Errata slip inserted at back / Bibliography: leaves 159-192 / vi, 142, [75] leaves : ill ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Summary: Primary object ... was to construct a pulsed field gel electrophoresis (PFGE) derived long range restriction map of this region by physically linking adjacent DNA probes to common high molecular weight genomic DNA fragments / Thesis (Ph.D.)--University of Adelaide, Dept. of Paediatrics, 1994

Errata attached to inside front cover. / Bibliography: leaves [156-188] / vi, 155, [33] leaves, [48] leaves of plates : ill. (some col.) ; 30cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Plant Science, 2001

Thesis (M.Sc.), Memorial University of Newfoundland, 1998. / Restricted until June 1999. Bibliography: leaves 85-97.

Trypanosoma brucei and T. congolense are protozoan parasites that infect humans, domestic livestock and wildlife in Africa. These parasites undergo complex morphological and biochemical changes, during the various stages of their life cycle. These changes correlate with alterations in the levels of trypanosomal lysosomal cysteine proteases, suggesting a role for transcriptional regulation of the cysteine protease in these parasites. The mechanism of this regulation is not yet understood nor have the promoter regions of the cloned trypanosome cysteine protease genes been investigated. This study involved an attempt to clone the T. brucei and T. congolense DNA fragments containing the promoter regions as the initial step in the investigation of the control elements of the cysteine protease gene. Trypanosomes were isolated from infected rat blood employing a combination of the methods of isopicnic isolation on Percoll gradients and DEAE-cellulose anion exchange resin chromatography. Approximately 5 x 10⁹ viable trypanosome cells were isolated from the infected rat blood and chromosomal DNA (approximately 500 μg) was extracted by alkaline-lysis method. Trypanosome genomic libraries were initially constructed in Eschericia coli HB101 employing the positive selection vector pEcoR251. The Trypanosoma brucei pEcoR251 library contained 6 000 recombinants and the Trypanosoma congolense library contained 15 000 recombinants. Plasmid DNA was then extracted from pools of recombinants, employing the alkaline-lysis method, digested with EcoRl restriction endonuclease and resolved by agarose gel electrophoresis. After Southern hybridisation, the pEcoR251 libraries did not reveal any putative clones containing the fragment of interest when probed with both an oligonucleotide probe and the PCR generated dsDNA probe. Genomic libraries were then constructed in the phagemid pUC119. The T. brucei and T. congolense genomic libraries contained 33 000 and 27 000 recombinants respectively. Recombinants from the T. brucei and T. congolense libraries were pooled in lots of 400 and 300 respectively. Of the 80 T. brucei plasmid pools that were screened 30 pools contained fragments that hybridised with the probe whilst 12 pools from the 90 T. congolense library pools that were screened contained fragments that hybridised with the probe. Putative clones identified appeared to contain inserts, ranging between two and seven kb in size. A partial T. congolense library consisting of approximately 12 pools was screened by colony hybridisation for identification of individual clones and 76 putative clones were identified. After confirmation of these putative clones on a dot blot using a DIG-labelled dsDNA probe, a selection of 30 putative clones were subjected to Southern hybridisation using a DIG-labelled DNA probe. Following Southern hybridisation 23 putative clones were identified to contain DNA inserts of interest in the range of two to seven kb. Five clones, designated pCPC1, pCPC2, pCPC3, pCPC4 and pCPC5 were then selected for further restriction mapping. Clone pCPC4 contains a seven kb fragment of T. congolense genomic DNA. A partial T. brucei library consisting of approximately 30 pools was screened by colony hybridisation for the identification of individual putative clones. Although plasmid pools containing putative clones were identified repeatedly by Southern blotting and DNA/DNA hybridisation, it was not possible to identify individual putative clones following transformation into E. coli MV1184 and colony hybridisation. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 1997.

Includes bibliographies. / xiv, 175, [96] leaves, [17] leaves of plates : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / This thesis reports on the generation of molecular tools for the analysis of chromosome 6R of rye and the application of these tools in structural analysis of 6RL. Results presented include physical and genetic maps of chromosome 6RL incorporating RFLP and PCR markers and CreR, the locus conferring resistance to cereal cyst nematode (CCN). The ability to detect small introgessions of rye chromatin in wheat is demonstrated. / Thesis (Ph.D.)--University of Adelaide, Dept. of Plant Science, 1997

Thesis (Ph. D.)--University of Missouri--Columbia, 1997. / Typescript. Vita. Includes bibliographical references (leaves : 117-131). Also available on the Internet.

Made available in DSpace on 2015-03-26T13:37:39Z (GMT). No. of bitstreams: 1 texto completo.pdf: 2474594 bytes, checksum: 6cdab11f886d77e7d0c8a3d024dde318 (MD5) Previous issue date: 2008-07-30 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / The genus Begomovirus (family Geminiviridae) includes viruses with a genome comprised of one or two molecules of circular, single-stranded DNA, transmitted to dicot species by the whitefly Bemisia tabaci. In Brazil, after the introduction of the B biotype of B. tabaci in the early 1990s, the incidence of begomoviruses in tomato has become frequent, with several reports of new viral species. Some of these species have become prevalent under field conditions, including Tomato severe rugose virus (ToSRV) and Tomato yellow vein streak virus (ToYVSV). The purpose of this study was to characterize two isolates of these species through the cloning of their whole genomes followed by molecular analysis, and the production of infectious clones for determination of their host ranges. Total DNA extracts of infected plants were used for whole genome amplification using the phi29 phage DNA polymerase. The amplification products were cloned into plasmids and completely sequenced. Sequence analysis indicated that the DNA-A and DNA-B of ToSRV-[BR:Pir1:05] and ToYVSV- [BR:Pda30:05] isolates had greater than 90% identities with other isolates of ToSRV and ToYVSV, respectively. The molecular analysis indicated that the DNA-A of the BR:Pir1:05 isolate may be the result of a recombination event, in which the virus acquired the rep ORF of Tomato rugose mosaic virus (ToRMV) and the cp ORF of an unidentified virus. Analysis of the DNA-B of the same isolate indicated the existence of a relationship with viruses that infect weed/wild hosts in Brazil, corroborating the hypothesis that viruses present in wild hosts led to the virus currently found in tomatoes. Additionally, because of the high identity observed between the DNA-B of ToSRV-[BR:Pir1:05] and ToRMV, it is possible that this genomic component is shared by the two viruses. In host range assays, plants showing latent infections were observed for both isolates. Such plants can act as natural reservoirs and serve as a primary source of inoculum for host plants of economic importance such as the tomato. The infectious clones of ToYVSV- [BR:Pda30:05] had low infectivity in the host range assays. It is possible that the inoculation method was not effective in the transmission of this isolate, or one or both clones could contain mutations that reduce the efficiency of their replication. / O gênero Begomovirus (família Geminiviridae) inclui vírus com genoma composto por uma ou duas moléculas de DNA fita simples, transmitidos a espécies de plantas dicotiledôneas pela mosca-branca Bemisia tabaci. No Brasil, após a introdução do biótipo B de B. tabaci no início da década de 1990, a incidência de begomovírus em tomateiro tornou-se freqüente, com vários relatos de novas espécies virais. Algumas dessas espécies tornaram-se prevalentes em condições de campo no Brasil, incluindo o Tomato severe rugose virus (ToSRV) e o Tomato yellow vein streak virus (ToYVSV). O objetivo deste trabalho foi caracterizar dois isolados dessas espécies, por meio da clonagem de seus genomas completos seguida de análise molecular, e da obtenção de clones infecciosos para determinação de suas gamas de hospedeiros. Extratos de DNA total de plantas infectadas foram utilizados para a amplificação do genoma viral completo utilizando-se a DNA polimerase do fago φ29. Os produtos da amplificação foram clonados em plasmídeos e completamente seqüenciados. A análise das seqüências do DNA-A e DNA-B dos isolados ToSRV- [BR:Pir1:05] e ToYVSV- [BR:Pda30:05] indicou valores de identidade acima de 90% com outros isolados de ToSRV e ToYVSV, respectivamente. A análise molecular indica que o DNA-A do isolado BR:Pir1:05 pode ser resultado de um evento de recombinação, no qual o vírus adquiriu a ORF rep do Tomato rugose mosaic virus (ToRMV) e a ORF cp de um vírus não identificado. A análise do DNA-B do mesmo isolado indica a existência de relacionamento com vírus que infectam plantas daninhas/silvestres no Brasil, reforçando a hipótese de que vírus presentes em hospedeiros silvestres deram origem aos vírus atualmente encontrados em tomateiro. Adicionalmente, devido à alta identidade observada entre os DNAs-B dos isolados BR:Pir1:05 e do ToRMV, é possível que este componente genômico seja compartilhado pelos dois vírus. Nos testes de gama de hospedeiros, plantas apresentando infecções latentes foram observadas para os dois isolados. Tais plantas podem atuar como reservatórios naturais e servir de fonte de inóculo primário para plantas hospedeiras de importância econômica, como o tomateiro. Os clones infecciosos do isolado BR:Pda30:05 apresentaram baixa infectividade nos testes de gama de hospedeiros. É possível que o método de inoculação não tenha sido eficiente na transmissão deste isolado, ou que os clones apresentem mutações que reduzam a eficiência de sua replicação.

Para a obtenção da seqüência completa do cDNA da α-amilase foi realizada a varredura de uma biblioteca de cDNA, utilizando-se iniciadores planejados a partir do consenso entre seqüências de DNA ou aminoácidos disponíveis em bancos de dados. Após a excisão do vetor pBK-CMV carregando o inserto, foram obtidos subclones e todos foram seqüênciados. Foi obtida uma seqüência clonada de 1971pb, contendo uma ORF de 1251pb, codificando uma proteina de 416 aminoácidos (aa). Esta proteína apresentou urn provável peptídeo sinal de 15 aa, peso molecular calculado de 45.080 Da e pI 5,84. A seqüência de bases obtida mostrou similaridade de 75% quando comparada corn genes de outras monocotiledôneas como arroz, milho, cevada e trigo. Para a seqüência de aminoácidos, deduzida a partir região codificadora, esta semelhança foi de ate 80%, mostrando-se altamente conservada. Os modelos das estruturas primária, secundária e terciária para a α-amilase de banana coincidem em diversos pontos com estruturas determinadas por cristalografia de raios X para α-amilases de outras espécies, incluindo a disposição espacial de resíduos catalíticos e de ligação de substrato e íons cálcio. Análises por northern e Southern blot sugerem que apesar da existência de uma família de genes corn múltiplas cópias, a expressão destes nas frutas não alcança níveis detectáveis por northern blot. / Abstract not available.

Submitted by Jaqueline Silva (jtas29@gmail.com) on 2016-09-14T17:40:15Z No. of bitstreams: 2 Dissertação - Isabella Inês Rodrigues Rosa - 2016.pdf: 1910068 bytes, checksum: 8f551746fe8c21347507aecf98f02609 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Jaqueline Silva (jtas29@gmail.com) on 2016-09-14T17:41:15Z (GMT) No. of bitstreams: 2 Dissertação - Isabella Inês Rodrigues Rosa - 2016.pdf: 1910068 bytes, checksum: 8f551746fe8c21347507aecf98f02609 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2016-09-14T17:41:15Z (GMT). No. of bitstreams: 2 Dissertação - Isabella Inês Rodrigues Rosa - 2016.pdf: 1910068 bytes, checksum: 8f551746fe8c21347507aecf98f02609 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2016-06-28 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Staphylococcus saprophyticus is a pathogenic bacterium of the urinary tract and the main etiological agent of urinary tract infections by Gram-positive bacteria. Although S. saprophyticus potentially can cause serious infections such as pyelonephritis, septicemia, endocarditis and nephrolithiasis, and also multidrug resistance has been reported, not much is known about the mechanisms used by this bacterium during infection. Secreted proteins might be essential on those mechanisms if their role is accomplished during phagocytosis by their assistance of an active infection in phagocytic cells, protecting against oxidative stress and increasing the persistence of bacterial cells within phagocytes, and / or causing lysis of the host cell. Recently our group identified the immunogenic protein SsaA in the secretome of S. saprophyticus. This protein had been previously identified in S. aureus proteome, and it appears to be controlled by regulatory systems for known virulence factors. It also presents similarities with lytic proteins and proteins that assist the persistence within phagocytic cells. However, no approach had analyzed the contribution of SsaA during infection, therefore, through the construction a cloning vector containing the S. saprophyticus gene ssaA, heterologous expression of the recombinant protein and the production of specific polyclonal antibodies, it was able to verify the interaction of SsaA and proteins from macrophages infected by bacterial cells. Through immunofluorescence microscopy, it was verified that the dispersion of SsaA is not limited to phagocytic cells but it was throughout their cytoplasm after internalization of the bacterium. These findings together with other evidence in the literature suggest that SsaA is used during infection by S. saprophyticus, more specifically during phagocytosis. Further approaches are required to confirm if SsaA has a lytic activity and also characterize this protein as a virulence factor, contributing to elucidate strategies used by S. saprophyticus during infection in the human host. / Staphylococcus saprophyticus é uma bactéria patogênica do trato urinário e principal agente etiológico de infecções urinárias por bactérias Gram-positivas. Apesar de potencialmente S. saprophyticus ocasionar infecções graves como pielonefrite, septicemia, nefrolitíase e endocardite, e relatos de multirresistência a antibióticos, relativamente pouco é conhecido sobre os mecanismos utilizados por esta bactéria durante infecção. Proteínas secretadas podem ser essenciais nesses mecanismos se seus papéis forem durante fagocitose auxiliando uma internalização ativa na célula fagocítica, protegendo contra o estresse oxidativo e aumentando a persistência de células bacterianas no interior de fagócitos, e/ou causando lise da célula hospedeira. Recentemente nosso grupo identificou a proteína imunogênica SsaA no secretoma de S. saprophyticus. Essa proteína já havia sido identificada em S. aureus como altamente imunogênica, e parece estar relacionada a fatores de virulência como o Antígeno Imunodominante A (IsaA), a proteína Urease, a hidrolase de peptideoglicano LytM, a Proteína Repressora de Toxinas (Rot) e a proteína de choque térmico HslU. Contudo, poucos estudos conseguem identificar a proteína SsaA secretada e nenhum analisa sua contribuição durante infecção por bactérias do gênero Staphylococcus. Através da construção de um vetor de clonagem contendo o gene ssaA de S. saprophyticus, expressão heteróloga da proteína recombinante e produção de anticorpos policlonais específicos, foi possível verificar a interação entre SsaA e proteínas de macrófagos infectados por células de S. saprophyticus. Através de microscopia de imunofluorescência, foi verificado que a secreção de SsaA não é limitada à fagossomos, mas esta proteína é dispersa em todo o citoplasma da célula fagocítica após internalização de células bacterianas. Os resultados encontrados sugerem que SsaA é utilizada por S. saprophyticus durante infecção, especificamente durante fagocitose. Estudos posteriores serão necessários para confirmar se SsaA possui atividade lítica e caracteriza-la como fator de virulência, contribuindo para elucidar estratégias utilizadas por S. saprophyticus durante infecção no hospedeiro humano.

Bacillus sp VLSH08 screened from sea wetland in Nam Dinh province produces an extracellular endo-1,4-beta-glucanase. According to the results of the classified Kit API 50/CHB as well as sequence of 1500 bp fragment coding for 16S rRNA gene of the Bacillus sp VLSH 08 strain showed that the taxonomical characteristics between the strain VLSH 08 and Bacillus amyloliquefaciene JN999857 are similar of 98%. Culture supernatant of this strain showed optimal cellulase activity at pH 5.8 and 60 Celsius degree and that was enhanced 2.03 times in the presence of 5 mM Co2+. Moreover, the gene encoding endo-1,4-beta-glucanase from this strain was cloned in Escherichia coli using pCR2.1 vector. The entire gene for the enzyme contained a 1500-bp single open reading frame encoding 500 amino acids, including a 29-amino acid signal peptide. The amino acid sequence of this enzyme is very close to that of an EG of Bacillus subtilis (EU022560.1) and an EG of Bacillus amyloliquefaciene (EU022559.1) which all belong to the cellulase family E2. A cocktail of enzyme containing this endo-1,4-beta-glucanase used for biomass hydrolysis indicated that the cellulose conversion attained to 72.76% cellulose after 48 hours. / Chủng vi khuẩn Bacillus sp VLSH08 được tuyển chọn từ tập hợp chủng vi khuẩn phân lập ở vùng ngập mặn tỉnh Nam Định có khả năng sinh tổng hợp enzyme endo-1,4-beta-glucanase ngoại bào. Kết quả phân loại chủng vi khuẩn Bacillus sp VLSH08 bằng Kit hóa sinh API 50/CHB cũng như trình tự gen mã hóa 16S rRNA cho thấy độ tương đồng của chủng Bacillus sp VLSH08 và chủng Bacillus amyloliquefaciene JN999857 đạt 98%. Dịch lên men của chủng được sử dụng làm nguồn enzyme thô để nghiên cứu hoạt độ tối ưu của enzyme ở pH 5,8 và nhiệt đô 60oC. Hoạt tính enzyme tăng 2,03 lần khi có mặt 5 mM ion Co2+. Đồng thời, gen mã hóa cho enzyme endo-1,4-betaglucanase cũng được tách dòng trong tế bào Escherichia coli sử dụng vector pCR 2.1. Gen mã hóa cho enzyme này có chiều dài 1500 bp, mã hóa cho 500 axit amin, bao gồm 29 axit amin của chuỗi peptid tín hiệu. So sánh cho thấy trình tự gen endo-1,4-beta-glucanase của chủng Bacillus sp VLSH08 có độ tương đồng cao với enzyme này của chủng Bacillus subtilis (EU022560.1) và của chủng Bacillus amyloliquefaciene (EU022559.1). Tất cả các enzyme nhóm này đều thuộc họ cellulase E2. Enzyme của chủng này cũng đã được phối trộn với các enzyme khác tạo thành cocktail để thủy phân sinh khối cho kết quả cellulose bị thủy phân 72,76% sau 48 giờ.