Thesis (Ph. D.)--Hong Kong University of Science and Technology, 2002. / Includes bibliographical references (leaves 419-420). Also available in electronic version. Access restricted to campus users.

Thesis (doctoral)--Lund University, 1995. / Added t.p. with thesis statement inserted.

Thesis (doctoral)--Lund University, 1995. / Added t.p. with thesis statement inserted.

No description available.

Archaeal protein trafficking is a poorly understood process that is only beginning to unfold. Integral to this process are the various signal peptidases. Two types of archaeal signal peptidases are identified thus far: signal peptidase I (SPI) and the preflagellin peptidase. SPI is responsible for processing the majority of secreted proteins. Sequence analysis of archaeal SPI enzymes indicates a Sec-11 type enzyme with two conserved Aspartic acid and a Histidine in place of the conserved Lysine in bacteria. Site directed mutagenesis and in vitro assays identified three conserved residues (Ser52, His122 and Asp148) critical for M. voltae SPI activity, distinguishing the archaeal enzyme from its bacterial and eukaryal counterparts. The archaeal preflagellin peptidase is a type IV prepilin peptidase-like enzyme, initially characterized for its essential role in preflagellin processing prior to flagellar filament assembly. Unusual substrates have been proposed for this enzyme, including preflagellins of peculiar signal peptide lengths in certain archaeal species, as well as sugar binding proteins with extremely short signal peptides in S. solfataricus. In this thesis, in vitro comparisons of the signal peptide length requirements for the two different preflagellin peptidases, FlaK in M. voltae and PibD in S. solfataricus, are presented. While a signal peptide length cut-off of 5 amino acids was found for FlaK below which preflagellins remained unprocessed, substrates with shorter (4 and 3 aa) signal peptides were recognized and properly cleaved by PibD, suggesting a diversification of the preflagellin peptidases among the archaeal species. The ability of FlaK and PibD to complement FlaK activity in an M. maripaludis flaK mutant was evaluated. M. maripaludis flaK is a markerless, stable mutant displaying pili as the sole cell surface appendage, providing the unique opportunity to closely study this structure without the interference of flagella. Here, purification and characterization of the archaeal pili is described for the first time in any archaeon. A putative pilus gene cluster with characteristics of type IV pilus genes was identified in M. maripaludis. In-frame deletion of the putative major pilin gene, MMP0237, resulted in a nonpiliated phenotype that could be restored by complementation, providing a direct link of this gene to piliation. / Thesis (Ph.D, Microbiology & Immunology) -- Queen's University, 2007-09-28 12:59:16.801

No description available.

Thesis (M.S.)--Oregon State University, 2010. / Printout. Includes bibliographical references (leaves 95-97). Also available on the World Wide Web.

Thesis (Ph. D.)--Ohio State University, 2005. / Title from first page of PDF file. Document formatted into pages; contains xiv, 110 p.; also includes graphics. Includes bibliographical references (p. 100-110). Available online via OhioLINK's ETD Center

Blood-feeding and digestion are crucial for the tick reproduction because they provide nutrients for anabolic processes such as vitellogenesis, molting and eggs production. Digestion in ticks is mediated by a network of cystein and aspartic peptidases. Therefore, tick digestive peptidases could be a relevant anti-tick vaccine candidates.

Submitted by RONIVALDO RODRIGUES DA SILVA (rds.roni@yahoo.com.br) on 2016-03-01T13:46:53Z No. of bitstreams: 1 Tese Doutorado RONIVALDO R. SILVA.pdf: 3318357 bytes, checksum: 82fadd527a2ede34e2a0a237a881e8f8 (MD5) / Approved for entry into archive by Ana Paula Grisoto (grisotoana@reitoria.unesp.br) on 2016-03-01T18:27:48Z (GMT) No. of bitstreams: 1 silva_rr_dr_sjrp.pdf: 3318357 bytes, checksum: 82fadd527a2ede34e2a0a237a881e8f8 (MD5) / Made available in DSpace on 2016-03-01T18:27:48Z (GMT). No. of bitstreams: 1 silva_rr_dr_sjrp.pdf: 3318357 bytes, checksum: 82fadd527a2ede34e2a0a237a881e8f8 (MD5) Previous issue date: 2016-02-12 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Aspártico (E.C. 3.4.23), cisteíno (E.C. 3.4.22) e serino peptidases (E.C. 3.4.21) são endopeptidases, cujos modos de ação são dependentes de resíduos de ácido aspártico, cisteína e serina presentes no sítio catalítico, respectivamente. Atualmente, vários estudos são realizados na busca por novas enzimas com relevantes propriedades bioquímicas para aplicação industrial. Neste contexto, nós propomos a produção de enzimas em bioprocesso submerso, purificação, estudo das propriedades bioquímicas e determinação da especificidade catalítica das peptidases secretadas pelos fungos filamentosos Rhizomucor miehei, Phanerochaete chrysosporium e Leptosphaeria sp. Inicialmente, após produção por bioprocesso submerso, estas enzimas foram purificadas utilizando cromatografias de exclusão molecular e troca iônica. Em ensaios de inibidores na atividade enzimática, notamos inibição das peptidases por pepstatina A (R. miehei), ácido iodoacético/N-Etilmaleimida (P. chrysosporium) e fluoreto de fenil metil sulfonila (Leptosphaeria sp), sendo então definidas como aspártico, cisteíno e serino peptidases, respectivamente. Por SDS-PAGE (12%), as massas moleculares foram estimadas em 37 kDa (aspártico), 23 kDa (cisteíno) e 35 kDa (serino). O máximo de atividade proteolítica foi alcançado em pH 5,5 e 55 ºC para peptidase aspártica secretada por R. miehei; pH 7 e faixa de temperatura 45-55 ºC para cisteíno peptidase secretada por P. chrysosporium, e pH 7 e 45 ºC para serino peptidase secretada por Leptosphaeria sp. Sob efeito de incubação a diferentes pH, a peptidase aspártica mostrou-se estável em condições ácidas (pH 3-5); cisteíno peptidase foi estável em ampla faixa de pH (pH 4-9), e serino peptidase mostrou-se mais estável em condições com tendências alcalinas e pH ligeiramente ácido (pH 5-9). Em todas estas faixas de pH citadas, as peptidases apresentaram atividade proteolítica acima de 80% por 1 hora de incubação. Quanto à estabilidade térmica, a cisteíno peptidase mostrou-se mais termoestável dentre as três enzimas e serino peptidase descreveu a menor tolerância à temperatura. Em incubação com agentes desnaturantes, observamos redução na atividade proteolítica sob efeito de surfactantes iônicos (0,02-1%): dodecil sulfato de sódio (SDS) e brometo de cetil-trimetil amônio (CTAB); íon cobre II (5 mM); Ditiotreitol (DTT) e guanidina (ambos na faixa de 10-200 mM) para todas as peptidases. Por último, em estudo de especificidade catalítica destas enzimas, observamos a preferência por aminoácidos aromáticos (F e W), básicos (K e R) e apolares (em particular, resíduo de metionina) para peptidase aspártica. Alta especificidade descrita por cisteíno peptidase, cuja preferência catalítica é notória por aminoácidos básicos (K, H e R), especialmente na posição P3 e lisina-dependência para catálise na posição P'3. Em serino peptidase, notamos maior aceitação por aminoácidos apolares (G, I, L, M e V), básicos (H e R) e polares neutros (N e Q) para as diferentes posições avaliadas no substrato. / Aspartic (EC 3.4.23), cysteine (EC 3.4.22) and serine peptidases (EC 3.4.21) are endopeptidases whose modes of action are dependent on aspartic acid, cysteine and serine residues present in the catalytic site, respectively. Currently, several studies are conducted in the search for new enzymes with relevant biochemical properties for industrial application. In this context, we propose the production of enzymes in submerged bioprocess, purification, the study of biochemical properties and determining the catalytic specificity peptidases secreted by the filamentous fungus Rhizomucor miehei, Phanerochaete chrysosporium and Leptosphaeria sp. Initially, after production submerged bioprocess, these enzymes have been purified using size-exclusion and ion exchange chromatographies. In the effect of inhibitors on enzyme activity, we note peptidase inhibition by pepstatin A (R. miehei), iodoacetic acid/ N-Ethylmaleimide (P. chrysosporium) and phenyl methyl sulfonyl fluoride (Leptosphaeria sp), suggesting that these enzymes are aspartic, cysteine and serine peptidases, respectively. For SDS-PAGE (12%), molecular weights were estimated at 37 kDa (aspartic), 23 kDa (cysteine) and 35 kDa (serine). Maximum proteolytic activity was achieved at pH 5.5 and 55 °C for aspartic peptidase secreted by R. miehei; pH 7 and temperature range 45-55 °C for cysteine peptidase secreted by P. chrysosporium and pH 7 and 45 °C for serine peptidase secreted by Leptosphaeria sp. Under incubation at different pH effect, aspartic peptidase was stable under acidic conditions (pH 3-5); cysteine peptidase was stable in wide pH range (pH 4-9), and serine peptidase was more stable under alkaline conditions and pH slightly acidic (pH 5-9). In all these pH ranges mentioned, peptidases showed proteolytic activity above 80% by 1 hour incubation. As regards the thermal stability, cysteine peptidase was more thermostable enzyme and serine peptidase described the lowest temperature tolerance. In incubation with denaturing agents, we observed a decrease in proteolytic activity under the effect of ionic surfactant (0.02-1%) sodium dodecyl sulfate (SDS) bromide and cetyl-trimethyl ammonium bromide (CTAB); copper (II) ion (5 mM); Dithiothreitol (DTT) and guanidine (both in the range of 10-200 mM) for all peptidases. Finally, the study of catalytic specificity of these enzymes, we found a preference for aromatic amino acids (F and W), basic (K and R) and nonpolar (in particular, methionine residue) to aspartic peptidase. High specificity described by cysteine peptidase, which a catalytic preference is notorious for basic amino acids (K, R and H), especially in position P3 and lysine-dependence for catalysis at position P'3. In serine peptidase, for different evaluated positions, we noticed greater acceptance by nonpolar amino acids (G, I, L, M and V), basic (M and R) and neutral polar (N and Q).