Cette thèse a pour objectif l’identification de nouveaux inhibiteurs de kinase et plus particulièrement de kinases dépendantes de cyclines (CDKs). Des inhibiteurs de CDKs sont en essais cliniques depuis une dizaine d’année. Un faisceau d’informations récentes montre que cette nouvelle classe pharmacologique pourrait prochainement occuper une place prépondérante dans la thérapie antitumorale. L’introduction de cette thèse décrit les principaux inhibiteurs de CDKs en se focalisant sur ceux dont le développement clinique est en cours et sur les structures les plus récemment divulguées (2009 à 2013). Trois molécules avancées en études cliniques s’avèrent intéressantes et s’approchent de la mise sur le marché : la Roscovitine (phase clinique IIb), le Dinaciclib (phase clinique III) et le Palbociclib (phase clinique III). D’un point de vue expérimental, cette thèse se décompose en deux parties principales. La première modulation a consisté à rechercher des nouveaux groupements qui pourraient sur des squelettes déjà connus comme les purines apporter un avantage en ce qui concerne l’activité des produits. Les structures cristallines des complexes inhibiteur-enzymes et notamment celles de Roscovitine-CDK2 et CR8-CDK2 ont guidé la conception des nouvelles molécules. C’est ainsi que sur les structures biaryliques déjà connues, un groupement phénol a été greffé sur l’un des cycles conduisant ainsi à de nouveaux inhibiteurs de kinases. Ces molécules sont plus puissantes que les produits non hydroxylés. L’augmentation de l’activité est particulièrement sensible au niveau de la kinase CDK2 qui est impliquée notamment dans régulation du cycle cellulaire. La seconde partie du travail porte sur la recherche de structures isostères des purines. Ainsi, le squelette thieno[3,2-d]pyrimidines a été développé de novo. Deux types d’intermédiaires produits ont été préparés afin de permettre la diversification des structures. En premier temps la voie de synthèse via l’intermédiaire thiométhyle a été conduite, néanmois cette voie présente certaines limites. Le deuxième intermédiaire trihalogéné a permi d’optimiser la préparation des molécules thieno[3,2-d]pyrimidines. Les évaluations des produits préparés ne sont pas totalement terminées. Ces molécules sont moins puissantes que les purines sur les CDKs mais agissent au niveau d’autres kinases. / In the introduction, the main functions of cyclin dependent kinases are detailed. Whenever it was possible the link with pathologies where these kinases are overexpressed is presented. This is followed by the description of the inhibitors which are actually undergoing clinical testing. Most of these clinical studies are targeting cancer and leukemia. Impressive clinical results have been disclosed for Dinaciclib, Palbociclib and Roscovitine. The synthesis of two series of compounds is then envisioned. The first series of products are purine derivatives bearing a hydroxybiarylmethyl group on the 6 position of the purine scaffold.  Two approaches were compared in the synthesis of the hydroxylbiarylmethylamino group. In both approaches the key step was the orthoformylation of phenols using magnesium chloride as catalyst. The prepared compounds were evaluated against kinases and a tumor cell line. They were found more potent than homologous products without hydroxyls. The second families of products are thieno[3,2-d]pyrimidines. A new general route to these products based on the preparation of 7-bromo-2,4-dichloro-thieno[3,2-d]pyrimidine which can allow the synthesis of a large diversity of trisubstituted derivatives.

In Africa, use of medicinal plants for the treatment of diabetes is very common. However, efficacy on co-administering of medicinal plants with therapeutic drugs hasn't been fully determined, especially for African medicinal plants. The current study focused on assessing the in vitro modulation effects of three popular African medicinal plants, namely: Aloe ferox, Sutherlandia frutescens and Prunus africana (including five commercial preparations containing these medicinal plants) on two of the most important anti-diabetic drug metabolising enzymes, Cytochrome P450 (CYP450) 2C9 and CYP3A4 and a key drug efflux transporter, P-glycoprotein (P-gp). Vivid® microsome-based screening kits were used to assess inhibitory potency of plants preparations on CYP2C9 and CYP3A4 enzymes activities. The study showed that P. africana was a more potent inhibitor of CYP2C9 and CYP3A4 activity than the corresponding positive controls Ginkgo biloba and St. John's wort, which are known to cause clinically significant drug-herb interactions. S. frutescens leaf extract demonstrated potent to moderate inhibition on both the tested CYP activities, while its commercial products (Promune® and Probetix®) possessed moderate to mild inhibitory effects on the activities of both CYPs. Potent inhibitory effect on CYP2C9 and CYP3A4 was seen with Aloe Ferox®. Prosit® and Aloes powder® showed potent to moderate inhibition on CYP2C9 activity and moderate to mild inhibition on CYP3A4 activity. In addition to CYP450 activity, the present study also investigated the effects of the selected medicinal plant products on the activity of the main drug efflux protein, P-gp. A screening assay was specifically developed to assess the potential for herbal remedies to interact with P-gp mediated drug absorption. The assay is based on the principle of the reversal of drug resistance in modified Caco-2 cells specifically altered to express high iv efflux protein activity. These cells display a multidrug resistance phenotype and the addition of a plant extract containing a P-gp inhibitor or substrate will inhibit or compete with any cytotoxic drug and consequently reverse the drug resistance. The suitability of the assay was confirmed using a known P-gp inhibitor. The study observed that the anti-proliferation effect of vinblastine was significantly enhanced in vinblastine-resistant Caco-2 cells, which have high P-gp expression, when they were exposed to the selected African herbal preparations. This observation indicates that the studied plant preparations may alter P-gp functionality and therefore lead to interference with the absorption of co-administered drugs. The outcomes of this study provide useful information on whether there are any potential drug-herb interactions between the commonly used African medicinal plants and oral anti-diabetic drugs, at the level of CYP and P-gp drug metabolism and could contribute to better therapeutic management of Type II diabetics. However these predicted interactions will need to be verified in a clinical setting.

Les aptamères peptidiques sont des protéines combinatoires capables de moduler spécifiquement une fonction de leur cible. Une sélection fonctionelle d’aptamères peptidiques capables d’inhiber la mort cellulaire Bax-dependante chez la levure et en cellules mammaifères a été effectuée. Deux aptamères peptidiques ont été sélectionnés (Apta-32 et Apta-34). L’objectif de ce travail de thèse a été de caractériser ces deux aptamères peptidiques et leur(s) cible(s) dans le contexte de la mort cellulaire Bax-dependante. La première partie est l’étude de l’Apta-34 qui cible une protéine (C34) contenant un domaine de mort et ayant des fonctions pro-apoptotiques. Nous avons montré que lors de l’induction de l’apoptose, C34 est transloquée du noyau (sa localisation principale) au cytoplasme. Dans les mêmes conditions, Apta-34 co-localise avec C34 dans le noyau, empêchant, ou du moins retardant, sa sortie du noyau. De plus nous avons identifié le site de liaison d’Apta-34 sur C34, qui est localisé dans les 215 amino acides en N-terminale de la protéine, une région qui contient un site prédictif d’export nucléaire. Finalement, nous avons montré que la délétion de l’homologue de C34 protège contre la mort induite par hBax en levure. La seconde partie est l’étude d’Apta-32 qui cible deux paralogues (C32a et b) d’une famille de protéine impliquée dans le traffic membranaire dans les voies de l’endocytose. Nous avons montré qu’Apta-32 se lie à un domaine fonctionnel de C32. Des études in silico de docking ont permis d’identifier trois sites distincts de liaison d’Apta-32 sur ce domaine. Le site dominant est composé d’acides aminés qui partagent des propriétés physico-chimiques communes entre les différents interacteurs d’Apta-32 (C32a, C32b et l’homologue levure) mais pas avec des homologues qui ne lient pas Apta-32. De plus un screening double hybride d’une banque de cDNA levure a permis d’identifier des cibles mevure d’Apta-32. Finalement, des études préliminaires chez l’embryons de drosophile, permettent de suggérer que l’expression d’Apta-32 peut entraîner un défaut de la phagocytose. Cette étude a permis d’identifier des régulateurs de la mort cellulaires impliqués dans deux processus cellulaires distincts. / Peptide aptamers are small combinatorial proteins able to specifically modulate a function of their target. A functional selection of peptide aptamers able to inhibit Bax-dependent cell death in yeast and mammalian systems has been performed. Two peptide aptamers have been selected (Apta-32 and Apta-34). The aim of this thesis project was to characterize those two inhibitory peptide aptamers and their targets in order to understand their function in the Bax-dependent cell death. The first part focuses on Apta-34 that targets a Death Domain-containing protein (T34) that has pro-apoptotic functions. We showed that during the induction of apoptosis T34 translocates from nucleus (its major localization site) to the cytoplasm. In the same conditions, Apta-34 co-localizes with T34 in the nucleus, inhibiting or at least delaying its exit from the nucleus. Moreover we identified that Apta-34 binds to the well conserved 215 N-terminal amino acids of T34 that contains a putative Nuclear Export Signal. Finally we showed that the deletion of its homologue prevents hBax-induced cell death in yeast. The second part focuses on Apta-32 that targets two paralogues (T32a and b) of a family of proteins involved in the endocytotic membrane trafficking. We showed that Apta-32 is binding to a functional domain of T32. By in silico docking studies we identified 3 distinct binding sites of Apta-32 on this domain. The dominant binding site is composed by amino acid that share physico-chemical properties between binders of Apta-32 (T32a, T32b and a yeast homologue) but not with homologues that do not bind Apta-32. Moreover we identified yeast targets of Apta-32 by yeast two hybrid yeast cDNA library screening. Finally preliminary observations on drosophila embryos expressing Apta-32 suggest that Apta-32 expression could lead to a defect on phagocytosis. This study leads to the identification of regulators of the cell death acting on two distinct pathways.

Det beräknas att ca 36 % av Sveriges befolkning påverkas negativt av någon i deras närhet som antingen dricker för mycket alkohol eller brukar narkotika (Sundin & Landberg et al., 2018). De närmaste anhöriga, det vill säga familjen, är de som lider mest av deras anhörigas missbruk/beroende/substansbruk. Anhöriga till personer med substansbruk utsätts för mycket stress på grund av oro och negativa konsekvenser (Woititz, 1995). Det finns många olika stödgrupper och olika typer av behandlingar för personer med substansbruk och för dess anhöriga. CRAFT är en metod som socialstyrelsen rekommenderar i arbetet med anhöriga (Socialstyrelsen, 2019). Min egen upplevelse har varit att mitt eget sätt att arbeta, jag kallar det för behandlingsmodellen, med anhöriga har varit mer verksamt än CRAFT. Jag ställde mig därför frågande till vad skillnaden är mellan CRAFT och Behandlingsmodellen samt vilka komponenter (moment/inslag i grupp sessionerna) som kan anses som verksamma i behandling av anhöriga. För att få svar på detta utförde jag en kvalitativ undersökning av fyra olika anhöriggrupper/föräldragrupper. I två av anhöriggrupperna använde jag och min kollega CRAFT och i de andra två grupperna Behandlingsmodell. Gruppdeltagarna var föräldrar vars barn har eller har haft någon form av substansbruk.  Tidigare forskning gällande anhörigstöd samt affektteori och inlärningsteori har hjälpt mig att hitta mönster och förstärka resultatet av insamlat material. I jämförelse med CRAFT gav Behandlingsmodellen bättre resultat i samtliga undersökta områden. Exempelvis visade det sig att 86 % av de anhöriga i behandlingsmodell-grupperna upplevde stor påverkan i sitt psykiska mående i jämförelse med CRAFT-grupperna där 55 % av anhöriga upplevde stor påverkan av deras psykiska mående. I avseende hur deras ungdomar/barn har påverkas till det bättre i relation till missbruk/beroende upplevde 72 % av de anhöriga i behandlingsmodell-grupperna en stor påverkan medan 37 % av anhöriga i CRAFT-grupperna upplevde stor påverkan på deras barn i relation till missbruk/beroende. Deltagarna i de grupper där vi använde Behandlingsmodellen var också generellt mer nöjda med aspekter så som övriga gruppdeltagare, behandlingens innehåll och gruppledarna. Studien belyste betydelsen av faktorer så som gruppens betydelse, kunskap om medberoende, öva på kommunikationsfärdigheter, föräldrafärdigheter, aktivt lyssna, bygga upp en positiv relation och prioritera gemensamt umgänge med ungdomen. Dessa faktorer genomsyrade grupperna i båda behandlingsformerna medan det i Behandlingsmodellen fanns större utrymme för ytterligare faktorer så som vägledning i känslor, den anhörigas vardagliga upplevelser samt att låta individers behov styra behandlingsinnehåll. / It is estimated that about 36 % of Sweden's population is negatively affected by someone close to them who either drinks too much alcohol or uses narcotics (Sundin & Landberg et al., 2018). The closest relatives, the family, are those who suffers the most from their relative’s substance abuse. Relatives of people with substance abuse are exposed to a lot of stress due to anxiety and negative consequences that often arise (Woititz, 1995). There are many different support groups and different types of treatments for people with substance abuse and for their relatives. CRAFT is a method that the Swedish National Board of Health and Welfare recommends as treatment of relatives (Socialstyrelsen, 2019). Through my own experience I have found that my own way of working with relatives, I call it the Treatment Model, has been more effective than CRAFT. I therefore asked myself what is the difference between CRAFT and the Treatment Model and which components can be considered effective in the treatment of relatives. To get an answer to this, I have done a qualitative survey of four different Support Groups/Parent Groups. In two of the support groups, my colleague and I used CRAFT and in the other two groups, the Treatment Model. The group participants were parents whose children have or had some form of substance use. Previous research on support/treatment of relatives as well as theory’s such as the Affect Theory and Learning Theory has helped me find patterns and reinforce the results of collected material from this study. In comparison with CRAFT, the Treatment Model gave better results in all areas studied. For example, it turned out that 86 % of the relatives in the Treatment Groups experienced a great effect in their mental state in comparison with the CRAFT Groups where 55 % of the relatives experienced a great effect in their mental state. In regards to how their children have been affected for the better in relation to addiction/dependence, 72 % of the relatives in the Treatment Groups experienced a large impact while  37 % of relatives in the CRAFT Groups experienced a large impact on their children in relation to abuse/addiction. The participants in the groups where we used the Treatment Model were also generally more satisfied with aspects such as other group participants, the content of the treatment and the group leaders. The study highlighted the importance of factors such as the group's importance, knowledge of co-dependency, practicing communication skills, parenting skills, actively listening, building a positive relationship and prioritizing joint interaction with the youth. These factors permeated the groups in both forms of treatment, while in the Treatment Model there was more room for additional factors such as guidance in emotions, the relative's everyday experiences and to let individuals' needs control treatment content.

For many enzymes to function correctly they must have the freedom to display a level of dynamics or communication during their catalytic cycle. The effects that protein dynamics and communication can have are wide ranging, from changes in substrate specificity or product profiles, to speed of reaction or switching activity on or off. This project investigates the protein dynamics and communication in two separate systems, a non-ribosomal peptide synthetase (NRPS), and a protein arginine methyltransferase (PRMT). PRMT1, the enzyme responsible for 80% of arginine methylation in humans, has been implicated in a variety of disease states when functioning incorrectly. For this reason, much focus has been placed on better understanding how PRMT1 determines which products it creates and at what times. This project aims to shed light on how dynamics and communication within PRMT1 dictate its activity. We have to this point developed a protocol for creating and purifying a linked PRMT1 construct which will enable us to conduct the necessary experiments capable of answering our larger questions about the PRMT1 catalytic mechanism. Our collaborators in the Zhan lab discovered the presence of a methyltransferase (Mt) in the two NRPS systems they study, which produce two different and medically relevant compounds, bassianolide and beauvericin. The Hevel lab is well suited to study methyltransferases and so were asked to help evaluate the role of these Mt domains and how they affect the production of the relevant natural products. Achieving a more complete understanding of these systems will move us closer toward the “holy grail” of being able to manipulate and harness NRPS systems for the engineering of novel medically relevant compounds. This project has found that the Mt domain substrate specificity is affected by the surrounding protein domains, or even small portions of them.

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第20633号 / 工博第4371号 / 新制||工||1679(附属図書館) / 京都大学大学院工学研究科合成・生物化学専攻 / (主査)教授 森 泰生, 教授 浜地 格, 教授 跡見 晴幸 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

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The main objective of the thesis was to construct a wide-field Kerr microscope to study all-optical helicity-dependent (AOHDS) switching in FePt nanograins. The wide- field Kerr microscope was successfully implemented into AOHDS experiments, was fully characterized and optimized for maximum image contrast. The real-time imaging and resolution of 2, 5µm enables the study of a wide range of magnetic materials and their dynamics. Moreover, a new light source, the High Lumen Density MODULE from CRY- TUR, spol. s r.o., was tested for future application in Kerr microscopy. The technical solution enabled to form a collimated beam with low divergence required for Kerr mi- croscopy. From the switching experiments on FePt nanograins, we observed a strong non-magnetic contribution to the magnetic signal, not reported in previous works. The experiments have also shown that the switching intensity depends on the laser spot size and total laser power, suggesting that the FePt grains are not entirely isolated. The grains' ensemble exhibits a more complex behavior than anticipated. 1

Dietary N-3 PUFA plays a key role in brain maturation, development, stress response and cognitive abilities (Weiser et al., 2016; Devarshi et al., 2019). As adolescent’s prefrontal cortex is maturating, the period becomes sensitive to external factors such as environment, nutrition, and stress (Petrovich et al., 2001; Calabro et al., 2020). In this thesis, we aim to expand our knowledge of the influence of external factors, such as dietary omega-3 supplementation and enriched environment, during this critical maturation period. By designing four distinct studies, we tested the hypothesis that visible sex-specific alterations would arise from adolescence targeted diet n-3 PUFA supplementation and enriched environment, which would act to modify physiological and stress responses, as well as socio-emotional and cognitive performance. Our first study characterized the impact n-3 PUFA and n-6 PUFA regimen on corticosterone secretion and behavioural responses in adolescent male rodents. Additionally, it assessed the effects of delivery method (gavage versus restricted feeding) during this sensitive maturation period to ensure using a method with limited stress-mediated outcomes. This study highlighted gavage to induce reduced effects on corticosterone (CORT) secretion, regardless of the provided supplementation. On the last day of feeding, CORT secretion was diminished in fish oil (FO) fed rats exposed to restricted feeding, suggesting FO diet to promote physiological adjustments. Data also demonstrated that FO and soybean (CSO) rich diets were able to reduce anxiety-like behaviour compared to a high-fat diet intake (Hydrogenated Vegetal Fat - HVF), highlighting the role of n-3 PUFA dietary supplementation during adolescence on stress regulation. Our second study assessed sex-specific impact of adolescence targeted dietary supplementation on brain Docosahexaenoic Acid (DHA), Arachidonic Acid (AA) and Linolenic acid (LA) concentrations immediately following supplementation and during adulthood. Our findings demonstrated overall elevated DHA, AA and LA brain tissue concentrations in female compared to male rats, regardless of dietary supplementation. Benefit of supplementation were most apparent in adolescent males, where FO led to higher DHA concentrations compared to soybean oil supplementation, supporting a positive influence of FO dietary supplementation in males during intensive hormonal fluctuation and brain maturation. However, adolescent male rats showed reduced ability to extract nutrient essential fatty acids compared to female counterparts. Our third study characterized sex-specific coping strategies, socioemotional responses, and glucocorticoid regulation following an n-3 PUFA rich diet and enriched environment (EE) during the adolescent period. While basal CORT secretions were not significantly altered by supplementation in males, a gradual increase in CORT was observed during supplementation, peaking at DAY21. Passive coping strategies was preferred in the FST in RC (Regular Cage)- housed females exposed to FO while RC-housed CSO-fed males opted for an active climbing coping strategy. Increase locomotion and anxiolytic behaviour were observed in CSO-supplemented males (exposed to EE), while CSO by itself promoted social recognition in males. In contrast, sociability was improved in FO EE exposed females, indicating possible synergic effects. Adulthood hippocampal GR-ir expression was reduced at the hippocampal CA3 region in FO/RC and CSO/EE rat groups, which could have influenced memory consolidation and stress resilience. Overall, results from this study provided insights on positive effects associated with short-term adolescent n-3 PUFA supplementation in females, while male appeared to most benefited from soybean diet supplementation. Our fourth and last study assessed age- and sex-dependent influences of dietary supplementation on cognitive performance in the Barnes Maze Test. Our results showcase a gradual decrease in latencies to the escape box, as well as progressive decrease in working memory errors (WME) in adult compared to adolescent rats. Over the testing period, the FO females and CSO males showed improved performance through reduction of WMEs on specific days, which could subtend sex-related effects of dietary supplementations. However, while discrete effects of n-3 PUFA were more apparent in female rats, short-term supplementation appeared insufficient to promote consistent enhancement of visuospatial performance or cognitive flexibility that could be observed throughout the testing period. In conclusion, our findings support the importance of studying single and combined factors to understand overall impact. We were able to consistently demonstrate beneficial effects on coping strategies, stress reactivity, sociability, and cognitive performance of adolescence-targeted fish oil supplementation, especially in female rodents.

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