"March 2003." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (p. 116-126). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Bacillus thuringiensis (Bt) is a gram positive spore forming bacterium which produces intracellular protein crystals toxic to a wide variety of insect larvae and is the most commonly used biological pesticide worldwide. More recently, Bt crystal proteins known as parasporins have been discovered, that have no known insecticidal activity but target some human cancer cells exhibiting strong cytocidal activities with different toxicity spectra and varied activity levels. Amongst these parasporins, parasporin-3 most closely resembles the commercially used insecticidal toxins and presents the narrowest activity spectrum, showing moderate cytotoxicity against only two cancer cell lines, HL-60 (Human promyelocytic leukemia cells) and HepG2 (Human liver cancer cells). Parasporin-3, also called Cry41Aa, has only been shown to exhibit cytocidal activity towards these two cell lines after being proteolytically cleaved. In order to understand this activation mechanism various mutations were made at the N- or C-terminal region of the protein and the toxicity against both HepG2 and HL-60 cell lines was evaluated. Our results indicate that only N-terminal cleavage is required for activation and that N-terminally deleted mutants show some toxicity without the need for proteolytic activation. Furthermore we have shown that the level of toxicity towards the two cell lines depends on the protease used to activate the toxin. Proteinase K-activated toxin was significantly more toxic towards HepG2 and HL-60 than trypsin-activated toxin. N-terminal sequencing of activated toxins showed that this difference in toxicity is associated with a difference of just two amino acids (serine and alanine at positions 59 and 60 respectively) which we hypothesize occlude a binding motif. Preliminary work carried out on binding showed a lack of correlation between binding and toxicity since toxin binds to both susceptible and non-susceptible cancer cell lines. In an attempt to better understand the mechanism of action of Cry41Aa against these cells, we evolved resistance in HepG2 through repeated exposure to increasing doses of the toxin. Morphological, physiological and genetic characteristics of the resistant cell line were compared with susceptible cells. Toxin was shown to bind to resistant HepG2 similarly to the susceptible line. RNA sequencing identified AQP9 as a potential mediator of resistance but extensive investigations failed to show a direct link. The involvement of certain intracellular signalling pathways were also investigated in order to understand cell responses to the toxin and showed that in response to the toxin p38, but not ERK1/2, is activated and in a dose dependent manner.

A regulação dependente de Ca2+ da atividade ATPásica da acto-miosina em concentrações fisiológicas de actina, tropomiosina e troponina ocorre exclusivamente na presença de troponina T (TnT). Nosso grupo demonstrou que um polipeptídeo correspondente aos primeiros 191 aminoácidos da TnT ativa a atividade ATPásica da acto-miosina na presença de tropomiosina e na ausência das outras duas subunidades do complexo troponina (TnI/TnC). Com o objetivo de mapear e caracterizar esse domínio ativatório da TnT, construímos fragmentos de TnT correspondentes às regiões compreendidas entre os resíduos de aminoácidos: 1-157 (TnTl-157), 1-76 (TnTl-76), 77-157 (TnT77-57), 77-191 (TnT77-191) e 158-191 (TnT158-191). Estudos das interações desses fragmentos com actina e tropomiosina demonstraram que: i) o fragmento TnTl-76 não se liga à tropomiosina ou a actina; ii) a região da TnT correspondente aos resíduos 158-191 liga-se à actina cooperativamente, mas não se liga à tropomiosina; iii) a região correspondente seqüência de aminoácidos 77-157 é necessária para a interação da TnT com o resíduo de aminoácido 263 da tropomiosina; iv) TnT77-191 ativa a atividade ATPásica da acto-miosina com a mesma intensidade que TnTl-191. Também observamos que TnTl-157, TnTl-76, TnT77-157, TnT158-91 e combinações de TnT158-191 com TnTl-157 e TnT77-157 não afetam a atividade ATPásica da acto-miosina. Concluímos que a região da TnT delimitada pelos aminoácidos 77 e 191 é essencial para a ativação da atividade ATPásica da actomiosina e que essa ativação é mediada pelas interações dessa região da TnT com a tropomiosina e a actina. / The Ca2+-regulation of the actomyosin ATPase activity at physiological ratios of actin, tropomyosin and troponin occurs only in the presence of troponin T. Our group has previously demonstrated that a recombinant polypeptide corresponding to the first 191 amino acids of TnT (TnTl-191) activates the aetomyosin Mg2+-ATPase activity in the presence of tropomyosin and in the absence of TnI/TnC. In order to further map and characterize this activation domain, we constructed a set of recombinant or synthetic TnT fragments, corresponding to amino acids 1-157 (TnTl-157), 1-76 (TnTl-76), 77-57 (TnT77-157), 77-191 (TnT77-191) and 158-191 (TnT158-191). Binding assays using these fragments demonstrated that: i) amino acids 1-76 of TnT do not bind to tropomyosin or actin; ii) amino acids 158-191 bind to actin cooperatively, but not to tropomyosin; iii) the sequence 77-157 is necessary for TnT\'s interaction with residue 263 of tropomyosin; iv) TnT77-191 on its own activates de actomyosin ATPase activity to the same extent as previously described for TnTl-191. TnT1-157; TnTl-76; TnT77-157; TnT158-191 and combinations of TnT158-191 with TnTl-157 or TnT77-157 showed no effect on the ATPase activity. We conclude that interactions of amino acids 77-191 of TnT with tropomyosin and actin are essential for the activation of actomyosin ATPase activity, and that this activation may be mediated in part by a direct interaction between TnT residues 158-191 and actin.

No description available.

Typewritten copy Includes bibliographical references.

Graduation date: 1969

A model of acute glutamate- and domoate-induced toxicity was developed and characterized in cultured rat cerebellar granule cells (CGCs) using experimental conditions which preserve the voltage-dependency of NMDA receptor function. Glutamate, which is normally non-toxic to CGCs in physiologic media (pH 7.4), was shown to induce a cytotoxic response after 2 hours when the exposure temperature was reduced from 37�� to 22��. Pharmacological characterization of this response demonstrated that cytotoxicity is mediated by the activation of NMDA receptors, while non-NMDA receptors produce a depolarizing stimulus that enhances release of the voltage-dependent Mg����� blockade of NMDA receptor ion channels. Reduced temperature was shown to facilitate NMDA receptor activation by compromising the ability of CGCs to maintain normal electrochemical gradients during glutamate-induced ion flux. When compared to glutamate, the non-NMDA receptor agonist, domoate, demonstrated an acute cytotoxic response in CGCs that was also mediated predominantly by NMDA receptors. NMDA receptor activation was produced secondary to a domoateinduced release of glutamate and aspartate from CGCs; therefore, domoate synergistically potentiates glutamate/aspartate-mediated neurotoxicity. Domoate-induced excitatory amino acid (EAA) release was investigated and found to occur almost exclusively through reversal of the high affinity Na+-coupled glutamate transporter and by osmoregulatory mechanisms. CGCs also responded to domoate-induced depolarization by releasing adenosine which suppresses exocytotic EAA release through A1 receptor activation. The functional and pharmacological characteristics of NMDA receptors were characterized in 12 DIC CGCs using the channel blocking compound [��H]MK-801 (dizocilpine). Kinetic analysis of [��H]MK-801 binding indicated the possible existence of at least two NMDA receptor populations on 12 DIC CGC membranes, and the equilibrium competition binding of MK-801 and other channel blocking compounds was consistent with the presence of high and low affinity binding sites. The neuroprotective potencies of NMDA receptor channel blockers correlated significantly with their affinities for the NMDA receptor derived from equilibrium competition analysis of [��H]MK-801 high-affinity binding. Thus, whereas 12 DIC CGCs express a pharmacologically heterogeneous population of NMDA receptors, it is the high-affinity component of [��H]MK-801 binding that mediates glutamate toxicity. / Graduation date: 1998

Thesis (Ph. D.)--Oregon State University, 2007. / Printout. Includes bibliographical references (leaves 218-225). Also available on the World Wide Web.

Thesis (M.Sc.)--Memorial University of Newfoundland, 2005. / Bibliography: leaves 105-133.

Abstract “Biomimetic Aminoacylation” Svetlana K. Tzvetkova Doctor of Philosophy, 2008 Graduate Department of Chemistry University of Toronto The accuracy of ribosomal protein synthesis depends on the fidelity of highly specific enzymes, aminoacyl tRNA synthetases, towards amino acid – tRNA pairs. These biological catalysts are responsible for activating the amino acids as aminoacyl adenylates and for their subsequent attachment to the 2’- or 3’-OH at the 3’-terminal of the correct tRNA to give aminoacyl-tRNA. Extended diversity in protein structure and function could be achieved if non-natural side chains can be introduced in protein synthesis. This requires that the acceptor stem of a tRNA molecule be synthetically aminoacylated. The most widely used methods for charging tRNA with non-natural amino acids involve multi-step synthesis of an aminoacyl-pCpA and its consequent enzymatic ligation to truncated tRNA. No direct route to these species has been reported. We have developed a method for direct biomimetic aminoacylation of the 3’-terminal hydroxyls of tRNA. Our approach shows to be promising in reactions leading to direct 2’- or 3’-O-aminoacylation of not only nucleosides and nucleotides but also RNA in general and tRNA in particular. The system we have developed provides: 1) efficient activation of the amino acids as aminoacyl phosphates, analogues of the enzymatic intermediates, and 2) specific recognition of the 3’-terminal of tRNA by lanthanide ions present in the reaction. The aminoacylating reagents used in our studies were carefully selected to provide handles to follow the reaction: UV absorbance, fluorescence spectroscopy and 19F NMR. Lanthanide (III) ions can play a role similar to a key part of the aminoacyl tRNA synthetases – they bring the aminoacyl close to the 3’-terminal of tRNA, in this case by forming a bis-bidentate complex with the aminoacyl phosphate and the 2’,3’-diol functionality of the 3’-terminal adenosine. This process relies on the specificity towards the unique 3’-terminal diol on tRNA, provided by the metal ion and the simultaneous complexation of the aminoacyl phosphate.