Yes / Objectives: Resistance-associated variants (RAVs) in Rv0678, a regulator of the MmpS5-MmpL5 efflux pump, have been shown to lead to increased MICs of bedaquiline (2- to 8- fold) and clofazimine (2- to 4-fold). The prevalence of these Rv0678 RAVs in clinical isolates and their impact on treatment outcomes are important factors to take into account in bedaquiline treatment guidelines. Methods: Baseline isolates from two bedaquiline MDR-TB clinical trials were sequenced for Rv0678 RAVs and corresponding bedaquiline MICs were determined on 7H11 agar. Rv0678 RAVs were also investigated in non-MDRTB sequences of a population-based cohort. Results: Rv0678 RAVs were identified in 23/347 (6.3%) of MDR-TB baseline isolates. Surprisingly, bedaquiline MICs for these isolates were high (>0.24 mg/L, n¼8), normal (0.03 0.24 mg/L, n¼11) or low(<0.03 mg/L, n¼4). A variant at position 11 in the intergenic region mmpS5–Rv0678 was identified in 39 isolates (11.3%) and appeared to increase the susceptibility to bedaquiline. In non-MDR-TB isolates, the frequency of Rv0678 RAVs was lower (6/ 852 or 0.7%). Competition experiments suggested that rifampicin was not the drug selecting for Rv0678 RAVs. Conclusions: RAVs in Rv0678 occur more frequently in MDR-TB patients than previously anticipated, are not associated with prior use of bedaquiline or clofazimine, and in the majority of cases do not lead to bedaquiline MICs above the provisional breakpoint (0.24mg/L). Their origin remains unknown. Given the variety of RAVs in Rv0678 and their variable effects on the MIC, only phenotypic drug-susceptibility methods can currently be used to assess bedaquiline susceptibility. / This work was supported by Janssen Pharmaceutica. N. C. was supported by a Baekeland PhD scholarship from the Flemish Institute for Scientific Technology (IWT 130308, Belgium). C. J. M., L. R. and B. d. J. were supported by a European Research Council Starting Grant INTERRUPTB (311725).

Die vorliegende Arbeit beschäftigt sich mit den molekularen Prozessen der Lipidanreicherung in der Gallenflüssigkeit. Leberzellen (Hepatozyten) sind polare Zellen, die für die Sekretion der Gallenflüssigkeit verantwortlich sind. Die Anbindung an den Blutkreislauf besteht über die basolaterale Membran. Durch die gegenüberliegende, sogenannte apikale Membran werden zwischen benachbarten Leberzellen tubuläre Stukturen (bile canaliculi, BC) gebildet, in die die Gallenflüssigkeit abgesondert wird. Daher wird diese Membran auch als Canalicularmembran (CM) bezeichnet. Die Gallenflüssigkeit besitzt hinsichtlich ihrer Lipidzusammensetzung eine bemerkenswerte Spezifität. Obwohl der Anteil von Phosphatidylcholin (PC) an den Phospholipiden der CM nur 35% beträgt, macht es 95% der Phospholipide der Gallenflüssigkeit aus. Mögliche Mechanismen, die zur Spezifität der Lipidsekretion in die Gallenflüssigkeit führen, werden untersucht und diskutiert. Phospholipide werden aus der äußeren Lamelle der CM durch Gallensalze herausgelöst. Die Wechselwirkung von Gallensalzen mit Phospholipiden ist kopfgruppenunspezifisch. Eine Solubilisierung von Phosphatidylserin (PS) und Phosphatidylethanolamin (PE) durch Gallensalze könnte durch die Wirkung einer Aminophospholipidtranslokase (APLT) verhindert werden, die diese Lipide aktiv auf die zytoplasmatische Seite der Membran pumpt. Zur Überprüfung dieser Hypothese wurden Versuche durchgeführt, um die Aktivität einer APLT in der CM nachzuweisen. Dabei wurde die Hepatomazelllinie HepG2 eingesetzt, die in der Lage ist, Canalicularvakuolen (BC) zu bilden. Zunächst wurde die Einwärtsbewegung einer Reihe fluoreszierender Lipidanaloga mit unterschiedlicher Affinität zur APLT charakterisiert. Dies geschah an der basolateralen Membran von HepG2 Zellen, wo eine APLT-Aktivität bereits bekannt ist. Die Aufnahme geeigneter APLT-Substrate konnte durch den APLT-Inhibitor Suramin reduziert werden. Ebenso wurde die Affinität eines Paares von PS-Analoga bestätigt, von denen Diether PS ein "schlechtes" und Diacyl PS ein "gutes" APLT-Substrat darstellt. Im zweiten Schritt wurde die Anreicherung der gleichen Analoga in BC von HepG2 Zellen untersucht. Es ergab sich eine auffallende Korrelation zwischen einer APLT vermittelten Aufnahme von Phospholipidanaloga an der basolateralen Membran und dem Fehlen dieser Analoga im Lumen der BC. Wenn Zellen mit Phospholipiden markiert wurden, die keine oder nur "schlechte" APLT-Substrate darstellen, erschienen die BC stark fluoreszierend. Diese Beobachtungen zeigen, dass eine APLT-Aktivität in der CM von Hepatozyten vorhanden ist, welche das Fehlen der Aminophospholipide in der Gallenflüssigkeit erklärt. Ein zweiter Schwerpunkt dieser Arbeit war die Untersuchung der Rolle von MDR-Proteinen (wie MDR3) bei der Lipidsekretion in die Gallenflüssigkeit. Aufgrund bisheriger Arbeiten wird vermutet, dass MDR3 daran als spezifischer Membrantransporter für PC beteiligt ist. In der vorliegenden Arbeit konnte jedoch gezeigt werden, dass verschiedene MDR-Inhibitoren die Anreicherung fluoreszierender Phospholipidanaloga in den BC von HepG2 Zellen nur wenig reduzieren. Diese Beobachtung kann unter der Annahme erklärt werden, dass MDR3 eher für die Exposition von PC an der lumenalen Seite der CM verantwortlich ist, als für den Tranport von PC über die Membran. Solche "Liftase"-Aktivität von MDR3 könnte endogenes PC der Detergenzwirkung von Gallensalzen zugänglich machen, ein Prozess, der für die hydrophileren fluoreszierenden PC-Analoga nicht nötig ist. Im dritten Teil wird die Rolle von Sphingolipiden und die Bildung von "Rafts" in der CM behandelt. Solche Membrandomänen sollten die Solubilisierung von Spingolipiden in die Gallenflüssigkeit verhindern. Eine Anreicherung fluoreszierender Sphingolipidanaloga in den BC wurde jedoch nachgewiesen, was darauf hindeutet, dass die verwendeten Analoga das Verhalten endogener Sphingolipide in der CM nicht korrekt wiederspiegeln. Im abschließenden Teil dieser Arbeit wurden die Grundlagen für eine Methode zur Aufklärung der physikochemischen Prozesse der Lipidsekretion an der Canalicularmembran gelegt. Die starke Umgebungsabhängigkeit der Fluoreszenzlebensdauer für verschiedene fluoreszierende Lipidanaloga wurde in einer Reihe von Modellumgebungen analysiert und deren Nutzbarkeit für die Vorhersage der Lipidorganisation geprüft. Insbesondere wurde die Wechselwirkung verschiedener Gallensalze mit Lipidanaloga und der Fluoreszenzresonanzenergietransfer zwischen verschiedenen Lipidanaloga charakterisiert. Diese Daten sind Ausgangsbasis für die mikroskopische Charakterisierung der Organisation von Lipidanaloga in den BC in vivo. / This thesis addresses the molecular processes which are important in the formation of bile fluid. The polar liver cells (hepatocytes) secrete the bile fluid at their apical (canalicular) membrane into tubular bile canaliculi (BC) which are formed between adjacent cells. The basolateral membrane of hepatocytes faces the blood vessel. Bile fluid possesses a remarkable specificity regarding its lipid composition. Even though phosphatidylcholine (PC) contributes to only 35% of the phospholipids in the canalicular membrane, it constitutes 95% of biliary phospholipids. In this thesis possible mechanism that might lead to the specificity in biliary lipid secretion are analysed and discussed. Phospholipids are secreted from the outer leaflet of the canalicular membrane into bile by the effect of bile salts. The interaction of bile salts with phospholipids was shown to be independent of the phospholipid headgroup. Solubilisation of phosphatidylserine (PS) and phosphatidylethanolamine (PE) by bile salts could be prevented by the action of an aminophospholipid translocase (APLT) which actively pumps these lipids to the cytoplasmic leaflet of the membrane. Experiments to demonstrate a canalicular APLT activity were performed to proof this hypothesis. For this, the hepatoma cell line HepG2 which is able to polarise and to form a canalicular vacuole (BC) was utilised. A panel of fluorescent lipid analogues with different affinities to this transporter was used and first characterised at the basolateral membrane of HepG2 cells, where an APLT activity was already demonstrated. The rapid APLT mediated uptake of aminophospholipid analogues representing appropriate substrates of APLT was reduced by applying the inhibitor suramin. The affinity of a pair of PS analogues with diether NBD-PS as a poor APLT substrate and diacyl NBD-PS representing a suitable substrate was confirmed. In a next step the enrichment of the same phospholipid analogues in the BC was investigated. There was a striking correlation between APLT mediated uptake of phospholipid analogues at the basolateral membrane and absence of these analogues from the BC. In the case of phospholipid analogues that were no or poor substrates of APLT the BC appeared highly fluorescent, indicating that indeed a canalicular APLT is responsible and sufficient for biliary absence of aminophospholipids. Further experiments were aimed on the investigation of the role of MDR proteins (as MDR3) in biliary lipid secretion. It has been proposed that MDR3, which is crucial for biliary phospholipid secretion, acts as a specific flippase for PC. However, different MDR inhibitors did not completely abolish the enrichment of fluorescent phospholipid analogues in the BC in this study. This observation can be explained assuming that MDR3 is responsible for the exposure of PC at the lumenal side of the canalicular membrane rather than for its transport across the membrane. Such a "liftase" activity of MDR could make endogenous PC accessible to the detergent bile salts which is not necessary for its more hydrophilic fluorescent analogues. The third part of this thesis addressed the role of sphingolipids and the formation of detergent resistant rafts in the canalicular membrane. Rafts are thought to prevent sphingolipid solubilisation into bile. Fluorescent sphingolipid analogues were found to enrich in the BC even at low temperatures, however. These experiments suggest that the applied analogues might not suitably represent the majority of sphingolipids in the canalicular membrane. The final part of this study provides the basis for a method to investigate the physico-chemical processes occurring during lipid secretion at the canalicular membrane. The sensitivity of fluorescence life times on environmental changes was analysed using fluorescent lipid analogues in a set of model environments and its utility for predicting biliary lipid organisation is discussed. Especially the interaction of different bile salts with lipid analogues and fluorescence energy transfer between distinct lipid analogues was characterised. These data can be utilised for characterisation of the organisation of biliary enriched lipid analogues in vivo at a microscopic level in future.

As concepções de moduladores da dinâmica dos microtúbulos, que levam ao bloqueio do ciclo celular, e de bloqueadores de canais de cálcio tipo L (Cav), tais como o 1,4-di-hidropiridinas e análogos, que diminuem a resistência do organismo humano aos tratamentos quimioterápicos através da inibição da proteína de transmembrana P-gp, são estratégias importantes tanto para terapias antitumorais quanto para leishmanicida. Esta abordagem tem mostrado resultados interessantes na diminuição da resistência à quimioterapia em câncer chamada de MDR (do inglês Multi Drug Resistence), além de também serem uma estratégia importante para controlar a fase inicial da leishmaniose. Diante desse contexto, e baseado no estudo de Ueki 2013 e colaboradores que, a partir de estudos anteriores, os quais relatam a superexpressão das enzimas estona deacetilase (HDAC) e catepsina L (CTSL) em células tumorais, propuseram um pró-fármaco seletivo, planejado a partir de um espaçador de lisina acetilada, que garante a liberação do fármaco seletivamente nas células tumorais, trabalhamos no desenvolvimento de uma nova proposta de pró-fármaco trifuncional. Nossa proposta foi desenvolvida a partir de estudos de triagem virtual, baseados em ligantes e em estrutura, predição das propriedades farmacocinéticas e toxicológicas (ADME/Tox) e também técnicas de bioinformática para a construção de um modelo de canal de cálcio, devido à inexistência de estruturas, do mesmo, que estivessem depositadas no banco de dados de proteínas PDB (Protein Data Bank). Paralelamente, nosso grupo de síntese colaborador sintetizou, através de técnicas de \"Click Chemistry\" e reações de Mitsunobu multicomponentes, uma biblioteca de novos híbridos trifuncionais, os quais, após estudos de atividade biológica, foram avaliados (in silico) frente à estrutura da tubulina, e os compostos mais promissores desta biblioteca serviram de base para novos estudos de triagem virtual. Para a obtenção dos nossos hits, executamos 4 estratégias de triagem virtual, separadas em 2 tarefas. Ao final, selecionarmos um total 59 hits, dos quais, 9 hits apresentam promissoras atividades bloqueadoras do canal de cálcio e 65 hits apresentam promissoras atividades moduladoras da tubulina. Estes hits seguem em estágio de compra e ensaios in vitro e após comprovada a eficácia dos mesmos, estes futuramente farão parte de uma nova proposta de pró-farmaco trifuncional. / The concepts of modulating microtubule dynamics, and calcium channel L-types (CAV) blockers are important strategies for anticancer and antileishmanial therapies. Microtubule modulators that blocks the cell cycle and the calcium channel blockers, such as, 1,4-dihydropyridines and analogues, reduce the resistance of the human body to chemotherapeutic treatments by inhibiting transmembrane P-gp protein. This approach has shown interesting results in reduced resistance to chemotherapy in cancer called MDR (Multi Drug Resistance), and an important strategy for controlling the early stage of leishmaniasis. In this context, we work to develop a new proposal for trifunctional prodrug. We have based on the study of Ueki 2013 and collaborators, which, from earlier studies with reported overexpression of both deacetylase estona enzymes (HDACs) and cathepsin L (CTSL) in tumor cells, proposed a selective prodrug. We considering an acetylated lysine link/spacer, which ensures the release of the drug selectively in tumor cells, have now designed this selective prodrug. Our proposal was developed from virtual screening studies, based on ligands and structure, prediction of pharmacokinetic and toxicological properties (ADME / Tox) and also bioinformatics techniques for the construction of a calcium channel model, due to the inexistence of Structures of the same that were deposited in the database of proteins PDB (Protein Data Bank). At the same time, our collaborating synthesis group synthesized, through Click Chemistry techniques and Mitsunobu multicomponent reactions, a library of new trifunctional hybrids, which, after studies of biological activity, were evaluated (in silico) against the structure of tubulin , And the most promising compounds from this library served as the basis for further virtual screening studies. To obtain our hits, we performed 4 virtual screening strategies, separated into 2 tasks. In the end, we selected 59 hits, of which 9 hits show promising calcium channel blocking activities and 65 hits show promising tubulin modulating activities. These hits follow in vitro purchase and testing, and after proven effectiveness, they will be part of a new tri prodrug proposal.

D’après un rapport de l’OMS, la tuberculose reste en 2015 l’une des 10 premières causes de décès à l’échelle mondiale. De ce fait, en matière de santé, éradiquer la maladie à l’horizon 2030 est un des objectifs majeurs fixés par les Nations Unies. La bactérie responsable de cette infection, Mycobacterium tuberculosis, est un pathogène obligatoire dont l’origine et l’évolution sont intrinsèquement liées à celles de son hôte principal, Homo sapiens. En effet, les souches actuelles de tuberculose présentent, tout comme l’homme, une forte structure phylogénétique, trace de leur origine géographique. Les pays pauvres et en développement sont les plus touchés par l’épidémie globale, favorisée par des systèmes de santé défaillants et une haute prévalence du VIH. Les pays occidentaux ne sont pas épargnés, menacés par l’émergence de souches de plus en plus résistantes aux antibiotiques provenant en grande partie de l’ex URSS. Au cours de cette thèse, j’analyse l’histoire évolutive, la propagation et l’acquisition de résistances aux antibiotiques de plusieurs épidémies de tuberculose en me basant sur des données génétiques et génomiques. Dans un premier temps je m’intéresse aux effets d’une campagne nationale de traitements en Asie Centrale sur le développement de souches multi-résistantes et met également en lumière le rôle clef de certaines mutations dans le succès des clones présentés. Ainsi cette campagne a été partiellement mise en échec par la présence de souches pré-résistantes, grâce à la survenue de mutations avant même la mise en place des traitements antibiotiques. Par la suite je me suis focalisé sur un clade particulier de souches multi-résistantes, le clone Russe W148. Je présente sa dispersion géographique et temporelle à travers l’Eurasie et démontre l’importance des mutations compensatoires dans son succès épidémique. De plus, la tuberculose ne touche pas seulement les hommes mais infecte également plusieurs autres mammifères. Afin d’appréhender les contraintes adaptatives accompagnants ces changements d’hôtes, j’ai effectué divers tests de sélection dans le but d’identifier les gènes impliqués. Pour finir, nous avons développé un indice souche spécifique, permettant de mesurer le succès épidémique de celles-ci à un niveau individuel. Dans le cadre d’études épidémiologiques, cette mesure peut être croisée avec des informations sur le patient, la souche ou même socio-économiques. / According to a 2015 WHO report, tuberculosis remains one of the top 10 causes of death worldwide. Despite considerable efforts by the United Nations to eradicate the disease by 2030, a global TB epidemic still persists. Its causative agent, the bacterium Mycobacterium tuberculosis, an obligate pathogen, has been plaguing humanity since it originated, and has coevolved with its main host, Homo sapiens, over thousands of years. Contemporary tuberculosis strains exhibit a structured phylogeographic pattern, carrying the genetic print of their geographic origin. The Koch bacillus infects and kills in large numbers, in poor and developing countries, where fragile health care systems, combined with high HIV prevalence, facilitate epidemic spread. In western countries, the major current threats are the multiplication and propagation of antibiotic resistant strains (MDR/XDR) coming predominantly from former Soviet republics. In this thesis, I unravel the evolutionary history, propagation, and acquisition of drug resistance-conferring mutations in different settings, by implementing multiple genetic and genomic data sets. First, focusing on Central Asia, using whole genome sequencing and Bayesian statistics, I assess the effects of a treatment campaign on the development of MDR strains and highlight key mutations in successful strains. More importantly, the success of DOTs campaigns was compromised by the genetic make-up of these outbreak clades (pre-treatment low frequency resistance SNPs). Special attention was also given to a particular outbreak of MDR strains, i.e. the Russian W148 clone. I present its westward spatial and temporal propagation at a continental scale during the last century, and underline the key contribution of compensatory mutations in its epidemic success. However, tuberculosis does not only infect humans, but also has experienced successive mammalian host jumps. To decipher the adaptive constraints accompanying such secondary events, a systemic gene screen with selection signature-detecting algorithms was implemented to identify putative targets during diversifying selection. Finally, novel mathematical tools and indices that reflect the epidemicity of a strain were developed, jumping from a population-driven approach to a strain specific one, with broader epidemiological applications. This allows us to correlate strain fitness with patient, lineage, and socio-economic information.

As concepções de moduladores da dinâmica dos microtúbulos, que levam ao bloqueio do ciclo celular, e de bloqueadores de canais de cálcio tipo L (Cav), tais como o 1,4-di-hidropiridinas e análogos, que diminuem a resistência do organismo humano aos tratamentos quimioterápicos através da inibição da proteína de transmembrana P-gp, são estratégias importantes tanto para terapias antitumorais quanto para leishmanicida. Esta abordagem tem mostrado resultados interessantes na diminuição da resistência à quimioterapia em câncer chamada de MDR (do inglês Multi Drug Resistence), além de também serem uma estratégia importante para controlar a fase inicial da leishmaniose. Diante desse contexto, e baseado no estudo de Ueki 2013 e colaboradores que, a partir de estudos anteriores, os quais relatam a superexpressão das enzimas estona deacetilase (HDAC) e catepsina L (CTSL) em células tumorais, propuseram um pró-fármaco seletivo, planejado a partir de um espaçador de lisina acetilada, que garante a liberação do fármaco seletivamente nas células tumorais, trabalhamos no desenvolvimento de uma nova proposta de pró-fármaco trifuncional. Nossa proposta foi desenvolvida a partir de estudos de triagem virtual, baseados em ligantes e em estrutura, predição das propriedades farmacocinéticas e toxicológicas (ADME/Tox) e também técnicas de bioinformática para a construção de um modelo de canal de cálcio, devido à inexistência de estruturas, do mesmo, que estivessem depositadas no banco de dados de proteínas PDB (Protein Data Bank). Paralelamente, nosso grupo de síntese colaborador sintetizou, através de técnicas de \"Click Chemistry\" e reações de Mitsunobu multicomponentes, uma biblioteca de novos híbridos trifuncionais, os quais, após estudos de atividade biológica, foram avaliados (in silico) frente à estrutura da tubulina, e os compostos mais promissores desta biblioteca serviram de base para novos estudos de triagem virtual. Para a obtenção dos nossos hits, executamos 4 estratégias de triagem virtual, separadas em 2 tarefas. Ao final, selecionarmos um total 59 hits, dos quais, 9 hits apresentam promissoras atividades bloqueadoras do canal de cálcio e 65 hits apresentam promissoras atividades moduladoras da tubulina. Estes hits seguem em estágio de compra e ensaios in vitro e após comprovada a eficácia dos mesmos, estes futuramente farão parte de uma nova proposta de pró-farmaco trifuncional. / The concepts of modulating microtubule dynamics, and calcium channel L-types (CAV) blockers are important strategies for anticancer and antileishmanial therapies. Microtubule modulators that blocks the cell cycle and the calcium channel blockers, such as, 1,4-dihydropyridines and analogues, reduce the resistance of the human body to chemotherapeutic treatments by inhibiting transmembrane P-gp protein. This approach has shown interesting results in reduced resistance to chemotherapy in cancer called MDR (Multi Drug Resistance), and an important strategy for controlling the early stage of leishmaniasis. In this context, we work to develop a new proposal for trifunctional prodrug. We have based on the study of Ueki 2013 and collaborators, which, from earlier studies with reported overexpression of both deacetylase estona enzymes (HDACs) and cathepsin L (CTSL) in tumor cells, proposed a selective prodrug. We considering an acetylated lysine link/spacer, which ensures the release of the drug selectively in tumor cells, have now designed this selective prodrug. Our proposal was developed from virtual screening studies, based on ligands and structure, prediction of pharmacokinetic and toxicological properties (ADME / Tox) and also bioinformatics techniques for the construction of a calcium channel model, due to the inexistence of Structures of the same that were deposited in the database of proteins PDB (Protein Data Bank). At the same time, our collaborating synthesis group synthesized, through Click Chemistry techniques and Mitsunobu multicomponent reactions, a library of new trifunctional hybrids, which, after studies of biological activity, were evaluated (in silico) against the structure of tubulin , And the most promising compounds from this library served as the basis for further virtual screening studies. To obtain our hits, we performed 4 virtual screening strategies, separated into 2 tasks. In the end, we selected 59 hits, of which 9 hits show promising calcium channel blocking activities and 65 hits show promising tubulin modulating activities. These hits follow in vitro purchase and testing, and after proven effectiveness, they will be part of a new tri prodrug proposal.

Tuberculosis has for decades been the leading cause of death, worldwide, originating from merely one infectious matter i.e. the bacilli Mycobacterium tuberculosis (MTB). It is killing approximately 1.5 million people every year. The World Health Organization (WHO) estimates that 2 billion people might be infected by this specific bacterium, and that there emerges about 9 million new, active cases of TB each year. The aim with this thesis is to elucidate the possibilities and the difficulties with two new drugs against susceptible and resistant, latent and replicating strains of M. tuberculosis. A search for relevant articles on Pub Med, through the university library, was undertaken and approximately 35 articles were found. These articles were read; facts were sorted out and used for this paper. Figures were found through Creative Commons. The two new drugs discussed in this paper are TMC 207 and PA-824. They have two different mechanisms of action, and additionally and most importantly, that differ from the drugs used as first-line and second-line options. TMC 207 targets the mycobacterial ATP synthase. PA-824, a pro-drug, targets the cell wall synthesis and in addition causes respiratory poisoning, through the release of nitric oxide. The different mechanisms are vital in order to combat emerging resistance, due to various missense mutations in specific genes in the bacilli. The bactericidal activity of the drugs against mycobacteria is high, promising a successful cure for MDR (multi drug resistant)/XDR (extensively drug resistant) patients. The median survival is 4.1 years for MDR patients and 2.9 years for XDR patients, with currently available treatments. This is noticeably worse than some cancer prognoses. Perhaps with these drugs there is even a possibility of shortening the treatment time. The adverse events are mild to moderate, for both drugs. This is another additive advantage, since the toxicity of the drugs will be minimal and hopefully tolerable, and most importantly will bring about treatment commitment and the sought elimination of the disease. However, as with all new drugs the outcomes are still to be proven in real settings, the complex fields (in low- and middle income countries, with difficult TB cases), and with complicated cases (e.g. HIV co-morbidity) during a longer period of time. Reporting and evaluating the outcomes is crucial, since whatever is displayed from the drug consumption and the effects of it can lead to alterations in regimens, doses and treatment commitment.

ABCG2 ou BCRP est une protéine membranaire de la famille des transporteurs ABC. Elle utilise l’énergie de l’hydrolyse de l’ATP pour exporter des composés endogènes et exogènes hors des cellules. Elle participe ainsi à la protection et la détoxication de l’organisme. En revanche, dans le cas des cellules cancéreuses, elle est surexprimée et participe donc au phénotype de résistance à de multiples médicaments (MDR : Multi Drug Resistance). En effet, lors de la surexpression de cette protéine, les agents anti-cancéreux sont exportés hors des cellules tumorales, ce qui diminue leurs concentrations intracellulaires sous leurs seuils de cytotoxicité et les rend inefficaces. De par l’importance d’ABCG2 dans la chimiorésistance, de nombreux efforts sont effectués pour concevoir des inhibiteurs afin de restaurer la sensibilité des cellules cancéreuses. Dans ce contexte, le projet de thèse vise à caractériser ABCG2 sur les plans structural et fonctionnel afin de comprendre son mécanisme d’action. La protéine ABCG2 exprimée chez E. coli, a été purifiée, sous forme stable et homogène et le rendement est de 1,5 mg de protéine par litre de culture. La caractérisation fonctionnelle de celle-ci témoigne de son repliement correct. En effet, il a été démontré qu’elle est capable de fixer différents substrats (naturels et agents anti-cancéreux) avec des affinités différentes. Des essais préliminaires de cristallisation ainsi que des observations par microscopie électronique révèlent des résultats encourageants pour la suite de la caractérisation structurale / ABCG2 or BCRP is a membrane protein that belongs to the ABC transporter family. It uses the hydrolysis energy of ATP to export endogenous and exogenous compounds out of cells. It is thus involved in the protection and detoxification of the body. However, it is overexpressed by cancer cells and participates in the multidrug resistance phenotype (MDR); in fact, anti-cancer agents are exported out of the tumor cells, which reduce their concentration below their cytotoxicity threshold and renders them ineffective. Because of its importance in chemoresistance, many efforts are made to design inhibitors to restore the sensitivity of cancer cells. In this context, the PhD project aims to characterize ABCG2 at structural and functional levels in order to understand its mechanism of action. We have succeeded in purifying ABCG2 expressed in E. coli, the protein obtained is stable and homogeneous, with a yield of 1.5 mg of protein per liter of culture. The functional characterization of ABCG2 demonstrates its correct folding. In fact, we have demonstrated that it is able to bind different substrates (natural and anti-cancer agents) with different affinities. Preliminary crystallization assays and electron microscopy observations reveal encouraging results for subsequent structural characterization

Europeiska unionen röstade igenom nya regelverk för medicintekniska produkter och för medicintekniska produkter för in vitrodiagnostik 2017. Regelverken implementerades 2021. De nya regelverken innebär striktare krav på tillsyn, klinisk prövning och spårbarhet, för att stärka patientsäkerheten. Målet med projektet var att undersöka om vårdcentralerna inom Stockholm läns sjukvårdsområde (SLSO) uppfyller de nya regelverken och sedan ta fram en analysmodell och analysera hur väl de anpassar sig till regelverken. För att säkerställa att SLSO:s vårdcentralerna uppfyller de nya regelverken utfördes i detta projekt fysiska inventeringar på tio slumpmässigt utvalda enheter som inte har befintliga serviceavtal med leverantörer eller servicegivare. Det visade sig att på samtliga enheter nyttjas konsumentprodukter till patienter, som inte är avsedda för medicinskt bruk. Flertalet enheter saknade även produkter som bör finnas tillgängliga. Överlag var enhetscheferna på vårdcentralerna inte insatta i de nya regelverken och visste inte att produkterna de använde inte är avsedda för sjukvård. De hade även flera produkter som var gamla och/eller trasiga som användes på daglig basis vilket inte uppfyller kraven på patientsäkerheten. Slutsatsen blev därför att SLSO:s vårdcentraler inte uppfyller de nya regelverken. / The European Union voted through new regulations regarding medical devices and medical devices for in vitro diagnostics in 2017. These new regulations were implemented in 2021. The new regulations entailed stricter supervision, clinical testing, and traceability requirements to strengthen patient safety. To ensure that care centers within Region Stockholm comply with the new regulations, physical inventories were carried out at ten randomly selected units that do not have existing service agreements with suppliers or service providers. To compare different units’ individual adaptations to the regulations, an analysis model was created in which the proportion of products that were not approved for medical use and the lack of certain necessary equipment were factors. In all units, consumer products are used for patients, which are not intended for medical use, and several units lack products that should be available. Overall, the unit managers at the care centers were unfamiliar with the new regulations and were unaware that the products they used were not intended for healthcare. The care centers also had several old and/or broken products that were used daily, which does not meet the requirements for patient safety. The conclusion was therefore that the care centers in Region Stockholm do not comply with the new regulations.

This study investigated how a QMS that is compliant with the Medical Device Regulation (MDR) can be constructed and what the most important aspects are when creating a functional QMS at a Swedish hospital. QMS:s are a fundamental part of a healthcare organization and are used to ensure safe and secure patient care. To establish qualitative healthcare, the Swedish National Board of Health and Welfare has created regulations for hospitals to construct their QMS concerning care processes and handling of medical devices. In addition to these, there are also regulations within the European Union, including the MDR. To facilitate QMS:s work, hospitals can also follow international standards created for quality work. The most common for medical device departments is ISO 13485. Regulations are often perceived as difficult to follow and can be hard to interpret. Swedish regulations mention that a hospital should have a “suitable” QMS based on the organization’s areas of activity. But what is considered appropriate is up to the organization to decide on, which to some extent has been difficult for Swedish hospitals. Furthermore, several studies depict challenges with hospital QMS implementation and getting the QMS to work functionally. A qualitative method has been used to answer the research question where the main source of data gathering has been semi-structured interviews. The interviews have been conducted with quality managers and with experts in regulations and standards for hospitals. The questions were mainly focused on how QMS:s that follow regulations should be constructed and how to effectively implement them in a hospital organization. The study concludes that it basically is impossible to construct a general QMS suitable for every hospital without having to adjust it to the hospital's different and specific work areas, which can look very different from hospital to hospital. Regardless, since ISO 13485 is harmonized with the QMS part of the MDR, it can be helpful for a medical technology department. The authors constructed a gap analysis tool to facilitate the development and implementation process for hospital QMS:s. When it comes to aspects that constitute a successful QMS implementation, the study found the most important aspects being culture, value creation, simplicity, and leadership. / Denna studie har undersökt hur ett kvalitetsledningssystem (KLS) som uppfyller kraven från Medical Device Regulation (MDR) ska konstrueras och implementeras i svenska sjukhus. KLS är en väsentlig del för att säkerhetsställa en säker och trygg patientvård. För att säkerhetsställa en kvalitativ vård har Socialstyrelsen upprättat föreskrifter för sjukhus att basera sina KLS på samt gällande hantering av medicintekniska produkter. Utöver dessa föreskrifter finns också regelverk gemensamma för Europeiska unionen bland vilket bland andra inkluderar MDR. För att underlätta arbetet med kvalitetsledningssystemet utnyttjar sjukhus i hög grad också standarder för sitt kvalitetsledningssystem. Den vanligaste är ISO 13485 för en medicinteknisk avdelning. Föreskrifterna som svenska sjukhus ska efterfölja kan ibland anses svårtolkade. Svenska föreskrifter nämner bland annat att svenska sjukhus ska ha ett “lämpligt” KLS utefter organisationens verksamhetsområden. Vad som anses lämpligt är upp till organisationen att definiera, vilket till viss del varit en svårighet för svenska sjukhus. Speciellt gällande medicintekniska produkter och hur dessa bör och ska hanteras. Hanteringen av medicintekniska produkter är en viktig faktor när KLS ska upprättas, vilket i vissa fall missats i svenska sjukhus. Utöver detta påpekar flera litterära studier på svårigheterna med implementeringen av KLS på sjukhus samt hur man får dessa att fungera optimalt i organisationen. För att besvara frågeställningen har en kvalitativ metod använts där främsta datainsamlingsmetoden varit semi-strukturerade intervjuer. Intervjuerna har genomförts med kvalitetsansvariga, samt med personer kunniga inom föreskrifter och standarder. Frågorna rörde i huvudsak hur ett föreskrifts efterföljande KLS ska konstrueras samt hur detta på ett effektivt sätt ska implementeras i organisationen. Studien kommer fram till att det i princip inte går att konstruera ett generellt KLS utan att detta måste anpassat till sjukhusets specifika arbetsområde vilket också kan skilja sig markant mellan olika avdelningar på sjukhuset. Oavsett så kan ISO 13485, som är harmoniserad mot KLS avsnittet i MDR, vara till hjälp för en medicinteknisk avdelning. Författarna konstruerade ett “gap-analysis tool” för att underlätta en första gapanalys som i studien visat sig vara ett första steg när KLS ska konstrueras. Gällande implementationen finner studien att viktigaste faktorerna är kultur, värdeskapande och enkelhet, samt ledarskap.

Au cours de chimiothérapies, les cellules cancéreuses parviennent fréquemment à échapper aux effets toxiques des médicaments en développant des mécanismes de chimiorésistance qui résultent souvent de la présence d’un système d’efflux de ces médicaments. Cette chimiorésistance est corrélée à un phénomène appelé « phénotype MDR » pour (MultiDrug Resistance) et associé à la surexpression d’ATPases membranaires appartenant aux transporteurs ABC (ATP Binding Cassette). Le transporteur ABCG2 fait partie de cette grande famille de protéines. Un alignement de séquence a permis l’identification chez ABCG2 une séquence spécifique (LSGGE) très semblable à la séquence signature (VSGGE) de tous les transporteurs ABC. La mutation ponctuelle des résidus de cette séquence en alanine a produit une perte importante de fonction des mutants L352A et S353A, observée au niveau du transport et de l’activité ATPasique. Des relations structure-activité établies à partir de différents composés de la famille des flavonoïdes ont permis d’identifier MBLI 97, boéravinone G, MHT et ABI comme des composés puissants et spécifiques, capables d’abolir la résistance à de multiples drogues et chimiosensibiliser la croissance cellulaire. Le ciblage de séquences spécifiques et l'utilisation d'inhibiteurs spécifiques de ces transporteurs constituent des stratégies destinées à contrer la chimiorésistance et augmenter l’efficacité des traitements chimiothérapeutiques. / During chemotherapy, cancer cells frequently succeed to escape the toxic effects of drugs by developing mechanisms of chemoresistance which often result from the presence of an efflux system of these drugs. Such a chemoresistance is correlated to the MDR (MultiDrug Resistance) phenotype and associated to overexpression of membrane ATPases belonging to the ABC (ATP-Binding Cassette) transporters. The ABCG2 transporter belongs to this large family of proteins. Sequence alignment allowed the identification of a specific (LSGGE) sequence in ABCG2, which is quite similar to the canonical sequence signature (VSGGE) of all ABC transporters. Point mutation of these residues into alanine produced a loss of function in L352A and S353A mutants, as observed in transport and on ATPase activity. Structure-activity relationships drawn from some compounds among the family of flavonoids allowed the identification of MBLI 97, boeravinone G, MHT and ABI as potent and ABCG2-specific inhibitors, able to revert multidrug resistance and chemosensitize cell growth. The study of specific sequences and use of specific inhibitors of these transporters constitute strategies to abolish cancer cell chemoresistance and to increase the efficiency of chemotherapeutic treatments.