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Se desarrolló cinco sistemas automatizados utilizando análisis por inyección secuencial (SIA) y análisis por inyección en flujo multijeringa (MSFIA). Se desarrolló un SI-analizador para la determinación de formaldehído utilizando la reacción de Hantzsch. El analizador fue aplicado a la monitorización en un cultivo de P. pastoris. Se desarrolló un SI-analizador para la determinación de glicerol y sorbitol. Se utilizó periodato para la reacción de Malaprade. El formaldehído generado fue cuantificado con la reacción de Hantzsch. El sistema incluyó la dilución de la muestra y procedimientos seleccionado por decisión inteligente del programa o por el usuario. Se lo aplicó a la monitorización en cultivos de P. pastoris.Se desarrolló un sistema de electroforesis capilar (CE) acoplado a un SIA aplicado a la separación de nitrofenoles. Se acopló por primera vez MSFIA con CE para pre-concentración en fase sólida y separación de nitrofenoles. Ambos sistemas mostraban recuperaciones satisfactorias para aguas ambientales. / Five analytical systems using Sequential Injection Analysis (SIA) and Multisyringe Flow Injection Analysis (MSFIA) were developed and applied with satisfactory analytical performance. A SI-analyzer for formaldehyde was developed automating Hantzsch reaction. It was successfully applied to formaldehyde monitoring in continuous medium filtrate of P. pastoris cultivation. A second SI-analyzer was developed for the determination of glycerol and sorbitol. Periodate was used as additional reagent to carry out Malaprade reaction and formaldehyde generated by polyalcohol oxidation was quantified. The system included automated sample dilution and procedures for two working ranges, selected by smart software decision or user-input. It was applied to monitoring of P. pastoris cultivations.A capillary electrophoresis (CE) system was developed and coupled to SIA. It was applied to the determination of nitrophenols. MSFIA and CE were coupled for the first time. Solid phase concentration and separation of nitrophenols were automated. The systems were applied to environmental water samples.

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Tuberculosis (TB) is an infectious and fatal disease that ranks as the second leading killer worldwide. It is caused by Mycobacterium tuberculosis (Mtb) which is an obligate intracellular parasite that colonizes the alveolar macrophages of the immune system. The major health concern associated with TB is its co-infection with HIV and the development of strains with multi-drug resistance. The elimination of TB has been hindered due to the lack of understanding of the survival strategies used by this pathogen. Thus, research towards discovering new effective antibacterial drugs is necessary and a group of Mtb kinase enzymes were targeted in this study because these enzymes are crucial for metabolism, pathogenesis and, hence, the survival of Mtb. Kinases are a group of structurally distinct and diverse proteins that catalyze the transfer of the phosphate group from high energy donor molecules such as ATP (or GTP) to a substrate. The phosphorylation of proteins modifies the activity of specific proteins which is subsequently used to control complex cellular processes within Mtb. The starting point of this research targeted eight specific Mtb kinases namely; Nucleoside diphosphokinase, Homoserine kinase, Acetate kinase, Glycerol kinase, Thiamine monophosphate kinase, Ribokinase, Aspartokinase and Shikimate kinase. The aim of this project was to subclone the gene sequences for these eight recombinant Mtb kinases and express them in Escherichia coli, to purify the proteins and determine their activity. In the effort to find new lead compounds, the final stage of this study focused on the basic screening of the TB kinases against an extract prepared from Pelargonium sidoides, a medicinal plant, to identify any inhibitory effects. Although this traditional medicinal plant has been broadly researched and extensively used to treat TB, there is still a lack of understanding of this plant’s scientific curative effect. Various molecular and biochemical methods were used to achieve the aims of this project. The putative gene sequence was obtained from the annotated genome of H37Rv, deposited at NCBI as NC_000962.2. The genes encoding the kinases were successfully PCR-amplified from genomic DNA, cloned into an expression vector in-frame with a C- or N-terminal 6-histidine-tag and expressed in E. coli BL21 (DE3). The purification of the protein was complex, but various different methods and techniques were explored to obtain sufficient amounts of protein. The functional characterization of the kinases involved an HPLC enzyme assay that showed that the recombinant kinases were active. These enzymes were then screened against the potential inhibitory compounds in P. sidoides using enzyme assays to generate dose-response curves. This allowed an effective comparison not only of the Mtb kinases’ activity under normal conditions but also the kinases’ activity in the presence of a potential inhibitor. Overall, the inhibition of the enzymes required the presence of higher concentrations of the P. sidoides extract. However, the SK enzyme results presented a significantly higher inhibition and the lowest IC50 value, in comparison to the other kinases, which makes this kinase an attractive potential drug target against TB. In summation, cloning and purification of SK was successful, resulting in a concentration of 2030 μg/ml of purified enzyme and its activity analysis demonstrated enzyme functionality. This activity was reduced to zero in the presence of 1 x 102 mg/ml dilution of P. sidoides plant extract. This research conducted has extended the quality of information available in this field of study. These interesting results, proposing and identifying SK as a suitable potential target can be a starting point to significantly contribute and progress in this field of research, with the eventual goal of developing a drug to combat this fatal disease. / Life Sciences / M. Sc. (Life Sciences)

Les milieux réactionnels issus de l’étude de la réactivité de deux types de polyols, le glycérol et le diglycérol par réaction d’estérification directe avec l’acide undécylénique, catalysée par l’acide dodécylbenzène sulfonique (ADBS). Les résultats montrent que les ystèmes polyol/acide undécylénique donnent une émulsion eau dans huile (E/H). L’ajout de l’ADBS et de l’eau formée in-situ aux systèmes polyol/acide undécylénique ont permis de réduire la taille des gouttelettes de 50 μm à moins de 1 μm et d’obtenir un système organisé (micro-réacteur). L’augmentation de la température contribue à favoriser le transfert de matière dans les systèmes émulsionnés et / ou gélifiés et d’obtenir un système monophasique, homogène et structurés. L’étude de la réaction de ces systèmes avec une approche site à site (site OH / site COOH) a montré que lorsque le nombre de sites acides carboxyliques est inférieur à celui des sites hydroxyles, la synthèse est totalement sélective en esters partiels des deux polyols (glycérol et diglycérol). Les rendements sont supérieurs respectivement à 60% en esters partiels de glycérol et à 70% en esters partiels de diglycérol. La modélisation de la cinétique de synthèses et la régression des données cinétiques ont montré que la réaction est réversible d’ordre 2 et athermique. Les énergies d’activation calculées sont de 17 kcal/mol et 16 kcal/mol respectivement pour le monoundécénoate de glycérol (MUG) et le diundécénoate de glycérol (DUG). De plus, la méthodologie de recherche expérimentale a montrée que les variables (concentration en catalyseur ADBS et température) permettent d’obtenir le MUG avec un rendement de plus de 60% et une sélectivité en MUG de 80%. Ensuite, l’étude de la réactivité de la double liaison terminale du MUG en présence de deux agents oxydants pour engendrer des molécules bolaamphiphiles simples a été réalisée par H2O2 / acide formique et acide métachloroperbenzoïque (m-CPBA). Les résultats ont montré le 10,11-dihydroxy-monoundécénoate de glycérol (MUGDiol) est obtenu par oxydation au H2O2 / acide formique et le 10,11-époxy-monoundécénoate de glycérol (MUGE) par réaction d’époxydation avec la m-CPBA. L’ouverture de la fonction époxyde par des molécules aminées permet l’observation de nouvelles molécules bolaamphiphiles : le 10-hydroxy-N-11-((2-hydroxyéthyl)amino)monoundécénoate de glycérol(bola éthanolamineglycérol) et le N,N-11-(diaminobutan)-10-hydroxymonoundecanoate de glycérol (bola diaminobutaneglycérol). L’étude des propriétés physico-chimiques de ces molécules amphiphiles et bolaamphiphiles a permis de monter que toutes ces molécules sont de solvo-surfactants actifs aux interfaces et elles réduisent la tension interfaciale de l’eau jusqu’à la limite de la solubilité dans l’eau. L’adsorption des molécules ne vérifie pas le modèle de Gibbs. Le MUG et le MUDG s’auto-assemblent dans l’eau et donnent des nano-objets (vésicules et agrégats plats) et s’adsorbent sur des surfaces polaires et solides (silice et ciment). Ces deux molécules retiennent 30% et 56% molécules d’eau et le nombre de molécules d’eau fortement liée aux têtes polaires est de 21 et 49 respectivement pour le MUG et le MUDG. Pour es molécules bolaamphiphiles pures (MUGE et bola éthanolamineglycérol), elles retiennent plus de 56% de molécules d’eau et se lient à plus 53 molécules d’eau. L’ensemble de ces propriétés physico-chimiques a permis de répondre aux problématiques industrielles et de formuler un produit de cure, un agent de démoulage et un produit anti-adhérent. / This study is of the reactivity of two types of polyols (glycerol and diglycerol) by direct esterification reaction with undecylenic acid from castor oil. This reaction was catalyzed by dodecylbenzene sulfonic acid (DBSA). The first step was to study of polyol / undecylenic acid reaction systems by physico-chemical approach. The result have shown that these systems give water-in-oil (W / O) emulsion. Adding DBSA and water formed in-situ in polyol/undecylenic acid systems have reduced droplet size from 50 microns to less than 1 μm and form an organized system (micro-reactor). Increasing temperature can simplify transfers in emulsified systems and / or melted gel and to get a monophasic and homogeneous system. The only systems and aided by water formed in-situ assists the organization and structuring of gels. The reaction study of these systems was analyzed by gas chromatography. This showed that when the number of carboxylic acid function sites is less than the hydroxyl function site, synthesis is totally selective to partial esters of the two polyols (glycerol and diglycerol). The yields are higher than 60% in partial glycerol esters and 70% in partial diglycerol esters. The kinetic modeling of this synthesis and regression of kinetic data by the software GEPASI showed that the reaction follows the reversible 2 order and it is athermic. The calculated activation energy is 17 kcal/mol for the synthesis of glycerol monoundecenoate (GMU) and 16 kcal/mol for glycerol diundecenoate (GDU), these values are close to the theoretical values and they show that the reaction is happening at room temperature. Moreover, the response of the surface methodology shows that the variables chosen for the present study are temperature and catalyst concentration have a positive effect on the yield of the GMU. This approach was used to determine the optimum conditions for producing the GMU. Second study performed was of the reactivity of the terminal double bond of the GMU in presence of two oxidizing agents H 2 O 2 / formic acid and metachloroperbenzoic acid (m-CPBA), for synthesized bolaamphiphiles molecules was performed. The H 2 O 2 /formic acid was used to oxidize the double bond of GMU in diol function of glycerol 10,11-dihydroxymonoundecenoate (GMUDiol). The m-CPBA epoxidizes the double bond of GMU to give glycerol 10,11-epoxymonoundécénoate (GMUE). The opening of the epoxide function by aminoalcohol molecules are used to generate the new molecules bolaamphiphiles molecules: the bola ethanolamineglycerol and the bola diaminobutaneglycerol. The third step was the stady of the physico-chemical properties of pure amphiphilic and bolaamphiphiles molecules. The result was shown that all molecules are solvo-surfactants molecules and they are active in the interfaces (liquid/air and liquid/solid). The curves of surface tension of water do not respect the Gibbs rule. GMU and DGMU self- assemble in water and give nano-objects (vesicles and aggregates) in diluted solutions. In hydrogel, the molecules self-assemble in lamellar phase. In this lamellar phase, the amount of water retained is 56% and the number of water molecules strongly linked to the polar heads is 49 moles of water/diglycerol monoundecenoate molecule (DGMU). All these physico-chemical properties have permit to respond to industrial problems such as water retention for the curing product, self-assembly for demoulding concrete and for surface anti-adhesion and adsorption and finally foaming required for the aged bitumen regeneration. For pure bolaamphiphiles molecules (GEMU and ethanolamineglycerol bola) reduce the interfacial tension of water to the limit of the solubility of this bola molecules in water but do not provide a critical aggregation concentration (CAC). They retain more water molecules respectively between 56% and 63% water and the number of water molecules strongly bound with two polar heads groups pure bolaamphiphiles molecules is between 42 and 53.

Monolithische Phasen werden seit ca. 20 Jahren entwickelt und sind in den letzten Jahren eine attraktive Alternative zu etablierten mit Partikeln gefüllten Säulen geworden. Sie werden in anorganische Phasen und organische Polymermonolithe unterteilt. Monolithe bestehen aus einem einzigen, durchgehenden Stück. Charakteristisch ist das sie durchziehende Porennetzwerk, durch das der Eluent mit geringerem hydraulischen Widerstand fließen kann und das somit schnellere Flussraten ermöglicht. Polymermonolithe werden vorwiegend für die Separation großer Biomoleküle aufgrund eines durch Konvektion beschleunigten Massentransfers eingesetzt. Zudem sind sie über einen breiten pH-Wert-Bereich stabil und können direkt (in situ) im gewünschten Format polymerisiert werden. In der vorliegenden Arbeit gelang die Herstellung neuartiger epoxidbasierter Phasen nach einem von Weller et al. entwickelten Konzept, die im Affinitätsexperiment angewendet wurden. Die Herstellung erfolgte durch Autopolymerisation polyfunktioneller Glycidylether. Für die Funktionalisierung wurden nicht polymerisierte Epoxide genutzt. Als Monomere dienten TEPIC, GE 100 sowie GE 500. Die Arbeiten konzentrierten sich vor allem auf die bei Raumtemperatur durchführbaren Synthesen mit dem höher funktionellen GE 500. Die Polymerisationsbedingungen wurden hinsichtlich Porogenmischung und -anteil optimiert. Eine mit 75 Vol.-% Porogen (Dioxan/ MTBE (2:3)) hergestellte und mit rProtein A funktionalisierte Kapillarsäule (66 %, 12 µm, 7m2/g) ergab im Affinitätsexperiment eine Kapazität von 0,44 mg/mL aus Kaninchenserum isolierbarem IgG. Durch Beimischung von 60 % BDE konnte der Epoxidgehalt vervierfacht und die Porengröße auf 400 nm bei 59 % Porosität reduziert werden. Die spezifische Oberfläche wurde verdreifacht und die Kapazität präparierter Disks auf 0,90 mg/mL etwa verdoppelt. Die in dieser Arbeit entwickelten Disks können zur Isolierung von IgG aus einer komplexen Probe, wie beispielsweise Blutserum, eingesetzt werden. / Monolithic supports have been developed since 20 years and have become an attractive alternative to well-established columns packed with particles over the past years. They are classified into inorganic media and organic polymer monoliths. Monoliths consist of a single, continuous piece with an integrated characteristic porous network through which the eluent can flow with lower hydraulic resistance and which consequently offers higher flow rates. Due to an accelerated mass transfer caused by convection polymer monoliths are mainly used for separation of large biomolecules. In addition, they are stable over a wide pH range and can be polymerized directly (in situ) in the desired format. In the present work the successful preparation of new epoxide-based supports according to a concept introduced by Weller et al. as well as their application in affinity chromatography are reported. Their preparation was carried out by self-polymerization of polyfunctional glycidyl ethers and for functionalization non-polymerized epoxide groups were used. As monomers TEPIC, GE 100 and GE 500 were utilized. The work has focused especially on the polymerization of the higher functional GE 500, which can be perfomed at room temperature and was optimized in terms of both composition and amount of porogen. The extraction of IgG from rabbit serum with a capillary column (66 %, 12 µm, 7m2/g) prepared by 75 vol.-% porogen (dioxane/ MTBE (2:3)) and functionalized with rprotein A resulted in a capacity of 0,44 mg/mL. By addition of 60 % BDE the epoxide content was quadrupled and the pore size reduced to 400 nm while maintaining consistently high porosity of 59 %. The specific surface area was tripled and the capacity of prepared disks approximately doubled to 0,90 mg/mL. The disks developed in this work can be applied for the isolation of IgG from complex samples such as serum.

La adaptación de la levadura Saccharomyces cerevisiae a condiciones de alta osmolaridad está mediada por la vía de HOG ((high-osmolarity glycerol). La activación de esta vía induce una serie de respuestas que van a permitir la supervivencia celular en respuesta a estrés. La regulación génica constituye una respuesta clave para dicha supervivencia. Se han descrito cinco factores de transcripción regulados por Hog1 en respuesta a estrés osmótico. Sin embargo, éstos no pueden explicar la totalidad de los genes regulados por la MAPK Hog1. En el presente trabajo describimos cómo el complejo transcripcional formado por las proteínas Rtg1 y Rtg3 regula, a través de la quinasa Hog1, la expresión de un conjunto específico de genes. Hog1 fosforila Rtg1 y Rtg3, aunque ninguna de estas fosforilaciones son esenciales para regulación transcripcional en respuesta a estrés. Este trabajo también muestra cómo la deleción de proteínas RTG provoca osmosensibilidad celular, lo que indica que la integridad de la vía de RTG es esencial para la supervivencia celular frente a un estrés osmótico. / In Saccharomyces cerevisiae the adaptation to high osmolarity is mediated by the HOG (high-osmolarity glycerol) pathway, which elicits different cellular responses required for cell survival upon osmostress. Regulation of gene expression is a major adaptative response required for cell survival in response to osmotic stress. At least five transcription factors have been reported to be controlled by the Hog1 MAPK. However, they cannot account for the regulation of all of the genes under the control of the Hog1 MAPK. Here we show that the Rtg1/3 transcriptional complex regulates the expression of specific genes upon osmostress in a Hog1-dependent manner. Hog1 phosphorylates both Rtg1 and Rtg3 proteins. However, none of these phosphorylations are essential for the transcriptional regulation upon osmostress. Here we also show that the deletion of RTG proteins leads to osmosensitivity at high osmolarity, suggesting that the RTG-pathway integrity is essential for cell survival upon stress.

L'activació sostinguda de Hog1 porta a una inhibició del creixement cel·lular. En aquest treball, hem observat que el fenotip de letalitat causat per l'activació sostinguda de Hog1 és parcialment inhibida per la mutació del complexe SCFCDC4. La inhibició de la mort causada per l'activació sostinguda de Hog1 depèn de la via d'extensió de la vida. Quan Hog1 s'activa de manera sostinguda, la mutació al complexe SCFCDC4 fa que augmenti l'expressió gènica depenent de Msn2 i Msn4 que condueix a una sobreexpressió del gen PNC1 i a una hiperactivació de la deacetilassa Sir2. La hiperactivació de Sir2 és capaç d'inhibir la mort causada per l'activació sostinguda de Hog1. També hem observat que la mort cel·lular causada per l'activació sostinguda de Hog1 és deguda a una inducció d'apoptosi. L'apoptosi induïda per Hog1 és inhibida per la mutació al complexe SCFCDC4. Per tant, la via d'extensió de la vida és capaç de prevenir l'apoptosi a través d'un mecanisme desconegut. / Sustained Hog1 activation leads to an inhibition of cell growth. In this work, we have observed that the lethal phenotype caused by sustained Hog1 activation is prevented by SCFCDC4 mutants. The prevention of Hog1-induced cell death by SCFCDC4 mutation depends on the lifespan extension pathway. Upon sustained Hog1 activation, SCFCDC4 mutation increases Msn2 and Msn4 dependent gene expression that leads to a PNC1 overexpression and a Sir2 deacetylase hyperactivation. Then, hyperactivation of Sir2 is able to prevent cell death caused by sustained Hog1 activation. We have also observed that cell death upon sustained Hog1 activation is due to an induction of apoptosis. The apoptosis induced by Hog1 is decreased by SCFCDC4 mutation. Therefore, lifespan extension pathway is able to prevent apoptosis by an unknown mechanism.