Submitted by Jos? Henrique Henrique (jose.neves@ufvjm.edu.br) on 2017-09-12T18:28:33Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) aline_bacelar_goncalves.pdf: 5988024 bytes, checksum: 24d09b3ca0a2e58cc7aee0ca7ca2528f (MD5) / Approved for entry into archive by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2017-09-18T12:57:54Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) aline_bacelar_goncalves.pdf: 5988024 bytes, checksum: 24d09b3ca0a2e58cc7aee0ca7ca2528f (MD5) / Made available in DSpace on 2017-09-18T12:57:54Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) aline_bacelar_goncalves.pdf: 5988024 bytes, checksum: 24d09b3ca0a2e58cc7aee0ca7ca2528f (MD5) Previous issue date: 2017 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (CAPES) / O tratamento das leucemias ? desafiador por v?rios aspectos, entre os quais podem ser destacados os efeitos adversos e a obten??o de op??es terap?uticas de alta qualidade e de custos razo?veis. A utiliza??o da enzima L-asparaginase como agente terap?utico, limita a fonte ex?gena de asparagina, da qual as c?lulas malignas dependem para o metabolismo celular e para a sobreviv?ncia. Essa ? uma op??o que oferece menores riscos ao paciente e ?s c?lulas sadias, que s?o capazes de sintetizar este amino?cido. Neste cen?rio o objetivo deste trabalho foi selecionar, entre fungos filamentosos, linhagens produtoras da enzima L-asparaginase. O estudo tamb?m buscou avaliar o efeito da varia??o da fonte de carbono e da raz?o carbono-nitrog?nio no crescimento e na express?o da atividade enzim?tica, a fim de desenvolver meios de cultivo para o processo produtivo. Realizou-se tamb?m um estudo do crescimento apical das tr?s linhagens selecionadas, duas do g?nero Penicillium sp. e uma do g?nero Fusarium sp., em diversos meios de cultivo. O conhecimento gerado sobre as linhagens produtoras e os demais estudos realizados permitiram a obten??o de um meio de cultivo que possibilitou a produ??o enzim?tica em at? 11,45 U.min-1.mL-1 com a linhagem de Fusarium sp. / Disserta??o (Mestrado) ? Programa de P?s-gradua??o em Ci?ncias Farmac?uticas, Universidade Federal dos Vales do Jequitinhonha e Mucuri, 2016. / The treatment of leukemia is challenging in many ways, including the adverse effects and obtaining treatment options of high quality and reasonable cost. The use of L-asparaginase enzyme as a therapeutic agent limits the exogenous source of asparagine, which the malignant cells depend for cellular metabolism and survival. This option offers lower risk to patients and healthy cells, which are able to synthesize this amino acid. Therefore, the objective of this work was to select among filamentous fungi, producing strains of L-asparaginase enzyme. The study also aimed at evaluating the effect of varying the carbon source and carbon-nitrogen ratio in the growth and expression of the enzymatic activity to develop culture media for the production process. It was also carried out a study of the apical growth of the three strains selected, two of the genus Penicillium sp. and one Fusarium sp., cultivated in various culture media. The knowledge about the growth of the strains studied in different nutritional sources and other studies allowed obtaining a culture medium that enabled the enzyme production of 11.45 U.min-1.mL-1 by Fusarium sp.

Made available in DSpace on 2016-02-05T18:30:04Z (GMT). No. of bitstreams: 0 Previous issue date: 2015-04-30. Added 1 bitstream(s) on 2016-02-05T18:34:11Z : No. of bitstreams: 1 000857377.pdf: 1866796 bytes, checksum: 83e072efd615d5585f2008373b9ff113 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / As coleções de culturas de micro-organismos apresentam importante contribuição para a preservação, manutenção e disponibilização de material microbiológico para uso acadêmico e em pesquisa. A Central de Recursos Microbianos da UNESP (CRM-UNESP) tem como missão dar suporte ao conhecimento da biodiversidade microbiana, bem como ao desenvolvimento científico e tecnológico, apoiando projetos de relevância. A CRM-UNESP possui um acervo associado de fungos filamentosos e leveduras de ambientes incomuns e/ou extremos. Neste contexto, o presente estudo tem como objetivo reorganizar o acervo de fungos marinhos da costa brasileira, marinhos e terrestres da Antártica e prospectar as enzimas xilanase e L-asparaginase. Para tanto, cerca de 696 fungos marinhos oriundos da costa brasileira e 185 fungos isolados de ambientes antárticos foram avaliados quanto à viabilidade e pureza, fotografados e preservados por dois métodos distintos: criopreservação e Castellani. Destes, 71% dos isolados foram considerados puros e viáveis e submetidos às triagens quantitavas e qualitativas para enzima xilanase e L-asparaginase. Na triagem qualitativa (meio sólido) 112 fungos produtores de xilanase foram isolados de ambiente marinho da costa brasileira e 19 de ambiente antártico. Estes foram submetidos à triagem quantitativa (meio liquído), na qual, o fungo LAMAI 31 identificado como Aspergillus tubingensis apresentou o melhor resultado de produção enzimática (49,41 U/mL). Para a enzima L-asparaginase além dos 622 isolados, 17 fungos filamentosos adicionais foram analisados na triagem qualitativa, totalizando 510 de origem marinha da costa brasileira e 129 de origem Antártica. Dentre estes, 407 fungos foram positivos para L-asparaginase em meio sólido e foram submetidos à triagem em meio liquido. A L-asparaginase foi produzida na faixa de 0,02 a 1,65 U/mL por 103 isolados. Delineamentos experimentais foram aplicados aos isolados que... / Microbial culture collections have an important contribution to the preservation, maintenance, and availability of the microbial material for academic and research pourpose. The mission of the Central of Microbial Resources of the UNESP (CRM-UNESP) is to provide support for the knowledge of the microbial biodiversity, as well as for the scientific and technological development, supporting relevant projects. CRM-UNESP has an associated collection of filamentous fungi and yeast from uncommon and/or extreme environments. In this context, the present study aims to reorganize the marine-derived fungal collection from brasilian coast and the marine and terrestrial collection from Antarctica and to prospect the enzymes xylanase and L-asparaginase. For that end, about 696 marine fungi originating from brasilian coast and 185 isolated from antarctic environments had their viability and purity assessed and were photographed and preserved by two different methods: criopreservation and Castellani. Among them, 71% isolated were considered pure and viable and were submitted to the quantitative and qualitative screening for the production of xylanase and L-asparaginase. In the qualitative quantitative screening (solid medium) 112 filamentous fungi able to produce xilanases were isolated from marine environment (brasilian coast) and 19 from the antartic environment. These isolates were submitted to quantitative screening (liquid medium), in which, the fungus LAMAI 31 indentified as Aspergillus tubingensis presented the best result of enzymatic production (49.41 U/mL). For L-asparaginase besides the 622 isolates, 17 additional filamentous fungi were analysed, being 510 originated from the brasilian marine coast and 129 from Antarctica. Among them, 407 fungi were positive for L-asparaginase in solid medium and were submitted to the screening in liquid medium. L-asparaginase was produced in a range of 0.02 to 1.65 U/mL by 103 isolated. Experimental designs ...

Acrylamide is an undesirable carcinogenic component of thermally processed foods being formed from reducing saccharides and asparagine. In this work, the effect of ammonium and sodium raising agents themselves or in their combination with L-asparaginase enzyme catalyzing the conversion of asparagine into aspartic acid resulting in the reduction of acrylamide in gingerbreads was studied. Also, the influence of selected inorganic salts on the content of acrylamide in a model matrix simulating a composition of cereal products was observed. Simultaneously, the impact of these salts on activity of L-asparaginase was examined to find optimal conditions for its application in cereal technology. Based on experiments it was found, that addition of L-asparaginase reduces acrylamide content by 40 % while inorganic salts addition decreases acrylamide content in the range of 30 - 99 % when the most effective compounds were NH4Cl and CaCl2.

Acrylamide (AA) is a probable human carcinogen and undesirable contaminant which is produced by the reaction of reducing sugars with asparagine in plant foods during their thermal treatment above 120 °C. AA is most often determined by GC-MS and LC-MS/MS in isolates from the matrix in a wide range of foods. According to our observations, AA intake from food is higher among young people (from 1.8 to 3.8 µg/kg bw/day), which is consistent with the estimations of JECFA FAO/WHO from the year 2006. Considering the health risk, it is recommended to reduce AA formation in food during its processing, in particular exploiting the available experience. The aim of this thesis was to extend the knowledge of the possibility of AA elimination in selected types of thermally processed foods. The study was focused on cereal foods that contribute significantly to AA exposure, especially bread and sweet biscuits. The whole AA content in the bread is in the crust, which represents 5-15% of the bread. Crust of home-made bread contains approximately 30-75 µg/kg, however the marketed bread contains 2 to 10 times more of AA. This is due to the composition of bread mix, preparation conditions and baking. For maintaining the quality of home-made bread during the dry mixture shelf-life, optimization of bread mixtures was designed by increasing of yeast content, which proved positive effect on the reduction of AA content at sufficiently high activity of the yeast. Monitoring of AA content in assortment of sweet bakery products showed higher levels of AA in diabetic biscuits containing fructose instead of sucrose. Three of them even exceeded the reference value (500 µg/kg) more than 1.5 times for commodity "cookies". Elimination of AA by applications of the enzyme asparaginase has been designed for minimal interference in technology of their production. The concentration of the enzyme and the appropriate method of its use in industrial environment have been tested previously in model systems. In optimized conditions of the enzyme application, AA content in diabetic biscuits was reduced by more than 40% without affecting the organoleptic properties of the final product. Effect of the antioxidants on AA formation was also part of the study. AA content in gingerbread was reduced efficiently by the use of fennel, anise, cloves, vanilla and white pepper (by about 9-21%). Conversely, coriander and cinnamon significantly increased its content (by 18-54%). Since correlations between the DPPH• radical quenching activity of the spice extracts and AA content was not observed, the final content of AA was probably influenced by the chemical composition of spices and reactivity of the individual components in the matrix. Investigated methods appear to be suitable ways of elimination AA in some foods; however their specific use must be optimized with regard to the composition of the food, processing and the technology used. Estimated impact of application of the above-mentioned methods to the overall elimination of AA exposure showed that its intake in high school students from the Czech and Slovak Republic can be reduced on average by 10%. This decrease is a success to reduce the possible risk of cancer disease by eating foods with a high AA content. It is also important piece of information for food producers for further development of relevant methods for AA elimination which would help to reduce the AA intake from foods even more.

La leucémie aiguë lymphoblastique (LAL) est le cancer pédiatrique le plus fréquent. Elle est la cause principale de mortalité liée au cancer chez les enfants due à un groupe de patient ne répondant pas au traitement. Les patients peuvent aussi souffrir de plusieurs toxicités associées à un traitement intensif de chimiothérapie. Les études en pharmacogénétique de notre groupe ont montré une corrélation tant individuelle que combinée entre les variants génétiques particuliers d’enzymes dépendantes du folate, particulièrement la dihydrofolate réductase (DHFR) ainsi que la thymidylate synthase (TS), principales cibles du méthotrexate (MTX) et le risque élevé de rechute chez les patients atteints de la LAL. En outre, des variations dans le gène ATF5 impliqué dans la régulation de l’asparagine synthetase (ASNS) sont associées à un risque plus élevé de rechute ou à une toxicité ASNase dépendante chez les patients ayant reçu de l’asparaginase d’E.coli (ASNase). Le but principal de mon projet de thèse est de comprendre davantage d’un point de vue fonctionnel, le rôle de variations génétiques dans la réponse thérapeutique chez les patients atteints de la LAL, en se concentrant sur deux composants majeurs du traitement de la LAL soit le MTX ainsi que l’ASNase. Mon objectif spécifique était d’analyser une association trouvée dans des paramètres cliniques par le biais d’essais de prolifération cellulaire de lignées cellulaires lymphoblastoïdes (LCLs, n=93) et d’un modèle murin de xénogreffe de la LAL. Une variation génétique dans le polymorphisme TS (homozygosité de l’allèle de la répétition triple 3R) ainsi que l’haplotype *1b de DHFR (défini par une combinaison particulière d’allèle dérivé de six sites polymorphiques dans le promoteur majeur et mineur de DHFR) et de leurs effets sur la sensibilité au MTX ont été évalués par le biais d’essais de prolifération cellulaire. Des essais in vitro similaires sur la réponse à l’ASNase de E. Coli ont permis d’évaluer l’effet de la variation T1562C de la région 5’UTR de ATF5 ainsi que des haplotypes particuliers du gène ASNS (définis par deux variations génétiques et arbitrairement appelés haplotype *1). Le modèle murin de xénogreffe ont été utilisé pour évaluer l’effet du génotype 3R3R du gène TS. L’analyse de polymorphismes additionnels dans le gène ASNS a révélé une diversification de l’haplotype *1 en 5 sous-types définis par deux polymorphismes (rs10486009 et rs6971012,) et corrélé avec la sensibilité in vitro à l’ASNase et l’un d’eux (rs10486009) semble particulièrement important dans la réduction de la sensibilité in vitro à l’ASNase, pouvant expliquer une sensibilité réduite de l’haplotype *1 dans des paramètres cliniques. Aucune association entre ATF5 T1562C et des essais de prolifération cellulaire en réponse à ASNase de E.Coli n’a été détectée. Nous n’avons pas détecté une association liée au génotype lors d’analyse in vitro de sensibilité au MTX. Par contre, des résultats in vivo issus de modèle murin de xénogreffe ont montré une relation entre le génotype TS 3R/3R et la résistance de manière dose-dépendante au traitement par MTX. Les résultats obtenus ont permis de fournir une explication concernant un haut risque significatif de rechute rencontré chez les patients au génotype TS 3R/3R et suggèrent que ces patients pourraient recevoir une augmentation de leur dose de MTX. À travers ces expériences, nous avons aussi démontré que les modèles murins de xénogreffe peuvent servir comme outil préclinique afin d’explorer l’option d’un traitement individualisé. En conclusion, la connaissance acquise à travers mon projet de thèse a permis de confirmer et/ou d’identifier quelques variants dans la voix d’action du MTX et de l’ASNase qui pourraient faciliter la mise en place de stratégies d’individualisation de la dose, permettant la sélection d’un traitement optimum ou moduler la thérapie basé sur la génétique individuelle. / Acute lymphoblastic leukemia (ALL) is the most frequent malignancy of childhood. It is the principal cause of cancer–related mortality in children due to a persistent group of patients who does not respond to standard anti-cancer treatment. Susceptible patients may also suffer from number of toxicities associated with intensive chemotherapy treatment. Pharmacogenetic studies of our group, showed that particular genetic variants of the folate dependent enzymes, particularly, dihydrofolate reductase (DHFR) and thymidylate synthase (TS), major targets of methotrexate (MTX), correlate both individually and combined with increased risk of relapse in patients with childhood ALL. Furthermore, variations of ATF5 gene involved in asparagine synthetase (ASNS) regulation and of ASNS gene were associated with higher risk of ALL relapse or with ASNase related toxicity in patients who received E.coli asparaginase (ASNase). The major goal of my doctoral research project was to further understand from the functional point of view the role of genetic variations underlying therapeutic responses of childhood ALL, by focusing on two major components of ALL treatment, MTX and ASNase. My specific goal was to analyze associations found in clinical setting using cellular proliferation assay in lymphoblastoid cell lines (LCLs, n=93) and xenograft mice model of ALL. Genetic variation in TS polymorphism (homozygosity for triple repeat allele, 3R) and of DFHR haplotype *1b (defined by particular allelic combination derived from six polymorphic sites in the major and minor promoter of DHFR), on MTX sensitivity was assessed using cellular proliferation assay. Similar in vitro assay in response to E.coli ASNase was used to access the T1562C variation in the ATF5 5’UTR and particular haplotypes of ASNS gene (defined by two genetic variation and arbitrarily named haplotype *1). Xenograft mouse model was used to access the effect of TS 3R3R genotype. Analysis of additional polymorphisms in ASNS gene revealed diversification of haplotype *1 of ASNS gene in 5 subtypes, two polymorphisms (rs10486009 and rs6971012,) defining particular subtypes correlated with in vitro sensitivity to ASNase and one of them (rs10486009) seems particularly important for reducing sensitivity to ASNase in vitro, possibly providing mechanistic explanation for lower sensitivity of haplotype *1 observed in clinical setting. No association between ATF5 T1562C variation and cellular proliferation assay in response to E.coli ASNase was found. We did not observe genotype-related association when in vitro sensitivity to MTX in LCLs was analyzed. In contrast, in vivo results using xenograft mouse model demonstrated the relationship between the TS 3R/3R genotype and the resistance to MTX treatment in dose-dependent manner. Obtained results provided function explanation for the significantly higher risk of relapse seen in 3R/3R ALL patients and suggest that these patients might benefit from increase dose of MTX. Through these experiments we also showed that xenogeneic mice model can serve as a preclinical tool to explore individualized treatment options. In conclusion, the knowledge acquired through my doctoral work confirmed and/or identified some functional variants in MTX and ASNase action pathway which may facilitate dose individualization strategies, allowing for optimal treatment selection or tailoring childhood ALL therapy based on individual genetics.

A L-Asparaginase II de Escherichia coli (EcA II) é uma enzima amplamente utilizada no tratamento da Leucemia Linfoblástica Aguda (LLA), atuando na depleção do aminoácido L-asparagina, o qual é fundamental para a multiplicação das células cancerosas. Contudo, o tratamento com a EcA II está associado a altos índices de hipersensibilidade, devido à formação de anticorpos anti-L-asparaginase e à clivagem da enzima pelas proteases sanguíneas asparagina endopeptidase (AEP) e catepsina B (CTSB). Também ocorre neurotoxicidade associada ao efeito L-glutaminase da enzima. O principal objetivo do presente trabalho é a obtenção de mutantes da EcA II (gene ansB) com equivalente eficiência catalítica, maior resistência à clivagem proteolítica e menor atividade glutaminase. Para este propósito, através da reação em cadeia da polimerase propensa a erro (epPCR) do gene ansB, foi construída uma biblioteca de 1128 clones expressos no vetor pET15b em BL21(DE3). Nenhum mutante com atividade asparaginásica equivalente à EcA II selvagem apresentou atividade glutaminásica inferior à esta. Dentre os clones triados obtivemos um mutante (T161I) resistente à clivagem proteolítica pela CTSB e dois mutantes (Q190L e P40S/S206C) resistentes à clivagem proteolítica por ambas AEP e CTSB. Estes três mutantes apresentaram atividade asparaginásica e glutaminásica equivalentes a EcA II selvagem. Nossos resultados mostram promissoras possibilidades de EcA II mutantes com maior estabilidade frente às proteases sanguíneas humanas e possivelmente menos imunogênicas. / Escherichia coli L-asparaginase (EcA II) is an enzyme widely used in the treatment of acute lymphoblastic leukemia (ALL), acting in the depletion of the amino acid L-asparagine, which is essential for cancer cells proliferation. However, treatment with L-asparaginase is associated with a high rate of hypersensitivity, due to formation of anti-L-asparaginase antibody and the enzyme cleavage by the serum proteases asparagine endopeptidase (AEP) and cathepsin B (CTSB). Furthermore, the neurotoxicity is associated with the effect of the enzyme L-glutaminase activity. The main aim of the current work is to obtain variants of EcA II (gene ansB) with an equivalent catalytic efficiency, greater resistance to proteolytic cleavage and a reduced glutaminase activity. For such purpose, through error-prone polymerase chain reaction (epPCR) of gene ansB, a library of 1128 clones was constructed in pET15b vector and expressed in BL21(DE3). None mutant with an asparaginase activity equivalent to EcA II wild type showed a reduced glutaminase activity. Among the screened clones, one mutant (T161I) was resistant to CTSB proteolytic cleavage and two mutants (Q190L e P40S/S206C) were resistant to both CTSB and AEP proteolytic cleavages. These three mutants were EcA II wild type equivalents in asparaginase and glutaminase activities. Our data show promising new possibilities of mutant EcA II presenting higher stability against human serum proteolytic cleavage and maybe lower immunogenicity.

A L-Asparaginase II de Escherichia coli (EcA II) é uma enzima amplamente utilizada no tratamento da Leucemia Linfoblástica Aguda (LLA), atuando na depleção do aminoácido L-asparagina, o qual é fundamental para a multiplicação das células cancerosas. Contudo, o tratamento com a EcA II está associado a altos índices de hipersensibilidade, devido à formação de anticorpos anti-L-asparaginase e à clivagem da enzima pelas proteases sanguíneas asparagina endopeptidase (AEP) e catepsina B (CTSB). Também ocorre neurotoxicidade associada ao efeito L-glutaminase da enzima. O principal objetivo do presente trabalho é a obtenção de mutantes da EcA II (gene ansB) com equivalente eficiência catalítica, maior resistência à clivagem proteolítica e menor atividade glutaminase. Para este propósito, através da reação em cadeia da polimerase propensa a erro (epPCR) do gene ansB, foi construída uma biblioteca de 1128 clones expressos no vetor pET15b em BL21(DE3). Nenhum mutante com atividade asparaginásica equivalente à EcA II selvagem apresentou atividade glutaminásica inferior à esta. Dentre os clones triados obtivemos um mutante (T161I) resistente à clivagem proteolítica pela CTSB e dois mutantes (Q190L e P40S/S206C) resistentes à clivagem proteolítica por ambas AEP e CTSB. Estes três mutantes apresentaram atividade asparaginásica e glutaminásica equivalentes a EcA II selvagem. Nossos resultados mostram promissoras possibilidades de EcA II mutantes com maior estabilidade frente às proteases sanguíneas humanas e possivelmente menos imunogênicas. / Escherichia coli L-asparaginase (EcA II) is an enzyme widely used in the treatment of acute lymphoblastic leukemia (ALL), acting in the depletion of the amino acid L-asparagine, which is essential for cancer cells proliferation. However, treatment with L-asparaginase is associated with a high rate of hypersensitivity, due to formation of anti-L-asparaginase antibody and the enzyme cleavage by the serum proteases asparagine endopeptidase (AEP) and cathepsin B (CTSB). Furthermore, the neurotoxicity is associated with the effect of the enzyme L-glutaminase activity. The main aim of the current work is to obtain variants of EcA II (gene ansB) with an equivalent catalytic efficiency, greater resistance to proteolytic cleavage and a reduced glutaminase activity. For such purpose, through error-prone polymerase chain reaction (epPCR) of gene ansB, a library of 1128 clones was constructed in pET15b vector and expressed in BL21(DE3). None mutant with an asparaginase activity equivalent to EcA II wild type showed a reduced glutaminase activity. Among the screened clones, one mutant (T161I) was resistant to CTSB proteolytic cleavage and two mutants (Q190L e P40S/S206C) were resistant to both CTSB and AEP proteolytic cleavages. These three mutants were EcA II wild type equivalents in asparaginase and glutaminase activities. Our data show promising new possibilities of mutant EcA II presenting higher stability against human serum proteolytic cleavage and maybe lower immunogenicity.

Le travail présenté dans cette thèse porte sur le développement de chimie de surface et de biocapteurs pouvant être utilisés dans le milieu hospitalier afin d’améliorer les méthodes de dépistages et de suivi de traitement de diverses maladies et cancers. Les méthodes actuelles de dépistage du cancer reposent principalement sur l’analyse histologique des cellules par des experts dans le domaine. Cela complique la transmission de l’information au patient et retarde le moment où un traitement approprié peut être entamé. Grâce à de nouvelles techniques d’analyses simples et peu coûteuses comme la spectroscopie de résonance des plasmons de surface (SPR), il est possible de développer des tests qui pourront être effectués par le personnel de l’hôpital, en peu de temps et à peu de frais. Afin de pouvoir utiliser la SPR en milieu clinique, une chimie de surface appropriée doit être développée afin d’empêcher les matrices biologiques de masquer le signal des analytes d’intérêt. En effet, qu’il s’agisse de lysat cellulaire, de sérum ou de tout autre fluide biologique, le contenu protéique et lipidique peut s’adsorber de façon non-spécifique aux surfaces d’analyse, compromettant ainsi les résultats obtenus. Afin de pallier ce problème, le développement de chimie de surface a été effectué. Des monocouches de peptides et de liquides ioniques ont été utilisées afin de réduire l’adsorption non-spécifique de lysat cellulaire non-dilué sur des capteurs SPR. Parmi les peptides, le plus efficace s’est avéré être le 3 MPA (His)2(Leu)2(Phe)2 OH, un peptide chargé positivement formé de 6 acides aminés plutôt hydrophobes. Grâce à cette surface, l’adsorption non-spécifique de lysat cellulaire a pu être réduite jusqu’à 159 ± 27 ng/cm2, par rapport à 929 ± 186 ng/cm2 sur une surface d’or non protégée. Une étude en spectrométrie de masse a permis de mieux comprendre le phénomène d’adsorption non-spécifique de lysat cellulaire et de confirmer que ce sont principalement des lipides qui s’adsorbent non-spécifiquement au capteur SPR lorsque celui-ci est exposé à du lysat cellulaire. Malgré la nette amélioration par rapport à un capteur non protégé, le phénomène d’adsorption non-spécifique était encore significativement présent avec les monocouches de peptides. L’adsorption non-spécifique de lysat cellulaire a ensuite été drastiquement réduite grâce aux liquides ioniques hydrophobes et chargés. Le liquide ionique le plus performant a montré une adsorption non-spécifique d’à peine 2 ± 2 ng/cm2. Par la suite, un biocapteur permettant la détection de HER2, un biomarqueur de cancer du sein présent dans environ 30% des cas de cancers du sein agressifs, a été développé. Cela a permis de démontrer que le liquide ionique pouvait être utilisé pour la construction d’un biocapteur, ouvrant ainsi la porte à un large domaine d’analyses en lysat cellulaire. Finalement, les défis de l’analyse SPR avec des échantillons cliniques ont été explorés par le développement d’un biocapteur pour l’anti-asparaginase, permettant de faire le suivi de traitement de patients atteints de leucémie. L’asparaginase est administrée aux patients leucémiques, en combinaison avec plusieurs autres composés chimiothérapiques, afin de combattre ce cancer. Toutefois, plusieurs patients ont une réaction allergique à cette protéine de source bactérienne, mais ne démontrent pas de symptômes physiques. Le biocapteur développé visait donc à détecter les réactions immunitaires des patients afin de modifier leur traitement lorsque cela s’avère nécessaire. Un biocapteur pour la détection de l’anti-asparaginase dans le sérum non-dilué de patients leucémiques a donc été développé. Des échantillons cliniques ont été étudiés et les résultats obtenus pour le nouveau biocapteur SPR ont montré une bonne concordance avec les résultats obtenus en ELISA. / This thesis describes the development of clinical biosensors. These biosensors were developed with the aim of improving diagnostic and treatment monitoring methods. Actual monitoring methods often rely on histological analysis performed by experts. This complicates the transmission of the information to the patient and delays the onset of an appropriate treatment. It is envisioned to develop simple experiments at low cost, which will allow untrained personnel to perform the testing on-site with biosensing technologies such as surface plasmon resonance (SPR). In order to perform SPR in clinical analysis, appropriate surface chemistry must be developed to prevent nonspecific adsorption. Nonspecific adsorption is the fouling of surfaces with biomolecules contained in the sample matrix such as proteins or lipids of biofluids. This leads to false positive signals preventing the correct measurement of the analyte concentration. Peptide and ionic liquid monolayers have been studied in this thesis to prevent nonspecific adsorption of undiluted cell lysate. The most efficient peptide was the 3 MPA (His)2(Leu)2(Phe)2 OH peptide, a 6 amino acids hydrophobic and positively charged peptide. The nonspecific adsorption of cell lysate was reduced to 159 ± 27 ng/cm2, compared to 929 ± 186 ng/cm2 on a bare gold surface. Also, mass spectrometry was performed to better understand the cell lysate nonspecific adsorption phenomenon. This study showed lipids were mostly adsorbed on the sensor when exposed to cell lysate. Despite a significant reduction of nonspecific adsorption with peptides, it remained unoptimal and should be improved. The newly developed hydrophobic and charged ionic liquids nearly eliminated the nonspecific adsorption of undiluted cell lysate, with only 2 ± 2 ng/cm2 of nonspecific material adsorbed on the surface. Then, a biosensor of an aggressive breast cancer biomarker, HER2, was developed. This proved that the ionic liquids could be used in the development of clinical biosensors. Finally, the challenges of the analysis of clinical samples with SPR sensing were explored with the development of an anti-asparaginase biosensor for leukemic patients. Asparaginase is a chemotherapeutic drug administered to patients in combination with various other drugs to treat leukemia. However, many patients suffer from silent allergic reactions due to the bacterial source of this drug. Therefore, a biosensor was developed to detect the antibodies in undiluted serum produced against the drug, which could ultimately serve to modify the patient’s treatment when necessary. Clinical samples from leukemia patients were studied and the results were in good agreement with ELISA experiments.

Le traitement de la leucémie lymphoblastique aiguë (LLA) de l’enfant, une affection d'origine maligne des cellules progénitrices lymphoïdes, s’est considérablement amélioré au cours des dernières décennies. En effet, le taux de succès du traitement a dépassé 90% dans des conditions favorables. Cependant, des toxicités liées au traitement peuvent être fatales et entrainer l’interruption ou la cessation du traitement. L'allergie, la pancréatite et la thrombose sont des complications fréquentes du traitement de la LLA et sont associées à l'utilisation de l'asparaginase (ASNase), tandis qu’une toxicité fréquente due à la vincristine (VCR) induit la neuropathie périphérique (VIPN). Étant donné que l’ajustement du schéma posologique afin d’augmenter l'efficacité et diminuer la toxicité est un processus sensible, ceci demeure un défi majeur dans plusieurs protocoles de traitement. La pharmacogénétique étudie comment des altérations de la composante génétique peuvent influer sur la variabilité interindividuelle observée dans la réponse au traitement. Une meilleure compréhension de la base moléculaire de cette variabilité pourrait améliorer considérablement les résultats du traitement, en permettant la personnalisation de ce dernier en fonction du profil génétique du patient. Des études récentes suggèrent l’avantage d’appliquer l’analyse de l’exome à la découverte de variants associés à des traits humains complexes ainsi qu’à des phénotypes de réactions médicamenteuses. L'objectif de notre travail était d'utiliser les données de séquençage pour réaliser des études d'association à l'échelle de l'exome, y compris des étapes de filtrage et de validation, afin d'identifier de nouveaux variants génétiques susceptibles de moduler le risque de développer des complications associées à ASNase et à VIPN. Douze SNP étaient associés à des complications due à l’ASNase dans la cohorte initiale, dont 3 étaient associés à une allergie, 3 à une pancréatite et 6 à une thrombose. Parmi ceux-ci, les variants rs3809849, rs11556218 et rs34708521 des gènes MYBBP1A, IL16 et SPEF2 respectivement ont été associés à des complications multiples et leur association à une pancréatite a été répliquée dans une cohorte de validation indépendante. En ce qui concerne la VCR, trois variantes ont été associées à la modulation du risque de VIPN: rs2781377 dans SYNE2, rs10513762 dans MRPL47 et rs3803357 dans BAHD1. Nous démontrons également le puissant effet combiné de la présence de plusieurs variants de risque pour chacune des toxicités étudiées et fournissons des modèles de prédiction du risque pour la pancréatite et le VIPN basés sur la méthode d’évaluation du risque génétique pondérée et qui ont été validés à l’interne. De plus, étant donné une association du polymorphisme du gène MYBBP1A avec de multiples issus de traitement, nous avons cherché à comprendre comment cette altération génétique se traduit par des variabilités de réponse aux traitements à l’ASNase. En utilisant la technique CRISPR-CAS9 pour induire l'inactivation de gènes dans des lignées cellulaires cancéreuses PANC1 (pancréatiques) nous avons testé la différence de viabilité entre les cellules inactivées et les cellules du type sauvage à la suite de la suppression du gène et du traitement par ASNase. Nos résultats suggèrent un rôle fonctionnel de ce gène dans la modulation de la viabilité, de la capacité de prolifération et de la morphologie des cellules knock-out, ainsi que dans leur sensibilité à l'ASNase, et plaident en outre pour que le gène influence l’issus du traitement de la LLA par ASNase. Le présent travail démontre que l’utilisation de l’approche de séquençage de l’exome entier dans le contexte d’une étude d’association à l’échelle de l’exome est une stratégie valide « sans hypothèse » pour identifier de nouveaux marqueurs génétiques modulant l’effet du traitement de la LLA de l’enfant, et souligne l’importance de l'effet synergique de la combinaison des locus à risque. / Treatment of childhood acute lymphoblastic leukemia (ALL), a malignant disorder of lymphoid progenitor cells has improved significantly over the past decades and treatment success rates have surpassed 90% in favorable settings. However, treatment-related toxicities can be life-threatening and cause treatment interruption or cessation. Allergy, pancreatitis and thrombosis are common complications of ALL treatment associated with the use of asparaginase (ASNase), while vincristine-induced peripheral neuropathy (VIPN) is a frequent toxicity of vincristine (VCR). It is a sensitive process and a constant struggle to adjust the dosing regimen to ensure maximum efficacy and minimum toxicity. Pharmacogenetics studies show alterations in the genetic component between individuals can influence the observed variability in treatment response. A better understanding of the molecular basis of this variability in drug effect could significantly improve treatment outcome by allowing the personalization of ALL treatment based on the genetic profile of the patient. Emerging reports suggest the benefit of applying exome analysis to uncover variants associated with complex human traits as well as drug response phenotypes. Our objective in this work was to use available whole-exome sequencing data to perform exome-wide association studies followed by stepwise filtering and validation processes to identify novel variants with a potential to modulate the risk of developing ASNase complications and VIPN. Twelve SNPs were associated with ASNase complications in the discovery cohort including 3 associated with allergy, 3 with pancreatitis and 6 with thrombosis. Of those, rs3809849 in MYBBP1A, rs11556218 in IL16 and rs34708521 in SPEF2 genes were associated with multiple complications and their association with pancreatitis was replicated in an independent validation cohort. As for VCR, three variants were associated with modulating the risk of VIPN: rs2781377 in SYNE2, rs10513762 in MRPL47 and rs3803357 in BAHD1. We also demonstrate a strong combined effect of harbouring multiple risk variants for each of the studied toxicities, and provide internally-validated risk-prediction models based on the weighted genetic risk score method for pancreatitis and VIPN. Furthermore, given the association of the polymorphism in MYBBP1A gene with multiple treatment outcomes, we aimed at understanding how this genetic alteration translates into differences in ASNase treatment response through cell-based functional analysis. Using CRISPR-CAS9 technology we produced gene knockout of PANC1 (pancreatic) cancer cell-lines and tested the difference in viability between the knockouts and wild-type cells following gene deletion and ASNase treatment. Our results suggest a functional role of this gene in modulating the viability, proliferation capacity and the morphology of the knockout cells as well as their sensitivity to ASNase and further advocates the implication of the gene in influencing the outcome of ALL treatment with ASNase. The present work demonstrates that using whole-exome sequencing data in the context of exome-wide association study is a successful “hypothesis-free” strategy for identifying novel genetic markers modulating the effect of childhood ALL treatment and highlights the importance of the synergistic effect of combining risk loci.