Les bicycles imidazo-imidazoles constituent une classe de composés hétérocycliques intéressants tant sur le plan chimique que pharmaceutique. Ils jouent un rôle très important dans la synthèse et la fonctionnalisation des composés à visé thérapeutique. Dans le cadre de la recherche de nouveaux candidats inhibiteurs de kinases, nous avons développé une voie de synthèse des imidazo[1,2-a]imidazoles mono- et bifonctionnalisés. Par la suite, nous avons mis au point une stratégie de synthèse rapide et efficace de bicycles imidazo[1,5-a]imidazolin-2-one et imidazo[1,5-a]imidazole. En outre, nous avons développé deux stratégies de fonctionnalisation via des réactions de couplage pallado-catalysées. Finalement nous avons synthétisé le motif imidazo[1,5-a]imidazole via la réaction de Groebke-Blackburn-Bienaymé (GBB). La potentialité de cette réaction a été exploitée dans des réactions decyclisation intramoléculaire! afin de préparer une nouvelle chimiothèque de composés polyhétérocycliques azotés. / The imidazo-imidazoles bicycles have received special attention among other nitrogen cycles due to their biologically interesting properties exploited in the medicine manufacturing. The imidazo-imidazole scaffold is one of the most representative nitrogen containing heterocycle, as it plays a significant role and possesses a major interest in drug synthesis and functionalization. In this work we report firstly a synthetic pathway to novel imidazo[1,2-a]imidazoles candidates for CKD inhibitors. Secondly we develop two strategies to prepareimidazo[1,5-a]imidazoles and their reactivity via pallado-catalyzed reactions. Finally, we disclose a fast and an efficient access to imidazo[1,5-a]imidazoles by using the Groebke-Blackburn-Bienaymé reaction (GBB), followed by a palladium catalysed intramolecular cyclization, affording thus new tetracyclic products with an elevated degree of molecular diversity.

La découverte récente du premier gène de résistance plasmidique à la colistine mcr-1 a mis en évidence le besoin urgent d'améliorer la détection des isolats résistant à la colistine dans les laboratoires de microbiologie clinique afin de pouvoir rapidement isoler les patients porteurs. L’objectif de ma thèse a ainsi été de répondre à cette problématique par le développement et l’évaluation d’outils spécifiques. Une revue a été rédigée visant à résumer les nouveaux outils développés pour détecter la résistance aux polymyxines, y compris la présentation des méthodes actuelles phénotypiques, antibiogrammes et génotypiques. Le milieu de culture LBJMR a été développé pour détecter toutes les bactéries à Gram négatif résistantes à la colistine et également les souches d’entérocoques résistants à la vancomycine qui représentent un autre problème majeur de santé publique. LBJMR est polyvalent et a permis de dépister 3 bactéries d'intérêt clinique à partir d’un même échantillon: E. coli et K. pneumoniae résistantes à la colistine, et E. faecium résistant à la vancomycine. Un brevet a été déposé. Le kit UMIC Colistine est un dispositif commercial de détermination de la CMI basé sur la méthode de référence. Évalué conformément à la norme ISO 20776, sa sensibilité était excellente pour la détection des souches porteuses du gène mcr-1. Le kit UMIC Colistine constitue ainsi une méthode rapide et fiable pour évaluer la CMI des isolats cliniques dans les laboratoires de microbiologie clinique. Enfin, mes travaux de thèse ont compris l’analyse d’une souche de K. pneumoniae hétérorésistante à la colistine et la description d’une nouvelle espèce bactérienne : Pseudomonas massiliensis. / The recent discovery of mcr-1, the first plasmid-mediated colistin resistance gene, highlighted the urgent need to implement the detection of colistin-resistant isolates in clinical microbiology laboratories, in order to isolate carrier patients. My thesis aimed to address this issue by developing and evaluating specific tools. A review was redacted to summarize the new tools developed to detect polymyxin resistance including the current phenotyping, susceptibility testing and genotyping methods. The LBJMR culture medium has been developed to detect all colistin-resistant Gram-negative bacteria and also the vancomycin-resistant enterococci which represent another major problem of public health. The LBJMR medium allowed the detection of different bacteria of interest from the same clinical sample: colistin-resistant E. coli and K. pneumoniae and also a vancomycin-resistant E. faecium. This project led to a patent filing. The UMIC Colistine kit is a ready-to-use device based on the reference method to determine the polymyxin MIC. Evaluated in accordance with ISO 20776 standard, its sensitivity was excellent for the detection of colistin-resistant strains harboring the mcr-1 gene. The UMIC Colistin system is a rapid and reliable method to determine colistin MIC of clinical isolates in clinical microbiology laboratories. My thesis project also included the analysis of a colistin-heteroresistant Klebsiella pneumoniae strain, and the description of a new bacteril species, Pseudomonas massiliensis. These studies highlight the need to improve the detection of colistin-resistant strains by using reliable methods, suitable for diagnosis in clinical microbiology laboratories.

Made available in DSpace on 2015-05-14T13:21:39Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 5619556 bytes, checksum: f89abc7c11080124052c031ad8d11131 (MD5) Previous issue date: 2014-08-01 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / With the increasing use of manufactured products such as pharmaceuticals, cosmetics and toiletries, a new category of water bodies pollutants, called emerging contaminants, is gaining prominence in the environmental issues and preservation scenario of the planet's fresh water. Among these are endocrine disruptors, such as estrogens that has concerned the scientific community that has been warning for need to establish maximum concentration levels of these types of substances in the drinking water. Parallel to the legislation s rises to control these pollutants is necessary the development of rapid analytical methodologies, robust and with low waste generation. Almost all the analytical methods for quantification of estrogens in water samples make use of liquid or gas chromatography coupled to mass spectra associated to long chromatographic runs. In this work a fast method is presented for simultaneous quantification of four estrogens (E1, E2, E3 and EE2) in surface water samples using HPLC-DAD and multiway calibration with the purpose to exploit the potentialities of the second order advantage to circumvent the problems of sample complexities. Second order calibration models based on PARAFAC, MCR-ALS, N and U-PLS/RBL methods were constructed using individual pattern of analytes (pure standard) solutions. These models were then validated using a set of synthetic mixtures of these analytes with two potential interferences (daizein and biochanin A) to simulate the organic load and to demonstrate second order advantage. Satisfying statistical validation parameters were obtained, especially for U and N-PLS/RBL models. Finally river and sewage samples fortified with four estrogens were subjected to a pre-concentration and sample clean up using SPE-C18 cartridge followed by chromatographic analysis. The application of the calibration model to the fortified waters' samples led to, most cases, a recovery between 70 and 120%, reflecting the good accuracy of the proposed method / Com o uso cada vez maior de produtos industrializados, como os farmacêuticos, os cosméticos e os de higiene pessoal, uma nova categoria de poluentes dos corpos aquáticos, chamados de contaminantes emergentes, vem ganhando destaque no cenário das questões ambientais e preservação da água potável do planeta. Entre estes estão os alteradores endócrinos, como é o caso dos estrógenos que têm preocupado a comunidade científica que já vem alertando para necessidade de se estabelecer limite máximo de concentração dessas substâncias nas águas de abastecimento. Paralelamente ao surgimento de legislação para controle desses poluentes é necessário que ocorra o desenvolvimento de metodologias analíticas rápidas, robustas e com baixa geração de resíduos. Em quase sua totalidade os métodos analíticos para quantificação de estrógenos em amostras de águas fazem de uso da cromatografia líquida ou gasosas acopladas a espectros de massa, associada a longas corridas cromatográficas. Neste trabalho é proposta uma metodologia rápida para quantificação simultânea de quatro estrógenos (E1, E2, EE2 e E3) em amostras de águas superficiais empregando HPLC-DAD e calibração multivias com propósito de explorar as potencialidades da vantagem de segunda ordem para contornar os problemas da complexidade da matriz. Modelos de calibração de segunda ordem baseados nos métodos PARAFAC, MCR-ALS, N e U-PLS/RBL foram construídos usando soluções padrão individual dos analitos (padrões puros). Estes modelos foram depois validados empregando-se um conjunto de misturas sintéticas destes analitos com mais dois potenciais interferentes (a daizeina e biochanina A) para simular a carga orgânica e demonstrar a vantagem de segunda ordem. Estas misturas sintéticas foram construídas usando planejamento Taguchi. Parâmetros estatísticos de validação satisfatórios foram obtidos, em especial para os modelos U e N-PLS/RBL. Por fim amostras de rio e esgoto fortificadas com os quatro estrógenos foram submetidas a uma pré-concentração e limpeza da amostra usando cartuchos SPE-C18, seguido da análise cromatográfica. A aplicação dos modelos de calibração as amostras de águas fortificadas levou para a maioria dos casos a uma recuperação entre 70 e 120%, refletindo a boa exatidão do método proposto

Bactérias multirresistentes (MRs) pertencentes ao grupo ESKAPE (i.e., Enterococcus faecium resistente à vancomicina, VRE; Staphylococcus aureus resistente à meticilina, MRSA; Klebsiella spp., e Escherichia coli produtoras de β-lactamases de amplo espectro; Acinetobacter baumannii, Pseudomonas aeruginosa e Enterobacter spp. resistentes aos carbapenêmicos) são importantes patógenos de infecções relacionadas à assistência à saúde (IRAS), onde a sua endemicidade e prevalência tem sido decorrente da seleção de linhagens clonais. Embora, o fenótipo MR decorra da expressão de mecanismos mediados por genes intrínsecos e/ou adquiridos, o crescimento bacteriano na forma de biofilme contribui para um importante fenômeno fisiológico de resistência, o qual é inespecífico quanto ao substrato antimicrobiano. O presente estudo teve como objetivo avaliar a eficácia de agentes físicos e químicos contra biofilmes produzidos por clones de bactérias MRs de importância clínica e epidemiológica no Brasil. Cerdas de poliamida foram utilizadas como modelo de superfície de adesão para o crescimento de biofilmes, os quais foram monitorados por microscopia eletrônica de varredura (MEV). In vivo, o modelo de biofilme foi avaliado pela inserção das cerdas na proleg de larvas de Galleria mellonella, enquanto que, diferentes tratamentos foram aplicados para inibir a formação do biofilme. Adicionalmente, mediante ao ensaio de bioluminescência, o modelo de biofilme produzido pela cepa de P. aeruginosa PAO1/lecA::lux foi avaliado na presença de soluções hipertônicas de cloreto de sódio (NaCl). In vitro, soluções hipertônicas de cloreto de sódio (> 6%) utilizadas de maneira profilática, apresentaram efeito bacteriostático (CIM90= 1,7 M) contra biofilmes produzidos por todos os isolados analisados. Além disso, através do uso profilático de soluções hipertônicas de NaCl, foi possível visualizar a inibição da motilidade dos isolados. Por outro lado, os compostos quaternários de amônio (CQAs) cloreto de benzalcônio (CBA) e cloreto de cetilpiridínio (CCP) apresentaram efeito bactericida (CBM90= 256 µg/mL) contra biofilmes previamente formados em 24h. A atividade de ambos os CQAs foi potencializada na presença de soluções salinas hipertônicas, como avaliado pela metodologia de checkerboard, tendo um efeito sinérgico contra E. coli (ST10, ST101) MCR-1 (∑FIC= 0,5); parcialmente sinérgico contra A. baumannii OXA-23 (ST79), E. cloacae CTX-M-8 (ST131), E. faecium VRE (ST478) e K. pneumoniae KPC-2 (ST340) (∑FIC= 0,75); e indiferente contra cepas de P. aeruginosa SPM-1 (ST277) e S. aureus MRSA (ST5). Adicionalmente, a CIM de carbapenêmicos, fluoroquinolonas e aminoglicosídeos contra biofilmes de bactérias Gram-negativas MRs foi potencializada na presença de solução salina hipertônica resultando em uma queda da CIM >=2. Finalmente, in vivo, para todas as espécies MRs estudadas, biofilmes formados em 08, 12 e 24h resultaram em 100% de morte das larvas de G. mellonella em até 96 horas pós-infecção. O mesmo comportamento foi observado para a cepa PAO1/lecA::lux, sendo possível detectar sinais intensos de bioluminescência nas larvas infectadas com os biofilmes. Entretanto, para os biofilmes previamente tratados com solução salina hipertônica, observou-se a diminuição dos sinais de bioluminescência em até 60%. Já para biofilmes formados em 24, 12 e 08h, o tratamento prévio em solução salina hipertônica e posteriormente com antibióticos resultou em um aumento de até 40, 70 e 80% da sobrevida de G. mellonella, respectivamente. / ESKAPE pathogens (ie, vancomycin-resistant (VRE) Enterococcus faecium; methicillin-resistant (MRSA) Staphylococcus aureus; extended spectrum β-lactamase-producing Klebsiella spp., and Escherichia coli; Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp. Resistant to carbapenems), represents an important group of multidrug-resistant (MDR) bacteria related to healthcare-associated infections (HAIs), whereas endemicity have been associated with selection and predominance of clones. Although the MR phenotype derives from the expression of mechanisms mediated by intrinsic and/or acquired genes, bacterial growth in the biofilm form contributes to an important physiological phenomenon of resistance, which is non-specific to the antimicrobial substrate. The present study aimed to evaluate the efficacy of physical and chemical agents against biofilms produced by clones of MDR bacteria of clinical and epidemiological importance, in Brazil. Polyamide bristles were used as adhesion surface model for the growth of biofilms, which were monitored by scanning electron microscopy (SEM). In vivo, the biofilm model was evaluated by the insertion of the bristles into the proleg of larvae of Galleria mellonella, while different treatments and physicochemical conditions were applied to inhibit biofilm formation. Additionally, the biofilm model produced by the P. aeruginosa PAO1/lecA::lux strain was evaluated in the presence of hypertonic solutions of sodium chloride (NaCl). In vitro, hypertonic solutions of sodium chloride presented a bacteriostatic effect (MIC90 = 1.7 M) against biofilm formation of all the isolates analyzed. Moreover, through the prophylactic use of hypertonic solutions of NaCl, it was possible to observe the inhibition of the motility of the isolates. On the other hand, the ammonium quaternary compounds (QACs) benzalkonium chloride (BAC) and cetylpyridinium chloride (CPC) had a bactericidal effect (CBM90 = 256 µg / mL) against previously formed biofilms in 24h. The activity of both QACs was potentiated in the presence of hypertonic saline solutions, as evaluated by the checkerboard methodology, having a synergistic effect against E. coli (ST10, ST101) MCR-1 (∑FIC = 0.5); (ST340) (∑FIC = 0.75), E. faecium VRE (ST478) and K. pneumoniae KPC-2 (ST340), E. cloacae CTX-M-8 (ST131); and indifferent effect against strains of P. aeruginosa SPM-1 (ST277) and S. aureus MRSA (ST5). Furthermore, the MIC of carbapenems, fluoroquinolones and aminoglycosides against biofilms of MDR Gram-negative bacteria was potentiated in the presence of hypertonic saline solution resulting in a decrease in MIC >=2-fold. Finally, for all MDR species studied, biofilms formed at 08, 12 and 24h resulted in 100% death of G. mellonella larvae within 96h post-infection. In fact, the same behavior was observed for the strain PAO1/lecA::lux, and it is possible to detect intense bioluminescence signals in the larvae infected with biofilms. However, for biofilms previously treated with hypertonic saline solution, bioluminescence signs decreased by up to 60%. As for biofilms formed at 24, 12 and 8h, pretreatment in hypertonic saline solution and later with antibiotics resulted in an increase of up to 40, 70 and 80% of the survival of G. mellonella, respectively.

INTRODUÇÃO: O aumento da incidência de infecções causadas por bactérias resistentes a múltiplos antimicrobianos limita cada vez mais as opções terapêuticas, dificultando o tratamento e aumentando os índices de morbidade e mortalidade, além dos gastos em saúde. Ao longo dos últimos cinco anos, essa limitação tem levado ao reestabelecimento do uso de antimicrobianos consideradas ultrapassados, como as polimixinas. Este grupo passou a ser utilizado com cada vez mais frequência no tratamento de infecções causadas por microrganismos gram-negativos resistentes aos carbapenêmicos. As enterobactérias, em particular a espécie Klebsiella pneumoniae, tem apresentado frequentemente esse perfil, porém, a resistência à polimixinas têm sido relatada, eliminando essa importante alternativa terapêutica. Apesar da importância do tema, são escassas as publicações sobre frequência de resistência às polimixinas em K. pneumoniae e a relação clonal entre isolados resistentes à polimixina B no Brasil. OBJETIVOS: Avaliar a relação genética, perfil de sensibilidade antimicrobiana e mecanismos de resistência às polimixinas em K. pneumoniae. MATERIAIS E MÉTODOS: A execução deste trabalho dividiu-se em duas partes principais: (i) levantamento de dados de culturas positivas para K. pneumoniae da rotina de pacientes hospitalizados em instituições atendidas pelo serviço de análises clínicas do Fleury Medicina e Saúde; (ii) confirmação das concentrações inibitórias mínimas (CIM) para polimixina B, avaliação da relação clonal por eletroforese em campos pulsados (PFGE),e sequenciamento de múltiplos loci (MLST), avaliação da integridade do gene mgrB e da presença do gene mcr-1 por PCR entre isolados resistentes à polimixina B e aos carbapenêmicos (CPRKp). RESULTADOS e CONCLUSÕES: Na análise de 3.085 isolados de K. pneumoniae obtidos de pacientes internados em 11 hospitais da Grande São Paulo entre os anos de 2011 e 2015, foi evidenciado um aumento estatisticamente significativo na resistência aos carbapenêmicos de 6,8% em 2011 para 35,5% em 2015. Em 2015, KPC foi detectada em 96,2% dos isolados resistentes aos carbapenêmicos. A distribuição das concentrações inibitórias mínimas de polimixina B entre todos os isolados de K. pneumoniae evidenciou uma distribuição bimodal com a CIM de 2 mg/L como o valor de ponto de corte para a susceptibilidade à polimixina B; assim, 3,6% do número total de isolados sensíveis aos carbapenêmicos foram interpretados como resistentes enquanto essa proporção foi de 22,5% entre as resistentes aos carbapenêmicos (CRKp). Entre esses últimos isolados também houve um aumento estatisticamente significativo na tendência anual de resistência à polimixina B, de 0% em 2011 para 27,1% em 2015. Estas taxas variaram de 0,7% em 2011 para 3,9% até junho de 2014 entre os sensíveis aos carbapenêmicos. Entre os antimicrobianos alternativos, a amicacina e a tigeciclina foram os compostos mais ativos. A análise por PFGE de 60 isolados de CPRKp obtidos de pacientes distintos nos anos de 2014 e 2015 evidenciou dois grandes grupos clonais: CPRKp1 e CPRKp2, os quais segundo a análise por MLST pertencem, respectivamente, aos grupos ST11 e ST437, ambos do complexo clonal 258. Foi observado o mesmo grupo ST entre isolados obtidos dentro de um mesmo hospital e também entre diferentes hospitais, públicos e privados. O mecanismo de resistência mais comum entre os isolados de CPRKp foi a presença de sequências de inserção interrompendo o gene mgrB. O gene mcr-1 não foi detectado em nenhum dos isolados. / INTRODUCTION: The increasing incidence of infections caused by bacteria resistant to multiple antimicrobials increasingly limits therapeutic options, making treatment difficult and increasing the morbidity and mortality and health spending. Over the past five years, this limitation has led to the reestablishment of the use of antimicrobials deemed outdated, such as polymyxins. This group is now used with increasing frequency to treat infections caused by carbapenem-resistant gram-negative microorganisms. Enterobacteria, especially Klebsiella pneumoniae, have often presented this profile, however, resistance to polymyxins have been also reported, eliminating this important therapeutic alternative. Despite the importance of this issue, the publications are scarce on the polymyxins resistance frequency in K. pneumoniae and clonal relationship among isolates resistant to polymyxin B in Brazil. OBJECTIVES: To evaluate the genetic relationship, antimicrobial susceptibility profile and polymyxin B resistance mechanisms in K. pneumoniae. MATERIALS AND METHODS: The execution of this work was divided into two main parts: (i) survey data on routine cultures positive for K. pneumoniae from patients hospitalized in institutions attended by the clinical analysis service of Fleury Health and Medicine; (ii) confirmation of to polymyxin B minimum inhibitory concentrations (MIC), evaluation of clonal relationship by electrophoresis pulsed field gel electrophoresis (PFGE), multilocus sequence typing (MLST), evaluation of the integrity of the mgrB gene and the presence of mcr-1 gene by PCR among isolates resistant to polymyxin B and carbapenems (CPRKp). RESULTS AND CONCLUSIONS: The analysis of 3,085 K. pneumoniae isolates obtained from inpatients from 11 hospitals in the São Paulo urban area between 2011 and 2015, has shown a statistically significant increase in carbapenem resistance from 6.8% in 2011 to 35.5% in 2015. In 2015, KPC was detected in 96.2% of isolates resistant to carbapenems. The polymyxin B MIC distribution of all Klebsiella pneumoniae showed a bimodal distribution with the MIC of 2 mg/L as the cutoff value for polymyxin B susceptibility; thus, 3.6% of the total number of isolates susceptible to carbapenems were interpreted as resistant while this proportion was 22.5% among carbapenem-resistant isolates (CRKp). Among these isolates there was also a statistically significant increase in the annual trend of polymyxin B resistance, from 0% in 2011 to 27.1% in 2015. These rates ranged from 0.7% in 2011 to 3.9% by June 2014 between carbapenem-susceptible isolates. Among alternative antimicrobials, amikacin and tigecycline were the most active compounds. The analysis by PFGE of 60 CPRKp isolates obtained from different patients in the years 2014 and 2015 showed two major clonal groups: CPRKp1 and CPRKp2, which according to the analysis by MLST belong respectively to ST11 and ST437 groups, both from clonal complex 258. We observed the same ST group of isolates obtained within a hospital and between different public and private hospitals. The most common mechanism of polymyxin B resistance among CPRKp isolates was the presence of insertion sequences interrupting the mgrB gene. The mcr-1 gene was not detected in any of the isolates.

La colistine est un vieil antibiotique, utilisé à la fois en médecine humaine et vétérinaire. Cependant, l'arsenal antibiotique étant de plus en plus limité, la colistine apparait comme un des derniers remparts dans la lutte contre les bactéries multi-résistantes chez l'Homme. Afin de préserver l'efficacité de la colistine, deux problématiques ont été abordées dans cette thèse : (i) les risques de sélection de résistance à la colistine en lien avec la découverte fin 2015 d'un gène porté par un plasmide (mcr-1). Ainsi, l'impact de l'usage de colistine par voie orale en production porcine a été évalué in vivo et une absence de sélection a été observée dans nos conditions expérimentales. De façon similaire, l'usage (minoritaire) de colistine en médecine humaine comme prophylaxie de décontamination digestive sélective (SDD) a été étudié chez des rats hébergeant un microbiote intestinal humain. Les résultats préliminaires ne montrent pas non plus d'effet de sélection. (ii) le développement d'un modèle pharmacocinétique basé sur la physiologie (PBPK) chez le porc pour l'usage par voie systémique de la colistine et de sa prodrogue, le colistine méthanesulfonate (CMS). Ce modèle a permis d'explorer la distribution tissulaire du CMS et de la colistine, notamment au niveau rénal où la toxicité est la plus fréquente. Comme application de ce modèle, l'estimation des temps d'attente avant abattage lors d'usage de CMS chez le porc a été effectué. Enfin, la capacité des modèles PBPK à réaliser des extrapolations intra et inter-espèces a été utilisé pour adapter ce modèle chez l'adulte et l'enfant, afin de pouvoir prédire les concentrations plasmatiques de colistine lors d'un traitement. / Colistin is an old antibiotic used in human and veterinary medicine. However, as less and less antibiotics are discovered, colistin is considered as a last-line antibiotic to fight against multi-drug resistant bacteria in human. In order to preserve the efficacy of colistin, two issues were investigated in this thesis:(i) Risks of selection of bacteria resistant to colistin, in conjunction with the discovery by the end of 2015 of a plasmid-mediated resistance gene (mcr-1). Thus, the impact of oral use of colistin in pigs was assessed in vivo and no selection was observed in our experimental conditions. Similarly, the use of colistin in human medicine for selective digestive decontamination was studied thanks to human flora‐associated rats. Preliminary results were also neither in favour of a selective effect of colistin.(ii) development of a physiologically-based pharmacokinetic model (PBPK) in pigs for the systemic use of colistin and its prodrug, the colistimethate sodium (CMS). This model provided a further insight into CMS and colistin tissue distribution, especially in kidneys where toxic effects are frequent. As a model application, the withdrawal period after use of CMS in pigs was estimated. Then, we used the ability of PBPK models to carry out intra and inter-species extrapolations in order to adapt this model in adults and children and eventually predict the plasmatic concentrations of colistin during a treatment with CMS.

Code review is a recurring activity at software companies where the source code, orparts of it, undergoes an inspection where the aim is to detect possible errors beforethe code is released for production. A variation of code review that is common today iscalled modern code review and is more lightweight practise than formal code review. Inmodern code review, the developers participate and continuously revise their colleagues’code.At a financial technology company in Stockholm, modern code review is applied. Thecompany has expressed a need to implement a tool that can facilitate the code reviewprocess. One suggestion from the company was to implement high-level conceptual codereview (HCCR), an idea of a tool where code changes are sorted automatically intodifferent commits with a specific message.In order to implement the tool, HCCR needs to be defined and concretised since it haspreviously existed solely as an idea. As a first step of the project, developers’ view ofwhat information is desirable in a commit needed to be examined. The project addressedthe following research questions: What information is desirable and needed by the developers of a medium-sizedcompany, to help them do code reviews in a pull-based environment?– What should the information consist of?– How should the information be presented?To answer these questions, interviews were conducted with software developers at thecompany, together with observations where the developers had to try out a first iterationof HCCR. The first iteration was developed using the company’s guidelines on howdevelopers contribute to code changes together with our company supervisor’s viewson how the tool can work. The interviews were recorded and transcribed, whereafteriia thematic analysis was applied. From the analysis, 13 concepts emerged, which weredivided into five categories. The developers wanted the commits to be atomic, compilableand testable in order to facilitate debugging. The developers also expressed a need toget clear information about both pull-requests (PRs) and commit messages. In theinterviews, a theme emerged that the messages should consist of: what has changed andwhy it has changed. Differences were also observed in the code review process as differentdevelopers use different strategies when reviewing code.Based on the information that emerged from the interviews and observations along withprevious research, a second iteration of HCCR was prepared. The report concludes bydiscussing possible implementations of the tool. / Kodgranskning är en vanligt förekommande aktivitet hos mjukvaruföretag där källkoden,eller delar av den, genomgår en granskning för att upptäcka möjliga fel innan kodensläpps till produktion. En variation av kodgranskning som är vanlig idag kallas modernkodgranskning och är en mindre formell kodgranskning där utvecklarna själva är medoch kontinuerligt reviderar sina kollegors kod.Ett finansiellt teknikbolag i Stockholm tillämpar modern kodgranskning. Företagethar uttryckt ett behov av att implementera ett verktyg som kan underlättakodgranskningsprocessen. Ett förslag från företaget var att implementera HCCR, enidé om ett verktyg där kodändringar automatiskt sorteras till olika, så kallade, commits1med ett specifikt meddelande.För att implementera verktyget behöver HCCR definieras och konkretiseras. Som ettförsta steg i projektet behövde vi undersöka utvecklarnas önskvärda information av huren commit bör utformas. Projektet behandlar följande forskningsfrågor: Vilken information är önskvärd och behövs av utvecklarna på ett medelstort företag,för att hjälpa dem att göra kodgranskningar i en pull-based miljö?– Vad ska informationen bestå av?– Hur skall informationen presenteras?För att svara på frågorna gjordes intervjuer med mjukvaruutvecklarna på företagettillsammans med observationer där utvecklarna fick prova på en första iteration avHCCR. Den första iterationen togs fram genom att använda företagets riktlinjer gällandehur utvecklare bidrar med kodändringar tillsammans med åsikter från handledaren påföretaget om hur verktyget kan fungera. Intervjuerna spelades in och transkriberades1När det kommer till ord och uttryck inom Git, (exempelvis commits, pull-request, push, pull) finnsdet ingen standardiserad översättning till svenska. Därför kommer dessa ord skrivas på engelska isammanfattningen.ivvarpå en tematisk analys genomfördes. Från analysen framträdde 13 koncept kringkodgranskning vilka delades in i fem kategorier. Utvecklarna önskade att varje commitskulle vara atomisk, kompilerbar samt testbar för att underlätta felsökning av buggar.Utvecklarna uttryckte också ett behov av att få tydlig information om både PR ochcommit-meddelanden. I intervjuerna framkom det att meddelandena borde bestå av:vad som har ändrats och varför det har ändrats. Det observerades även skillnader ikodgranskningsprocessen då olika utvecklare använder olika strategier när de granskarkod.Baserat på den information som framträdde från intervjuerna och observationernatillsammans med tidigare forskning utarbetades en andra iteration av HCCR. Rapportenavslutas med att diskutera möjliga implementationer av verktyget.

Le durcissement des normes d'émission, tout autant que l'impératif d'économie,poussent à étudier de nouveaux modes de combustion pour les moteurs. L'autoallumage decharges homogènes à basse température offre de bonnes perspectives quant au rejet de NOx,suies, et CO2. Cependant son control reste délicat, car il est extrêmement sensible à latempérature et la cinétique de l'hydrocarbure. L'assistance par plasma hors-équilibre pourraitfournir une solution. Les expériences sont menées dans une MCR avec des mélanges pauvresd'isooctane/air et un prototype d'allumeur Renault. La combustion obtenue identifiée commeSICI se déroule en deux phases: la propagation d'une flamme comprime les gaz restantjusqu'à leur autoallumage. Le réchauffement du système expérimental est intégré dans leprotocole d'exploitation, afin de quantifier l'effet SICI relativement à l'autoallumage pur.L'effet du plasma semble avant tout dépendre de l'énergie déposée, bien qu'il convergerapidement, quel que soit l'avance du déclenchement. Le comportement asymptotique à hauteénergie s'explique par la thermalisation des filaments, soulignée par comparaison avec l'effetSICI d'un arc classique. A l'inverse, le seuil minimal d'énergie nécessaire semble lié à lacapacité à générer un noyau de flamme viable, rapprochant le phénomène d'un problèmeclassique d'allumage en conditions difficiles. La propagation de la flamme détermine ledéclenchement de l'autoallumage selon une caractéristique linéaire particulièrementremarquable, car indépendante des conditions thermodynamiques du mélange. L'existenced'une flamme froide est mise en avant par des acquisitions de PLIF formaldéhyde. Lapréréaction semble accélérer la propagation du front de flamme.

Aquest treball s'integra en una de les línies d'investigació del grup de recerca "Quimiometria" del Departament de Química Analítica de la Universitat de Barcelona. Aquesta línia d'investigació es centra en el desenvolupament de mètodes quimiomètrics d'anàlisi multivariant de dades, i en la seva aplicació a l'estudi analític dels canvis de conformació i/o de les interaccions entre biomolècules.Actualment és possible enregistrar l'espectre sencer d'una mostra en poc temps. Aquest augment del nombre i de la complexitat de les dades adquirides ha portat a l'aparició de mètodes que tenen com a finalitat la obtenció d'informació d'interés físico-químic a partir d'aquests conjunt de dades. Amb aquesta finalitat es poden trobar dues aproximacions: a) els mètodes de modelatge rígid que exigeixen la postulació d'un model químic o cinètic al qual ajustar les dades experimentals, i b) els mètodes de modelatge flexible que no necessiten la postulació d'un model.El treball realitzat en aquests tesi doctoral es pot dividir en tres blocs.En primer lloc, s'ha desenvolupat una interfície gràfica en l'entorn de programació MATLAB pel mètode de resolució multivariant de corbes mitjançant mínims quadrats alternats (MCR-ALS). Aquesta interfície millora notablement la interacció entre l'usuari i el programa, i potencía la seva utilització generalitzada per part d'usuaris no acostumats a treballar amb eines pròpies de la Quimiometria.En segon lloc, s'ha dut a terme la validació de diversos mètodes d'anàlisi multivariant, és a dir, s'ha estudiat la fiabilitat de les solucions obtingudes per aquest tipus de mètodes quimiomètrics. Així, pel mètode MCR-ALS, s'ha analitzat la influència i la propagació de l'error experimental i les possibles repercusions sobre les ambigüetats matemàtiques existents en les solucions obtingudes. Aquest estudi s'ha realitzat tant en el cas de l'anàlisi individual de matrius de dades obtingudes en un únic experiment, com en el cas de l'anàlisi simultani de matrius de dades obtingudes en diversos experiments. En el cas dels mètodes de modelatge rígid s'ha estudiat l'ambigüetat existent al ajustar mecanismes cinètics complexos. En aquest cas s'ha observat l'aparició de mínims locals múltiples amb el mateix valor d'ajust en la superfície de desposta associada.Finalment, s'han aplicat els mètodes quimiomètrics de modelatge flexible i de modelatge rígid a l'estudi dels equilibris en solució dels àcids nucleics. Aquestes són biomolècules que tenen una organització jeràrquica començant en la seqüència de nucleòtids a les cadenes fins a estructures complexes d'ordre superior com els tríplexs o quadruplexs. Els canvis conformacionals o les interaccions amb d'altres biomolècules s'han estudiat tradicionalment mitjançant experiments seguits amb tècniques espectroscòpies. En aquest treball es seguiran aquests processos mitjançant lectures a moltes longituts d'ona (aproximació multivariant) i s'aplicaran mètodes quimiomètrics adients de tractaments de dades multivariants. Els procesos estudiants en aquesta Tesi són bàsicament els canvis conformacionals provocats en variar condicions del medi, com el pH, la temperatura, la concentració d'altres ions... S'han emprat tècniques espectroscòpiques com l'absorció molecular a l'UV-visible, la fluorescència, el dicroisme circular i la ressonància magnètica nuclear. Una altra aplicació, ha estat l'anàlisi de micromatrius d'ADN. L'aparició d'aquesta la tecnologia ha permès obtenir informació sobre els nivells de l'expressió gènica per un gran nombre de gens en un únic experiment. La generació de grans quantitats de dades requereix la utilització d'eines mitjançant les quals es pugui extreure la informació biològica. En aquest treball s'ha aplicat el mètode MCR-ALS a l'anàlisis de diversos conjunts de dades per tal de poder determinar la relació entre les mostres que presenten diferents tipus de càncer i els gens estudiats. / OF THE PHD THESIS: This PhD Thesis has been developed in the framework of the Chemometrics group at the Universitat de Barcelona. The work deals with the development and validation of Multivariate Curve Resolution (MCR) methods (both hard- and soft-modelling), and with their application to bioanalytical problems. The work has been organized into three blocks:First, a graphical interface has been developed for the program running the MCR-ALS (Multivariate Curve resolution Alternating Least Squares) method in the MATLAB® environment. This interface improves the interaction between the user and the program and facilitates the use of multivariate curve resolution to little experineced potential users.Secondly, validation of multivariate resolution methods of data analysis has been carried out. For the MCR-ALS method, effects of rotational ambiguities and of propagation of experimental noise have been studied. These studies have been performed in the analysis of a single experiment and in the case of analyzing multiple experiments simultaneously. In the case of hard-modelling kinetic data fitting methods, ambiguities in the analysis of kinetic experiments have been studied and methods to overcome this ambiguity have been proposed.Third, multivariate resolution methods have been applied to the study of conformational equilibria of nucleic acids. These are biomolecules that have a hierarchic organization from the nucleotide sequence to higher order structures such as triplex or quadruplex. Traditionally, conformational changes or interactions of nucleic acids with other biomolecules have been spectroscopically monitored at just one wavelength. In this work, these processes have been followed at multiple wavelengths and suitable multivariate resolution methods for the data treatment have been applied. Processes studied during this Thesis have been DNA conformational changes induced by pH, temperature or salinity. Spectroscopic techniques such as molecular absorption in the UV-visible, circular dichroism or nuclear magnetic resonance have been used for this purpose. Finally, data obtained using DNA microarrays have been analyzed. This technique allows highthroughput analysis of relative gene expressions of thousands of genes of an organism that generates large amounts of data. This has caused a need for statistical methods that can extract useful information for further research. In this PhD Thesis, the MCR-ALS method has been proposed for the analysis of this kind of data with very promising results.

Kolistin är ett gammalt polypeptidantibiotikum och används som sista utväg för behandling av allvarliga infektioner orsakad av multiresistenta gramnegativa bakterier. Nya studier har påvisat kolistinresistensgener, mcr (mobil colistin resistance), hos extended spectrum beta-lactamase (ESBL)-producerande Enterobacteriaceae. Mcr-genen ligger i plasmider som kan överföras mellan bakterier, vilket innebär att det är mycket svårare att behandla människor och djur vid infektion orsakad av patogen som erhållit denna resistens. Syfte med detta projekt var att utveckla en PCR-baserad metod för detektion av mcr-1 och mcr-3 gener. I denna studie optimerades en PCR-metod och sedan screenades 60 ESBL-isolat från Kristianstads vattenrike. Därefter undersöktes PCR-produkt med hjälp av agarosgelselektrofores. Resultatet visade att 51oC är den optimala annealingtemperaturen vid multiplex-PCR för detektion av mcr-1 och mcr-3. Ingen av mcr-generna kunde detekteras i de 60 ESBL-isolaten. Positiva kontrollstammar med specifika primers kunde detekteras i PCR-analyser som genomfördes i denna studie, vilket tyder på att den optimerade PCR metoden kan vara pålitlig för detektion av mcr-1 och mcr-3 generna.