Ingeniera Civil en Biotecnología / Ingeniera Civil Química / La enzima Monoamino oxidasa A (MAO-A) participa en las vías de degradación de diferentes neurotransmisores, entre ellos la serotonina, razón por la cual es punto de interés de investigadores para el desarrollo de antidepresivos inhibidores de la enzima. Estos comenzaron a desarrollarse en la década del 60, sin embargo paulatinamente se dejaron de utilizar debido a sus efectos secundarios, generados por el poco conocimiento específico que se tenía sobre la relación enzima/sustrato. En este estudio se realiza una búsqueda en la estructura de la MAO-A para encontrar posibles sitios donde podría ocurrir la desprotonación del sustrato, paso previo necesario para que pueda desarrollarse la catálisis de la enzima. Para esto se pretende identificar y caracterizar el campo electrostático de la proteína y el carácter ácido/base de los aminoácidos, para luego determinar en la estructura los posibles sitios de desprotonación y caracterizarlos. Así, se estudiaron las interacciones electrostáticas a partir de una dinámica molecular convencional de 10[ns], con el fin de definir la capacidad de desprotonación de cada aminoácido. Los factores que se consideraron fueron el valor del pKa de cada residuo y el campo electrostático de la proteína completa. En base a esto se cree que el sitio de desprotonación está en la superficie de la enzima y no en la cavidad del sitio activo, ya que éste es en su mayoría hidrofóbico. Además este sitio estaría en la cara frontal de la proteína, ya que la cara reversa posee un campo electrostático positivo muy alto que repelería al grupo amino protonado del sustrato. En particular, se encontraron dos zonas donde podría ocurrir la desprotonación del sustrato: en el aminoácido ARG-79, que se encuentra relativamente cerca de la región reportada como la entrada al sitio activo y posee una capacidad de desprotonación estable (desviación estándar = 0.5) y alta (pKamax = 17); o los residuos LYS-90 y ARG-109, que se encuentran en el área reportada como entrada y por ello deberían interactúan directamente con el sustrato cuando éste ingresara al canal. El siguiente paso en esta línea de investigación es validar estos resultados en laboratorio mediante mutagénesis dirigida. El desarrollo de este trabajo da pie a una línea de investigación que permita reunir el conocimiento químico y biológico de la interacción enzima/sustrato de la proteína MAO-A. Estos resultados se podrían aplicar en la modificación de la via de degradación de la serotonina, por medio de fármacos que interfieran en su relación con la enzima MAO-A, desarrollando así antidepresivos específicos para este sustrato.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

The current environment for drug discovery and disease treatment relies heavily on genomic analysis, structural biology and chemical biology techniques. With the enormous advances in genomic analysis and structural biology, the use of and desire for targeted therapies has increased. However, as more genomic data for cancer disease state pathology becomes available we must ask increasingly difficult questions and even produce new technologies, such as activity-based probes, to answer these questions. In particular, targeted kinase inhibitors for the treatment of cancer has become a mainstay for drug development for both industry and academia, but it is evident that the genomic data is not always indicative of protein expression. Additionally, protein expression alone does not completely characterize functional activity. Therefore, in order to more accurately validate drug targets and predict drug efficacy, we must not only identify possible targets but also determine their activity in vivo. The goal of this work was to develop a probe for Protein Kinase A that would act by alkylating a conserved cysteine in the substrate-binding pocket of the enzyme. We hypothesized that by targeting the substrate-binding pocket we could effectively utilize the natural substrate selectivity filters as well as take into account multiple endogenous regulatory mechanisms. We produced probes utilizing portions of the pseudosubstrate inhibitor PKI that demonstrate the ability to label the catalytic subunit of Protein Kinase A in an activity-dependent manner, thus making it an important first step in a new class of activity-based probes for the kinome.

This study was conducted to characterize cellular changes occurring during the progression of status epilepticus (SE) that could lead to the maintenance of increased membrane excitability. SE was induced by injection of pilocarpine after which rats were monitored both electrographically and behaviorally. After various lengths of time in SE, specific brain regions were isolated for biochemical study. SE resulted in an early maintenance of PKA activity in both cortical homogenate and crude synaptoplasmic membrane (crude SPM) fractions. At subsequent stages of SE there was a significant increase in PKA activity in both homogenate and crude SPM fractions. Wester blot analysis showed that alteration of PKA protein expression was not responsible for the increase in PKA activity. These results show that SE has a significant duration-dependent effect on PKA activity. Combined with other cellular changes these findings, could represent a mechanism for the formation for potentiated seizure states like epilepsy.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Membrane depolarization is an effective neurotrophic stimulus, with its trophic effect on spiral ganglion neurons (SGNs) even surpassing that of neurotrophins. Thus, SGN cultures are a favorable system to investigate pro-survival signal transduction downstream of depolarization. Depolarization promotes SGN survival by recruiting three distinct kinase pathways: cyclic AMP-dependent protein kinase (PKA), Ca2+/calmodulin-dependent protein kinase II (CaMKII) and CaMKIV. CaMKIV mediates the pro-survival effect of depolarization by activating CREB in nucleus. However, the mechanisms by which PKA and CaMKII promote survival are still not clear. By targeting constitutively active PKA or a PKA inhibitor (PKI) to the outer mitochondrial membrane (OMM), we showed that PKA activity at the OMM is sufficient to support SGN survival in the absence of other trophic factors and necessary for cAMP-dependent SGN survival. It has been suggested that PKA can promote survival by inactivating pro-apoptotic protein Bad. By cotransfection of SGNs with OMM-PKA and wild-type Bad, we showed that this was the case. We further showed that Ser112 and Ser136 in Bad, but not Ser155, a hypothetical PKA target, were necessary for functional inactivation of Bad by PKA. CaMKII mediates the third depolarization-dependent pro-survival pathway. A specific pro-survival target for CaMKII was identified through a separate investigation of the pro-apoptotic JNK-Jun signaling pathway, which we had identified as active in apoptotic SGNs in vivo. By measuring anti-phosphoJun immunofluorescence, we could quantify JNK-Jun activation in SGNs under different conditions. We showed that JNK inhibition or genetic deletion of JNK3 reduces SGN death after neurotrophic factor withdrawal. Neurotrophins have been shown to suppress JNK activation via their receptor protein tyrosine kinases (PTKs). By expressing constitutively active and dominant negative forms of candidate protein kinases, we identified a novel signaling pathway linking depolarization to JNK: Ca2+ entry - CaMKII - FAK/Pyk2 - PI-3-OH Kinase - Protein Kinase B - inhibition of MLKs (upstream activators of JNK). Thus, depolarization also recruits PTKs - the nonreceptor PTKs FAK and Pyk2 - to suppress JNK activation, implying a conserved PTK-PI3K-PKB pathway for suppression of pro-apoptotic JNK activation by neurotrophic stimuli.

The positive symptoms of schizophrenia primarily result from an excess of high affinity D2-like receptors (i.e. D2High receptors). First-generation antipsychotics, such as haloperidol, are D2-like antagonists that can cause severe extrapyramidal effects. Aripiprazole, a dopamine and serotonin partial agonist, has fewer side effects, making it tolerable for adults and children. Extrapyramidal effects (e.g. Parkinsonism, dystonia, and akathisia) are among the most problematic side effects produced by antipsychotic compounds, which likely result from an excess of D2-like receptors in the dorsal striatum. In order to examine the effects of repeated antipsychotic treatment on dopamine system functioning, this thesis compared the molecular effects of repeated haloperidol and aripiprazole administration on D2High receptors, as well as various indices of dopamine second messenger system functioning. Preadolescent and adult rats were pretreated with haloperidol or aripiprazole for 11 consecutive days. After either a 4- or 8-day drug abstinence period dorsal striatal tissue was extracted. [35S]GTPγS binding assays were conducted to assess the effects of repeated haloperidol and aripiprazole treatment on the efficacy and potency of D2-like receptors. PKA subunits and components of the Akt pathway were measured using Western Blots. Results showed that repeated treatment with haloperidol or aripiprazole did not significantly affect D2-like receptor efficacy or potency in young or adult rats. In both age groups, haloperidol significantly increased the expression of PKA-Cα, PKA-Cβ, and PKA-RII, but not p-PKA. Haloperidol also significantly increased PKA-Cβ and PKA-RII levels relative to aripiprazole. Repeated administration of haloperidol significantly increased p-GSK-3β levels in young and adult rats, but neither haloperidol nor aripiprazole significantly affected GSK-3β, Akt, or p-Akt levels. Overall, the results of this thesis indicate that repeated aripiprazole and haloperidol treatment differentially affects D2 signaling pathways in the dorsal striatum. Aripiprazole has less extreme or prolonged effects on D2 receptor signaling pathways than haloperidol, as evidenced by the lack of post-treatment upregulation in the cAMP and Akt pathways. Upregulation of D2-like receptors and, in turn, upregulation of proteins in the cAMP and Akt pathways may be partially responsible for the side effects induced by long-term antipsychotic treatment.

This study concerns the isolation, characterisation and physio-chemical properties of 8,8-methylmethine-(epi)catechin-malvidin-3-glucoside compounds found in red wines. 8,8-Methylmethine-(epi)catechin-malvidin-3-glucoside compounds were isolated via chromatographic methods developed in this study. The compounds were characterised via nuclear magnetic resonance spectrometry which, with the aid of molecular modelling, afforded their possible 3-dimensional structures. Their physio-chemical properties including ionisation and hydration constants, colour parameters and chemical stabilities were determined. The formation of 8,8-methylmethine-flavan-3-ol-malvidin-3-glucoside compounds and other pigments in wines was also studied. 8,8-Methylmethine-(epi)catechin-malvidin-3-glucoside compounds were also synthesised by condensing malvidin-3-glucoside with (epi)catechin in the presence of acetaldehyde. Diastereomers of 8,8-methylmethine-(epi)catechin-malvidin-3-glucoside pigments were isolated from the reaction using size-exclusion liquid chromatography followed by cation-exchange liquid chromatography. The structures of the four 8,8-methylmethine-catechin (and epicatechin)-malvidin-3-glucoside diastereomers were determined using mass spectrometry and one and two-dimensional nuclear magnetic resonance spectroscopy. It was found that for all four compounds, the methylmethine bridge occurs at the 8-positions of malvidin-3-glucoside and (epi)catechin and that the 3-dimensional structural differences between the diastereomers is the positioning of the (epi)catechin moiety with respect to the glucoside group. One diastereomer has the (epi)catechin on the same side, with respect to the malvidin entity whilst it is on the opposite side for the other diastereomer. The proposed structures also afforded the malvidin entity protection from nucleophilic attack via steric hindrance by the (epi)catechin moiety. 8,8-Methylmethine-(epi)catechin-malvidin-3-glucoside pigments have greater colour stability with regards to changes in pH and SO2 bleaching compared to malvidin-3-glucoside providing evidence that little or no hydration in aqueous solutions is occurring for these compounds. Further evidence for little or no hydration occurring is the presence of isosbestic points in the UV-vis spectra observed for the 8,8-methylmethine-(epi)catechin-malvidin-3-glucoside in the pH range 2 to 7. Although the 8,8-methylmethine-(epi)catechin-malvidin-3-glucoside pigments have greater colour stability to pH, SO2 and oxidation, compared to malvidin-3-glucoside, they have lower temporal stabilities and under both aerobic and anaerobic conditions they have significantly higher degradation rate constants than malvidin-3-glucoside. The ionisation constants of the 8,8-methylmethine-(epi)catechin-malvidin-3-glucoside compounds were determined using high voltage paper electrophoresis (HVPE) and UV-visible spectroscopy. The first ionisation constants (pKa1) of the 8,8-methylmethine-(epi)catechin-malvidin-3-glucoside compounds were found to be higher than that of malvidin-3-glucoside whereas the second and third ionisation constants (pKa2 and pKa3) were found to be lower. The correlation of the ionisation constants between HVPE and UV-visible spectroscopy supports the proposal that there is little or no occurrence of hydration for the 8,8-methylmethine-(epi)catechin-malvidin-3-glucoside compounds in the pH range investigated. 8,8-Methylmethine-flavan-3-ol-malvidin-3-glucoside compounds were the major pigments formed during fermentations of chemically defined grape juice media containing malvidin-3-glucoside and various flavan-3-ols. The yeast strain used for fermentation had a major influence on the levels and rates of formation of these pigments during fermentation. The yeast strain used also has an important influence on wine pigment composition, concentration and evolution during maturation thereby affecting the colour density and hue of the resultant wines. The initial formation of 8,8-methylmethine-flavan-3-ol-malvidin-3-glucoside compounds and their subsequent gradual degradation during maturation, allowed a pool of malvidin-3-glucoside to be available for the formation of other colour stable and more temporally stable pigments. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1339479 / Thesis (Ph.D.) - University of Adelaide, School of Agriculture, Food and Wine, 2008

ABCA1 is believed to affect macrophage inflammatory responses, but the mechanism by which ABCA1 may impact cytokine secretion in macrophages has yet to be fully defined. We observed that the induction of ABCA1 expression in three different cell lines, namely BHK, RAW 264.7 macrophages, and primary bone marrow derived macrophages (BMDMs), results in a significant increase in phosphorylated CREB, a known protein kinase A (PKA) substrate. In RAW macrophages, induction of ABCA1 expression by the LXR-agonist T0901317 is correlated with a decrease in LPS-stimulated secretion of proinflammatory cytokines IL-6 and TNF-α. Additionally, the secretion of anti-inflammatory cytokine IL-10 was increased upon ABCA1 induction. A similar trend was observed in BMDMS: ABCA1-expressing BMDMs released less TNF-α and more IL-10 compared to ABCA1-knockout BMDMs. We speculated that the inflammation modulating effects of ABCA1 in macrophages could be a result of PKA activation. Indeed, we found that the LXR-induced ABCA1 phenotype can be mimicked by cAMP in macrophages. 8-bromo-cAMP, a PKA activator, dose-dependently suppressed inflammatory cytokine secretion while promoting IL-10 release in the absence of ABCA1 expression. Finally, we found that the T0901317-induced ABCA1 expression is correlated with higher expression levels of MKP-1, a downstream target of PKA known to suppress inflammatory responses. Together, our results suggest that ABCA1 expression may activate PKA and CREB and that such activation may contribute to the inflammatory modulating effects of ABCA1.