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Alzheimer's disease (AD) accounts for around two thirds of all dementia cases and an increase in life expectancy of the population has resulted in a substantial increase in dementia cases and with that a rise in AD. AD is a debilitating and ultimately fatal neurodegenerative disorder of the elderly, and despite being identified over a century ago, the current treatments do not treat the underlying causes behind the disease, instead they help to mask the symptoms of the disease and prolong the brain's remaining function. It is therefore vital that an effective, disease modifying treatment for this disease is established as soon as possible. Soluble intracellular forms of amyloid β (peptide Aβ), a hallmark of AD have been identified and intracellular targets of Aβ are being investigated as potential drug targets for the disease. Two key intracellular, mitochondrial proteins investigated as potential drug targets: amyloid binding alcohol dehydrogenase (ABAD) and cyclophilin D (CypD) are the focus of the work reported in this thesis. To begin identifying potential inhibitors of the ABAD-Aβ interaction, a two-pronged approach was taken. Firstly, a series of analogues based on a known inhibitor of the interaction were tested using a variety of biophysical assays, for their therapeutic affect on the interaction, and secondly a fragment based screening approach was used to identify new small molecule binding partners of ABAD which could potentially be modified to produced inhibitors of the ABAD-Aβ interaction. Three different CypD constructs have been successfully expressed and purified, and taken into crystal trials. It is hoped that these constructs can be used to significantly aid the progress of identifying any potential inhibitors and binding partners of CypD that may produce therapeutic effects, and in the future could lead to the identification of an effective disease modifying drug in the treatment of AD. The work reported in this thesis has built upon previously reported findings and the groundwork has also been established for several in vitro biophysical assays, these include for example: measuring ABAD enzyme activity, and the novel morphology specific Aβ aggregation assay, which can be used as screening tools to help identify potential inhibitors of these interactions. Both the ABAD-Aβ interaction, and the blockade of CypD are known to be drug targets in the treatment of AD, and by elucidating the molecular mechanisms behind these interactions, through implementing biophysical assays, this will help in the identification and design of potential new therapeutic agents for the treatment of AD.

A incidência do câncer de cabeça e pescoço (CCP) vem crescendo substancialmente nos últimos anos, inclusive no Brasil. Esse aumento está em parte relacionado com o consumo de álcool e tabaco, mas a susceptibilidade genética individual também deve ser considerada. O objetivo desse trabalho foi avaliar a freqüência de polimorfismos em genes que codificam as enzimas de metabolização do álcool em pacientes com câncer de cabeça e pescoço do Hospital Heliópolis da cidade de São Paulo, comparados com um grupo de pacientes do mesmo hospital, sem diagnóstico de câncer. Foram investigados polimorfismos genéticos das enzimas álcool desidrogenase (ADH1B Arg48His, ADH1B Arg370Cys, ADH1C Ile350Val) e do citocromo P450 (CYP2E1 PstI), pela técnica PCR-RFLP, em 451 indivíduos, sendo 207 pacientes com CCP (confirmados histopatologicamente, 184 homens e 23 mulheres, idade média 54,3 ± 7,8 anos) e 244 controles (225 homens e 19 mulheres, idade média 53,6 ± 9,3 anos). O hábito de fumar foi relatado por 80% dos pacientes com CCP e 50% dos controles o que aumentou mais de dez vezes o risco de câncer (OR=11,1; 95% IC; 4,89-25,19). Apenas 7% dos pacientes com CCP relataram nunca haver consumido álcool em comparação com 22,5% dos controles hábito que aumentou mais de quatro vezes o risco de CCP (OR=4,39 95% IC; 2,35-8,22). Verificou-se que o consumo diário acima de 30,655g/L/dia de álcool (72,5% dos pacientes com CCP e 35,2% dos controles) estava associado ao maior risco de CCP (Curva de ROC). A análise dos polimorfismos genéticos revelou que o genótipo mutado ADH1B Arg48His em homozigose ou heterozigose foi mais freqüente nos controles (12,7%) do que nos pacientes com CCP (5,8%) conferindo proteção à doença (OR=0,42; 95% IC; 0,21-0,85). Resultados similares foram observados para os indivíduos com os haplótipos ADH1B*2 (OR=0,41; 95% IC; 0,20-0,82) ou ADH1B*2/ADH1C*1 (OR=0,32; 95% IC; 0,13-0,79). Análise de regressão múltipla escalonada revelou que os indivíduos com o genótipo mutante ADH1B Arg48His que consomem quantidades de álcool inferiores a 30g/L/dia mantém o risco diminuído de CCP (OR=0,12; 95% IC; 0,03-0,52). Entretanto, quando o consumo diário de bebidas alcoólicas supera 30,655g/L/dia o risco de CCP é aumentado independente da presença (OR=4,42; 95% IC; 1,21-16,11).ou não do genótipo ADH1B Arg48His com o alelo mutado (OR= 3,01; 95% CI, 1,90-4,78). Conclusão: Os genótipos de metabolização rápida do álcool podem proteger contra o CCP quando a quantidade de álcool ingerida for menor que 30,655 g/l/dia. / Garcia, S.M.N. Alcohol metabolizing genes and the risk of head and neck cancer. 2009. Dissertação (Mestrado)- Faculdade de Medicina, Universidade de São Paulo, São Paulo. The incidence of head and neck cancer (HNC) has increased substantially in the last years, including in Brazil. This increase is associated to alcohol and tobacco consumption, but genetic susceptibility also should be considered. The aim of this study was to evaluate the frequency of the polymorphism in genes of alcohol metabolizing enzymes in patients with head and neck cancer (HNC) of the Heliópolis Hospital in São Paulo, compared with a group from the same hospital, without the diagnosis of cancer. The genetic polymorphisms of the alcohol desydrogenase enzyme (ADH1C Ile350Val, ADH1B Arg48His, ADH1B Arg370Cys) and of the P450 citochrome enzyme (CYP2E1 PstI) was investigated by PCR-RFLP, in 451 individuals, being 207 histopathologically confirmed HNC patients (184 male and 23 female, mean age 54,3 ± 7,8 years) and 244 controls (225 male and 19 female, mean age 53,6 ± 9,3 years) selected in the same hospital. The smoking habit was revealed by 80% of the patients with HNC and 50% of the controls, the difference between the groups increased the HNC risk more than ten times (OR=11.1; 95% IC; 4.89-25.19). Just 7% of the patients reported never alcohol use against 22.5% of the controls, increasing more than four times the risk of HNC (OR=4.39 95% IC; 2.35-8.22). The daily consumption of alcohol above 30.655g/L/day (72.5% of the patients with HNC and 35.2% of the controls) was associated with increased risk of the HNC. The analysis of the genetic polymorphisms revealed that the mutate genotype ADH1B Arg48His was more frequent in the controls (12.7%) than in the patients with HNC (5.8%) conferring protection to the disease (OR=0.42; 95% IC; 0.21-0.85). Similar results were observed for individuals with ADH1B*2 (OR=0.41; 95% CI; 0.20-0.82) or ADH1B*2/ADH1C*1 (OR=0.32; 95% CI; 0.13-0.79) haplotypes. Multiple regression analyses showed that the mutant genotype ADH1B Arg48His was associated to HNC protection for those that consumed alcohol lower than 30 g/l/day (OR=0.12; 95% IC; 0.03- 0.52).However, when the daily alcohol consumption exceeded 30.655g/L/day the HNC risk was higher in the presence (OR=4.42; 95% IC; 1.21-16.11) or not of the genotype ADH1B Arg48His with the mutate allele (OR= 3.01; 95% CI, 1.90-4.78).The fast alcohol metabolizing genotypes seams to prevent HNC when the amount of alcohol intake is lower than 30.655 g/L/day.

<p>Enzymes are useful tools as specific analytical reagents. Two different analysis methods were developed for use in the separate fields of protein science and organic synthesis. Both methods rely on the substrate specificity of enzymes. Enzyme catalysis and substrate specificity is described and put in context with each of the two developed methods.</p><p>In <strong>paper I </strong>a method for C-terminal peptide sequencing was developed based on conventional Carboxypeptidase Y digestion combined with matrix assisted laser desorption/ionization mass spectrometry. An alternative nucleophile was used to obtain a stable peptide ladder and improve sequence coverage.</p><p>In paper<strong> II </strong>and <strong>III</strong>, three different enzymes were used for rapid analysis of enantiomeric excess and conversion of O-acylated cyanohydrins synthesized by a defined protocol. Horse liver alcohol dehydrogenase,<em> Candida antarctica</em> lipase<strong> </strong>B<strong> </strong>and pig liver esterase were sequentially added to a solution containing the O-acylated cyanohydrin. Each enzyme caused a drop in absorbance from oxidation of NADH to NAD⁺. The conversion and enantiomeric excess of the sample could be calculated from the relative differences in absorbance.</p>

In order to investigate the effect of food restriction on fat mobilization in horses with impaction in left ventral colon during treatment, serum triglycerides, NEFA and total bilirubine (TB) were measured before and after treatment. On another side, the determination of alcohol dehydrogenase (ADH) activity in serum could facilitate the distinguishing of the non-strangulating intestinal obstruction from the potential fatal strangulation obstruction and could submit a new prognostic biochemical parameter for intestinal strangulation. With the intention of giving a highlight over the analgesic effect of Buscopan® compositum in horses with colic, it was attempted to investigate the effect of Buscopan® compositum on the intestinal motility of healthy conscious horses in different regions of intestine. A significant elevation of NEFA and TB was observed in horses with impaction in left ventral colon at admission. By relieving the impaction, there was a significant elevation of triglycerides in comparison to its level at admission. There was a significant increase in ADH activity in all horses with acute intestinal obstruction. ADH activity was significantly higher in horses with strangulation in comparison to non-strangulation obstruction. There was only a significant correlation between ADH and lactate in horses with non-strangulation obstruction and colon torsion. Only AST and GLDH were significantly increased in horses with colon torsion. ADH activity > 20 U/l had 80.56% specificity and 80.49% sensitivity for discriminating horses with intestinal strangulation from non-strangulation obstruction. ADH activity < 80 U/l had 94.44% specificity and 66.67% sensitivity for survival. Buscopan® compositum had an immediate, rapid and significant (p< 0.05) reduction of duodenal, cecal and left ventral colon contractions after application. Cecal and left ventral colon contractions restored rapidly their normal contractions after 30 min, while duodenal contractions returned to the normal rate after 120 min of Buscopan® compositum administration. The horses with impaction in left ventral colon are susceptible to fat mobilization during the period of treatment as a result of food restriction. It was characterized by a revisable hypertri-glyceridemia and hyperbililrubinemia. Serum ADH activity could have a useful clinical value in detecting the intestinal strangulation and predicting the prognosis in horses with intestinal strangulation. Buscopan® compositum at its therapeutic dosage has an immediate, potent, short-lived reductive effect on cecum and left ventral colon contractions but a minor, longer effect on the duodenal contractions. Therefore, it is thought to be more effective in treatment of spasmodic colic than in large colon impaction. / Um den Effekt der Nahrungskarenz auf die Fettmobilisation bei Pferden mit Verstopfung der linken ventralen Längslagen des Kolons während der Behandlung zu untersuchen, wurden Triglyceride (TG), freie Fettsäuren (FFS) und Gesamtbilirubin (GB) bestimmt. Andererseits ermöglicht die Bestimmung der Aktivität der Alkoholdehydrogenase (ADH) im Serum die Unterscheidung zwischen einer nichtstrangulierenden intestinalen Obstruktion und einer potentiell tödlichen Strangulation. ADH kann somit als ein neuer prognostischer biochemischer Parameter für die intestinale Strangulation eingesetzt werden. Um den spasmolytischen Effekt von Buscopan compositum bei Pferden mit Kolik zu untersuchen, wurde der Effekt von Buscopan compositum auf die intestinale Kontraktion von gesunden Pferden in verschiedenen Regionen des Darmes getestet. Eine signifikante Erhöhung der FFS und des GB wurde bei Aufnahme von Pferden mit einer Verstopfung in der linken ventralen Längslagen festgestellt. Nach der Behandlung der Verstopfung konnte eine signifikante Erhöhung der Konzentration von TG, bezogen auf die TG Konzentration bei Aufnahme in die Klinik, festgestellt werden. Bei Pferden mit akuter intestinaler Obstruktion wurde eine signifikante Erhöhung der Aktivität der ADH beobachtet. Die Aktivität der ADH war bei Pferden mit einer Strangulation signifikant höher als bei Pferden, die eine nichtstrangulierende Obstruktion des Darmes hatten. Bei Pferden mit einer nichtstrangulierenden Obstruktion oder einer Kolontorsion wurde eine positive Korrelation zwischen der ADH-Aktivität und der Laktatkonzentration im Serum festgestellt. Nur bei Pferden mit Kolontorsion waren die Aktivitäten von AST und GLDH signifikant erhöht. Für die Unterscheidung zwischen Pferden mit einer intestinalen Strangulation oder einer nichtstrangulierenden Obstruktion wurde für die ADH- Aktivität größer als 20 U/l eine Spezifität von 80,56% und eine Sensitivität von 80,49% ermittelt. Eine ADH-Aktivität kleiner 80 U/l zeigt, mit einer Spezifität von 94,44% und einer Sensitivität von 66,67%, eine günstige Prognose für das Überleben des Pferdes an. Nach Gabe von Buscopan® compositum trat eine sofortige schnelle und signifikante (p<0,05) Reduktion der Kontraktionen im Duodenum, Zäkum und den linken ventralen Längslagen ein. Die Kontraktionen des Zäkums und der linken ventralen Längslagen normalisierten sich schnell innerhalb von 30 min, wogegen die Kontraktionen des Duodenums erst 120 min nach der Applikation von Buscopan® compositum den Normalzustand erreichten. Pferde mit einer Verstopfung in der linken ventralen Längslagen des Kolons sind während der medizinischen Behandlung anfällig für Fettmobilisation aufgrund der reduzierten Futter-aufnahme. Dies ist gekennzeichnet durch eine reversible Hypertriglyceridämie und eine Hyperbilirubinämie. Die Aktivität von ADH im Serum kann ein nützlicher klinischer Parameter sein, um eine intestinale Strangulation zu identifizieren und bietet sich auch als prognostischer Marker bei intestinaler Strangulation an. Die Applikation von Buscopan® compositum in der therapeutischen Dosierung hat eine sofortige, potente und kurzzeitige Reduktion der Kontraktionen des Zäkums und der linken ventralen Längslage aber einen geringen und länger anhaltenden Effekt auf die duodenalen Kontraktionen zur Folge. Daraus folgt, dass Buscopan® compositum bei der Behandlung von Krampfkoliken effektiver ist als bei Verstopfungen des großen Kolons.

The increasing demands for biopharmaceuticals to treat different diseases have raised concerns about controlling the quality and efficacy of such pharmaceuticals. The design and formulation of a stable protein or peptide based biopharmaceutical runs into the limitation that at high concentrations (> 100 mg/ml) or during long storage process the drug undergoes aggregation. During synthesis, purification, storage or packaging of these drugs different kinds of stresses like chemical, oxidative, thermal, shear, etc. are encountered. These stresses promote the non-native aggregation of protein and peptide based drugs. Injection or administration of such drugs if contaminated with aggregates causes patient discomfort or development of an antibody which can adversely affect patient’s conditions. This brings out the necessity of finding a way so that such aggregation is avoided. Nanoparticles have been used as vehicles for drug delivery and diagnostic agents in biology for a while. The surface of the nanoparticles is known to adsorb small as well as large molecules with different kinetics and energetics of interaction. I have used nanoparticles to adsorb proteins to protect them against aggregation when they are subjected to denaturing conditions. The effectiveness of the nanoparticles in stopping protein aggregation, recovery of the proteins and reversibility of the adsorption process, the catalytic activity of the proteins before and after adsorption on the surface have all been studied in details. The work described here has been divided in 8 chapters and the contents of each chapter are described below. In Chapter 1 I have provided a brief introduction to the protein aggregation problem. The motivation and scope of the current work has been presented in this chapter. Materials and methods have been described in Chapter 2. Synthesis of gold and silica nanoparticles, their characterization and stability under experimental conditions have been illustrated in this chapter. The spectroscopic assays and techniques which I have used to study the effect of gold and silica nanoparticles on protein aggregation have been discussed at lengths in this chapter. In Chapter 3 I have demonstrated the effect of gold nanoparticles on thermal aggregation of alcohol dehydrogenase (ADH). The size of the nanoparticle was varied in the range of 15-60 nm and the effect was measured by various spectroscopic assays and techniques. I have observed that gold nanoparticles prevent thermal aggregation of ADH and the efficiency is high. Gold nanoparticles in nanomolar or even picomolar concentrations are capable of preventing the aggregation of ADH at micromolar concentrations. In Chapter 4 the role of gold nanoparticles as suppressor of protein aggregation was extended to another protein, insulin. Chemically induced aggregation of insulin using dithiothreitol (DTT) in the presence of gold nanoparticles was studied in the same manner as was done for ADH. Similar prevention property of gold nanoparticles was established by making the observation independent of the method of denaturation or the type of protein used in the prevention experiments. In Chapter 5 huge second harmonic light scattering (SHS) signal from pure gold nanoparticles has been used to measure the free energy of interaction of ADH and insulin with nanoparticles in solution, for the first time. The change in the second harmonic scattered signal was monitored which decreased steadily as a function of added protein concentration to the aqueous solution of gold nanoparticles. The fitting of the second harmonic signal decay was done with a modified Langmuir adsorption isotherm to extract the free energy change in the interaction and the number of protein molecules adsorbed on the surface. In Chapter 6 I have demonstrated a way to recover the adsorbed ADH and insulin from the gold nanoparticle surface and tested the activity of ADH by an assay. The structure of the proteins in the adsorbed state has been probed by CD spectroscopy and described in this chapter. It is found that ADH retains its activity in the adsorbed state. Both the proteins retain the native secondary structures in their adsorbed state. However, the structures change drastically under denaturing conditions. In Chapter 7 the effect silica nanoparticles which are known to have hydrophilic surface has been examined on the aggregation of ADH and insulin in pretty much the same way as was done with gold nanoparticles. The efficiency of silica nanoparticle was found to be lower compared to gold nanoparticles. In addition, the size dependency of prevention efficiency of silica and gold nanoparticles was found to be completely opposite to each other. In Chapter 8 I have presented the overall summary and possible future directions of this work

A incidência do câncer de cabeça e pescoço (CCP) vem crescendo substancialmente nos últimos anos, inclusive no Brasil. Esse aumento está em parte relacionado com o consumo de álcool e tabaco, mas a susceptibilidade genética individual também deve ser considerada. O objetivo desse trabalho foi avaliar a freqüência de polimorfismos em genes que codificam as enzimas de metabolização do álcool em pacientes com câncer de cabeça e pescoço do Hospital Heliópolis da cidade de São Paulo, comparados com um grupo de pacientes do mesmo hospital, sem diagnóstico de câncer. Foram investigados polimorfismos genéticos das enzimas álcool desidrogenase (ADH1B Arg48His, ADH1B Arg370Cys, ADH1C Ile350Val) e do citocromo P450 (CYP2E1 PstI), pela técnica PCR-RFLP, em 451 indivíduos, sendo 207 pacientes com CCP (confirmados histopatologicamente, 184 homens e 23 mulheres, idade média 54,3 ± 7,8 anos) e 244 controles (225 homens e 19 mulheres, idade média 53,6 ± 9,3 anos). O hábito de fumar foi relatado por 80% dos pacientes com CCP e 50% dos controles o que aumentou mais de dez vezes o risco de câncer (OR=11,1; 95% IC; 4,89-25,19). Apenas 7% dos pacientes com CCP relataram nunca haver consumido álcool em comparação com 22,5% dos controles hábito que aumentou mais de quatro vezes o risco de CCP (OR=4,39 95% IC; 2,35-8,22). Verificou-se que o consumo diário acima de 30,655g/L/dia de álcool (72,5% dos pacientes com CCP e 35,2% dos controles) estava associado ao maior risco de CCP (Curva de ROC). A análise dos polimorfismos genéticos revelou que o genótipo mutado ADH1B Arg48His em homozigose ou heterozigose foi mais freqüente nos controles (12,7%) do que nos pacientes com CCP (5,8%) conferindo proteção à doença (OR=0,42; 95% IC; 0,21-0,85). Resultados similares foram observados para os indivíduos com os haplótipos ADH1B*2 (OR=0,41; 95% IC; 0,20-0,82) ou ADH1B*2/ADH1C*1 (OR=0,32; 95% IC; 0,13-0,79). Análise de regressão múltipla escalonada revelou que os indivíduos com o genótipo mutante ADH1B Arg48His que consomem quantidades de álcool inferiores a 30g/L/dia mantém o risco diminuído de CCP (OR=0,12; 95% IC; 0,03-0,52). Entretanto, quando o consumo diário de bebidas alcoólicas supera 30,655g/L/dia o risco de CCP é aumentado independente da presença (OR=4,42; 95% IC; 1,21-16,11).ou não do genótipo ADH1B Arg48His com o alelo mutado (OR= 3,01; 95% CI, 1,90-4,78). Conclusão: Os genótipos de metabolização rápida do álcool podem proteger contra o CCP quando a quantidade de álcool ingerida for menor que 30,655 g/l/dia. / Garcia, S.M.N. Alcohol metabolizing genes and the risk of head and neck cancer. 2009. Dissertação (Mestrado)- Faculdade de Medicina, Universidade de São Paulo, São Paulo. The incidence of head and neck cancer (HNC) has increased substantially in the last years, including in Brazil. This increase is associated to alcohol and tobacco consumption, but genetic susceptibility also should be considered. The aim of this study was to evaluate the frequency of the polymorphism in genes of alcohol metabolizing enzymes in patients with head and neck cancer (HNC) of the Heliópolis Hospital in São Paulo, compared with a group from the same hospital, without the diagnosis of cancer. The genetic polymorphisms of the alcohol desydrogenase enzyme (ADH1C Ile350Val, ADH1B Arg48His, ADH1B Arg370Cys) and of the P450 citochrome enzyme (CYP2E1 PstI) was investigated by PCR-RFLP, in 451 individuals, being 207 histopathologically confirmed HNC patients (184 male and 23 female, mean age 54,3 ± 7,8 years) and 244 controls (225 male and 19 female, mean age 53,6 ± 9,3 years) selected in the same hospital. The smoking habit was revealed by 80% of the patients with HNC and 50% of the controls, the difference between the groups increased the HNC risk more than ten times (OR=11.1; 95% IC; 4.89-25.19). Just 7% of the patients reported never alcohol use against 22.5% of the controls, increasing more than four times the risk of HNC (OR=4.39 95% IC; 2.35-8.22). The daily consumption of alcohol above 30.655g/L/day (72.5% of the patients with HNC and 35.2% of the controls) was associated with increased risk of the HNC. The analysis of the genetic polymorphisms revealed that the mutate genotype ADH1B Arg48His was more frequent in the controls (12.7%) than in the patients with HNC (5.8%) conferring protection to the disease (OR=0.42; 95% IC; 0.21-0.85). Similar results were observed for individuals with ADH1B*2 (OR=0.41; 95% CI; 0.20-0.82) or ADH1B*2/ADH1C*1 (OR=0.32; 95% CI; 0.13-0.79) haplotypes. Multiple regression analyses showed that the mutant genotype ADH1B Arg48His was associated to HNC protection for those that consumed alcohol lower than 30 g/l/day (OR=0.12; 95% IC; 0.03- 0.52).However, when the daily alcohol consumption exceeded 30.655g/L/day the HNC risk was higher in the presence (OR=4.42; 95% IC; 1.21-16.11) or not of the genotype ADH1B Arg48His with the mutate allele (OR= 3.01; 95% CI, 1.90-4.78).The fast alcohol metabolizing genotypes seams to prevent HNC when the amount of alcohol intake is lower than 30.655 g/L/day.

La récente définition de Brachypodium distachyon comme modèle des graminées en fait un organisme de choix pour l’étude de leur paroi cellulaire, en particulier dans le cadre de leur utilisation comme matière première renouvelable pour le bioéthanol de seconde génération. Les lignines, dont les trois unités (H, G et S) proviennent de la polymérisation des monolignols, sont associées aux acides hydroxycinnamiques dans la paroi des céréales et représentent l’obstacle majeur à l’exploitation industrielle de la biomasse lignocellulosique. L’acquisition de connaissances sur les mécanismes dirigeant leur mise en place et leur organisation permettrait d’identifier des facteurs modulant les rendements de production qui y sont associés. Quatre familles de gènes ont été étudiées et l’implication dans la voie de biosynthèse des monolignols de trois gènes a été montrée : BdF5H2 possède une activité férulate-5-hydroxylase permettant la synthèse des précurseurs des unités S des lignines, BdCOMT3 est l’isoforme principale des acide cafféique O-Méthyltransférases et sa perte partielle de fonction cause une diminution de la quantité de lignine, la modification du rapport S/G et une baisse de quantité d’acide p-coumarique dans deux lignées mutantes indépendantes. Enfin, BdCAD1 est l’isoforme principale des alcools cinnamylique déshydrogénases : sa perte de fonction dans deux lignées indépendantes cause la diminution de la quantité globale de lignine et d’acide p-coumarique, une baisse du rapport S/G ainsi que l’accumulation de sinapaldéhyde. Par ailleurs ces deux lignées présentent des rendements de saccharification augmentés de plus d’un quart par rapport au sauvage. / Brachypodium distachyon was recently adopted as an experimental model for grass species. As such, it is used to study grass cell wall, in particular in the context of their use as renewable feedstock for the production of second generation bioethanol. Lignins are polymers of three main units (H, G and S) originating from the polymerization of monolignols, and are linked to hydroxycinnamic acids in grasses. They constitute the main bottleneck to industrial processes targeting lignocellulosic biomass and improving the understanding of the mechanisms directing their structure and deposition could lead to the identification of the factors modulating associated production yields. Four gene families were studied and the involvement of three genes in the monolignols biosynthetic pathway was shown: BdF5H2 displays a ferulate-5-hydroxylase activity enabling the synthesis of the S lignin units, BdCOMT3 is the main caffeic acid O-methyltransferase and its partial loss of function in two independent mutant lines leads to the reduction of lignin content, the modification of the S/G units ratio and a decrease in p-coumaric acid accumulation. BdCAD1 is the main cinnamyl alcohol dehydrogenase isoform: its loss of function in two independent mutant lines results in a decrease in lignin content and of the S/G ratio and the accumulation of sinapaldehyde. Moreover, these two lines display significatively increased saccharification yields.

Enzymes are useful tools as specific analytical reagents. Two different analysis methods were developed for use in the separate fields of protein science and organic synthesis. Both methods rely on the substrate specificity of enzymes. Enzyme catalysis and substrate specificity is described and put in context with each of the two developed methods. In paper I a method for C-terminal peptide sequencing was developed based on conventional Carboxypeptidase Y digestion combined with matrix assisted laser desorption/ionization mass spectrometry. An alternative nucleophile was used to obtain a stable peptide ladder and improve sequence coverage. In paper II and III, three different enzymes were used for rapid analysis of enantiomeric excess and conversion of O-acylated cyanohydrins synthesized by a defined protocol. Horse liver alcohol dehydrogenase, Candida antarctica lipase B and pig liver esterase were sequentially added to a solution containing the O-acylated cyanohydrin. Each enzyme caused a drop in absorbance from oxidation of NADH to NAD+. The conversion and enantiomeric excess of the sample could be calculated from the relative differences in absorbance. / QC 20101108

Une attention croissante est portée aux cascades multi enzymatiques pour l’élaboration de procédés de synthèse plus efficaces. Cependant, le contrôle de l’expression hétérologue de plusieurs gènes dans un même hôte s’avère difficile et peut mener à un déséquilibre du flux réactionnel. Pour exploiter au mieux les avantages d’une cascade in vivo, il est nécessaire d’ajuster les activités de chaque étape, et de construire des catalyseurs cellulaires capables de programmer la stœchiométrie des enzymes. Nous avons développé dans ce projet une approche originale pour moduler le ratio de deux enzymes in cellulo en jouant sur le nombre de copies de plasmides par cellule (PCN). Nous avons choisi comme modèle un système autosuffisant associant une Alcool Déshydrogénase (ADH) et une Baeyer-Villiger MonoOxygenase (BVMO), NADP(H)-dépendantes. Plusieurs plasmides recombinants portant les deux gènes ont été conçus et combinés dans E. coli. Les souches de co-expression construites ont été comparées en termes de PCN, de production d’enzymes et d’activité. Nous avons montré l’importance d’un choix judicieux de la combinaison de plasmides ainsi que l’existence d’une corrélation entre ratios d’enzymes et activité. Nos biocatalyseurs s’étendent sur une gamme allant du système inactif à un système affichant un TTN d’environ 6000. Ce système a permis la synthèse de lactones d’intérêt industriel, la dihydrocoumarine et la caprolactone, à partir d’indanol et de cyclohexanol. Enfin, sur ce modèle de combinaison de plasmides, trois nouveaux biocatalyseurs cellulaires, associant l’ADH à diverses BVMOs, ont été créés afin d’élargir la gamme d’esters et de lactones synthétisables à partir d’alcools. / Growing attention is paid to multienzymatic cascades to develop more efficient synthetic processes. However, in in cellulo process, the control of the simultaneous heterologous expression of several genes in the same host is often difficult and can lead to imbalances in the reaction flow. To exploit the benefits of cascades, activities of each step have to be adjusted and thus, cellular biocatalysts capable of programming enzymes stoichiometry have to be constructed. In this work, to modulate the stoichiometry of two enzymes in vivo, we developed an original approach based on the copy number per cell of plasmids (PCN) used as vectors. The PCN is regulated in bacteria by three main mechanisms leading, according to the replicon, to low, medium or high PCN. As proof of concept, we chose a self-sufficient system combining an Alcohol Dehydrogenase (ADH) and a Baeyer-Villiger MonoOxygenase (BVMO), both NADP(H)-dependent. Several recombinant plasmids harboring both genes were designed and combined in E. coli. Coexpression strains constructed were compared in terms of PCN, enzyme production and activity. We showed the importance of a judicious choice of plasmids combination and the existence of a correlation between enzymes ratios and activity. Our biocatalysts ranged from an inactive system to a system with a TTN of about 6000. This system allowed the synthesis of lactones of industrial interest, dihydrocoumarin and caprolactone, via double oxidation of indanol and cyclohexanol. Finally, based on this plasmids combination model, three new cellular biocatalysts combining ADH with various BVMOs were designed to broaden the range of esters and lactones synthesizable from alcohols.