Cette thèse est dédiée à l'approche pharmacogénétique des effets anti-inflammatoires de la thérapie par les thiénopyridines. Prenant en compte que les plaquettes activées jouent un rôle central dans les états inflammatoires et que des polymorphismes du cytochrome P450 (CYP) 2C19 ont été montré responsable de différences inter individuelle dans la réponse de l'effet antiplaquettaire de thiénopyridines, nous avons émis l'hypothèse que CYP2C19 *2 ou *17 sont également associés à la variabilité interindividuelle du potentiel antiinflammatoire des thiénopyridines. Les marqueurs d'inflammation utilisés pour suivre l'effet des thiénopyridines sont : la CRP, l'haptoglobines et l'orosomucoïde. Nous avons démontré que pour interpréter les valeurs de l'haptoglobine il était nécessaire de tenir compte du statut génétique et obtenir des valeurs de référence stratifiés. D'abord dans une population saine, nous n'avons pas trouvé d'association entre les valeurs de base des marqueurs inflammatoires et les polymorphismes fréquents de CYP époxygenases. Dans une population après intervention coronarienne percutanée qui était composée de 1128 sujets traités par clopidogrel ou prasugrel, le niveau de CRP observé a montré une interaction significative entre le tabac et le polymorphisme de CYP 2C19 ; cet effet est indépendant du niveau d'agrégation plaquettaire. Dans une 3ème population, sur plus de 1000 sujets hospitalisés à Coimbra, nous avons identifié une interaction entre le clopidogrel CYP2C19 et les médicaments bloqueurs des canaux calciques. En résumé, tous ces résultats obtenus sur plusieurs populations laissent envisager que les marqueurs d'inflammation pourraient être un moyen intéressant de suivi des patients lors de la thérapeutique par les thiénopyridines / The main part of the thesis is devoted to pharmacogenetic approach to the anti-inflammatory effects of thienopyridine therapy. Taking into the account that activated platelets play a central role in the inflammatory responses and that CYP2C19 gain- and loss-of-function polymorphisms (*2 and *17) are sources of inter-individual difference in response to the anti-platelet effects of thienopyridines, we hypothesized that *2 and/or *17 alleles are also associated with inter-individual variability in the potential inflammation-reducing effects of thienopyridines. The following markers were used to test the hypothesis: CRP, haptoglobin and orosomucoid acid. To be reliably interpretable in daily medical practice, genetic status should be considered for partitioning the reference values of haptoglobin. In a small healthy population, no significant association was observed between *2 allele and changes in levels of inflammatory markers from baseline to 7 days after administration of clopidogrel and our findings did not support the notion that the genetic variations of CYP epoxygenases are associated with the level of inflammatory markers. Also, in post-PCI population consisting of 1128 on-clopidogrel or on-prasugrel patients, CRP levels were observed to be regulated with a significant interaction between smoking and CYP2C19 polymorphisms; this effect was independent to the level of platelet aggregation. Additionally, in a large population of 1000 on-clopidogrel patients, whether there is a potential interaction between clopidogrel and calcium channel blockers. Collectively, we demonstrated in this thesis that inflammatory markers might be alternative tools for the prediction of response to thienopyridines

Ce projet de thèse portait sur le développement d’outils microfluidiques pour la toxicologie et la recherche contre le cancer. En permettant l’analyse simultanée d’un très grand nombre de réactions biologiques ou chimiques réalisés dans des compartiments indépendants (ie. gouttelettes), la microfluidique de gouttes offre une sensibilité de détection et une précision sans précédent pour l’analyse de molécules biologiques, telles que l’ADN ou les Anticorps, en comparaison des expériences réalisées conventionnellement en tubes ou en microplaques (essais en « bulk » ou volume). Ce format permet également de réaliser des expériences à très haut débit et est particulièrement pertinent pour la toxicologie, où des analyses robustes de l’effet des médicaments sont nécessaires. De même, ces procédures sont également très adaptées à l’analyse de cellules uniques pour le séquençage ADN ou ARN et l’épigénomique. Tout cela fait de la microfluidique en goutte un outil puissant pour la toxicologie et la recherche sur le cancer. En premier temps, une méthode du comptage précise des cellules encapsulée dans des microgouttelettes, nommée « hémocytométrie microfluidique », a été développée. Un nouvel algorithme de comptage a été proposé. Des cellules bactériennes (Escherichia Coli) et des cellules de 2 lignées humaines différentes (HL60 and H1975) ont été testées. Le nombre de chaque type de cellules a été déterminé avec une haute corrélation entre la théorie (basée sur la distribution de Poisson) et les résultats expérimentaux. Avec ces résultats robustes, un protocole de microfluidique en goutte a été mis en place pour interroger la viabilité cellulaire et la prolifération des 2 lignées humaines. Ces résultats sont en concordance avec ceux de la littérature. Pour la toxicologie, 3 différents modèles, y compris des microsomes (extrait de cellules d’insectes infectées par un baculovirus exprimant le cytochrome P450 3A4 humain, CYP3A4), HepG2-CYP3A4 (modifiée génétiquement pour exprimer le gène CYP3A4 humain), et HepaRG, une lignée hépatique, ont été évaluées pour l’activité enzymatique du CYP3A4, une enzyme largement utilisée en routine pour le criblage de médicament candidat. Les microsomes ont permis de développer un essai fluorogénique permettant de mesurer l’inhibition du CYP3A4. Cependant, ni l’utilisation des microsomes ni des cellules HepG2 exprimant CYP3A4 n’a donné de résultats satisfaisants en microgouttelettes. L’utilisation des cellules HepaRG, une lignée cellulaire qui conserve la majorité de l’expression des cytochromes P450 et des récepteurs nucléaires nécessaire à leur expression, a montré des résultats encourageant à la fois sur les tests de mesure de l’activité enzymatique et d’analyse de l’induction du CYP3A4. Pour la recherche sur le cancer, 4 essais originaux de PCR digitale en gouttes ont été mis en place pour la détection et la quantification de mutations (NRAS, DNMT3A, SF3B1 and JAK2) importante pour les syndromes myélodysplasiques, un groupe hétérogène de maladies touchant les cellules souches hématopoïétiques caractérisées par une hématopoïèse inefficace et des cytopénies périphériques. Finalement, un essai de PCR sur cellule unique encapsulées au sein de billes agarose a été proposé. / This thesis project consists in developing droplet-based microfluidic tools for toxicology and cancer research. Owing to its large numbers of discretized volumes, sensitivity of detection of droplet-based microfluidics for biological molecules such as DNA and antibody is much higher than bulk assays. This high throughput format is particularly suitable for experiments where a robust dose-response curve is needed, as well as for single cell analysis with applications in genomic or sequencing and epigenetics. All above makes droplet-based microfluidics a powerful tool for toxicology and cancer research. In a first part of the work, an accurate cell counting method, named “microfluidics hemocytometry”, has been developed. A new counting algorithm was proposed to count the cells within each droplet. Escherichia Coli and two different human cell lines (HL60 and H1975) were used to validate our strategy. The number of each type of cells in droplets was determined with a high consistency between theory (Poisson distribution) and experimental results. With these robust results, a droplet-based microfluidic protocol has then been established to inquiry both cell viability and proliferation for the two human cell lines. The results are in good agreement with the one of the literature. For the toxicology, 3 different biological models, including microsomes (extracted from baculovirus-infected insect cell expressing human CYP3A4), HepG2-CYP3A4 (genetically modified to express the human CYP3A4 gene) and HepaRG liver cells lines were evaluated for enzymatic activity of cytochromes P450 (CYP3A4), a routinely used enzyme for drug candidate screening. Microsome-based assays were used to validate a fluorogenic inhibition assay. However neither microsome-based assay nor the assay using CYP3A4 expressing HepG2 gave satisfying results in droplet-based format. However, HepaRG cells, a hepatic function-conserved cell line with most cytochrome and related nuclear receptors, demonstrated high relevance both for enzymatic activity testing and CYP3A4 expression induction study. For cancer research, 4 different picoliter droplet-based PCR assays were developed for the detection and quantification of mutations (NRAS, DNMT3A, SF3B1 and JAK2) present in Myelodysplastic syndromes, a heterogeneous group of clonal bone marrow hematopoietic stem cell disorders characterized by ineffective hematopoiesis and peripheral cytopenias. Furthermore, a single cell multistep PCR assay using encapsulation of target DNA in agarose droplets was proposed.

Xiao, Yajie. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 156-190). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Lau Pui Man Irene. / Thesis submitted in: July 2003. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 162-172). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abbreviations --- p.ii / Abstract --- p.v / Abstract in Chinese --- p.vii / List of Figures --- p.ix / List of Tables --- p.xii / Contents --- p.xiii / Chapter Chapter 1. --- Introduction --- p.1 / Chapter 1.1 --- Introduction --- p.2 / Chapter 1.1.1. --- Therapeutic uses of nucleic acids --- p.2 / Chapter 1.1.1.1 --- Antisense oligonucleotides --- p.2 / Chapter 1.1.1.2 --- RNA interference --- p.4 / Chapter 1.1.1.3 --- Aptamer --- p.6 / Chapter 1.2 --- Selection of Aptamer --- p.7 / Chapter 1.2.1 --- SELEX 'Systematic Evolution of Ligands by Exponential enrichment' --- p.7 / Chapter 1.2.1.1 --- In vitro selection --- p.8 / Chapter 1.2.1.2 --- Amplification --- p.8 / Chapter 1.2.1.3 --- Monoclonal Aptamer --- p.10 / Chapter 1.2.2 --- Photo-SELEX --- p.10 / Chapter 1.3 --- Examples of target molecules of aptamers --- p.12 / Chapter 1.4 --- Applications of aptamer --- p.14 / Chapter 1.4.1 --- Detection of Aptamer --- p.14 / Chapter 1.4.2 --- Examples of diagnostic use Contents --- p.15 / Chapter 1.4.2.1 --- Aptamer against theophylline with high specificity --- p.15 / Chapter 1.4.2.2 --- Aptamer chip --- p.16 / Chapter 1.4.3 --- Examples of therapeutic use --- p.18 / Chapter 1.4.3.1 --- Vascular endothelial growth factor (VEGF) --- p.18 / Chapter 1.4.3.2 --- Aptamer as a reversible antagonists of coagulation factor IXa is another example to show the potential case of aptamers as therapeutic agents --- p.20 / Chapter 1.4.4 --- Problem faced by aptamer --- p.21 / Chapter 1.4.4.1 --- Stability --- p.21 / Chapter 1.4.4.2 --- Clearance from blood --- p.22 / Chapter 1.5 --- Comparison between aptamer and antibody --- p.24 / Chapter 1.5.1 --- General comparison between aptamer and antibody --- p.24 / Chapter 1.5.1.1 --- Diversity --- p.24 / Chapter 1.5.2 --- Specificity --- p.26 / Chapter 1.5.3 --- Disadvantages of antibody --- p.26 / Chapter 1.5.4 --- Advantages of aptamer --- p.27 / Chapter 1.6 --- Project Objectives --- p.29 / Chapter Chapter 2. --- Materials and Methods --- p.31 / Chapter 2.1 --- Materials --- p.32 / Chapter 2.1.1 --- Chemicals --- p.32 / Chapter 2.1.2 --- Buffers --- p.36 / Chapter 2.1.2.1 --- Buffers commonly used --- p.37 / Chapter 2.1.2.2 --- Reagents for molecular work --- p.37 / Chapter 2.1.3 --- Bacterial Culture --- p.38 / Chapter 2.1.4 --- Culture of cell --- p.38 / Chapter 2.1.4.1 --- "TNF-α Sensitive Cell Line, L929" --- p.38 / Chapter 2.1.4.2 --- Medium for cell culture --- p.38 / Chapter 2.1.5 --- Reagent for Western blotting --- p.39 / Chapter 2.1.5.1 --- Protein extraction --- p.39 / Chapter 2.1.5.2 --- SDS-PAGE --- p.40 / Chapter 2.1.5.3 --- Electro-blotting --- p.41 / Chapter 2.2 --- Methods --- p.42 / Chapter 2.2.1 --- Conjugation of protein to solid support --- p.42 / Chapter 2.2.1.1 --- Conjugation of protein on PVDF membrane --- p.42 / Chapter 2.2.4.2 --- Conjugation of protein on Sepharose --- p.42 / Chapter 2.2.4.3 --- Conjugation of protein on magnetic bead --- p.42 / Chapter 2.2.2 --- SELEX --- p.43 / Chapter 2.2.2.1 --- Selection --- p.43 / Chapter 2.2.2.2 --- Photo-selection --- p.44 / Chapter 2.2.2.3 --- PCR --- p.45 / Chapter 2.2.3 --- Separation of oligonucleotides --- p.46 / Chapter 2.2.3.1 --- Separate short length double-stranded oligonucleotides by using polyacrylamide gel --- p.46 / Chapter 2.2.3.2 --- Separate short length single-stranded oligonucleotides by using denaturing polyacrylamide gel --- p.47 / Chapter 2.2.3.3 --- Extract the DNA from polyacrylamide gel --- p.48 / Chapter 2.2.3.4 --- Estimate the amount of DNA in solution after extraction --- p.49 / Chapter 2.2.3.5 --- Agarose Gel Electrophoresis --- p.49 / Chapter 2.2.4 --- Cloning of selected polyclonal aptamer --- p.50 / Chapter 2.2.4.1 --- Restriction cutting --- p.50 / Chapter 2.2.4.2 --- Ligation --- p.50 / Chapter 2.2.4.3 --- Preparation of the competent cells --- p.50 / Chapter 2.2.4.4 --- Transformation of plasmid into competent cell --- p.51 / Chapter 2.2.4.5 --- Plasmid extraction from bacterial culture --- p.51 / Chapter 2.2.5 --- Cell culture --- p.52 / Chapter 2.2.5.1 --- Cell culture of L929 --- p.52 / Chapter 2.2.5.2 --- Preservation of cells --- p.52 / Chapter 2.2.5.3 --- Treatment with TNF-α --- p.53 / Chapter 2.2.5.4 --- Fixation of cells --- p.53 / Chapter 2.2.6 --- Western blotting analysis --- p.54 / Chapter 2.2.6.1 --- Preparation of proteins from cells --- p.54 / Chapter 2.2.6.2 --- SDS polyacrylamide gel electrophoresis (SDS-PAGE) --- p.54 / Chapter 2.2.6.3 --- Electroblotting of protein --- p.55 / Chapter 2.2.6.4 --- Probing antibodies or aptamers for proteins --- p.55 / Chapter 2.2.6.5 --- Enhanced chemiluminescence (ECL) Assay --- p.56 / Chapter Chapter 3. --- Results --- p.57 / Chapter 3.1 --- Selection of aptamer against cytochrome c dotted on membrane with counter selection against BSA on membrane --- p.58 / Chapter 3.1.1 --- Selection process --- p.58 / Chapter 3.1.1.1 --- PCR cycles --- p.59 / Chapter 3.1.1.2 --- Polyclonal aptamer --- p.61 / Chapter 3.1.1.3 --- Monoclonal aptamer Contents --- p.63 / Chapter 3.1.2 --- Binding test of cy-1 to cy-4 to cytochrome c --- p.65 / Chapter 3.1.3 --- Binding of cy-3 to the cytochrome c dotted on PVDF membrane --- p.67 / Chapter 3.1.4 --- Test the binding of cy-3 with cytochrome c by ELISA --- p.68 / Chapter 3.1.5 --- Competitive binding between monoclonal aptamer cy-3 and anti-cytochrome c antibody --- p.70 / Chapter 3.1.6 --- Western blotting of pure cytochrome c by cy-3 --- p.71 / Chapter 3.1.7 --- Western blotting of pure cytochrome c from different species --- p.73 / Chapter 3.1.8 --- Cell lysate SDS-PAGE labeled with cy-3 --- p.75 / Chapter 3.1.9 --- Cell lysate labeled with cy-1 to cy-9 after SDS-PAGE --- p.77 / Chapter 3.2 --- Selection of cytochrome c-specific aptamer with counter selection against cytosolic protein --- p.79 / Chapter 3.2.1 --- Selection of aptamer against cytochrome c with counter selection against cytosolic cell lysate --- p.79 / Chapter 3.2.2 --- Selection of aptamer against cytochrome c by fixed cell followed by cytochrome c elution --- p.82 / Chapter 3.2.3 --- Selection of aptamer from cytochrome c band --- p.84 / Chapter 3.3 --- Primers Testing --- p.86 / Chapter 3.3.1 --- Cell lysate labeled with primers after SDS-PAGE --- p.86 / Chapter 3.3.2 --- Cell lysate labeled with cy-3 without primers --- p.87 / Chapter 3.3.3 --- Test the effect of sense oligonucleotide --- p.89 / Chapter 3.3.4 --- Sequence of monoclonal aptamer --- p.90 / Chapter 3.3.5 --- Cell lysate labeled with aptamers without primer ends --- p.92 / Chapter 3.3.6 --- Test of the aptamers after mutations --- p.93 / Chapter 3.3.7 --- Test for other biotinylated primers --- p.96 / Chapter 3.4 --- Elimination of non-specific binding --- p.98 / Chapter 3.4.1 --- Different types of cell lysate --- p.98 / Chapter 3.4.2 --- Heating effect on the non-specific binding --- p.99 / Chapter 3.4.3 --- Using milk as a blocking agent --- p.101 / Chapter 3.4.3.1 --- Milk blocked membrane --- p.101 / Chapter 3.4.3.2 --- Milk prevented the binding of aptamer to cytochrome c --- p.102 / Chapter 3.4.3.3 --- Cell lysate labeled with cy-3 after SDS-PAGE by using milk as blocking agent --- p.104 / Chapter 3.4.3.4 --- Aptamer selection against cytochrome c in the presence of milk --- p.105 / Chapter 3.4.4 --- Using DNA as a Blocking agent --- p.107 / Chapter 3.4.4.1 --- DNA blocked the non-specific binding --- p.107 / Chapter 3.4.4.2 --- Cell lysate labeled with cy-3 after SDS-PAGE by using DNA as blocking agent --- p.109 / Chapter 3.4.4.3 --- Selection against cytochrome c blocked by DNA --- p.110 / Chapter 3.4.4.4 --- "Labeling of cell lysate treated with DNase, RNase or both after SDS-PAGE" --- p.112 / Chapter 3.5 --- Photo-SELEX --- p.114 / Chapter 3.5.1 --- Selection process --- p.114 / Chapter 3.5.2 --- Cell lysate labeled with photo-aptamer --- p.116 / Chapter 3.5.3 --- Testing by immunoprecipitation --- p.118 / Chapter 3.6 --- Application --- p.120 / Chapter 3.6.1 --- Detection of the cytochrome c in cytosolic proteins after treatment of TNF-α --- p.120 / Chapter 3.6.2 --- Detection of the cytochrome c in total cell lysate after treatment of TNF-α --- p.123 / Chapter 3.6.3 --- Detection of cytochrome c in different cellular compartments after treatment of TNF-α --- p.125 / Chapter Chapter 4. --- Discussion --- p.130 / Chapter 4.1 --- General information --- p.131 / Chapter 4.1.1 --- The pool of oligonucleotide --- p.131 / Chapter 4.1.2 --- Design of oligonucleotides --- p.131 / Chapter 4.1.3 --- SELEX --- p.133 / Chapter 4.1.3.1 --- Buffer condition of selection --- p.133 / Chapter 4.1.3.2 --- Binding equilibrium --- p.134 / Chapter 4.1.3.3 --- Prevalence of matrix-binding species --- p.134 / Chapter 4.2 --- Selection --- p.135 / Chapter 4.2.1 --- Cycle numbers of PCR --- p.135 / Chapter 4.3 --- Assay of aptamers selected --- p.137 / Chapter 4.3.1 --- The use of biotin-streptavidin for recognition --- p.137 / Chapter 4.3.2 --- Polyclonal aptamers --- p.137 / Chapter 4.3.3 --- Monoclonal aptamer --- p.137 / Chapter 4.3.4 --- Cy-3 shows the highest affinity to cytochrome c --- p.138 / Chapter 4.3.5 --- The presence of non-specific binding --- p.138 / Chapter 4.4 --- Counter selection against cell lysate --- p.140 / Chapter 4.5 --- Primer testing --- p.143 / Chapter 4.6 --- Sequences and secondary structures of monoclonal aptamers --- p.145 / Chapter 4.7 --- Elimination of non-specific binding Contents --- p.147 / Chapter 4.7.1 --- Non-specific binding may be mediated by sequence-independent recognition --- p.147 / Chapter 4.7.2 --- Elimination of non-specific binding by milk --- p.147 / Chapter 4.7.3 --- Eliminate the non-specific binding by using DNA --- p.149 / Chapter 4.8 --- Photo-aptamer --- p.151 / Chapter 4.9 --- Application of the monoclonal aptamer cy-3 --- p.153 / Chapter 4.9.1 --- Aptamer can label cytochrome c as antibody does --- p.153 / Chapter 4.10 --- Conclusion I --- p.158 / Chapter 4.11 --- Conclusion II --- p.159 / Chapter Chapter 5 --- References --- p.161

Des inhibiteurs du complexe bc1 de la chaîne respiratoire mitochondriale ont été développés comme agents antimicrobiens pour lutter contre des pathogènes de l’Homme et de plantes. Ces drogues ciblent les poches catalytiques Qo et Qi formées par le cytochrome b. La comparaison de séquences de cette protéine montre que les sites Qo et Qi sont bien conservés entre les organismes mais qu’il existe toutefois des variations qui pourraient expliquer leur différence de sensibilité aux drogues. A l’aide du modèle levure S. cerevisiae, nous avons étudié les déterminants de la résistance/sensibilité naturelle à deux antipaludiques se liant au site Qo de Plasmodium: l’atovaquone et RCQ06. Nous avons notamment montré que le résidu 275 joue un rôle clé dans ce phénomène. Une approche similaire est actuellement utilisée pour identifier les facteurs de la sensibilité différentielle à deux drogues ciblant le site Qi des oomycètes. Malheureusement, des cas de résistance acquise à ces antimicrobiens ont été rapportés et ont pour origine des mutations dans le cytochrome b. De ce fait, de nouvelles molécules sont requises pour court-circuiter ces résistances. Au cours de ma thèse, nous avons mis au point un test qui permet de cribler des molécules capables d’inhiber la fonction respiratoire. Nous avons ainsi pu identifier un nouvel inhibiteur du complexe bc1 : D12. Nous avons ensuite déterminé le mode de liaison de cette molécule ainsi que celui d’un composé capable d’inhiber la prolifération de Plasmodium, HDQ, grâce à une collection de mutants des poches catalytiques. HDQ s’est avéré être un inhibiteur du site Qi. Il pourrait être utilisé avec un inhibiteur du site Qo afin de limiter l’apparition de mutations de résistance. D12 est un inhibiteur du site Qo qui est capable notamment de court-circuiter la mutation de résistance à des fongicides du site Qo G143A. Cette dernière a été trouvée chez de nombreux phytopathogènes, mais n’est cependant pas apparue chez des champignons possédant un intron immédiatement après le codon codant pour la glycine 143. En utilisant la levure, nous avons montré que la mutation empêche l’épissage de l’intron en altérant la structure exon/intron. Nous avons également identifié des mécanismes de « by-pass » qui permettent de restaurer la fonction respiratoire du mutant et qui pourraient apparaître chez les pathogènes. Les mutants créés au cours de ma thèse pourront aider à identifier, concevoir et caractériser de nouveaux antimicrobiens et à étudier l’apparition de mutations de résistance. / Inhibitors of the mitochondrial respiratory chain bc1 complex are currently used against human and plant pathogens. These drugs bind to Qo and Qi pockets of the mitochondrially-encoded cytochrome b. Comparison of the cytochrome b sequences shows that the Qo and Qi sites are well conserved between organisms. However, there are variations that could explain the differential sensitivity to respiratory inhibitors. In order to investigate the determinants of resistance / sensitivity to the antimalarial compounds, atovaquone and RCQO6, we used S.cerevisiae as a model. We showed that residue 275 plays a central role in the sensitivity to these drugs. We are now using a similar approach to identify the determinants of sensitivity towards two drugs targeting the oomycete Qi site. Unfortunately, cases of acquired resistance to these antimicrobial agents have been reported. They are caused by mutations in the cytochrome b. Thus, new molecules are required to bypass resistance. During my PhD, we developed a test to screen chemical libraries and identify inhibitors of the respiratory function. We identified a novel inhibitor of bc1 complex: D12. We determined the binding mode of D12 as well as of HDQ, a compound capable of inhibiting the proliferation of Plasmodium. To do this, we used a collection of mutants with alterations of the catalytic pockets. We showed that HDQ targets the Qi site. This finding suggests that HDQ could be used with an inhibitor of the Qo site to limit the emergence of resistance mutations. D12 is an inhibitor of Qo site and fully active against the enzyme harbouring the fungicide resistance mutation G143A. This mutation has been reported in many plant pathogenic fungi but has not evolved in fungi that harbour an intron immediately after the codon for G143. Using yeast, we showed that the mutation hinders the splicing of this intron by altering the exon / intron structure needed for efficient intron excision. We also identified by-pass mechanisms that restore respiratory function of the G143A mutant. These mechanisms identified in yeast could potentially arise in pathogenic fungi. Mutants created during my PhD will help to identify, design and characterize new drugs and to study the emergence of resistance mutations.

Thesis (Ph. D.)--West Virginia University, 2001. / Title from document title page. Document formatted into pages; contains viii, 199 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references (p. 176-195).

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 169-182).

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 165-195).

Thesis (Ph. D.)--West Virginia University, 2004. / Title from document title page. Document formatted into pages; contains xiii, 133 p. : ill. Vita. Includes abstract. Includes bibliographical references (p. 110-125).

Most administered drugs are metabolised in the liver by Phase I enzymes and more importantly by the cytochrome P450 (CYP) system. The extent of first-pass metabolism is important in determining whether the drug will have therapeutic or adverse effects after being administered to a patient. To date the CYP family has been shown to consist of 74 families denoted as CYPl to CYP118, and only a few families are significantly involved in drug metabolism. CYP3A4 is the most important isoenzyme followed by CYP2D6, CYP2C9, and CYP2C19 with a small contribution by CYP2E1, CYP2A6, and CYPlA4. CYP2D6 and CYP3A4 enzyme isoforms have been well established to exhibit interethnic and interindividual variability with regard to drug metabolising capacity. Mutation on the gene coding for a metabolising enzyme is a major cause of variation in drug metabolism. This mutation gives rise to allelic variants producing enzymes with altered metabolising activity. The presence of an allele with decreased metabolic activity in an individual gives rise to the poor metabolising (PM) phenotype. When the PM phenotype occurs at a frequency of more than 1% within a given population, then the term genetic polymorphism applies. The aberrant metabolic capacity translates into variable drug responses of more than 20-fold, leading to different susceptibility to sub-therapeutic effects or adverse drug reactions. A significant number of drugs, such as the B-adrenergic blockers, antidepressants, antipsychotic and antiarrhythmic agents, are entirely or partly metabolised by CYP2D6 and CYP3A4. Genetic polymorphism is especially important for drugs with a narrow therapeutic/toxicity window. Phenotyping involves the use of a probe drug that is administered to the subject, followed by determination of the parent drug and its metabolites in the urine. The aim of this study was to develop and validate an HPLC method for phenotypic determination of the CYP3A4 and CYP2D6 enzymes, followed by the application of the assay in a random heterogeneous population of males. Dextromethorphan (DXM) was used as an in vivo probe for simultaneous determination of the phenotypic expression of CYP2D6 and CYP3A4. An HPLC method coupled with a fluorescence detector was developed for the phenotypic determination of CYP2D6 and CYP3A4 iso-enzymes as determined by the concentration of dextromethorphan/dextrophan (DXM/DX) and dextromethorphan/3methoxy-morphinan (DXM/3MM) metabolic ratios respectively. The compounds were separated on a phenyl column (150 x 4,6 mm, 5-um particle size) serially connected to nitrile column (250 x 4,6 mm, 5-um particle size) using mobile phase of 80% (1.5% glacial acetic acid and 0.1% triethyl amine in distilled water) and 20% acetonitrile. Solid phase extraction was used to extract the analytes from urine samples using silica cartridges. The suitability of the method was demonstrated in a preliminary study with sixteen healthy Caucasian males. After a single oral 30 mg DXM dose, the volunteers were required to collect all urine samples voided 8 hours post oral dose. DXM/3HM and DXM/DX metabolic ratios were determined from collected urine samples. The method was validated for DXM and DX at a concentration range of 0.25 - 30 ug/ml, and at 0.025 - 3 ug/ml for 3MM. Calibration curves were linear with R2 values of at-least 0.999 for all compounds of interest. Recoveries were 97%, 93%, and 65% for DX, DXM and 3MM, respectively. The method was reproducible with intra-day precision having coefficients of variation percentage (CV%) of less than 17% for all analytes. Inter-day precision had a CV% of less than 14% for all analytes. The limit of detection was 30 ug/ml for all compounds. All volunteers were classified with an extensive metaboliser (EM) phenotype. In conclusion the method described is suitable for polymorphic determination of CYP2D6 and CYP3A4 in a population study, and may have value in further studies planned at investigating the critical issue of racial genetic polymorphism in ethnic groups in South Africa. / Thesis (M.Sc. (Pharm.))--North-West University, Potchefstroom Campus, 2004.