Thesis advisor: Jia Niu / Nucleic acid aptamers are promising alternatives to antibodies for a wide array of diagnostic and therapeutic applications. However, state-of-the-art aptamers suffer from poor pharmacokinetics and diversity, limiting their affinity and specificity for many therapeutically relevant targets. The emerging field of glycoscience provides opportunities to improve the utility of aptamers over antibodies. Combining synthetic chemistry with modern molecular biology and polymer science, the synthesis of Xeno Nucleic Acid monomers and chemoenzymatic polymerization via engineered polymerase enzymes allows the production of nucleic acid drugs with superior resistance to endogenous nucleases. The modular structure of nucleic acids provides for the design of sequence defined polymers capable of post-synthetically appending complex synthetic glycans, extending the catalytic geometry of aptamers. Our SELEX inspired FACS based particle display approach allows for high-throughput screening. Additionally, we expect this method has the capability of screening aptamers in human serum. Our synthetic approach utilizes a Sonogashira cross-coupling reaction to install a flexible alkyne to the major groove of 2′-deoxy-2′-fluoro-arabinose uracil base. By incorporated recent advances in nucleic acid synthesis, one-pot nucleobase activation and sugar glycosylation is achieved and bis-oxybenzyl phosphoamidite synthesis can afford gram scale HPLC-free purification of the triphosphates. The FANA C8-alkyl-uridine triphosphate will be incorporated by an engineered Tgo DNA polymerase to allow systematic introduction of alkynyl conjugation handles into a DNA-templated FANA polymer. Subsequent conjugation with azido-modified glycans via the Huisgen coppercatalyzed alkyne-azide cycloaddition (CuAAC) click reaction will generate sequence controlled nucleic acid-carbohydrate hybrid molecules amendable for directed evolution. / Thesis (MS) — Boston College, 2019. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

京都大学 / 新制・課程博士 / 博士(エネルギー科学) / 甲第23998号 / エネ博第434号 / 新制||エネ||82(附属図書館) / 京都大学大学院エネルギー科学研究科エネルギー基礎科学専攻 / (主査)教授 森井 孝, 教授 片平 正人, 教授 佐川 尚 / 学位規則第4条第1項該当 / Doctor of Energy Science / Kyoto University / DFAM

A crucial step in the drug discovery and development process is the assessment of membrane permeability properties of new chemical entities and researchers are constantly searching for cost-effective, high through-put models with as high as possible predictive value. In addition, a thorough understanding of the membrane permeability pathways and metabolism mechanisms are required when evaluating drug disposition and pharmacokinetics. Various in vitro methods/techniques are available to measure the rate of permeation of compounds across epithelial cell membranes to estimate oral drug absorption in humans. The aim of this study is to compare three in vitro models (i.e. excised rat intestinal tissue, excised pig intestinal tissue and Caco-2 human cell cultures) in terms of drug permeability characteristics by means of different techniques including the Ussing type Sweetana-Grass diffusion chamber apparatus, everted sac glass apparatus and the Transwell® plate apparatus. The transport of abacavir sulphate was determined in two directions (i.e. apical-to-basolateral or AP - BL and basolateral-to-apical or BL - AP) across excised rat intestinal tissue, excised pig intestinal tissue and Caco-2 cell monolayers. The test solution was applied to the donor side and samples (200 μl) were drawn from the acceptor side at 20 min intervals for a period of 2 h. The concentration of abacavir in the samples was then measured by means of a validated high performance liquid chromatography (HPLC) method. The transepithelial electrical resistance (TEER) was measured before and after each transport experiment to give an indication of the integrity of the cell membranes. The apparent permeability coefficient (Papp) and efflux ratio (ER) values were calculated and used to compare the different methods and techniques in terms of drug permeation characteristics. All three of the in vitro methods, in all of the techniques employed, showed higher transport of abacavir in the BL - AP direction than in the AP - BL direction. This indicates that all three in vitro methods had intact active efflux transporters over the entire study period. The excised rat intestinal method showed similar drug permeability characteristics in both techniques compared to that of the Caco-2 cell monolayers. In contrast, the excised pig intestinal method only showed similar drug permeability characteristics in the Sweetana-Grass diffusion apparatus when compared to the Caco-2 cell monolayers. This phenomenon can possibly be explained by the relatively large surface area of the pig tissue used in the everted sac technique where the role of physiological and other factors take effect. These factors may include the thickness of the membrane and mucus layer as well as variables such as diet, age, gender and size of the pigs obtained from the abattoir that cannot be controlled. / MSc (Pharmaceutics), North-West University, Potchefstroom Campus, 2015

A crucial step in the drug discovery and development process is the assessment of membrane permeability properties of new chemical entities and researchers are constantly searching for cost-effective, high through-put models with as high as possible predictive value. In addition, a thorough understanding of the membrane permeability pathways and metabolism mechanisms are required when evaluating drug disposition and pharmacokinetics. Various in vitro methods/techniques are available to measure the rate of permeation of compounds across epithelial cell membranes to estimate oral drug absorption in humans. The aim of this study is to compare three in vitro models (i.e. excised rat intestinal tissue, excised pig intestinal tissue and Caco-2 human cell cultures) in terms of drug permeability characteristics by means of different techniques including the Ussing type Sweetana-Grass diffusion chamber apparatus, everted sac glass apparatus and the Transwell® plate apparatus. The transport of abacavir sulphate was determined in two directions (i.e. apical-to-basolateral or AP - BL and basolateral-to-apical or BL - AP) across excised rat intestinal tissue, excised pig intestinal tissue and Caco-2 cell monolayers. The test solution was applied to the donor side and samples (200 μl) were drawn from the acceptor side at 20 min intervals for a period of 2 h. The concentration of abacavir in the samples was then measured by means of a validated high performance liquid chromatography (HPLC) method. The transepithelial electrical resistance (TEER) was measured before and after each transport experiment to give an indication of the integrity of the cell membranes. The apparent permeability coefficient (Papp) and efflux ratio (ER) values were calculated and used to compare the different methods and techniques in terms of drug permeation characteristics. All three of the in vitro methods, in all of the techniques employed, showed higher transport of abacavir in the BL - AP direction than in the AP - BL direction. This indicates that all three in vitro methods had intact active efflux transporters over the entire study period. The excised rat intestinal method showed similar drug permeability characteristics in both techniques compared to that of the Caco-2 cell monolayers. In contrast, the excised pig intestinal method only showed similar drug permeability characteristics in the Sweetana-Grass diffusion apparatus when compared to the Caco-2 cell monolayers. This phenomenon can possibly be explained by the relatively large surface area of the pig tissue used in the everted sac technique where the role of physiological and other factors take effect. These factors may include the thickness of the membrane and mucus layer as well as variables such as diet, age, gender and size of the pigs obtained from the abattoir that cannot be controlled. / MSc (Pharmaceutics), North-West University, Potchefstroom Campus, 2015

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

L’incidence du cancer du rein et sa mortalité associée se sont accrues au cours des dernières années. Le type de cancer rénal le plus fréquent est celui nommé Cancer Rénal à Cellules Claires (CRCC) où le plus souvent, le gène suppresseur de tumeur Von Hippel Lindau (VHL) est inactivé. Malgré une détection plus précoce, l’évolution de la pathologie demeure incertaine, en particulier quand les patients développent des métastases ou acquièrent une résistance au traitement (25-30% des patients). De nouvelles thérapies ciblant des kinases (Sunitinib, Sorafenib ou Temsirolimus) bien que très prometteuses conduisent très souvent à l’acquisition de résistance. Dans ce contexte, il est urgent de développer de nouveaux modèles prédictifs de la réponse des patients aux traitements et d'identifier de nouvelles cibles thérapeutiques.Ma thèse de science visait trois objectifs complémentaires : 1) Identifier par criblage chimio-génomique des kinases comme cibles thérapeutiques combinées. 2) Etablir deux modèles de culture 3D de cancer du rein qui intègrent le microenvironnement d’une tumeur: les sphéroïdes et la culture organotypique de coupe de tissus. 3) Etudier la chimio-sensibilité de ces modèles à une combinaison de molécules identifiées dans le criblage.Un criblage cellulaire a été réalisé sur la plateforme de Criblage de Molécules BioActives (CEA- Grenoble). Il a consisté à tester 80 molécules inhibitrices de protéine-kinases en combinaison avec l’extinction génique par interférence ARN (shRNAs lentiviraux) de 36 cibles potentielles connues pour leur implication dans divers cancers. La lignée cellulaire choisie (786-O) est dérivée d’une tumeur rénale à cellules claires radio et chimio-résistante et dépourvue de VHL. Parmi les touches qui compromettent la viabilité des cellules 786-O, la combinaison choisie pour son efficacité cible deux kinases importantes dans le contrôle de la survie cellulaire et de la réparation de l’ADN CK2 et ATM. Le statut VHL des cellules module de façon dramatique leur sensibilité à cette combinaison, l’association de ces deux inhibiteurs étant plus efficace sur les cellules 786-O (VHL -) que sur les mêmes cellules dans lesquelles VHL a été réintroduit (VHL+). Au sein d’une tumeur, les différents niveaux d’oxygénation constituent une variation environnementale supplémentaire créant des susceptibilités ou des résistances aux traitements thérapeutiques. Pour déterminer l’impact de nos molécules dans ce contexte, nous avons testé la viabilité des cellules 786-O VHL+ et VHL- dans des conditions normoxiques (21% O2) ou hypoxiques (1,5% O2), en présence des molécules seules ou en combinaison. En normoxie, une diminution synergique de la viabilité des cellules 786-O VHL- est observée en présence de la combinaison, alors que cet effet n’a pas lieu sur les cellules 786-O VHL+. Cette synergie est potentialisée en condition hypoxique. Au niveau mécanistique, les voies de signalisation de stress cellulaires sont d’avantage activées dans les cellules VHL- en présence de la combinaison de molécules comparé au traitement avec chacune des molécules seules. Dans les sphéroïdes tumoraux multicellulaires reproduisant l’organisation d’une micro-tumeur, nos résultats montrent que notre combinaison de molécules induit d’avantage l’apoptose des cellules VHL- que les molécules seules, alors que les cellules VHL+ ne sont sensibles à aucun des traitements.Ces résultats montrent que l’action de nos molécules combinées est clairement plus efficace dans un modèle 3-D. Ils démontrent également qu’il est possible d’objectiver une pharmaco-modulation de la viabilité de cultures organotypiques de tumeur du rein par des combinaisons d’inhibiteurs chimiques de protéine-kinases. Les perspectives de ce travail sont la validation de cette combinaison sur des tumeurs humaines et l’exploitation des cultures organotypiques comme test personnalisé de réponse aux traitements. / Renal cell carcinoma accounts for 3% of all malignant diseases in adults making it the 10th most common cancer in France. The most frequent type of Kidney cancer is Clear Cell Renal Cell Carcinoma (CCRCC). Almost all CCRCC show an inactivation of the Von Hippel Lindau tumour suppressor gene (VHL). Between 25-30% of the patients will develop metastatic renal cell carcinoma (mRCC) by the time they are diagnose or become unresponsive to all treatments and in these cases, the disease has a rapid progression. Over the past years, kinase-targeted therapies (Sunitinib, Sorafenib, Temsirolimus) have become the mainstay of treatment for mRCC, however, most, if not all, patients acquire resistance to these approaches over time.In this context my PhD had 3 goals: a) to find a new combinatory targeted therapy through a High Throughput Screening; b) to establish 3D models mimicking the real environment of the tumours (spheroids, Tissue Slice Culture); c) to validate the Hits through different molecular and cellular biology studies.We conducted a synthetic lethal screen on the CMBA platform (CEA-Grenoble), choosing 36 potential genes targets and 80 kinases inhibitors drugs. Each of the target gene was silenced by a transduction with shRNA Lentivirus into the 786-O cell line derived from ccRCC that lacks the tumour suppressor VHL, is radio- and chemo-therapy resistant, has increased mobility and is highly metastatic. Among the hit combinations that affect cell viability, one of them was chosen because it targets two important kinases involved in cell survival and DNA repair: CK2 and ATM. Moreover, this combination is specifically more active in the 786-O VHL- cells than in 786-O VHL+ cell line. We evaluated the effect of our drugs on the viability of our 786-O VHL+ and VHL- cells in normoxic (21% O2) or hypoxic (1.5% O2) conditions that reflect different environments that are present in a tumour. Surprisingly, in normoxia, we found a synergetic effect of the drug mix only on the 786-O VHL- cells but not on 786-O VHL+ cells. Furthermore, this effect was even stronger in conditions of Hypoxia (up to 20% of synergism).Mechanistically, an up-regulation of the stress pathways was much stronger in the VHL- cells in the presence of the combination than with the drugs alone. No apoptosis was detected in this 2D models. In Multi-Cellular Tumour Spheroid (MCTS) where the organization of a micro-tumour is reproduced, our drugs are even more effective in inducing cell apoptosis than in 2D monolayers of 786-O VHL- cells. These results also demonstrate that pharmaco-modulation of viability of renal tumour organotypic culture by chemical combination targeting protein kinases can be studied. Perspectives of this work are the validation of this drug combination on human renal tumours and the use of organotypic culture as a test for personalized treatment response.

Soil represents the largest global reservoir of microbial diversity for the discovery of novel genes and enzymes. Both stable-isotope probing (SIP) and metagenomics have been used to access uncultured microbial diversity, but few studies have combined these two methods for accessing the biotechnological potential of soil genetic diversity and fewer yet have employed functional metagenomics for recovering novel genes and enzymes for bioenergy or bioproduct applications. In this research, I demonstrate the power of combining functional metagenomics and SIP using multiple plant-derived carbon substrates and diverse soils for characterizing active soil bacterial communities and recovering glycosyl hydrolases based on gene expression. Three disparate Canadian soils (tundra, temperate rainforest and agricultural) were incubated with five native carbon (12C) or stable-isotope labelled (13C) carbohydrates (glucose, cellobiose, xylose, arabinose and cellulose). Sampling at defined time intervals (one, three and six weeks) was followed by DNA extraction and cesium chloride density gradient ultracentrifugation. Denaturing gradient gel electrophoresis (DGGE) of all gradient fractions confirmed the recovery of labeled nucleic acids. Sequencing of original soil samples and labeled DNA fractions demonstrated unique heavy DNA patterns associated with all soils and substrates. Indicator species analysis revealed many uncultured and unclassified bacterial taxa in the heavy DNA for all soils and substrates. Among characterized taxa, Salinibacterium (Actinobacteria), Devosia (Alphaproteobacteria), Telmatospirillum (Alphaproteobacteria), Phenylobacterium (Alphaproteobacteria) and Asticcacaulis (Alphaproteobacteria) were the bacterial ???indicator species??? for the heavy substrates and soils tested. Both Actinomycetales and Caulobacterales (genus Phenylobacterium) were associated with metabolism of cellulose. Members of the Alphaproteobacteria were associated with the metabolism of arabinose and members of the order Rhizobiales were strongly associated with the metabolism of xylose. Annotated metagenomic data suggested diverse glycosyl hydrolase gene representation within the pooled heavy DNA. By screening only 2876 inserts derived from the 13C-cellulose heavy DNA, stable-isotope probing and functional screens enabled the recovery of six clones with activity against carboxymethylcellulose and methylumbelliferone-based substrates.