Heart transplantation is often the only therapeutic option for patients with end stage heart disease. Allograft organs are in short supply. Thus, preserving the life of a grafted organ is extremely important. Cardiac allograft vasculopathy (CAV) is an expression of chronic rejection that accounts for the greatest loss of graft function in transplanted hearts. Peri-transplant ischemia/reperfusion (I/R)-injury occurs during transplantation when blood flow is stopped to remove the heart from the donor and then is reinstated upon implantation of the donor heart into the recipient. This oxidative injury contributes to vascular dysfunction and CAV. In this dissertation, I hypothesize that prevention and/or reduction of I/R during transplantation reduces post-transplant vascular dysfunction and CAV. In this regard, myself and my colleagues examined the roles of apoptosis repressor with caspase recruitment domain (ARC) and cytochrome p450 (CYP) 2C enzymes in UR-induced vascular dysfunction and CAV. ARC expression was detected in endothelial cells (ECs) and smooth muscle cells (SMCs); however, increased levels of ARC do not protect against oxidant injury. ARC overexpression did protect against oxidant-induced cell death in H9c2 rat embryonic myoblasts. We observed that ARC-overexpression prevented H9c2 differentiation into muscle cells. With our focus on vascular injury, we turned our attention to the CYP 2C enzymes. Both endothelium-dependent and independent vascular function was impaired following I/R. Pre-treatment with the CYP 2C inhibitor sulfaphenazole (SP) restored endothelial sensitivity to acetylcholine, but did not restore sensitivity to endothelium-independent vasodilators. Rat heterotopic heart transplants were performed with rats being treated with SP or vector control prior to surgery. Rats treated with SP showed significantly reduced luminal narrowing and had decreased SMC proliferation, oxidant and interferon-y levels. No differences were detected in immune infiltration or apoptosis. Complementary studies in cultured vascular cells revealed that CYP 2C9 expression decreased viability and increased ROS production following hypoxia and re-oxygenation in ECs but not in SMCs. In summary, we did not detect protection of vascular cells by ARC, but did discover a novel role for ARC in differentiation. CYP 2C contributes to post-ischemic vascular dysfunction and CAV through increased oxidative stress and endothelial dysfunction.

Heart transplantation is often the only therapeutic option for patients with end stage heart disease. Allograft organs are in short supply. Thus, preserving the life of a grafted organ is extremely important. Cardiac allograft vasculopathy (CAV) is an expression of chronic rejection that accounts for the greatest loss of graft function in transplanted hearts. Peri-transplant ischemia/reperfusion (I/R)-injury occurs during transplantation when blood flow is stopped to remove the heart from the donor and then is reinstated upon implantation of the donor heart into the recipient. This oxidative injury contributes to vascular dysfunction and CAV. In this dissertation, I hypothesize that prevention and/or reduction of I/R during transplantation reduces post-transplant vascular dysfunction and CAV. In this regard, myself and my colleagues examined the roles of apoptosis repressor with caspase recruitment domain (ARC) and cytochrome p450 (CYP) 2C enzymes in UR-induced vascular dysfunction and CAV. ARC expression was detected in endothelial cells (ECs) and smooth muscle cells (SMCs); however, increased levels of ARC do not protect against oxidant injury. ARC overexpression did protect against oxidant-induced cell death in H9c2 rat embryonic myoblasts. We observed that ARC-overexpression prevented H9c2 differentiation into muscle cells. With our focus on vascular injury, we turned our attention to the CYP 2C enzymes. Both endothelium-dependent and independent vascular function was impaired following I/R. Pre-treatment with the CYP 2C inhibitor sulfaphenazole (SP) restored endothelial sensitivity to acetylcholine, but did not restore sensitivity to endothelium-independent vasodilators. Rat heterotopic heart transplants were performed with rats being treated with SP or vector control prior to surgery. Rats treated with SP showed significantly reduced luminal narrowing and had decreased SMC proliferation, oxidant and interferon-y levels. No differences were detected in immune infiltration or apoptosis. Complementary studies in cultured vascular cells revealed that CYP 2C9 expression decreased viability and increased ROS production following hypoxia and re-oxygenation in ECs but not in SMCs. In summary, we did not detect protection of vascular cells by ARC, but did discover a novel role for ARC in differentiation. CYP 2C contributes to post-ischemic vascular dysfunction and CAV through increased oxidative stress and endothelial dysfunction.

Heart transplantation is often the only therapeutic option for patients with end stage heart disease. Allograft organs are in short supply. Thus, preserving the life of a grafted organ is extremely important. Cardiac allograft vasculopathy (CAV) is an expression of chronic rejection that accounts for the greatest loss of graft function in transplanted hearts. Peri-transplant ischemia/reperfusion (I/R)-injury occurs during transplantation when blood flow is stopped to remove the heart from the donor and then is reinstated upon implantation of the donor heart into the recipient. This oxidative injury contributes to vascular dysfunction and CAV. In this dissertation, I hypothesize that prevention and/or reduction of I/R during transplantation reduces post-transplant vascular dysfunction and CAV. In this regard, myself and my colleagues examined the roles of apoptosis repressor with caspase recruitment domain (ARC) and cytochrome p450 (CYP) 2C enzymes in UR-induced vascular dysfunction and CAV. ARC expression was detected in endothelial cells (ECs) and smooth muscle cells (SMCs); however, increased levels of ARC do not protect against oxidant injury. ARC overexpression did protect against oxidant-induced cell death in H9c2 rat embryonic myoblasts. We observed that ARC-overexpression prevented H9c2 differentiation into muscle cells. With our focus on vascular injury, we turned our attention to the CYP 2C enzymes. Both endothelium-dependent and independent vascular function was impaired following I/R. Pre-treatment with the CYP 2C inhibitor sulfaphenazole (SP) restored endothelial sensitivity to acetylcholine, but did not restore sensitivity to endothelium-independent vasodilators. Rat heterotopic heart transplants were performed with rats being treated with SP or vector control prior to surgery. Rats treated with SP showed significantly reduced luminal narrowing and had decreased SMC proliferation, oxidant and interferon-y levels. No differences were detected in immune infiltration or apoptosis. Complementary studies in cultured vascular cells revealed that CYP 2C9 expression decreased viability and increased ROS production following hypoxia and re-oxygenation in ECs but not in SMCs. In summary, we did not detect protection of vascular cells by ARC, but did discover a novel role for ARC in differentiation. CYP 2C contributes to post-ischemic vascular dysfunction and CAV through increased oxidative stress and endothelial dysfunction. / Medicine, Faculty of / Pathology and Laboratory Medicine, Department of / Graduate

Southern Magnolia (Magnolia grandiflora) is a primitive tree species that has attracted attention because of its horticultural distinctiveness, the wealth of natural products associated with it, and its evolutionary position as a basal angiosperm. Terpenoid constituents were determined from Magnolia leaves and flowers. Magnolia leaves constitutively produced two major terpenoids, andamp;acirc;-cubebene and germacrene A. However, upon wounding Magnolia leaves biosynthesized a significant array of monoand sesquiterpenoids, including andamp;acirc;-pinene, trans-andamp;acirc;-ocimene, andamp;aacute;-gurjunene, andamp;acirc;-caryophyllene and andamp;acirc;-cubebene, along with fatty acid derivatives such as cis-jasmone, for up to 19 hours after treatment. Flowers were also examined for their emission of terpene volatiles prior to and after opening, and also in response to challenge by Japanese beetles. Opened and un-opened flowers constitutively emitted a blend of monoterpenes dominated by andamp;acirc;-pinene and cis-andamp;acirc;-ocimene. However, the emission levels of monoterpenes such as verbenone, geraniol, and citral, and sesquiterpenes such as andamp;acirc;-cubebene, andamp;aacute;-farnesene, and andamp;acirc;-caryophyllene were significantly elevated in the emissions of the beetle-challenged flowers. Three cDNAs corresponding to terpene synthase (TPS) genes expressed in young Magnolia leaves were isolated and the corresponding enzymes were functionally characterized in vitro. Recombinant Mg25 converted FPP (C15) predominantly to andamp;acirc;-cubebene, while Mg17 converted GPP (C5) to andamp;aacute;-terpineol. Efforts to functionally characterize Mg11 were unsuccessful. Transcript levels for all 3 genes were prominent in young leaf tissue and significantly elevated for Mg25 and Mg11 mRNAs in stamens. A putative N-terminal signal peptide of Mg17 targeted the reporter GFP protein to both chloroplasts and mitochondria when transiently expressed in epidermal cells of Nicotiana tabacum leaves. Phylogenetic analyses indicated that Mg25 and Mg11 belonged to the angiosperm sesquiterpene synthase subclass TPS-a, while Mg17 aligned more closely to the angiosperm monoterpene synthase subclass TPS-b. Unexpectedly, intron/exon organizations for the three Magnolia TPS genes were different from one another and from other well characterized terpene synthase gene sets. The Mg17 gene consists of 6 introns arranged in a manner similar to many other angiosperm sesquiterpene synthases, but Mg11 contains only 4 introns, and Mg25 has only a single intron near the 5 terminus of the gene. Our results suggest that much of the structural diversity observed in the Magnolia TPS genes may have occurred by means other than intron-loss from a common ancestor TPS gene. Costunolide is a sesquiterpene lactone widely recognized for its diverse biological activities, including its bitter taste in lettuces, and as a precursor to the more potent pharmacological agent parthenolide. A lettuce EST database was screened for cytochrome P450 genes that might be associated with sesquiterpene hydroxylation. Five ESTs were selected based on sequence similarity to known sesquiterpene hydroxylases and three of them (Ls7108, Ls3597 and Ls2101) were successfully amplified as fulllength cDNAs. To functionally characterize these cDNAs, they were co-expressed along with a germacrene A synthase and a cytochrome P450 reductase in yeast. Based on product profile comparisons between the three different lines to the control line, only the Ls7108-harboring line produced unique compounds. Neither of the other lines showed a new product peak. The more abundant, polar product generated by the Ls7108-containing line was purified and identified as a 12-acetoxy-germacrene by NMR analysis. In vitro studies using Ls7108 microsomal proteins did not yield the 12-acetoxy-germacrene A, but the putative germacra-1(10),4,11(13)-trien-12-ol intermediate. Catalytic activity of the Ls7108 microsomal enzyme was NADPH, pH and time dependent. Our results demonstrate that Ls7108 is a lettuce cytochrome P450 which catalyzes the hydroxylation of a methyl group of the isopropenyl substituent of germacrene A, generating germacra-1(10),4,11(13)-trien-12-ol, and that when this mono-hydroxylated sesquiterpene is synthesized in yeast, an endogenous yeast enzyme further modifies the germacrenol compound by acetylation of the alcohol group at the C-12 position.

Les plantes sont des organismes sessiles qui ne peuvent fuir des conditions souvent défavorables et doivent par conséquent s’adapter à un environnement hostile pour survivre. La cutine partie intégrante de la cuticule qui joue un rôle de barrière pour la plante est un polymère lipidique constitué principalement d’acides gras en C16 et C18 hydroxylés et époxydés reliés entre eux par des liaisons ester mettant en jeu les fonctions carboxyl et ω-hydroxyl des acides gras. La cutine ne joue pas seulement un rôle de barrière physique mais joue un rôle de réservoir de molécules possédant des propriétés physiologiques fondamentales. Grâce à des approches biochimiques et génétiques, nos travaux ont permis de mettre en évidence AtEH1, une époxyde hydrolase responsable de la formation des diols incorporés dans la cutine d’Arabidopsis thaliana. Ces diols sont décrits dans la littérature comme intervenant dans les interactions plante-pathogène. Nous avons également montré que ces composés ainsi que d’autres dérivés d’acides gras sont perçus par la plante. Nous avons identifié et caractérisé CYP77B1, une époxygénase d’acide gras qui a un rôle potentiel à jouer dans la formation d’acides gras polyhydroxylés incorporés dans la cutine. / Plants are sessile organisms that are not able to escape from difficult environmental conditions and therefore have to adapt to multiple abiotic and biotic stress to survive. Cutin is a part of the cuticle which plays a major role as a barrier for the plant. It’s a lipid polymer composed mainly by hydroxylated and epoxidized C16 and C18 fatty acids linked together by ester links involving the carboxyl and ω-hydroxyl functions of those fatty acids. Cutin plays also a role as a reservoir of molecules with fundamental physiological properties. With biochemical and genetic approaches, we characterized AtEH1, an epoxide hydrolase responsible for the formation of diols incorporated in Arabidopsis thaliana cutin. These diols are described as being involved in plant-pathogen interactions. We also showed that these compounds as well as others fatty acids derivatives are perceived by plants. We have also identified and characterized CYP77B1, an epoxidase that has a potential role in the formation of polyhydroxylated fatty acids incorporated in cutin.

Sclerotinia homoeocarpa (F.T. Bennett) is one of the most economically important pathogens on high amenity cool-season turfgrasses where it causes dollar spot. Due to decades of over-reliance and repeated chemical treatments, S. homoeocarpa has developed resistance and insensitivity to multiple classes of fungicides. To understand the genetic mechanisms of fungicide resistance, the whole genomes of two strains with varying resistance levels to fungicides, were sequenced. In unpublished data (Sang et al.), a RNA-sequencing analysis revealed three CYP450s that were validated to play a functional role in S. homoeocarpa’s resistance against different fungicide classes. We also identified CYP450 metabolic action on the multi-site mode of action fungicide chlorothalonil. Chlorothalonil is an extensively used contact fungicide and has been known to be persistent in soils. Yet, S. homoeocarpa resistance to chlorothalonil has not been reported in the field. High Performance Liquid Chromatography (HPLC) indicated faster rates of chlorothalonil biotransformation by CYP450 overexpression strains when compared to the wild-type. We show by GC-MS that the primary transformation intermediate found in soils, 4-hydroxy-2,5,6 trichloroisophthalonitrile is produced by CYP450s’ metabolism.

La fusariose des épis est l’une de maladies les plus dommageables des céréales tempérées et est principalement causée par le champignon toxinogène Fusarium graminearum (Fg). Ces dix dernières années, de nombreuses études ont rapporté l’induction transcriptionnelle de gènes de la plante codant pour des cytochromes P450 (P450) en réponse l’infection par Fg. Les P450s constituent une famille enzymatique impliquée dans de nombreuses voies métaboliques, certaines avec des intérêts potentiels dans la résistance face aux maladies. Nous avons utilisé la petite graminée modèle Brachypodium distachyon (Bd) pour caractériser fonctionnellement le premier gène codant pour un P450 induit chez la plante au cours de la fusariose des épis par l’utilisation de lignées altérées dans la séquence ou l’expression du gène Bradi1g75310 codant le P450 BdCYP711A29. Nous avons montré qu’en plus d’être un facteur de sensibilité à la maladie, le gène Bradi1g75310 est impliqué dans une voie de biosynthèse hormonale chez Bd, celle des strigolactones (SLs). En effet, en plus de complémenter génétiquement les phénotypes aériens de la lignée mutante max1-1 d’Arabidopsis thaliana altérée dans le gène homologue MAX1 (AtCYP711A1), une lignée de Bd surexprimant Bradi1g75310 (lignée OE) exsude davantage d’orobanchol, une SL spécifique, que la lignée sauvage ou mutante. Une analyse préliminaire de l’impact direct de l’orobanchol sur la croissance de Fg semble indiquer une activation des étapes précoces du développement du champignon (germination) qui pourrait être à l’origine de l’induction plus rapide de gènes de défenses observée chez une lignée OE de Bradi1g75310. Nous avons également montré que les 4 paralogues de Bradi1g75310 chez Bd, qui codent également pour des CYP711A, sont tous capable de complémenter la lignée max1-1 et avons généré du matériel végétal fondamental pour la poursuite de l’étude de la diversification des SLs chez la plante monocotylédone modèle Bd. Au global, ce projet constitue une première étape dans la caractérisation de l’implication des P450 dans la réponse de la plante face à l’infection par Fg en plus de donner de nouveaux indices concernant le rôle des SLs dans les interactions plante-pathogène. Les résultats obtenus au cours de ce travail de thèse pourront permettre l’amélioration de caractères tant développementaux que de résistance à la fusariose chez les céréales cultivées. / Fusarium Head Blight (FHB) is one of the most important diseases of temperate cereals and is mostly caused by the toxin producing-fungus Fusarium graminearum (Fg). This last decade, several studies reported the transcriptional activation of cereal cytochrome P450-encoding genes (P450s) in response to Fg infection. P450s constitute an enzymatic family participating in very diverse metabolic pathways with potential interest for disease resistance. We used the model temperate cereal Brachypodium distachyon (Bd) to functionally characterize the first FHB-induced P450- encoding gene using Bd lines altered in the locus or gene expression of the Bradi1g75310 gene encoding the BdCYP711A29 P450. We showed that in addition to be a plant susceptibility factor towards the disease, the Bradi1g75310 gene is involved in the hormonal biosynthetic pathway of strigolactones (SLs) in Bd. Indeed, in addition to genetically complement the shoot phenotypes of the Arabidopsis thaliana mutant line for the homologous gene MAX1 (AtCYP711A1, max1-1 line), a Bd linewhich overexpresses the Bradi1g75310 gene (OE) exudes more orobanchol, a specific SL, compared to wild-type or mutant lines. Preliminary analysis of the direct impact of orobanchol on Fg growth suggests an activation of early fungal development (germination) likely to induce faster induction of defense-related genes during FHB, observed in Bradi1g75310 OE line. We showed that the four paralogs of Bradi1g75310 encoding BdCYP711A P450s are all able to genetically complement max1-1 line and provide important plant material for studying SLs diversification in the model monocot B. distachyon. Overall, this project constitutes a first step in the characterization of P450s involvement in plant response towards Fg infection in addition to give new evidences about the role of SLs in plant-pathogen interactions. Results obtained during this Ph.D. project will allow the improvement of both developmental and FHB-related traits in cereal crops.

Genome sequencing has resulted in the identification of >55,000 cytochrome P450 enzymes, many of which have an unknown function and regulation. In mammals, CYP3 genes appear in only one subfamily (CYP3A), which metabolize >50% of pharmaceuticals and some steroids in humans. Unlike mammals, fish contain genes in the CYP3A, CYP3B, CYP3C and CYP3D subfamilies. While it is commonly assumed that fish and mammalian CYP3A are functional similar, the function and regulation of fish CYP3 remains largely unknown. In this thesis, the receptors and compounds that regulate CYP3C genes in zebrafish were assessed. The induction of CYP3C genes in response to the aryl hydrocarbon (AHR) and estrogen receptor (ER) ligands, β-naphthoflavone and 17β-estradiol, was measured using quantitative PCR in intestine, liver and gonads. Zebrafish CYP3C genes were inducible by β-naphthoflavone and 17β-estradiol, implicating the aryl hydrocarbon and estrogen receptor in CYP3C gene regulation and suggesting that regulation of CYP3 genes in fish differs from that in mammals. To define the function of zebrafish CYP3A65 and CYP3C1, fluorogenic compounds which are specific markers of CYP1 and CYP3A activity in humans, were screened for metabolism by CYP3A65 and CYP3C1. Both CYP3A65 and CYP3C1 had the capacity to metabolize several of these compounds and the substrate profile overlapped with zebrafish CYP1A, suggesting that these compounds are not specific in fish. A high throughput approach was employed to screen ~4000 small biologically and pharmacologically active compounds for metabolism by CYP3A65 and CYP3C1, using NADPH consumption to assess catalytic activity. The substrate profiles of CYP3A65 and CYP3C1 largely overlapped and were different than mammalian CYP3A4. CYP3A65 and CYP3C1 appeared to have a bias for quinone-based compounds but further studies are required to confirm quinones as substrates and to assess a strong structure-activity relationship. Overall, this study provides insight on the regulation, function and evolution on CYP3 genes in fish. / Dissertation / Doctor of Philosophy (PhD) / Cytochrome P450 (CYP) enzymes break down compounds such as hormones and pharmaceuticals. While mammals have genes in the CYP3A subfamily, fish have unique subfamilies not found in mammals. The function and regulation of the CYP3 family in fish is unknown, but commonly assumed to be like human CYP3. The purpose of this thesis was to identify what receptors and compounds regulate CYP3C enzymes in zebrafish. We found that regulation of CYP3C enzymes in zebrafish is different than humans. Zebrafish CYP3C genes are regulated by the aryl hydrocarbon receptor and estrogen receptor, while human CYP3A is regulated by the pregnane-x-receptor. I used a high throughput approach to screen thousands of compounds to identify the function of CYP3A65 and CYP3C1 from zebrafish. CYP3A65 and CYP3C1 metabolize several plant-based and pharmaceutical compounds. CYP3A65 and CYP3C1 are more functionally similar to each other than to CYP3A in humans.