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African trypanosomosis is a parasitic disease in man and animals caused by protozoan parasites of the genus Trypanosoma. T. congolense, T. vivax and T. brucei brucei cause nagana in cattle. The variable nature of the parasite surface coat has hindered the development of an effective vaccine. An option for developing vaccines and chemotherapeutic agents against trypanosomosis is to target pathogenic factors released by the parasite during infection, namely an “anti-disease” approach. Two pathogenic factors released during infection are oligopeptidase B (OPB) and TcoCATL (congopain). TcoCATL, a major lysosomal cysteine peptidase, is a member of the papain family C1 cysteine peptidases. RNA interference (RNAi) was used to down-regulate the expression of TcoCATL in T. congolense IL3000 TRUM183:29-13 parasites in vivo during mouse infections. TcoCATL RNAi was monitored in infected mouse blood by comparing the hydrolysis of Z-Phe-Arg-AMC and parasitaemia between mice in which RNAi was induced and control mice. Mice infected with parasites induced for TcoCATL RNAi had lower parasitaemia when compared to control mice. An attempt was also made at deleting the entire CATL gene array in both T. congolense IL3000 and T. brucei 427 Lister strains. The second pathogenic factor studied, OPB, is a cytosolic trypanosomal peptidase that hydrolyses peptides smaller than 30 amino acid residues, C-terminal to basic residues. In order to evaluate the role that OPB play during disease, RNAi was also applied to knock-down the expression levels of OPB in T. brucei T7T and T. congolense IL3000 TRUM183:29-13 strains (TbOPB and TcoOPB respectively). Oligopeptidase B null mutant strains (Δopb) were also generated in T. brucei brucei Lister 427. An attempt was also made to generate OPB null mutants in T. congolense IL3000 parasites. Western blot analysis of the knock-down experiments using chicken anti-TcoOPB peptide IgY showed that only TbOPB levels were reduced in T. brucei T7T parasites induced for RNAi when compared to TcoOPB RNAi induced cultures. Quantitative assessment of a fourteen day induction experiment for OPB RNAi in T. brucei showed an 87% reduction in TbOPB levels when compared to levels on day one. There was no growth effect observed in T. brucei parasites cultured in vitro and induced for TbOPB RNAi. It was concluded that TbOPB is not necessary for the in vitro survival of T. brucei parasites, thus making the generation of OPB null mutants possible. Δopb T. brucei parasites were successfully generated and grew normally in vitro and were as virulent as wild type strains during infection in mice. Immunohistopatholgy of infected mouse testes revealed Δopb parasites in extra vascular regions showing that T. brucei OPB (TbOPB) is not involved in assisting T. brucei parasites to cross microvascular endothelial cells. Gelatin gel analysis of Δopb null mutants and wild type strains showed an increase in cysteine peptidase activity. Enzymatic activity assays were carried out to identify how closely related oligopeptidases are affected by knocking out TbOPB, and a significant increase of T. brucei prolyl oligopeptidase (TbPOP) activity was observed. However, western blot analysis did not show any increase of TbPOP protein levels in Δopb parasites, suggesting that either TbOPB is responsible for generating an endogenous inhibitor for TbPOP or that another POP-like enzyme might compensate for a loss in OPB activity in Δopb null mutants. This study made a significant contribution to an understanding of the interplay between different trypanosomal peptidases that are important pathogenic factors in trypanosomosis. It highlights the need to simultaneously target several trypanosomal peptidases to develop an effective vaccine or chemotherapeutic agents for African animal trypanosomosis. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2011.

The proteinase, cathepsin L, has been strongly implicated in the processes of tumour invasion and metastasis. A new purification method, three-phase partitioning, characterised in terms of the parameters which affected its fractionation of proteins, was found to simplify the purification of cathepsin L from sheep liver. This method, together with a novel cation-exchange step on S-Sepharose and molecular exclusion chromatography, enabled the enzyme to be purified to homogeneity, in a single-chain form. A further enzyme fraction was isolated as a proteolytically active complex with the endogenous inhibitor of cysteine proteinases, cystatin. Studies on the proteolytically active complex revealed that approximately 60% of it was covalently bound and proteolytically active, while the other 40% was non-covalently bound and proteolytically inactive, in the manner normally found for the binding of cystatin to cysteine proteinases. A cystatin fraction from sheep liver containing variants of cystatin B, was shown to be able to form complexes with free cathepsin L in vitro in a pH-dependent, rapid process, which was mildly stimulated by a reducing agent. Cathepsin L was also isolated from human spleen, but only as a protcolytically inactive complex, presumably also with cystatin(s). The complexed and free cathepsin L from sheep liver were analysed for their pH-dependent characteristics, and it was found that both forms of the enzyme were more active and stable at, or near, neutral pH, than would have been expected from published values. Specific polyclonal antibodies to pure sheep cathepsin L were raised in rabbits and chickens. The chicken egg yolk antibodies were of a much higher titre and were immunoinhibitory towards the enzyme, which the rabbit antibodies were not. Anti-peptide antibodies, raised in rabbits against a peptide sequence selected from the active site of human cathepsin L, were highly specific for cathepsin L and immunoinhibitory towards the enzyme. Together with the polyclonal anti-cathepsin L antibodies, they show promise for immunoinhibitory and immunocytochemical studies on the enzyme, and as potential anti-tumour drugs. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1990.

Chiral structure formation is ubiquitous in surface self-assembly. Molecules that do not undergo chiral recognition in solution or fluid phases can do so when their configurational freedom is restricted in the two-dimensional field of a substrate. The process holds promise in the manufacture of functional materials for chiral catalysis, sensing or nonlinear optics. In this thesis, we investigate the influence of surface attraction and geometry on adsorption-induced chiral separation in several model molecules, as well as the relationships between molecular features, specifically molecular geometry and charge distribution, and chiral recognition at surface self-assembly. Simple model molecules embody the fundamental interactions involved in supramolecular structure formation in experimental systems, and allow the in-depth investigation of key parameters. Chiral pattern formation at the surface self-assembly is a complex problem, even in cases where very small organic molecules are considered. Even though the adsorption behaviour of small organic molecules on gold surfaces has been investigated extensively so far experimentally and theoretically, much of their chiral behaviour is yet to be understood at a molecular level. Theoretical investigations of chiral self-assembly of sulfur containing amino acids onto achiral and chiral gold surfaces is also presented in this thesis. By understanding chiral self-assembly on solid surfaces, one may control and direct it towards creating materials with desired functionality. / Graduate / 0494 / tp.popa@gmail.com

Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 103-117).

Thesis (M.S.)--Brigham Young University. Dept. of Physiology and Developmental Biology, 2007. / Includes bibliographical references (p. 38-40).

Thesis (Ph. D.)--University of Washington, 2005. / Vita. Includes bibliographical references (leaves 94-133).

Oι ιντεγκρίνες είναι κυτταρικοί υποδοχείς, οι οποίοι αλληλεπιδρούν με την εξωκυττάρια ύλη. Μέχρι σήμερα έχουν ανακαλυφθεί 18α και 8β υπομονάδες συνδυασμός των οποίων δημιουργεί 24 διαφορετικές ιντεγκρίνες . Ο ρόλος τους είναι η σύνδεση της εξωκυττάριας με την ενδοκυττάρια ύλη, καθώς ενεργοποιούν ενδοκυττάρια σηματοδοτικά μονοπάτια που εμπλέκονται στην επιβίωση, μετανάστευση, πολλαπλασιασμό και απόπτωση των κυττάρων, λειτουργίες ζωτικής σημασίας για τον οργανισμό. Ιδιαίτερο ενδιαφέρον παρουσιάζει η α4β1 ή VLA-4 ιντεγκρίνη, η οποία αποτελείται από μία α4 (155 kDa) και μία β1 (150 kDa) υπομονάδα. Εκφράζεται στα κύτταρα του μυελού των οστών εκτός από τα ουδετερόφιλα και έχει δύο κύριους φυσικούς προσδέτες, το αγγειακό μόριο κυτταρικής προσκόλλησης-1 (VCAM-1) και την φιμπρονεκτίνη. Ο ρόλος της α4β1 ιντεγκρίνης είναι ζωτικός στις φλεγμονώδεις διαταραχές ενώ φαίνεται να εμπλέκεται και στην αγγειογένεση. Στόχος της παρούσας διατριβής είναι η ανάπτυξη νέων α4β1 κυκλικών πεπτιδικών προσδετών και η μελέτη της δράσης αυτών ως αναστολείς της φλεγμονής και της αγγειογένεσης. Τα συντεθέμενα ανάλογα έχουν μία βασική κυκλική δομή, ενώ τροποποιήσεις έχουν πραγματοποιηθεί τόσο στην Ν-τελική όσο και στην C-τελική αλληλουχία. Η σύνθεση των αναλόγων πραγματοποιήθηκε με την Fmoc/But μεθοδολογία επί στερεάς φάσεως, χρησιμοποιώντας ως στερεό υπόστρωμα τις ρητίνες 2-χλωροτρίτυλο και την Rink Amide MBHA, για την παραλαβή C-τελικού καρβοξυλίου ή αμιδίου αντίστοιχα. Η βασική κυκλική δομή επιτεύχθηκε μέσω σχηματισμού δισουλφιδικής γέγυρας χρησιμοποιώντας ως οξειδωτικό μέσο διμεθυλοσουλφοξείδιο (DMSO) είτε σε υγρή, είτε σε στερεά φάση. Στην παρούσα φάση, πραγματοποιείται μελέτη των συντεθέντων αναλόγων in vivo στη χοριοαλλαντοϊκή μεμβράνη του εμβρύου όρνιθας (CAM) ως αναστολείς της αγγειογένεσης, ενώ βιολογικά πειράματα θα διεξαχθούν για την πιθανή χρήση αυτών ως αντιφλεγμονώδεις παράγοντες. Επιπρόσθετα, μελετάται η διαμόρφωση των παραπάνω πεπτιδικών αναλόγων, μέσω τεχνικών NMR φασματοσκοπίας και μοριακής μοντελοποίησης. / Integrins are cell surface receptors, which interact with the extracellular matrix. They are heterodimers consisting of α and β subunits. Until now, 18 α subunits and 8 β subunits have been discovered that form 24 different integrins. Their role is the connection of the extracellular matrix with the intracellular cytoskeleton, as they activate intracellular signaling pathways regulating the migration, proliferation, survival and apoptosis of the cells, functions of vital importance for the organism. An integrin with particular interest is α4β1 or VLA-4 integrin, which consists of a α4 (155 kDa) and a β1 (150 kDa) subunit. It is expressed on bone marrow derived cells, except on neutrophils, and has two main natural ligands, fibronectin and vascular cell adhesion molecule-1 (VCAM-1). The role of α4β1 is vital for the inflammation process, while it also seems to participate in tumor angiogenesis. The aim of this research is the development of α4β1 cyclic peptide antagonists and their study as inflammation and tumor angiogenesis inhibitors. The above analogues have a basic cyclic peptide structure, while modifications have been achieved at the N-terminus and C-terminus sequences. The analogues were synthesised by Fmoc/But solid phase methodology utilizing Rink Amide MBHA and 2-chlorotrityl-chloride resin to provide C-terminal amide and carboxylic acid, respectively. The basic cyclic unit of the above analogues was achieved through the formation of a disulfide bridge, using as oxidant dimethylsulfoxide (DMSO), in either solution or solid phase methodology. At present, the above analogues are tested in vivo in chick embryo chorioallantoic membrane (CAM) model as anti-angiogenic agents, while biological experiments will be performed for their potential use as anti-inflammatory agents. Furthermore, the conformation of the above peptide analogues is studied in solution environment, by NMR spectroscopy techniques and molecular modeling.

Pyruvate kinase catalyses the final step in glycolysis and is responsible for net ATP production. There are four pyruvate kinase isoforms expressed in humans; LPYK, RPYK, M1PYK and M2PYK. The allosteric enzyme M2PYK plays an important role in cancer cell metabolism and is subject to complex regulation by numerous naturally occurring small-molecule metabolites. Post-translational modifications have also been found to play a key role in the regulation of M2PYK, among these cysteine oxidation. This thesis describes the production and characterisation of M2PYK cysteine point mutants in order to investigate the mechanism of regulation by cysteine modification. From a total of ten cysteines present in M2PYK, five were chosen for mutation based on a combination of the results from the cysteine oxidation prediction program (COPP) web interface and published experimental evidence for cysteine modification of M2PYK. Eight point mutants of these five cysteines were produced and characterised. Low resolution gel filtration of all the mutants shows that mutation of these cysteines has an effect on tetramer:dimer:monomer equilibrium of M2PYK suggesting that cysteine modifications could regulate M2PYK activity by affecting oligomeric state. Activity assays show that none of the cysteine point mutations are sufficient to protect M2PYK from oxidation by H2O2 indicating that more than one cysteine is involved in the regulation of M2PYK by oxidation. Nitric oxide (NO) imbalance has recently emerged as playing a key role in numerous diseases including cancer. NO regulates the function of target proteins through the addition of a nitroso moiety from NO-derived metabolites to a reactive cysteine, a process known as protein S-nitrosylation. M2PYK has been found to be S-nitrosylated in vivo. Using the biotin-switch assay in vitro combined with mass spectrometry I have shown that a likely candidate for the target of S-nitrosylation of M2PYK is C326. This thesis also describes the structures of two cysteine point mutants; M2PYK C424A and M2PYK C358S. The structures show that these mutations have very little effect on the overall conformation of M2PYK with only very subtle localised changes. The structure of the mutant M2PYK C358S shows some interesting features including varying occupation of the active site resulting in differing conformations of the B domains within the same tetramer, and an unusual B factor distribution which could be indicative of a perturbation in cooperativity within the tetramer caused by the mutation.

Methylmercury (MeHg) is related to several deleterious effects on the vertebrate tissues, mainly on central nervous system, and part of these effects are through of interaction with sulfhydryl group found in cellular proteins. MeHg interacts with low and high molecular weight thiols in the blood and tissues and this fact, in some cases will allow a better absorption and tissue uptake of mercury. In this regard, the purpose of this study was to examine the effect of MeHg-Cysteine (MeHg-Cys) complex administration on cerebral areas, liver and kidney on Hg-uptake and to analyze possible behavioral changes associated with mercury accumulation in adult s mice. Adult male Swiss albino mice were divided into four groups; control (1 mL/Kg distilled water), MeHg (2 mg/kg), Cys (2 mg/kg) and MeHg-Cys complex (2 mg/kg equimolar concentration). All the animals received one injection per day (i.p.), for 60 consecutives days. The initial set of experiments was designed to analyze possible neurobehavioral changes (locomotor performance and/or exploratory activity) caused by treatments. Administration of MeHg or MeHg-Cys complex caused a significant reduction on total locomotor activity in adult s mice, when compared to control group. In contrast to locomotor activity, rearing frequency was decreased only in MeHg group. The final set of experiments was designed to determine the mercury concentration into the brain areas (cortex and cerebellum), liver and kidney in adult s mice. Treatment with MeHg significantly increased mercury concentrations in all tissues analyzed, when compared to control group. The accumulation of mercury on cerebral areas and in liver was further increased in animals treated with MeHg-Cys complex, when compared to MeHg alone group. However, the concentration of mercury found in kidney was lower in the MeHg-Cys treated group, than in the group treated only with MeHg. In conclusion, the present study shows, for the first time, that treatment with MeHg-Cys complex allow better absorption and tissue uptake of mercury than the treatment with MeHg alone, in the cerebral areas and in liver of mice. Furthermore, this study reinforces the view that MeHg causes impairment in motor performance and exploratory activity and suggests that the different forms of MeHg exposure affect its distribution in the tissues of mice, as well as, it can leads to the distinct neurobehavioral consequences. / O metilmercúrio (MeHg) é relatado por ter vários efeitos deletérios sobre tecidos de vertebrados, principalmente no sistema nervoso central, e parte desses efeitos está relacionado a sua capacidade de interagir com grupos sulfidrílicos encontrados em proteínas. O MeHg reage com tióis de baixo e alto peso molecular no sangue e outros tecidos permitindo, em alguns casos uma melhor absorção e captação de mercúrio pelo tecido. Nesse contexto, o objetivo desse trabalho foi examinar os efeitos da administração do complexo MeHg-Cisteína (MeHg-Cys) na captação de mercúrio sobre áreas cerebrais, fígado e rim; e analisar possíveis mudanças comportamentais associadas ao acúmulo de mercúrio em camundongos adultos. Camundongos machos Swiss albino foram divididos em quatro grupos: Controle (1 mL de água destilada), MeHg (2 mg/kg), Cys (2 mg/kg) e Complexo MeHg-Cys (2 mg/kg concentração equimolar). Todos os animais receberam uma injeção (i.p. por dia) durante 60 dias consecutivos. A parte inicial dos experimentos foi designada para analisar possíveis mudanças neuro-comportamentais (desempenho locomotor e/ou atividade exploratória) causadas pelos tratamentos. A administração do MeHg ou do complexo MeHg-Cys reduziu significativamente a atividade locomotora total dos camundongos adultos quando comparado ao grupo controle. Em contraste à atividade locomotora, a freqüência de levantar-se (rearing) diminuiu apenas no grupo que recebeu MeHg. A parte final dos experimentos foi designada para determinar a concentração de mercúrio nas áreas cerebrais (córtex e cerebelo), fígado e rins dos camundongos tratados. O MeHg aumentou significativamente a concentração de mercúrio em todos os tecidos analisados, quando comparado ao grupo controle. O acúmulo de mercúrio sobre as áreas cerebrais e fígado foi acentuadamente aumentado nos animais que receberam o complexo MeHg-Cys, quando comparado ao grupo que recebeu apenas MeHg. Entretanto, a concentração de mercúrio encontrada no rim foi menor no grupo tratado com o complexo MeHg-Cys quando comparado ao grupo tratado apenas com MeHg. Concluindo, o presente estudo mostrou, pela primeira vez, que o tratamento com o complexo MeHg-Cys permite maior absorção e captação de mercúrio pelos tecidos cerebrais e hepático, do que o tratamento apenas com MeHg. Além disso, esse estudo reforça a idéia que o MeHg causa prejuízo na performance motora e atividade exploratória dos animais e também sugere que diferentes formas de exposição ao MeHg afetam a sua distribuição nos tecidos de camundongos, bem como pode levar a conseqüências neuro-comportamentais distintas.