The development of the metabolic syndrome (MetS) and cardiovascular diseases have been suggested to be influenced more by the quality than the amount of dietary fat. The FA composition of serum lipids may be used as biomarkers of dietary fat quality. FAs can, however, also be endogenously synthesized by lipogenic enzymes such as elongases and desaturases. Three desaturases are important in humans: Stearoyl-CoA-desaturase (SCD), ∆6-desaturase (D6D) and ∆5-desaturase (D5D) and surrogate measures of desaturase activities can be estimated as product-to-precursor FA ratios. In this thesis, we demonstrated that high SCD, D6D and low D5D estimated activities predicted MetS 20 years later, as well as cardiovascular and total mortality during a maximum of 33.7 years. The relation between D5D and MetS was independent of lifestyle and BMI, while the relation between SCD, D6D and MetS was confounded by BMI. Serum proportions of palmitic (16:0), palmitoleic (16:1) and dihomo-γ-linoleic acids were higher and the serum proportion of linoleic acid (LA) lower at baseline in those individuals who developed MetS. Further, LA was inversely related to mortality, while palmitic, palmitoleic and dihomo-γ-linoleic acids were directly associated with mortality. We also demonstrated that a diet rich in saturated fat “induced” a similar serum FA pattern (including estimated desaturase activities) that was associated with MetS, cardiovascular disease and mortality. We also propose that the SCD ratio [16:1/16:0] might be a novel and useful marker of dietary saturated fat, at least in Western high-fat diets. Finally, genetic variations in the human SCD1 gene were linked to obesity and insulin sensitivity, results that agree with data in SCD1 deficient mice. This thesis suggests that dietary fat quality and endogenous desaturation may play a role in the development of metabolic and cardiovascular diseases and the results support current dietary guidelines.

Problem – The expansion of global trade and supply chain integration has put great emphasison logistics, particularly in the intermediary sector, freight forwarders. Whilst in developedcountries freight forwarders benefit from competitive markets and trade facilitatingpolicies, this sector in West coast Africa exhibits low logistics performance levels. Inorder to address such issues, one needs to analyse the problem and identify the causes; thisthesis focuses on identifying the bottlenecks in the freight-forwarding sector in west coastAfrica.Purpose – The main purpose of this study is to identify the bottleneck/s within thefreight-forwarding industry in west coast Africa, namely: Angola, Cameroon, DR of Congo,Gabon, and Nigeria.Method – This thesis employs a pre-study and case study method, to ensure sufficient collectionof relevant material, taking into account the lack of research in this subject. We usedthe material obtained from the interviews and the secondary source, to structure our purpose,research questions, and to define the case of our study.Results – The study concludes with a series of interesting findings; First, the activity of aFreight Forwarder depends on a series of factors that do not depend on the Freight Forwarderper se. And second, Freight Forwarders in order to accomplish their tasks, have accessto services that are shared by all providers, and that are beyond their control. To conclude,the study identifies infrastructure as a major bottleneck in the Freight Forwarding sector.

Stearoyl-CoA desaturase1 (SCD1) catalyzes the synthesis of conjugated linoleic acid (CLA) and mono-unsaturated fatty acids (MUFA) in the mammary gland of ruminant animals. We hypothesized that single nucleotide polymorphisms (SNPs) in the coding region, 5' and 3' untranslted regions (UTRs) of the SCD1 gene would influence the activity of SCD1 enzyme and consequently account for some within-breed variations in milk CLA and MUFA. Sequence analysis of the coding region of the SCD1 gene of Jerseys and Holsteins revealed c.702A→G, c.762T→C and c.878C→T SNPs in exon 5 in both breeds and c.435G→A in exon 3 in Holsteins. The SNPs resulted in: A (G435A702T 762C878), A1 (A435A702T 762C878), B (G435G702C 762T878) and B1 (A435G702C 762T878) coding variants in Holsteins and only variants A and B in Jerseys. Only SNP 878C→T resulted in a non-synonymous codon change resulting in p.293Ala and p.293Val protein variants or alleles at the SCD1 locus. Subsequent association studies found significantly higher C10 index, C12 index and C14 index and consequently higher concentrations of C10:1 and C12:1 in p.293AA cows compared to the p.293VV cows in both breeds. The SCD1 genotype had no influence on concentrations of C141, C16:1, C18:1 and CLA in both breeds. / Sequence analysis of the 5' and 3' UTRs revealed no SNPs in the 5'UTR and a total of 14 SNPs in the 3'UTR of both breeds. The SNPs were in complete linkage disequilibrium resulting in 3 haplotypes or regulatory variants: H1 (G1571G1644C1763C2053A2584 A3007C3107G3208 T3290G 3497G3682A4399C4533G4881), H2 (G1571G1644A1763C2053A 2584G3007 C3107G3208T3290G3497G 3682A4399C4533G4881) and H3 (T 1571C1644A1763 T2053G2584G3007T 3107A3208C3290A3497A3682T 4399T4533A4881) in Holsteins and only H1 and H3 variants in Jerseys. A subsequent association study involving 862 Holstein cows, found the H1 regulatory variant to be associated with higher C10 and C12 desaturase indices and consequently with higher concentrations of C10:1 and C12:1 compared with the H3 variant. The effects of the H2 variant were intermediate to those of H1 and H3. 3'UTR genotype had no influence on the concentrations of C14:1, C16:1, C18:1 and CLA. The concentrations of C10:1 and C12:1 in milk fat could therefore be due to effects of SNPs in the open reading frame and the 3'UTR regions of the SCD1 gene. These results indicate that SNPs in the coding and 3'UTR regions of the SCD1 gene could be used as markers for genetic selection for increased C10:1 and C12:1 contents of milk.

Les sécrétines de l’hormone de croissance (GHRPs) sont de petits peptides synthétiques capables de stimuler la sécrétion de l’hormone de croissance à partir de l’hypophyse via leur liaison au récepteur de la ghréline GHS-R1a. Le GHRP hexaréline a été utilisé afin d’étudier la distribution tissulaire de GHS-R1a et son effet GH-indépendant. Ainsi, par cette approche, il a été déterminé que l’hexaréline était capable de se lier à un deuxième récepteur identifié comme étant le récepteur scavenger CD36. Ce récepteur possède une multitude de ligands dont les particules oxLDL et les acides gras à longue chaîne. CD36 est généralement reconnu pour son rôle dans l’athérogénèse et sa contribution à la formation de cellules spumeuses suite à l’internalisation des oxLDL dans les macrophages/monocytes. Auparavant, nous avions démontré que le traitement des macrophages avec l’hexaréline menait à l’activation de PPARƔ via sa liaison à GHS-R1a, mais aussi à CD36. De plus, une cascade d’activation impliquant LXRα et les transporteurs ABC provoquait également une augmentation de l’efflux du cholestérol. Une stimulation de la voie du transport inverse du cholestérol vers les particules HDL entraînait donc une diminution de l’engorgement des macrophages de lipides et la formation de cellules spumeuses. Puisque CD36 est exprimé dans de multiples tissus et qu’il est également responsable du captage des acides gras à longue chaîne, nous avons voulu étudier l’impact de l’hexaréline uniquement à travers sa liaison à CD36. Dans le but d’approfondir nos connaissances sur la régulation du métabolisme des lipides par CD36, nous avons choisi des types cellulaires jouant un rôle important dans l’homéostasie lipidique n’exprimant pas GHS-R1a, soient les adipocytes et les hépatocytes. L’ensemble de mes travaux démontre qu’en réponse à son interaction avec l’hexaréline, CD36 a le potentiel de réduire le contenu lipidique des adipocytes et des hépatocytes. Dans les cellules adipeuses, l'hexaréline augmente l’expression de plusieurs gènes impliqués dans la mobilisation et l’oxydation des acides gras, et induit également l’expression des marqueurs thermogéniques PGC-1α et UCP-1. De même, hexaréline augmente l’expression des gènes impliqués dans la biogenèse mitochondriale, un effet accompagné de changements morphologiques des mitochondries; des caractéristiques observées dans les types cellulaires ayant une grande capacité oxydative. Ces résultats démontrent que les adipocytes blancs traités avec hexaréline ont la capacité de se transformer en un phénotype similaire aux adipocytes bruns ayant l’habileté de brûler les acides gras plutôt que de les emmagasiner. Cet effet est également observé dans les tissus adipeux de souris et est dépendant de la présence de CD36. Dans les hépatocytes, nous avons démontré le potentiel de CD36 à moduler le métabolisme du cholestérol. En réponse au traitement des cellules avec hexaréline, une phosphorylation rapide de LKB1 et de l’AMPK est suivie d’une phosphorylation inhibitrice de l’HMG-CoA réductase (HMGR), l’enzyme clé dans la synthèse du cholestérol. De plus, la liaison d'hexaréline à CD36 provoque le recrutement d’insig-2 à HMGR, l’étape d’engagement dans sa dégradation. La dégradation de HMGR par hexaréline semble être dépendante de l’activité de PPARƔ et de l’AMPK. Dans le but d’élucider le mécanisme d’activation par hexaréline, nous avons démontré d’une part que sa liaison à CD36 provoque une déphosphorylation de Erk soulevant ainsi l’inhibition que celui-ci exerce sur PPARƔ et d’autre part, un recrutement de l’AMPK à PGC-1α expliquant ainsi une partie du mécanisme d’activation de PPARƔ par hexaréline. Les résultats générés dans cette thèse ont permis d’élucider de nouveaux mécanismes d’action de CD36 et d'approfondir nos connaissances de son influence dans la régulation du métabolisme des lipides. / Growth hormone releasing peptides (GHRPs) are small synthetic peptides aimed at stimulating GH release from the pituitary through their binding to ghrelin receptor known as growth hormone secretagogue receptor 1a (GHS-R1a). Using the GHRP, hexarelin to study tissue distribution of GHS-R1a and its GH-independent effect, it was observed that hexarelin was capable of binding to a second receptor identified as scavenger receptor CD36. While having multiple ligands, CD36 is mainly known for binding and internalizing oxLDL and long chain fatty acids. CD36 is thought to play a detrimental role in macrophage derived foam cell formation and development of atherosclerosis. Previously, we have shown that in macrophages, expressing both GHS-R1a and CD36, hexarelin promoted an activation of PPARƔ via GHS-R1a but also through its binding to CD36. This activation led to the induction of the LXRα-ABC transporters pathway and an increase in cholesterol efflux, reducing lipid-laden macrophage content. This positive effect on macrophages was reproduced in apolipoprotein E-null mice on a high fat diet treated with hexarelin. A significant reduction in the size of atherosclerotic lesions was observed while similar increases in the expression of PPARƔ, LXRα and ABC transporters occurred in isolated peritoneal macrophages. CD36 also plays a role in fatty acid uptake, and to further investigate the impact of the interaction of hexarelin with CD36, we aimed at evaluating the role of CD36 in regulating lipid metabolism in cells devoid of GHS-R1a such as adipocytes and hepatocytes. In the present thesis, we demonstrated through its interaction with hexarelin, the ability of CD36 to decrease intracellular lipid content in both adipocytes and hepatocytes. In adipocytes, hexarelin was able to increase the expression of several genes involved in fatty acid mobilization, fatty acid oxidation but also to induce the expression of the thermogenic markers, PGC-1α and UCP-1. In addition, hexarelin increased the expression of genes involved in mitochondrial biogenesis which was accompanied by mitochondrial morphological changes in agreement with what is usually seen in highly oxidative cells. In support of these findings, we also observed an increase in the activity of cytochrome c oxidase (a component of the respiratory chain) which could reflect an increase in oxidative phosphorylation. The results generated with cultured white adipocytes suggest the ability of hexarelin to promote changes toward a brown fat-like phenotype which also occurred in vivo and was dependent on the presence of CD36. In hepatocytes, CD36 was capable of regulating cholesterol metabolism by rapidly phosphorylating LKB1 and AMPK which subsequently resulted in the inactivating phosphorylation of HMG-CoA reductase, the rate-limiting enzyme in cholesterol synthesis. Hexarelin via CD36 also induced the recruitment of insig-2 to HMGR, the committed step in HMGR degradation while lifting the exerted inhibitory effect of Erk on nuclear receptor PPARƔ activity, and promoting the recruitment of AMPK to PPARƔ coactivator PGC-1α, suggesting an enhanced transcriptional potential of PPARƔ. The results generated during my graduate studies represent unique and novel mechanisms by which CD36 is capable of regulating lipid metabolism.

La stéaroyl Co-A désaturase (SCD) est l'enzyme clé du métabolisme des acides gras mono-insaturés (AGMI). Son activité 9 désaturase introduit une double liaison cis en position 9 des acides gras saturés (AGS), formant des AGMI. Une altération de la voie de biosynthèse des AGMI est impliquée dans de nombreuses pathologies, telles que le cancer et les maladies cardiovasculaires. Les cellules cancéreuses présentent une synthèse de novo en acide gras accrue avec une accumulation d'AGMI. Ce changement dans le métabolisme des acides gras est associé à la surexpression de la SCD1. Plusieurs études ont démontré que l'inhibition de SCD1 conduit au blocage de la prolifération et l'induction de l'apoptose dans les cellules cancéreuses. Néanmoins, les mécanismes d'activation mort cellulaire restent à être mieux compris. Dans cette étude, nous avons démontré que l'extinction de SCD1 par siRNA, inhibiteur synthétique ou naturel induit l'abolition de la synthèse de novo AGMI dans les cellules cancéreuses ou non. L'activation de la mort cellulaire par apoptose lors de l'inhibition de SCD1 n'est observée que dans les cellules cancéreuses. En outre, la déplétion en SCD1 induite un stress du réticulum endoplasmique, ces caractéristiques étant l'épissage de l'ARNm XBP1, la phosphorylation de eIF2α et augmentation de l'expression CHOP. Toutefois, l'activation du stress du RE lors de l'abolition de SCD1 est particuliers puisque nous ne mettons pas en évidence de modification de l'expression de la protéine chaperonne GRP78, une autre caractéristique du stress du RE. Enfin, nous avons montré que l'induction de CHOP participe à l'activation de la mort cellulaire lors de l'extinction de SCD1. En effet, la surexpression de constructions dominants négatifs et anti-CHOP restaure partiellement la viabilité des cellules cancéreuses déplétées en SCD1. Pour conclure, ces résultats suggèrent que l'inhibition de la synthèse de novo en AGMI via l'extinction de SCD1 pourrait être une cible thérapeutique prometteuse contre le cancer en induisant la mort cellulaire par l'activation de la voie du stress du réticulum endoplasmique et du facteur de transcription CHOP. Nous nous sommes également intéressés à la régulation de SCD par différents AGMI dans un modèle cellulaire en lien avec la pathologie athéromateuse. De nombreux facteurs de risque participent au développement de cette pathologie, parmi lesquels les acides gras trans (AGT). En effet, des études épidémiologiques ont mis en corrélation la consommation d'AGT d'origine industrielle et le risque de maladie cardiovasculaire. Les AGT pourraient jouer leurs effets athérogènes par l'altération du métabolisme lipidiques des cellules vasculaires. L'accumulation de lipides dans les cellules musculaires lisses vasculaires (CML) est une caractéristique de l'athérosclérose et une conséquence de la lipogenèse accrue. L'expression de la SCD est associée à l'induction de la lipogenèse et développement de l'athérosclérose. Nous nous sommes intéressés à la régulation de l'activité SCD1 dans les CML exposés à des isomères d'AGMI en C18 [l'acide cis-9oléique(OL), l'acide trans-11 vaccénique (TVA) ou l'acide trans-9 élaïdique (ELA)]. Nous avons montré que la SCD, présente dans les CML était régulée différemment selon l'isomère en C18 :1. En effet, nous observons une augmentation de l'expression et de l'activité de SCD1 sous l'effet d'un traitement par ELA et une diminution importante pour le traitement par OL. L'effet du TVA sur l'expression et l'activité dans les CML reste modeste mais une diminution est néanmoins trouvée. Nous avons corrélé l'activité de SCD avec son niveau d'expression protéique. En effet, celle-ci est augmentée par l'ELA et diminuée par l'OL. Cette régulation n'est pas post-traductionnelle et l'expression de SCD1 lors des traitements par l'OL et l'ELA est moduler au niveau transcriptionnel.Pour conclure, nous avons démontré une modulation de l'activité SCD par des AGMI (C18: 1) de configuration cis et [...]

Chronic myeloid leukemia (CML) is a clonal hematopoietic stem cell disorder associated with the Philadelphia chromosome (Ph) that arises from a reciprocal translocation between chromosomes 9 and 22, thereby resulting in the formation of the chimeric BCR-ABL oncogene encoding a constitutively activated tyrosine kinase. BCR-ABL tyrosine kinase inhibitors (TKIs) induce a complete hematologic and cytogenetic response in the majority of chronic phrase CML patients. However, TKIs cannot efficiently eradicate leukemia stem cells (LSCs) because of the insensitivity of LSCs to TKIs. Therefore, developing new strategies to target LSCs is necessary and critical for curing CML, and success of this approach depends on further understanding the molecular mechanisms by which LSCs survive and are maintained. In Chapter I, I briefly introduce CML disease, BCR-ABL oncoprotein, and TKIs. I also describe the identification and features of LSCs. Several key pathways in LSCs including Wnt/ß-catenin, hedgehog, FoxO, Bcl6 and HIF1, are discussed. I also propose our strategy to identify unique molecular pathways that are important for LSCs but not their normal stem cell counterparts. In Chapter II, I describe our finding about the function of the positive regulator, HIF1α, in CML development and LSC survival. I show that loss of HIF1α impairs the maintenance of CML through impairing cell cycle progression and inducing apoptosis of LSCs, and I also report that p16Ink4a and p19Arf mediate the effect of HIF1α on LSCs, as knockdown of p16Ink4a and p19Arf rescues the defective colony-forming ability of HIF1α-/- LSCs. As detailed in Chapter III and IV, through comparing the global gene expression profiles of LSCs and HSCs, I find two novel regulators, Blk and Scd1, which act as tumor suppressors in CML development. In Chapter III, I show that Blk is markedly down-regulated by BCR-ABL in LSCs, and that c-Myc and Pax5 mediate this down-regulation. Deletion of Blk accelerates CML development; conversely, Blk overexpression significantly delays the development of CML and impairs the function of LSCs. I also demonstrate that p27, as a downstream effector, is involved in the function of Blk in LSCs. Blk also functions as a tumor suppressor in human CML stem cells, and inhibits the colony-forming ability of human CML cells. In Chapter IV, I investigate the function of another negative regulator, Scd1, in CML LSCs, and find that expression of Scd1 is down-regulated in mouse LSCs and human CML cells. We report that Scd1 acts as a tumor suppressor in CML, as loss of Scd1 causes acceleration of CML development and overexpression of Scd1 delays CML development. Using a colony-forming assay, I demonstrate that Scd1 impairs the maintenance of LSCs due to the change of expression of Pten, p53 and Bcl2. Importantly, I find that both Blk and Scd1 do not affect normal hematopoietic stem cells (HSCs) or hematopoiesis. Taken together, our findings demonstrate that HIF1α is required for the maintenance of CML LSCs, and conversely that Blk and Scd1 suppress the function of LSCs, suggesting that combining TKI treatment with specific targeting of LSCs will be necessary for curing CML.

Organic nitrates, such as nitroglycerine (NTG), are widely used for their potent vasodilator capacity in the management of coronary artery disease and heart failure. Unfortunately, their beneficial effect is rapidly lost due to the development of nitrate tolerance, which is translated by an impaired vasorelaxation to NTG and an increased oxidative stress production. Although the mechanisms of the development of nitrate tolerance are still not fully elucidated, much interest has been focused in treating nitrate-receiving patients together with other drugs in order to overcome the development of nitrate tolerance. The Nitric Oxide generating enzyme, eNOS, and the superoxide anion generating enzyme, NAD(P)H oxidase, have been suggested to play a role in the development of nitrate tolerance. The aim of this study was to analyse the underlying mechanism by which ramipril, an ACE inhibitor and rosuvastatin, a new molecule of the statin class, are able to protect against the development of nitrate tolerance in the aortas isolated from rats, wild-type (wt) and eNOS-/- mice. <p>These results show that ramipril as well as rosuvastatin are able to protect against the development of nitrate tolerance in the wt and eNOS-/- mice aortas suggesting that eNOS is not necessary for their protective effect. The aortas from nitrate tolerant rats and mice showed a significant increase in the NAD(P)H oxidase activation compared to the aortas from the control and from the co-treated ramipril+NTG or rosuvastatin+NTG animals. In line with these findings were the results obtained by RT-PCR analysis: the mRNA expression of the different subunits of the NAD(P)H oxidase, such as gp91phox, p22phox, were significantly decreased after rosuvastatin or ramipril treatment in wt and eNOS-/- mice aortas. Apocynin, the NAD(P)H oxidase inhibitor was also able to inhibit the development of nitrate tolerance in the rat and mouse aortas. <p>In conclusion, these results suggest that rosuvastatin and ramipril are able to protect against the development of nitrate tolerance by counteracting the nitrate-induced oxidative stress. The mechanism of protection involves a direct interaction with the NAD(P)H oxidase pathway and seems to be completely independent of the eNOS pathway. <p> / Doctorat en sciences pharmaceutiques / info:eu-repo/semantics/nonPublished

Le métabolisme de la cellule cancéreuse s’adapte aux besoins en macromolécules de cette cellule en prolifération et en réponse aux signaux du microenvironnement tumoral. Ainsi, la biosynthèse des acides gras monoinsaturés (AGMI), est augmentée dans les cellules cancéreuses coliques et associée à une augmentation de l’activité de la stéaroyl-CoA désaturase (SCD), enzyme limitante de cette synthèse. Les acides gras polyinsaturés (AGPI) comme les isomères conjugués de l’acide linoléique (CLA), c9,t11 CLA et t10,c12 CLA possèdent à la fois un effet inhibiteur sur l’activité SCD et un effet anti-tumoral dont les mécanismes moléculaires restent à préciser. Dans ce contexte, les objectifs de ce travail furent d’évaluer le rôle de SCD-1 dans la survie de la cellule cancéreuse colique (CCC) et les mécanismes de régulation sous-jacents mais également d’apporter des éléments nouveaux sur les régulations à l’origine de l’effet anti-prolifératif des CLA. Nous avons tout d’abord montré que l’extinction de l’expression de SCD-1 conduit à l’apoptose des CCC dépendante de CHOP. En revanche l’extinction de SCD 1 n’affecte en rien les cellules non cancéreuses. Par ailleurs, nous avons étudié les effets sur la viabilité des CCC de deux isomères de l’acide linoléique le c9,t11-CLA et le t10,c12-CLA in vitro. Nos résultats montrent que seul le t10,c12-CLA induit une mort cellulaire par apoptose des CCC sans affecter la survie de cellules coliques non transformées. Il apparait aussi être le seul isomère à réduire la biosynthèse des AGMI dans les CCC. La mort induite par le t10,c12 CLA dans les CCC est dépendante de l’activation d’un stress du RE via la production d’espèces réactives de l’oxygène.Nos travaux apportent des éléments nouveaux dans la compréhension du rôle de SCD 1 dans la survie des cellules cancéreuses coliques et les mécanismes d’action du t10,c12 CLA. Notre étude soutient l’hypothèse de faire de la biosynthèse des AGMI une cible thérapeutique possible dans le traitement des cancers colorectaux / Cancer cells adapt their metabolism in response to signals from the microenvironment and proliferation. Thus, MonoUnsaturated Fatty Acid (MUFA) synthesis is increased in colon cancer cells, and associated with increased Stearoyl-CoA Desaturase (SCD) activity, the rate limiting enzyme of MUFA biosynthesis. Polyunsaturated Fatty Acid (PUFA), as isomers of conjugated linoleic acid (CLA), exert inhibitor activity on SCD-1 and have anti-cancer properties, but their mechanisms are not yet clear. In this context, the aim of this work was first to evaluate the role of SCD-1 in the proliferation of colon cancer cells (CCC) and to define the underlying mechanisms. In a second time, we provide new information about regulation of the anti-proliferative effect of CLA. In a first time we showed that extinction of SCD-1 induces CCC apoptosis through CHOP expression. In contrast, the extinction of SCD-1 has no effect on viability of non cancerous cells. In addition, we studied effects of two isomers of CLA, c9,t11-CLA and t10,c12 CLA, on CCC viability in vitro. We showed that only t10,c12 CLA induces apoptotic CCC death without affecting survival of untransformed colon cells. t10,c12 CLA seems to be also the only to repress MUFA synthesis. It is also shown that cell death induced by t10,c12 CLA is ER stress dependent through reactive oxygen species generation. This work provides new information about SCD 1 role in colon cancer cells survival and mechanism of t10,c12 CLA. This study supports the hypothesis to consider MUFA biosynthesis as a potential therapeutic target in colorectal cancer treatment.

Bioluminescence is the chemical production of light that results when a luciferase enzyme catalyzes the luminogenic oxidation of a small-molecule luciferin substrate. The numerous luciferases and luciferins nature has evolved can be used to illuminate biological processes, from in vitro assays to imaging processes in live animals. However, we can improve the utility of bioluminescence through modification of these enzymes and substrates. My thesis work focuses on developing reporters that expand the bioluminescent toolkit and improving our understanding of how bioluminescence works on a molecular level. The first part of my thesis focuses on characterizing luciferases and luciferins that improve bioluminescence imaging in vivo. Some of our luciferins can outperform the natural D-luciferin substrate in live mouse imaging, while others are selectively utilized by mutant luciferases in live mouse brain. We also engineered luciferins that can selectively report on endogenous enzymatic activity in live mice. The second part of my thesis focuses on determining the molecular details of how enzymes related to firefly luciferase, long-chain fatty acyl-CoA synthetases (ACSLs), can function as latent luciferases. I have determined the structure for one of these enzymes and improved its bioluminescent activity with synthetic luciferins enough to image in live mouse brain. I also characterized the selectivity in chimerized enzymes that combine firefly luciferase and ACSLs. In summary, my work improves the utility of bioluminescence for in vivo use and informs us about how evolutionarily-related enzymes function as luciferases on a molecular level.

No description available.