L'hexanitrohexaazaisowurtzitane ou CL-20 est un nouveau composé énergétique présentement à l'étude pour fin d'utilisation militaire. Le RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine) et le HMX (octahydro-1,3,5,7-tétranitro-1,3,5,7-tétrazocine) sont des contaminants couramment retrouvés dans le sol des sites militaires. La toxicité de ces deux derniers nitramines sur les espèces de vertébrés terrestres, tel que les mammifères, les amphibiens et les oiseaux, est de plus en plus documentée. Le CL-20 est un nitramine polycyclique caractérisé par des liens N-NO₂, ce qui est comparable au RDX et au HMX qui sont deux composés nitramines monocycliques. Étant donné leurs structures chimiques similaires, nous avons émis l'hypothèse que les effets du CL-20 sur les oiseaux seraient aussi semblables à ceux du RDX. Au cours de la présente étude, nous avons évalué les effets toxiques du CL-20 sur un gallinacé, la caille japonaise (Coturnix coturnix japonica) exposée à des doses journalières et répétées de CL-20. En premier lieu, un test sub-aigu de 14 jours a été réalisé, durant lequel les oiseaux ont subi des gavages répétés pendant les premiers 5 jours suivis de 10 jours sans exposition au CL-20 (diluant seulement). Dans un deuxième temps, un test nutritionnel sub-chronique a été réalisé sur une période de 42 jours. Au cours de ces deux études, aucun effet toxique n'a été observé chez les oiseaux exposés au CL-20. Par contre, une augmentation du poids du foie, des niveaux du sodium plasmique et de la créatinine ont été observés chez les oiseaux ayant reçu les doses de CL-20 les plus élevées. L'analyse du plasma et de certains organes vitaux des cailles (foie, cerveau, coeur et rate) a montré que le CL-20 n'était pas présent dans ces tissus, suggérant que le CL-20 pourrait être biotransformé in vivo. Au cours de l'étude nutritionnelle sub-chronique, le poids des embryons a significativement et proportionnellement diminué en fonction de la dose de CL-20. Les embryons des oiseaux exposés au CL-20 présentaient plusieurs malformations congénitales (autant crâniennes que faciales), telles que des courbures du bec, l'hypertrophie du cerveau moyen et le développement d'un seul côté du visage couplé à une micro-opthalmie (comparaison non statistique avec les embryons contrôles). Le nombre d'oeufs pondus par femelle tendait aussi à diminuer avec l'augmentation de la dose. Ces résultats suggèrent que le CL-20 aurait un effet sur le foie des femelles adultes. Le développement des embryons d'oiseaux diffère de celui du foetus de mammifères en ce sens que 90% de l'énergie requise par l'embryon d'oiseau provient de la β-oxydation des acides gras dérivés des lipides vitellins. Les lipides vitellins sont produits par le foie maternel et transmis aux oocytes en croissance, servant de source d'acides gras pour l'embryon. Ainsi, il est possible que les effets du CL-20 sur le foie maternel (via le métabolisme des acides gras et des composés xénobiotiques) conduisent à des effets nuisibles sur le développement de l'embryon qui est intimement lié au vitellus maternel. Notre hypothèse principale de travail est que le foie de caille pourrait contenir une ou des enzymes capable de biotransformer le CL-20. Nos résultats avec le cytosol total indiquent que la disparition du CL-20 est inhibée in vitro par l'acide éthacrynique ou un produit analogue au GSH, le s-octylglutathione, qui est une enzyme GST. La purification et la caractérisation de la glutathione S-transférase cytosolique (GST) a été effectuée à partir du foie de caille. L'analyse partielle de la séquence N-terminale a montré que les deux classes de GST alpha et mu de la caille ont une homologie de 100% avec la GST du poulet. Cette enzyme purifiée a pu bio transformer le CL-20 en présence de la glutathione (GSH), un co-facteur obligatoire. Nos résultats suggèrent que la protéine purifiée a bio transformé le CL-20, tel que mis en évidence par la formation concomitante de nitrite (NO₂) et la disparition du CL-20. Nos résultats suggèrent que la biotransformation du CL-20 par GST in vitro, pourrait expliquer la faible toxicité et l'absence de CL-20 in vivo. ______________________________________________________________________________ MOTS-CLÉS DE L’AUTEUR : Caille japonaise, CL-20, GST, acide éthacrynique.

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 91-106).

Background and Objective: Severe inflammation resulted from small-for-size liver graft injury provides favorable environment for tumor growth. The oxidative stress not only accelerates the inflammatory response, but also stimulates the proliferation of cancer cells. Therefore, attenuating oxidative stress after liver surgery may not only ameliorate liver injury, but also suppress tumor growth and metastasis. Glutathione peroxidase 3 (GPx3) is an anti-oxidant which has been reported to be down-regulated in several types of cancer. Here, we aimed to investigate the clinical significance of GPx3 and characterize the role of GPx3 in liver graft injury and hepatocellular carcinoma (HCC). Furthermore, we intended to explore the therapeutic value of GPx3 using hiPSC-MSCs as a delivery vehicle in hepatic ischemia-reperfusion injury and HCC. Materials and methods: To investigate the clinical significance of GPx3, the HCC patients underwent liver transplantation (106 recipients) or hepatectomy (113 patients) were recruited to study the correlation of GPx3 with clinical parameters. To explore the mechanism of GPx3 in liver graft injury, simulated IR injury model and rat liver transplantation model were applied. To examine the effect of GPx3 on HCC, rGPx3 administration and forced-expression of GPx3 within HCC cells were performed in vitro and in vivo. To explore the therapeutic value of GPx3, engineered hiPSC-MSCs delivering GPx3 was established and applied in mice hepatic IR injury model and nude mice liver cancer model. Results: I. The role of GPx3 in graft injury. The intra-graft GPx3 expression was significantly down-regulated in small-for-size graft accompanied with severe graft injury in a rat liver transplantation model. Clinically, the lower plasma GPx3 was mainly observed in the recipients with small-for-size liver graft. Furthermore, the lower plasma GPx3 significantly correlated with higher tumor recurrence post-transplantation. The down-regulation of GPx3 was associated with hepatic senescence in small-for-size graft. GPx3 treatment delivered by hiPSC-MSCs could significantly ameliorated hepatic IR injury through inhibition of macrophages activation followed by decreased production of ROS, TNFα and IL-1. II. The role of GPx3 in HCC. Down-regulation of GPx3 in liver tumor was observed in half of HCC patients (56/113). It significantly correlated with advanced pTNM stage (P = 0.024), presence of venous infiltration (P =0.043) and high AFP level (P = 0.006). The one year (P = 0.038) and five year (P = 0.019) recurrence rate were significantly higher in the patients with lower GPx3 expression. In functional study, rGPx3 administration and over-expression of GPx3 significantly suppressed proliferation and invasiveness of HCC cells in vitro and in vivo. The tumor suppressive activity of GPx3 was mediated by inhibition of EMT through Erk-NFκB-SIP1 pathway. The GPx3 treatment delivered by hiPSC-MSCs could significantly inhibit proliferation of MHCC97L. Conclusions: I. Down-regulation of GPx3 was associated with small-for-size graft injury. Low circulating GPx3 at early phase after transplantation predicted higher tumor recurrence of HCC recipients. II. Down-regulation of GPx3 indicated poor prognosis of HCC patients. GPx3 suppressed tumor growth and invasiveness by inhibition of EMT through Erk-NFκB-SIP1 pathway. III. Engineered hiPSC-MSCs delivering GPx3 may possess therapeutic value in liver graft injury and HCC. / published_or_final_version / Surgery / Doctoral / Doctor of Philosophy

In neurons, modification by the lipid palmitate regulates trafficking and function of signaling molecules, neurotransmitter receptors and associated synaptic scaffolding proteins. HIP14 (huntingtin interacting protein 14) is the first identified and characterized mammalian palmitoyl transferase that regulates this process. I have shown that HIP14 has striking effects on modulating trafficking and function of many proteins important for synapse formation and plasticity such as PSD-95, a postsynaptic scaffolding molecule. The importance of the finding that HIP14 is a neuronal palmitoyl transferase is further emphasized by our recent discovery that huntingtin protein folding, trafficking and function are regulated by the enzyme HIP14. Expansion of the polyglutamine tract in huntingtin as seen in Huntington Disease (HD) results in reduced association with HIP14 and decreased palmitoylation of huntingtin, which contributes to the formation of inclusion bodies and enhanced neuronal toxicity. By manipulating HIP14 levels through expression or knockdown, we can manipulate the number of huntingtin inclusion bodies and neuronal cell viability. Overall, these discoveries offer novel mechanism for HD pathogenesis and provide new approaches to therapy for HD. The tight association of HIP14 with wild-type huntingtin, which differs from other known enzyme-substrate interactions, indicates that huntingtin serves other functions beyond being a substrate of HIP14. I have discovered that, in vitro, wild-type huntingtin may facilitate activity of HIP14 to palmitoylate other neuronal substrates such as SNAP25, PSD95 and GAD65. By contrast, mutant htt does not act this way, probably due to lack of interaction with HIP14. Furthermore, immunoprecipitated HIP14 from huntingtin+/- mice also exhibits less enzyme activity in palmitoylating GST-SNAP25 in vitro, suggesting that decreased huntingtin expression compromises HIP14 activity. In vivo, using Acyl Biotin Exchange assay, I have also found that palmitoylation of a number of presynaptic and postsynaptic proteins that are involved in neurotransmission are reduced in huntingtin+/- mice. This study not only ascribes an important biochemical function to wild-type huntingtin, but also suggests that defects in protein palmitoylation in general due to mutant huntingtin lack of ability to facilitate HIP14 activity may contribute to the pathogenesis of HD.

N-glycosylation is a metabolic process found in all three domains of life. It is the attachment of a polysaccharide glycan to asparagine (Asn) residues within the amino acid motif, Asn-Xaa-Ser/Thr. In the archaeon, Methanococcus maripaludis, a tetrasaccharide glycan was isolated from purified flagella and its structure determined by mass spectrometry analysis. The linking sugar to the protein is surprisingly, N-acetylgalactosamine (β-GalNAc), with the next proximal sugar a derivative of N-acetylglucosamine (β-GlcNAc), being named β-GlcNAc3Ac, and the third sugar a derivative of N-acetylmannosamine (β-ManNAc), with an attached threonine residue on the C6 carbon (β-ManNAc3NAm). The terminal sugar is an unusual diglycoside of aldulose ((5S)-2-acetamido-2,4-dideoxy-5-O-methyl-α-L-erythro-hexos-5-ulo-1,5-pyranose). Previous genetic analyses identified the glycosyltransferases (GTs) responsible for the transfer of the second and third sugars of the glycan, as well as the oligosaccharyltransferase (OST) which attaches the glycan to protein. Left unidentified were the first and fourth GTs, the flippase as well as any genes involved in glycan sugar biosynthesis and modification. In this work, genes suspected to be involved in the biosynthesis of N-linked sugars, as well as those that might encode the missing GTs and flippase were targeted for in-frame deletion. Mutants with a deleted annotated GT gene (MMP1088) had a small decrease in flagellin molecular weight as determined by immunoblotting. Mass spectrometry (MS) analysis confirmed that the N-linked glycan was missing the terminal sugar as well as the threonine found on the third sugar of wildtype cells. Mutants with a deleted gene annotated to be involved in acetamidino synthesis (a functional group that is present on the third sugar), also had a decrease in flagellin molecular weight. MS analysis determined that the N-linked glycan was missing the acetamidino group on the third sugar as well as its attached threonine, along with the terminal sugar. Both mutants were able to assemble functional flagella but had impaired motility compared to wildtype cells in mini-swarm agar. Deletions were also constructed in four other GT genes considered candidates in assembly of the linking sugar. However, none of these mutants had the expected decrease in flagellin molecular weight. With the work done in this study, the glycosyl transferase that attaches the last sugar of the M. maripaludis N-linked assembly pathway has been identified as well as a gene involved in the biosynthesis and modification of the glycan sugars. / Thesis (Master, Microbiology & Immunology) -- Queen's University, 2009-07-07 15:45:19.052

In neurons, modification by the lipid palmitate regulates trafficking and function of signaling molecules, neurotransmitter receptors and associated synaptic scaffolding proteins. HIP14 (huntingtin interacting protein 14) is the first identified and characterized mammalian palmitoyl transferase that regulates this process. I have shown that HIP14 has striking effects on modulating trafficking and function of many proteins important for synapse formation and plasticity such as PSD-95, a postsynaptic scaffolding molecule. The importance of the finding that HIP14 is a neuronal palmitoyl transferase is further emphasized by our recent discovery that huntingtin protein folding, trafficking and function are regulated by the enzyme HIP14. Expansion of the polyglutamine tract in huntingtin as seen in Huntington Disease (HD) results in reduced association with HIP14 and decreased palmitoylation of huntingtin, which contributes to the formation of inclusion bodies and enhanced neuronal toxicity. By manipulating HIP14 levels through expression or knockdown, we can manipulate the number of huntingtin inclusion bodies and neuronal cell viability. Overall, these discoveries offer novel mechanism for HD pathogenesis and provide new approaches to therapy for HD. The tight association of HIP14 with wild-type huntingtin, which differs from other known enzyme-substrate interactions, indicates that huntingtin serves other functions beyond being a substrate of HIP14. I have discovered that, in vitro, wild-type huntingtin may facilitate activity of HIP14 to palmitoylate other neuronal substrates such as SNAP25, PSD95 and GAD65. By contrast, mutant htt does not act this way, probably due to lack of interaction with HIP14. Furthermore, immunoprecipitated HIP14 from huntingtin+/- mice also exhibits less enzyme activity in palmitoylating GST-SNAP25 in vitro, suggesting that decreased huntingtin expression compromises HIP14 activity. In vivo, using Acyl Biotin Exchange assay, I have also found that palmitoylation of a number of presynaptic and postsynaptic proteins that are involved in neurotransmission are reduced in huntingtin+/- mice. This study not only ascribes an important biochemical function to wild-type huntingtin, but also suggests that defects in protein palmitoylation in general due to mutant huntingtin lack of ability to facilitate HIP14 activity may contribute to the pathogenesis of HD.

Diss. (sammanfattning) Uppsala : Univ., 2002. / Härtill 6 uppsatser.

Thesis (M.Sc.)--University of Adelaide, Dept. of Physiology, 1977.

Thesis (Ph. D.)--University of Washington, 1998. / Vita. Includes bibliographical references (leaves [48]-59).

Thesis (M.S.)--Brandeis University, 2009. / Title from PDF title page (viewed on June 29, 2009). Includes bibliographical references.