)