Characterization of PfPKA and PfPK5 substrates, as well as the proteins they interact with, will help us to develop innovative therapies targeting binding sites.; Malaria is a devastating disease that results in almost one million deaths annually. Most of the victims are children under the age of five in Sub-Saharan Africa. Malaria parasite strains throughout developing countries are continually building resistance to available drugs. Current therapies such as mefloquine, chloroquine, as well as artemisinin are becoming less effective, and this underscores the urgency for therapeutics directed against novel drug targets. In order to identify new drug targets, the molecular biology of the malaria parasite Plasmodium needs to be elucidated. Plasmodium exhibits a unique cell cycle in which it undergoes multiple rounds of DNA synthesis and mitosis without cytokinesis. Thus, cell cycle regulatory proteins are likely to be promising pathogen-specific drug targets. It is expected that fluctuating activity of key proteins, such as protein kinases, play an essential role in regulating the noncanonical life cycle of Plasmodium. Consequently, malarial kinases are a prime target for therapy. One way to better understand the role of malarial kinases in Plasmodium cell cycle regulation is to identify putative protein kinase substrates and interacting proteins. Two malarial kinases that have been implicated in regulating malaria parasite cell cycle stages were investigated in this study: P. falciparum CDK-like Protein Kinase 5 (PfPK5) and cAMP-Dependent Protein Kinase A (PfPKA). A transgenic P. falciparum line was created for the expression of epitope-tagged PfPK5 for pull-down analysis. Phospho-substrate antibodies were used to identify physiological substrates of both PfPK5 and PfPKA. Immunoblotting with these antibodies identified several potential substrates. Identities of the PfPKA physiological substrates were determined from the global P. falciparum phosphoproteome dataset that has recently been generated in our laboratory.

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<p>The prediction and characterisation of chemical and biochemical processes are fundamental tasks in computational chemistry. Small chemical systems can be characterised by the stationary points on potential energy surface and reaction paths linking them. For large biological systems, statistical sampling is required to characterising their average properties.</p> <p>This thesis presents my Ph.D. work on developing new methods to predict and characterise chemical and biological processes. Two path-finding methods for finding the minimum energy reaction path and alternative reaction paths for small gas-phase reactions have been elucidated with examples, and molecular dynamic simulations have been used to characterise the binding affinity of protein-ligand complex and the free energy of protonation processes in a protein.</p> <p>Specifically, the fast marching method (FMM) has been used to find the minimum energy path (MEP) on the potential energy surface (PES) for small gas-phase reactions. In this thesis, FMM is shown to be one of the most general and reliable surface-walking algorithms for finding the MEP. However, it is an expensive method. Some improvements have been illustrated in chapter 2 and chapter 3.</p> <p>I also proposed a new method (called QSM-NT) for finding all stationary points, accordingly all alternative reaction paths on the PES. Unlike other path-finding methods, QSM-NT overcomes the need of an initial guess of the path, and it can find all stationary points on the PES. QSM-NT has been proven to be efficient and reliable through applications on analytical PES and real chemical reaction. The difficulties and pitfalls associated with QSM-NT have been elucidated with examples.</p> <p>Molecular dynamic (MD) simulation and associated postprocessing procedures have been used to study the binding properties of caffeine-A_2A complex. The binding affinities of different binding modes have been calculated using MM/PBSA method. The binding pocket has been characterised with MM/GBSA energy decomposition. Our computational work provides significant insight to the targeted drug design of the adenosine A_2A receptor.</p> <p>The pH-dependent properties of a protein play important roles in the fundamental biological processes. The protonation states, namely, the pK_a values of ionisable residues, especially active-site residues are the prerequisites to understanding of the mechanisms of many biological processes. In this thesis, acetoacetate decarboxylase (AADase) is used as a test case for studying different types of pK_a prediction methods. Our computational results have shown that the site-site interactions from other ionisable residues are crucial to the pK_a prediction of the target residue.</p> <p>This thesis covers the range from small gas phase reaction prediction to large complex biological systems characterisation using quantum mechanical and molecular mechanical methods.</p> / Doctor of Philosophy (PhD)

Yes / Ca2+ release from the Golgi apparatus regulates key functions of the organelle, including vesicle trafficking. However, the signaling pathways that control this form of Ca2+ release are poorly understood and evidence of discrete Golgi Ca2+ release events is lacking. Here, we identified the Golgi apparatus as the source of prolonged Ca2+ release events that originate from the nuclear ‘poles’ of primary cardiac cells. Once initiated, Golgi Ca2+ release was unaffected by global depletion of sarcoplasmic reticulum Ca2+, and disruption of the Golgi apparatus abolished Golgi Ca2+ release without affecting sarcoplasmic reticulum function, suggesting functional and anatomical independence of Golgi and sarcoplasmic reticulum Ca2+ stores. Maximal activation of β1-adrenoceptors had only a small stimulating effect on Golgi Ca2+ release. However, inhibition of phosphodiesterase (PDE) 3 or 4, or downregulation of PDE 3 and 4 in heart failure markedly potentiated β1-adrenergic stimulation of Golgi Ca2+ release, consistent with compartmentalization of cAMP signaling within the Golgi apparatus microenvironment. β1-adrenergic stimulation of Golgi Ca2+ release involved activation of both Epac and PKA signaling pathways and CaMKII. Interventions that stimulated Golgi Ca2+ release induced trafficking of vascular growth factor receptor-1 (VEGFR-1) from the Golgi apparatus to the surface membrane. These data establish the Golgi apparatus as a juxtanuclear focal point for Ca2+ and β1-adrenergic signaling, which functions independently from the sarcoplasmic reticulum and the global Ca2+ transients that underlie the primary contractile function of the cell.

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Nous étudions le ribozyme VS de Neurospora, en tant que système modèle, pour augmenter nos connaissances sur la relation entre la structure et la fonction chez les ARNs, ainsi que pour mieux comprendre le mécanisme de clivage de ce ribozyme. Il a été proposé précédemment que la boucle interne A730 dans la tige-boucle VI (SLVI) contient le site actif du ribozyme et lie un ou plusieurs ions métalliques qui pourraient participer au mécanisme réactionnel. Nous avons déterminé par spectroscopie RMN la structure de la tige-boucle SLVI contenant la boucle A730 afin d’éclaircir ce mécanisme. La structure obtenue est en accord avec les études biochimiques antérieures et présente un ou plusieurs sites de liaison au magnésium associé à la boucle interne. Suite à des études de cinétique et de mutagenèse, il a été proposé qu’une adénine localisée dans le site actif, A756, participe à la catalyse par acide/base générale. Des études de pH effectuées précédemment ont identifié un pKa catalytique (5.2-5.8) qui correspond probablement à l’équilibre de protonation du A756. À l’aide de méthodes utilisant le carbone-13, nous avons identifié un pKa modifié appartenant au A756, ce qui supporte le rôle de ce résidu dans la catalyse par acide/base générale. Les études structurales présentées ici aident donc à augmenter notre compréhension du mécanisme de clivage chez le ribozyme VS. / We are studying the Neurospora VS ribozyme as a model system to increase our knowledge of the structure-function relationship in RNA and to better understand the mechanism of the cleavage reaction. It has been previously postulated that the A730 internal loop of stem-loop VI (SLVI) forms the active site of the VS ribozyme and binds magnesium ion(s) that may participate in catalysis. To get insights into the catalytic mechanism, we have determined by NMR spectroscopy the structure of a SLVI fragment containing the A730 loop. The structure we obtained is in agreement with previous biochemical studies and contains one or more magnesium-ion binding sites in the active site. Based on kinetic and mutagenesis studies, it has been proposed that an adenine in the A730 loop, A756, is important for catalysis and may participate in general acid/base catalysis. Previous pH-dependent enzymatic studies identified a catalytic pKa of 5.2-5.8, which likely corresponds to the protonation equilibrium of this A756 adenine in the A730 loop. Using 13C NMR methods, we have identified a shifted pKa for A756, which gives additional support to the role of this residue in the general acid/base mechanism. The NMR studies presented here therefore increase our understanding of the cleavage reaction in the VS ribozyme.

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