Introduction 52Mn (t½ =5.59 d, β+ = 29.6%, Eβmax = 0.58 MeV) has great potential as a long lived PET isotope for use in cell tracking studies, observation of immunologic response to disease states, or as an alternative to manganese-based MRI contrast agents. Its favorable max positron energy leads to superb imaging resolution, comparable to that of 18F.[1] Manganese is naturally taken up by cells via a multitude of pathways including the divalent metal transporter (DMT1), ZIP8, transferrin receptors (TfR), store-operated Ca2+ channels (SOC-Ca2+), and ionotropic glutamate receptor Ca2+ channels (GluR).[2] These natural transport mechanisms make 52Mn an attractive isotope for applications necessitating non-perturbative cell uptake. In particular, cell tracking is critical to the development and translation of stem cell therapies in regenerative medicine. Alternative-ly, 52Mn could be used in immunotherapy techniques such as adoptive cellular therapy (ACT) to evaluate the ability of external immune cells to reach their intended target. Material and Methods 52Mn was produced by natCr(p,x)52Mn using 16 MeV protons. The average thick target production yield was 0.23 mCi/µA-h with less than 0.25% co-production of 54Mn. Small amounts of 51Cr were observed in the target, but were absent from the radiochemically separated product. Target construction consisted of a water jet cooled chromium disc (3/4” diameter, 0.4” thick). Targets were purchased from Kamis Inc, and are 99.95% pure. Targets withstood beam currents of 30 µA with no visible aberration. Chromium targets were etched by concentrated HCl following bombardment. Mn2+ ions were extracted from 9M HCl to 0.8M trioctylamine in cyclohexane leaving the bulk chromium in the aqueous phase. After isolating the organic phase, 0.001M NH4OH was used to back-extract the Mn2+ ions to aqueous phase. This purification cycle was conducted a total of three times for each 52Mn production. Results and Conclusion For a starting bulk chromium mass of 456 ± 1 mg, a post-separation chromium mass of 5.35 ± 0.04 ng was measured by microwave plasma atomic emission spectrometry (MP-AES). This mass reduction corresponds to an average separation factor of 440 for a single purification cycle. Each purification cycle had a 52Mn recovery efficiency of 73 ± 7 % (n = 6), resulting in an overall separation efficiency of approximately 35 %. These efficiencies and separation factors agree reasonably well with the work conducted by Lahiri et. al.[3] Prior to use, the product was passed through a C-18 Sep-Pak to remove any residual organic phase. After four target irradiations and etchings, some pitting became noticeable on the target face. These have not yet compromised the o-ring seal with the target deplater, but it is possible that target replacement after every 6–9 52Mn productions will be necessary moving forward. Following the successful separation of 52Mn from chromium, in vitro experiments were conducted to demonstrate the uptake of 52Mn by human stem cells and mouse tumor cells. A linear uptake response was observed as a function of the amount of activity exposed to the cells for both cell models. These experiments have shown great promise for 52Mn as a long-lived PET isotope in cell tracking studies. Details will be presented.

Soft biological tissues, being live and due to their physiological structures, display considerably complex mechanical behaviors. For a better understanding and use in various applications, first study to be carried out is the tests made particularly as in vivo. An indenter test device developed for this purpose in the METU, Department of Mechanical Engineering, Biomechanics Laboratory is operational. In this study, in order to carry out precise and dependable tests, initially, various tests and improvements were conducted on the device and the software controlling the device. At the end of this study, displacement and load measurement accuracies and precisions were improved. Better algorithms for filtering the noisy data were prepared. Some test protocols within the software were improved and new protocols were annexed. To be able to conduct more dependable tests a new connection system was attached to the device. In order to study the anisotropic behavior of soft tissues ellipsoid tips were designed and produced. In the second phase of the study, tests on medial forearm were carried out. In these tests, hysteresis, relaxation and creep behaviors displaying the viscoelastic v properties of the soft biological tissues were observed. In addition to viscoelastic behaviors, preconditioning (Mullin&amp / #8217 / s) effect and anisotropic response were examined. By using the results of the relaxation and creep tests, parameters of the Prony series capable of modelling these data were determined. With this study, some important conclusions regarding the soft biological tissues were drawn and thus the behaviors of the soft biological tissues were better understood. Besides, the difficulties inherent to in-vivo tests were recognized and actions to reduce these difficulties were explained. Finally, clean experimental data, to be used in the computer simulations, were obtained.

Les campylobactérioses sont les infections intestinales bactériennes d’origine alimentaire les plus fréquemment rapportées au sein de l’Union Européenne et sont principalement associées à la consommation de viande de volailles. Une diminution de la colonisation intestinale des volailles par Campylobacter de 2 à 3 log10 UCF/g permettrait de réduire l’incidence des cas humains de 76 à 100 %. La vaccination aviaire constitue un moyen de lutte potentiel mais, malgré de nombreuses études, aucun vaccin commercial n’est actuellement disponible. L’objectif de ce projet a été d’identifier de nouveaux antigènes vaccinaux contre Campylobacter en appliquant la stratégie de la vaccinologie inverse et d’évaluer leurs pouvoirs immunogène et protecteur contre la colonisation intestinale des volailles. Sur la base de leur localisation subcellulaire, leur antigénicité, leur densité en épitopes B et leur homologie de séquence avec l’ensemble des souches de C. jejuni et C. coli, quatorze antigènes ont été sélectionnés. Six d’entre eux ont été produits et testés in vivo en appliquant un protocole vaccinal optimisé. Quatre antigènes ont montré des diminutions significatives de la charge intestinale des oiseaux de 2 à 4,2 log10 UFC/g associées à l’induction de réponses humorales spécifiques. L’immunogénicité de ces candidats vaccins et l’efficacité protectrice de deux antigènes ont été observées à nouveau. Ces premiers résultats montrent l’intérêt et la fiabilité de la vaccinologie inverse. L’évaluation du potentiel vaccinal de ces nouveaux antigènes doit être poursuivie et approfondie lors de futures expérimentations. / Campylobacteriosis is the most prevalent bacterial foodborne gastroenteritis reported in the European Union and is mainly associated to consumption of poultry meat. Reducing the intestinal colonization of broilers by Campylobacter from 2 to 3 log10 CFU/g could decrease human cases incidence by 76 to 100%. Vaccination of poultry could be a potential strategy but despite many studies, no efficient vaccine is available yet. The aim of this project was to identify new vaccine antigens against Campylobacter using the reverse vaccinology strategy and to assess their immune and protective powers against the avian intestinal colonization. Based on their sub-cellular localization, immunogenicity, B-epitopes density and their sequence conservation among C. jejuni and C. coli strains, fourteen antigens were selected. Six out of them were produced and in vivo tested according to an optimized avian vaccine protocol. Four antigens showed intestinal load decreases from 2 to 4.2 log10 CFU/g correlated with the induction of specific humoral responses. Vaccine candidates’ immunogenicity and the protective efficiency of two antigens were observed again. These first results highlight the interest and reliability of the reverse vaccinology. The assessment of these new antigens vaccine potential needs to be continued and deepened in next experiments.

Introduction 52Mn (t½ =5.59 d, β+ = 29.6%, Eβmax = 0.58 MeV) has great potential as a long lived PET isotope for use in cell tracking studies, observation of immunologic response to disease states, or as an alternative to manganese-based MRI contrast agents. Its favorable max positron energy leads to superb imaging resolution, comparable to that of 18F.[1] Manganese is naturally taken up by cells via a multitude of pathways including the divalent metal transporter (DMT1), ZIP8, transferrin receptors (TfR), store-operated Ca2+ channels (SOC-Ca2+), and ionotropic glutamate receptor Ca2+ channels (GluR).[2] These natural transport mechanisms make 52Mn an attractive isotope for applications necessitating non-perturbative cell uptake. In particular, cell tracking is critical to the development and translation of stem cell therapies in regenerative medicine. Alternative-ly, 52Mn could be used in immunotherapy techniques such as adoptive cellular therapy (ACT) to evaluate the ability of external immune cells to reach their intended target. Material and Methods 52Mn was produced by natCr(p,x)52Mn using 16 MeV protons. The average thick target production yield was 0.23 mCi/µA-h with less than 0.25% co-production of 54Mn. Small amounts of 51Cr were observed in the target, but were absent from the radiochemically separated product. Target construction consisted of a water jet cooled chromium disc (3/4” diameter, 0.4” thick). Targets were purchased from Kamis Inc, and are 99.95% pure. Targets withstood beam currents of 30 µA with no visible aberration. Chromium targets were etched by concentrated HCl following bombardment. Mn2+ ions were extracted from 9M HCl to 0.8M trioctylamine in cyclohexane leaving the bulk chromium in the aqueous phase. After isolating the organic phase, 0.001M NH4OH was used to back-extract the Mn2+ ions to aqueous phase. This purification cycle was conducted a total of three times for each 52Mn production. Results and Conclusion For a starting bulk chromium mass of 456 ± 1 mg, a post-separation chromium mass of 5.35 ± 0.04 ng was measured by microwave plasma atomic emission spectrometry (MP-AES). This mass reduction corresponds to an average separation factor of 440 for a single purification cycle. Each purification cycle had a 52Mn recovery efficiency of 73 ± 7 % (n = 6), resulting in an overall separation efficiency of approximately 35 %. These efficiencies and separation factors agree reasonably well with the work conducted by Lahiri et. al.[3] Prior to use, the product was passed through a C-18 Sep-Pak to remove any residual organic phase. After four target irradiations and etchings, some pitting became noticeable on the target face. These have not yet compromised the o-ring seal with the target deplater, but it is possible that target replacement after every 6–9 52Mn productions will be necessary moving forward. Following the successful separation of 52Mn from chromium, in vitro experiments were conducted to demonstrate the uptake of 52Mn by human stem cells and mouse tumor cells. A linear uptake response was observed as a function of the amount of activity exposed to the cells for both cell models. These experiments have shown great promise for 52Mn as a long-lived PET isotope in cell tracking studies. Details will be presented.

A la suite des nombreux travaux sur l’inhibition de la glycogène phosphorylase (GP) menés au laboratoire et au travers de diverses collaborations, cette thèse décrit en cinq chapitres suivis d’une partie expérimentale détaillée, les dernières avancées en termes de synthèse et d’évaluation biologique des inhibiteurs du site catalytique de la GP. La chapitre I de ce manuscrit est consacrée à la présentation des diabètes et plus particulièrement du diabète de type II dont le traitement, motivation première de ce projet, repose sur la connaissance des mécanismes complexes régulant la glycémie. Les différents inhibiteurs synthétisés sont classés par famille selon leur structure qui associe un aglycone hétérocyclique, susceptible d’affinité pour le canal β proche du site actif de l’enzyme, avec un motif glycopyranosidique, ou glycopyranosylidène dans le cas des motifs spiro. Le chapitre II est consacré aux inhibiteurs spiro-bicycliques tels que les glucopyranosylidène-spiro-1,4,2-oxathiazoles et les glucopyranosylidène-spiro-isoxazolines. Le chapitre III décrit la synthèse de C- et N-glycosyles hétérocycles, principalement des glycopyranosyl-1,2,3-triazoles. Enfin le chapitre IV décrit la fonctionnalisation de 5-halogéno-1,2,3-triazoles 4-substitués par couplages pallado-catalysés qui ont constitué un développement imprévu mais original des travaux. Pour terminer, le chapitre V décrit l’évaluation des molécules préparées en tant qu’inhibiteurs de la glycogène phosphorylase. Les expériences et résultats d’enzymologie, de cristallographie ainsi que les tests cellulaires in vitro et in vivo sur le rat sont présentés / Following many studies lead on the inhibition of glycogen phosphorylase (GP) in our laboratory an trough several collaborations, this thesis describes in five chapters and a detailed experimental section, the most recent advances in the areas of synthesis and biological evaluation of GP’s catalytic site inhibitors. Chapter I is dedicated to the description of diabetes and especially type 2 diabetes of which treatment, the main goal of this project, requires knowledge of the complex mechanisms that regulates glycemia. Synthesized inhibitors are broken down into families according to their structure which associates an heterocyclic aglycon, prone to binding in the β pocket lining the active site, with a glycopyranoside or glycopyranosylidene moiety in the case of spiro compounds. Chapter II focuses on spiro-bicyclic inhibitors such as glucopyranosilidene-spiro-1,4,2-oxathiazoles and glucopranosylidene-spiro-isoxazolines. Chapter III describes the synthesis of C- and N-glycosyl-heterocycles, mainly glycopyranosyl-1,2,3-triazoles. Finally, chapter IV studies the palladium-mediated cross coupling fonctionalization of 4-substituted-5-halogenated-1,2,3-triazoles that represents an unexpected but interesting development of the project. To conclude, chapter V gathers the evaluation of synthesized molecules as GP inhibitors. Enzymology and crystallography as well as in vitro and in vivo experiments are presented