A mancha-marrom de Alternaria causada pelo fungo Alternaria alternata f.sp. citri patótipo \"tangerina\" é uma das principais doenças que acometem a cultivo de tangerinas (Citrus reticulata) e alguns de seus híbridos. A infecção ocorre principalmente em folhas e frutos jovens que senescem prematuramente. Para realização de estudos com a finalidade de obtenção de cultivares resistentes, ou para a compreensão dos processos de interação planta-patógeno, é necessária a produção massal de esporos in vitro. Este trabalho, no primeiro momento, buscou desenvolver um método eficaz de obtenção de esporos do fungo em meio de cultura. Para isso, comparou os efeitos de diferentes meios de cultura (BDA e CaCO3-A) e tipos de iluminação (LED e NUV). Além do número de esporos, características como germinação, pigmentação, formação de apressórios e patogenicidade foram avaliadas. Descobriu-se que o melhor método de produção de esporos consistiu da manutenção de colônias em meio CaCO3-A por 3 dias sob LED e em seguida por 4 dias sob NUV, com fotoperíodo de 12 h. A partir de então, no segundo momento, realizaram-se testes com técnicas de microscopia e bioquímica fitopatológica para analisar a interação entre o patógeno e folhas das cultivares suscetíveis Ponkan e Murcott e da resistente Fremont. Notou-se por meio de microscopia eletrônica de varredura (MEV) que, independentemente da cultivar, o fungo germinou sobre as folhas, formou apressórios e entrou em cavidades estomáticas. Não foi observada, porém, a colonização de tecidos por meio de microscopia de luz. As cultivares suscetíveis responderam à inoculação com acúmulo de peróxido de hidrogênio e reação de hipersensibilidade. A cultivar resistente, por sua vez, não apresentou reação de hipersensibilidade e aumentou a síntese de enzimas relacionadas ao estresse oxidativo (SOD e CAT) e eliminação de espécies reativas de oxigênio. Como se trata de um fungo necrotrófico capaz de detoxificar espécies reativas de oxigênio, presumiu-se que a reação de hipersensibilidade e a morte celular nas cultivares suscetíveis favorecem a infecção e colonização do patógeno, enquanto que na cultivar resistente a ausência de morte celular e detoxificação de espécies reativas de oxigênio podem estar envolvidas na sua capacidade de defesa. Nas cultivares suscetíveis, verificou-se, por meio de microscopia eletrônica de transmissão (MET), plasmólise e fragmentação de membranas plasmáticas, local indicado como sítio de ação da toxina ACT produzida pelo patógeno. As células da cultivar resistente não apresentaram plasmólise ou alterações nas membranas plasmáticas em decorrência da inoculação do patógeno. / Alternaria brown spot caused by Alternaria alternate f.sp. citri pathotype \"tangerine\" is a important disease that affects tangerine (Citrus reticulate), and some of its hybrids. The infection happens mainly in young leaves and fruits and may cause premature senescence of tissues. To carry out studies in order to obtain resistant cultivars, or to understand the processes of plant-pathogen interaction, it is necessary abundant in vitro spores production. This work, in the first moment, aimed to develop an efficient method for in vitro spore production of the fungus. It was compared the effects of different culture media (BDA and CaCO3-A) and light (LED and NUV). Besides the number of spores, characteristics such as germination, pigmentation, apressoria formation and pathogenicity were investigated. It was found that the best method of spore production consisted of maintaining colonies on CaCO3-A medium for 3 days under LED and then for 4 days under NUV, with photoperiod of 12 h. Thus, in the second moment, assays were carried out using microscopy and phytopathological biochemistry techniques to analyze the interaction between the pathogen and leaves of the susceptible cultivars Ponkan and Murcott, and the resistant Fremont. It was noted by scanning electron microscopy (SEM) that independently of the cultivar, the fungus germinated on the leaves, formed apressoria and grew into stomatal cavities. However, colonization of tissues was not observed by light microscopy. Susceptible cultivars responded to inoculation with accumulation of hydrogen peroxide and hypersensitivity reaction. The resistant cultivar did not show hypersensitivity reaction, but increased the synthesis of enzymes (CAT and SOD) related to oxidative stress and elimination of reactive oxygen species. As a necrotrophic pathogen able to detoxify reactive oxygen species, it was assumed that the hypersensitivity reaction and cell death in the susceptible cultivars contribute to infection and colonization of the pathogen. On the other hand, the absence of cell death and detoxification of reactive oxygen species verified in the resistant cultivar may be involved on its capacity of defense. Cell plamolysis and plasma membranes fragmentation, indicated as the site of action of the ACT toxin produced by the pathogen, were verified in the susceptible cultivars by transmission electron microscopy (TEM). The cells of the resistant cultivar did not show plasmolysis or alterations in the plasma membranes due to pathogen inoculation.

Vírus da raiva provenientes de 23 morcegos de espécies hematófagas, frugívoras e insetívoras foram caracterizados geneticamente pelo seqüenciamento completo da região que codifica a nucleoproteína N. A análise filogenética das seqüências, incluindo lyssavirus e isolados de morcegos do Chile e Estados Unidos, mostrou que os diferentes isolados do vírus da raiva foram de modo geral segregados em quatro grupos genéticos distintas: morcegos hematófagos, morcegos insetívoros 1, 2 e 3. Os morcegos insetívoros 1 constituiram-se por isolados de Eptesicus furinalis: BR-EF1, BR-EF2, BREF3, BR-EF-4, BR-EA1 e BR-NL2; os morcegos insetívoros 2 consistiram de isolados de Molosssus spp: BR-MM1, BR-MM2 e BR- MA1 e os morcegos insetívoros 3 isolados de Nictinomops laticaudatus: BR-NL1 e BR-NL3. A homologia de nucleotídeos entre cada grupo de morcegos insetívoros 1, 2 e 3 foi maior que 99%, 97% e 99%, respectivamente. O grupo de morcegos hematófagos foi representado pelos isolados de: 3 morcegos hematófagos Desmodus rotundus (BR-DR1, BR-DR2 e BR-DR3); 5 morcegos frugívoros Artibeus lituratus BR-AL1, BR-AL2, BR-AL3, BR-AL4 e Artibeus planirostris BRAP1; 2 morcegos insetívoros (BR-MR1 e BR-EA2) e 2 de espécies não identificadas (BR-BAT1 e BR-BAT2). Entre as amostras seqüenciadas foram selecionadas cinco (BR-EF1, BR-NL1, BR-AL3, BR-MM1, BR-DR1) e um isolado de cão (BR-C) para os estudos de patogenicidade em camundongos albinos suíços inoculados pela vias intracerebral (IC) e intramuscular (IM). Todas as amostras quando inoculadas em camundongos pela via IC apresentaram-se patogênicas, provocando a morte dos mesmos num período de 4 a 14 dias pós-inoculação. No entanto, 500DLIC50 das mesmas amostras inoculadas pela via IM levaram a uma mortalidade de camundongos de: 60% (BR-DR1); 50% (BR-C, BR-NL); 40% (BR- AL3); 9,5% (BR-MM1); 5,2% (BR-EF10). As mesmas amostras foram utilizadas para a verificação de proteção cruzada, conferida por vacina comercial de uso animal, de camundongos que receberam uma ou duas doses de vacina pela via subcutânea (SC) e desafiados pelas vias IC e IM. Camundongos inoculados com duas doses de vacina foram protegidos quando desafiados pela via IC, com todas as amostras testadas. Quando os camundongos receberam uma dose da mesma vacina houve proteção parcial daqueles desafiados com as amostras de vírus PV e BR-C. Houve proteção de 100% dos camundongos desafiados pela via IM, com exceção daqueles vacinados com uma dose de vacina e desafiados com a amostra PV que apresentaram um índice de 66% de sobreviventes. Os resultados indicam a possibilidade de existir variantes do vírus da raiva espécies específicas circulando em morcegos. Sugerem ainda, que espécies de morcegos hematófagos, frugívoros e insetívoros compartilham o mesmo polimorfismo de vírus. A vacina comercial contra a raiva contendo vírus inativado e de uso veterinário protegeu os camundongos contra o desafio com as diferentes amostras testadas, sugerindo que as vacinas usualmente utilizadas são efetivas no tratamento profilático da raiva transmitida por morcegos, apesar da marcada diferença de neurovirulência dos diferentes isolados quando inoculados em camundongos pela via IM. / Twenty-three rabies viruses isolated from hematophagous, frugivorous and insectivorous bats were characterized genetically by complete sequencing of the region coding the nucleoprotein N. The phylogenetic analysis of the sequences, including the lyssavirus and the bat isolates from Chile and USA revealed that the isolates were segregated into four distinct genetic lineages: those related to the vampire bats and to the insectivorous bats 1, 2 and 3. The isolates related to the insectivorous bats 1 were from the Eptesicus furinalis: BR-EF1, BR- EF2, BREF3, BR-EF-4, BR-EA1 e BR-NL2; those of the insectivorous bats2 included the isolates from Molosssus spp: BR-MM1, BR-MM2 and BR-MA1 and the group 3, by the isolates from the Nictinomops laticaudatus: BR-NL1 and BR-NL3. The homology among each group of the insectivorous bats 1, 2 and 3 were greater than 99%, 97% and 99%, respectively. The lineage related to vampire bats was represented by three isolates from the D. rotundus (BR-DR1, BR-DR2 e BR-DR3); five from the fruit bats Artibeus lituratus (BR-AL1, BR-AL2, BR-AL3, BR-AL4) and Artibeus planirostris (BRAP1); two from insectivorous bats (BR-MR1 and BR-EA2) and two from unidentified species (BR-BAT1 and BR-BAT2). Among the sequenced amples, five bat isolates (BR-EF1, BR-NL1, BR-AL3, BR-MM1, BR- DR1) and one dog isolate (BR-C) were selected for the study of their pathogenicity in Swiss mice, inoculating through intracerebral (IC) and intramuscular (IM) routes. All the isolates, when inoculated via IC, were pathogenic, provoking death in 4 - 14 post inoculation days. However, mice inoculated with 500ICLD50 of the same isolates through IM route were found with different death rates: 60.0% (BR-DR1); 50.0% (BR-C, BR-NL); 40.0% (BR-AL3); 9.5% (BR-MM1) and 5.2% (BR-EF10). The same isolates were used for the assessment of cross protection conferred by a commercial vaccine of veterinary use. The mice were vaccinated subcutaneously, receiving either one or two shots of vaccine, and challenged through IC and IM routes. Mice receiving two shots were protected against all the isolates, when challenged intracerebrally. Mice receiving one shot were found only partially protected against the challenge with the fixed PV strain and BR-C isolate. Mice challenged intramuscularly showed 100.0% of protection, with the exception of those vaccinated with one dose and challenged with PV strain, which were found with 66.0% of survivors. These results indicate the possibility of the existence of rabies virus variants circulating in different species of bat population. The data also suggest that the vampires, frugivorous and insectivorous bats share the same lineage of rabies viruses. The commercial vaccine has protected the mice against the challenge with different rabies virus isolates, suggesting that the vaccines usually employed in the field are effective, although some marked difference in neurovirulence by IM inoculation was found among the isolates tested.

Xylella fastidiosa (Xf) é o agente causal de doenças em várias culturas economicamente importantes. Recentemente foi identificada como sendo o agente causal da clorose variegada dos citros (CVC), doença que representa um grande problema para os citricultores paulistas, pois vem causando perdas econômicas significativas. Trata-se de uma bactéria gram-negativa, fastidiosa e restrita ao xilema das plantas hospedeiras. Um fato observado na interação Xf - citros, é que plantas infectadas por Xf podem demonstrar sintomas da doença um longo tempo após a infecção. Isto faz supor que a expressão de fatores de patogenicidade e/ou virulência ocorre somente após a população de Xf na planta atingir altas densidades celulares. Como fatores de patogenicidade/virulência de algumas bactérias são regulados por sensores de quorum, seria interessante determinar se esse mecanismo de regulação gênica ocorre em Xf, e quais genes bacterianos são regulados por densidade celular. O objetivo deste trabalho foi verificar se genes de patogenicidade putativos de Xf são modulados pela densidade celular em meio de cultura. Para tal, Xf foi cultivada em PW líquido, células no início da fase exponencial (baixa densidade celular) e na fase estacionária (alta densidade celular) foram coletadas, e RNA total foi extraído. Fragmentos de 20 genes de Xf relacionados à patogenicidade putativos foram transferidos em arranjos ordenados para membranas de náilon, e hibridizados, sob condições de alta estringência, com sondas complexas de primeiras fitas de cDNA marcadas com fosfatase alcalina. A detecção foi feita por quimioluminescência. Sob as condições experimentais utilizadas, os genes fur (XF-2344), gumC (XF-2369), protease-serina (XF-1851) e rsmA (XF-0125) tiveram sua expressão significativamente suprimida sob condições de alta densidade celular (p < 0,05). Já a expressão de rpfF (XF-1115) foi induzida significativamente (p < 0,06) sob condições de alta densidade celular. Os genes gumD (XF-2367), gumJ (XF-2362) e rpfA (XF-0290) tiveram a expressão detectada somente sob condições de alta densidade celular, enquanto a expressão de rpfB (XF-0287) foi detectada somente sob condições de baixa densidade celular. A expressão de celulase (XF0818), mdoH (XF-1623), pluxR (XF-0972), protease-RE (XF-1823), rpoN (XF-1408), xpsK (XF-1523) e xpsL (XF-1524) não foi afetada significativamente pela densidade populacional em meio PW, enquanto a expressão dos genes luxR/UHPA (XF-2608), poligalacturonase (XF-2466), rpfC (XF-1114) e rsmB (XF-0928) não foi detectada em ambas as condições. Usando RT-PCR, os transcritos de todos os genes, exceto gumC, foram detectados sob condições de baixa e alta densidade celular, indicando que a expressão de alguns genes relacionados à patogenicidade putativos em meio de cultura é muito baixa. Os resultados obtidos sugerem que os EPS são sintetizados por Xf predominantemente em condições de alta densidade celular em meio PW. Adicionalmente, os dados sugerem que Xf sintetiza uma molécula sinal similar à de Xcc em meio PW sob condições de alta densidade celular, e que a disponibilidade de ferro pode ser importante para a regulação gênica em condições de alta densidade celular. É possível que o padrão de expressão gênica observado possa ser alterado em função do meio de cultivo ou in planta. / Xylella fastidiosa (Xf) is the causal agent of several economically important plant diseases. Recently, Xf has been identified as the causal agent of citrus variegated chlorosis (CVC), a disease that represents a major economic problem for citrus growers in the São Paulo State. Xf is a gram-negative, fastidious and xylem-limited bacterium. Based on the fact that plants may show disease symptoms a long time after infection in Xf-citrus interaction, we hypothesize that the expression of pathogenicity/virulence factors in Xf occurs after the bacterial population reach high cell densities. Since pathogenicity/virulence factors of some bacteria are quorum-sensing regulated, it would be interesting to determine whether this mechanism of genetic regulation occurs in Xf, and which genes are regulated by cell-density. In order to determine whether Xf putative pathogenesis-related genes are modulated by cell-density in culture media, Xf was grown in liquid PW and cells were sampled at early log (low cell density) and stationary phase (high cell density) and total RNA was extracted. Fragmentos of 20 putative pathogenicity-related genes from Xf were hybridized on ordered arrays nylon membranes to alkaline phosphatase-labeled first strand cDNA from low and high celldensity conditions as probes, at high stringency conditions. Detection was performed using chemiluminescence. Our results indicate that the following putative pathogenicity-related genes are significantly suppressed (p < 0.05) at high cell-density conditions: fur (XF-2344), gumC (XF-2369), serine-protease (XF-1851) and rsmA (XF-0125). The expression of rpfF (XF-1115) was significantly induced (p < 0.06) at high cell-density conditions. Expression of gumD (XF-2367), gumJ (XF-2362) and rpfA (XF-0290) was detected only at high cell-density conditions, whereas expression of rpfB (XF-0287) was detected only at low cell-density conditions. The expression of cellulase (XF0818), mdoH (XF-1623), pluxR (XF-0972), protease-RE (XF-1823), rpoN (XF-1408), xpsK (XF-1523) and xpsL (XF-1524) was not affected by the Xf population density in PW medium, whereas expression of luxR/UHPA (XF-2608), polygalacturonase (XF-2466), rpfC (XF-1114) and rsmB (XF-0928) was not detected under our experimental conditions. Using RT-PCR, transcripts for all genes, except gumC, were detected under low or high cell densities, indicating that the expression of some putative pathogenesis-related genes in culture medium is very low. Our results suggest that EPS may be synthesized by Xf mainly at high cell density conditions in PW medium. Additionally, our data suggest that Xf may synthesize a signal molecule similar to the Xcc in PW medium at high cell density conditions, and that iron availability may be important for gene regulation at high cell density conditions. It is possible that gene expression patterns observed under our experimental conditions may be altered with the culture medium used or when Xf grows in planta.

O cancro cítrico, causado pela bactéria Gram negativa Xanthomonas axonopodis pv. citri (Xac) é considerado uma das principais doenças da citricultura nacional e mundial, devido à susceptibilidade do hospedeiro e por não existirem métodos de controle eficientes. Dados da literatura relatam que alguns genes de bactérias fitopatogênicas possuem uma seqüência consenso de nucleotídeos (TTCGC...N15...TTCGC) denominada “PIP box" ou “plant-inducible-promoter box", localizada na região promotora e responsável pela ativação da expressão de fatores de patogenicidade e virulência quando o patógeno entra em contato com a planta hospedeira. O objetivo deste trabalho foi mapear e investigar no genoma da Xac a expressão de genes contendo seqüências do tipo “PIP box", a 5’ da ORF ou internamente na região codificadora, através da técnica de macroarranjo. Com auxílio de uma ferramenta de bioinformática (script PERL), 208 seqüências consenso para o elemento cis-regulatório “PIP box" foram mapeados no genoma da Xac, e clones contendo seqüências codificadoras associadas aos promotores do tipo “PIP box" foram recuperados do banco de clones gerado pelo projeto de seqüenciamento da bactéria e validados por seqüenciamento. Os DNAs plasmidiais representando cada clone foram preparados e imobilizados em membranas de náilon (macroarranjo), as quais foram hibridadas com sondas de cDNAs marcadas com [a33P] dCTP e obtidas a partir de RNA total extraído da bactéria em estádio não infectante (cultivada em meio de cultura Caldo Nutriente – meio CN) e da bactéria infectante (cultivada por 12 horas ou 20 horas em meio XAM1 indutor de patogenicidade e virulência, ou coletada por exsudação 3 dias ou 5dias após inoculação em folhas de laranjeira). A análise da expressão dos genes contendo promotores “PIP box" putativos revelou 67 genes diferencialmente expressos quando a bactéria teve o seu programa de infecção disparado (indução “in vitro" ou “in vivo"). A validação dos resultados de macroarranjo foi realizada para alguns dos genes por meio de RT-PCR semi-quantitativo e a funcionalidade da seqüência “PIP box" para alguns promotores foi demonstrada através do gene repórter GUS. Os genes com expressão diferencial foram classificados segundo a função biológica das respectivas proteínas codificadas e estão envolvidos em processos tão distintos quanto sistema de secreção tipo III, metabolismo de membrana e parede celular, sistema de captação de ferro, metabolismo de açúcares e degradação da parede vegetal, transdução de sinais, metabolismo de flagelo, formação de biofilme e adesividade, metabolismo de ácidos nucléicos, transportadores de membrana, metabolismo energético e resposta a estresse ambiental. O possível papel destas modificações para a interação planta-patógeno e a instalação do cancro cítrico é discutida. / The citrus canker, caused by the bacterial pathogen Xanthomonas axonopodis pv citri (Xac), is considered worldwide as one of the most important disease of citrus crop due to the lack of resistance in citrus plants and to the absence of efficient methods to control the disease. It was already shown that several genes involved in virulence and pathogenicity in plant pathogenic bacteria have a consensus nucleotide sequence (TTCGC…N15…TTCGC) named plant-inducible-promoter box or PIP box, located in the promoter region of the genes, which are responsible to activate the expression of genes during the host-bacteria interaction. The aim of this work was to map in Xac genome the genes containing the PIP box sequence in the promoter region, or internal to the ORF sequence, and analyze their expression during plant infection using the macroarray technique. Using a PERL script, 208 PIP box consensus sequence associated to genes were found in the Xac genome and clones containing all these genes and their PIP box sequences were recovered from a Xac genome clone bank and reconfirmed by sequencing. The plasmidial DNA containing the gene and the PIP box from each clone were purified, immobilized onto a nylon membrane (macroarray) and hybridized with [a33P] dCTP-labeled cDNAs synthesized from Xac total RNA isolated from bacteria in non-infecting (grown in NB nutrient medium) and infecting (grown for 12 or 20 hours in XAM1 medium that mimics the environment of the plant intercellular space (“in vitro") or for 3 or 5 days in orange plant leaves (“in vivo") conditions. The gene expression profile resulting from the array analysis revealed that 67 genes were differentially expressed during the temporal course of the infection. The results were validated through the analysis of expression profile for some genes using semi-quantitative RT-PCR and the PIP box functionality for some promoters was demonstrated using GUS as reporter gene. The differentially expressed genes were classified regarding the biological function of the encoded protein and related to several relevant processes as type III secretion system, membrane and cellular wall metabolism, iron uptake, sugar metabolism and host cell wall degradation, signal transduction, flagellum metabolism, biofilm formation and adhesiveness, nucleic acid metabolism, membrane transporters, energetic metabolism and stress response. The possible roles of these modifications for the bacteria-plant interaction and disease installation are discussed.

Pela utilização de métodos bioquímicos, biofísicos, de tipagem sorológica e de visualização das proteínas totais bacterianas, isolados de surubim Pseudoplatystoma corruscans, tilápia Oreochromis niloticus e pacu Piaractus mesopotamicus, foram caracterizados, identificados e sua virulência determinada. Dentre as linhagens de referência, o isolado de surubim caracterizou-se como sendo Plesiomonas shiguelloides e os demais isolados de tilápia e pacu foram identificados como Aeromonas hydrophila, todos pertencentes à família Vibrionaceae. Os isolados de tilápia e pacu caracterizaram-se como linhagens virulentas, resistentes aos antibióticos ampicilina, amoxicilina, lincomicina, novobiocina, oxacilina, penicilina, rifampicina e trimetoprim+sulfametoxazol, em ensaios de antibiograma realizados em meio YEA que evidenciaram que as linhagens isoladas de peixes são resistentes a oito das dezessete substâncias antimicrobianas testadas pelo método de difusão em disco. Essas características são compatíveis com as apresentadas pelo espécime tipo de A. hydrophila. Ambas as linhagens quando cultivadas em meio YEA compartilharam a mesma banda de aproximadamente 33,61kDa com o espécime tipo para A. hydrophila. Em meio enriquecido com glucose, a banda compartilhada entre elas teve peso molecular aproximado de 144,28kDa. Os testes de aglutinação sorológica evidenciaram nestas duas linhagens a presença de antígenos estáveis ao calor do tipo O. A técnica de dupla imunodifusão de Ouchterlony demonstrou que o antígeno preparado a partir do isolado de tilápia é a linhagem de A. hydrophila mais indicada para ser utilizada em estudos visando o desenvolvimento de uma vacina polivalente. / Bacteria isolated from surubim Pseudoplatystoma corruscans, tilapia Oreochromis niloticus e pacu Piaractus mesopotamicus, were characterized and identified by biochemical, biophysical, serology, and SDS-PAGE, and their virulence observed. The strain isolated from surubim was characterized as Plesiomonas shigelloides. The other strains isolated from tilapia and pacu were Aeromonas hydrophila. The isolated A. hydrophila strains presented virulence and resistance against the follow antibacterial substances: ampicillin, amoxicillin, lincomicin, novobiocin, oxacillin, penicillin, rifampin and trimetoprim+sulfametoxazole. Both strains when cultivated in YEA medium shared with the A. hydrophila type strain a similar protein band of 33,61kDa. In a medium supplemented with glucose, only one protein exhibiting relative molecular mass of 144.28 kDa, was shared by the type strains isolated from fish and the type strain. The serology tests revealed that all isolated strains presented heat-stable O-antigens. The Ouchterlony double-immunodiffusion showed that the antigen prepared from the tilapia strain possessed surface antigens similar to A. hydrophila type strain and the strains isolated from pacu. This suggested the possibility of development and usage of a common or polyvalent vaccine for A. hydrophila among tilapia and pacu or other freshwater fish species.

by Shuk-kei Lau. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 141-148). / Abstracts in English and Chinese. / Acknowledgement --- p.i / Table of Contents --- p.ii / Abstract --- p.vi / 論文摘要 --- p.viii / Abbreviations --- p.ix / List of Figures --- p.x / List of Tables --- p.xii / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- General introduction --- p.1 / Chapter 1.2 --- HBV and its role in hepatocarcinogenesis --- p.3 / Chapter 1.2.1 --- Current situation of HBV infection and the HCC incidencein the world --- p.3 / Chapter 1.2.2 --- Current situation of HBV infection and the HCC incidencein Hong Kong --- p.4 / Chapter 1.2.3 --- Genetic organization of HBV --- p.4 / Chapter 1.2.4 --- Principle of hepatocarcinogenesis induced by HBV --- p.5 / Chapter 1.2.4.1 --- Role of chronic hepatitis in hepatocarcinogenesis --- p.5 / Chapter 1.2.4.2 --- Role of HBV in hepatocarcinogenesis --- p.6 / Chapter 1.2.5 --- Current screening tests for HCC --- p.7 / Chapter 1.2.6 --- Current therapies for HCC --- p.9 / Chapter 1.3 --- Aim of the present study --- p.13 / Chapter 1.4 --- "Combining Expressed Sequence Tag (EST), Suppression Subtractive Hybridization and cDNA microarray for rapid differentially by expressed genes screening" --- p.14 / Chapter 1.4.1 --- Expressed Sequence Tag (EST) --- p.14 / Chapter 1.4.2 --- cDNA subtraction --- p.15 / Chapter 1.4.3 --- cDNA microarray --- p.16 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- PCR-select cDNA subtraction --- p.17 / Chapter 2.1.1 --- Amplification of subtracted cDNA clones by PCR --- p.17 / Chapter 2.1.2 --- Cycle sequencing of subtracted cDNA clones --- p.18 / Chapter 2.1.3 --- Sequence analysis using BLAST server and Stanford Online Universal Resource for Clones and ESTs (SOURCE) --- p.19 / Chapter 2.2 --- cDNA microarray analysis --- p.20 / Chapter 2.2.1 --- Array fabrication --- p.20 / Chapter 2.2.1.1 --- Amplification of cDNA clones by PCR --- p.20 / Chapter 2.2.1.2 --- Purification of PCR products --- p.21 / Chapter 2.2.1.3 --- Cycle sequencing for clones checking --- p.22 / Chapter 2.2.2 --- Microarray printing --- p.22 / Chapter 2.2.2.1 --- Preparation of cDNA target --- p.22 / Chapter 2.2.2.2 --- Arraying --- p.22 / Chapter 2.2.3 --- Screening of differentially expressed genes in hepatocellular carcinoma and its surrounding normal counterpart by cDNA microarray --- p.23 / Chapter 2.2.3.1 --- Extraction of RNA --- p.23 / Chapter 2.2.3.2 --- RNA labeling --- p.24 / Chapter 2.2.3.3 --- Microarray hybridization --- p.26 / Chapter 2.2.3.4 --- Collection of data --- p.27 / Chapter 2.2.3.5 --- Data normalization and analysis --- p.28 / Chapter 2.3 --- Molecular cloning and characterization of a novel cDNA clone differentially expressed in HCC --- p.30 / Chapter 2.3.1 --- Tissue distribution of T2L522 gene --- p.30 / Chapter 2.3.1.1 --- Northern hybridization --- p.30 / Chapter 2.3.1.2 --- Reverse-transcriptase polymerase chain reaction (RT-PCR) --- p.33 / Chapter 2.3.2 --- Expression level of T2L522 in HCC and its surrounding normal counterpart --- p.33 / Chapter 2.3.3 --- Identification of interacting partner of T2L522 using yeast two-hybrid assay --- p.35 / Chapter 2.3.3.1 --- "Cloning of T2L522 gene into the yeast two-hybrid DNA-BD vector, pGBKT7" --- p.35 / Chapter 2.3.3.2 --- Transformation of yeast competent cells --- p.39 / Chapter 2.3.3.3 --- Mating of T2L522-BD with pretransformed human liver cDNA library --- p.40 / Chapter 2.3.3.4 --- Colony lift p-galactosidase filter assay --- p.42 / Chapter 2.3.4 --- Subcellular localization of T2L522 gene by tagging with green fluorescence protein (GFP) --- p.43 / Chapter 2.3.4.1 --- "Cloning of T2L522 gene into the eukaryotic GFP expression vector, pEGFP-Cl" --- p.43 / Chapter 2.3.4.2 --- Transfection of pEGFP-T2L522 into HepG2 cell --- p.43 / Chapter Chapter 3 --- Results / Chapter 3.1 --- PCR-select cDNA subtraction --- p.45 / Chapter 3.1.1 --- The sequencing results of subtracted-HCC cDNA clones --- p.45 / Chapter 3.1.2 --- Categorization of ESTs sequenced from subtracted-HCC library --- p.45 / Chapter 3.2 --- Microarray analysis --- p.49 / Chapter 3.2.1 --- Array fabrication --- p.49 / Chapter 3.2.1.1 --- Amplification of cDNA microarray targets --- p.49 / Chapter 3.2.2 --- Microarray printing --- p.52 / Chapter 3.2.3 --- Microarray analysis of differentially expressed genesin hepatocellular carcinoma and its surrounding normal counterpart --- p.55 / Chapter 3.2.4 --- Data collection --- p.57 / Chapter 3.2.5 --- Image processing: spots finding and quantitation --- p.61 / Chapter 3.2.6 --- Data normalization and analysis --- p.61 / Chapter 3.3 --- Molecular cloning and characterization of a novel cDNA clone differentially expressed in HCC --- p.73 / Chapter 3.3.1 --- Tissue distribution of T2L522 --- p.77 / Chapter 3.3.1.1 --- Northern hybridization --- p.77 / Chapter 3.3.1.2 --- Reverse-transcriptase polymerase chain reaction (RT-PCR) --- p.79 / Chapter 3.3.2 --- Expression level of T2L522 in hepatocellular carcinoma and its surrounding normal counterpart --- p.81 / Chapter 3.3.3 --- Identification of interacting partner of T2L522 using yeast two-hybrid assay --- p.85 / Chapter 3.3.4 --- Subcellular localization of GFP tagged T2L522 --- p.87 / Chapter Chapter 4 --- Discussion / Chapter 4.1 --- EST analysis on subtracted-HCC cDNA library --- p.89 / Chapter 4.2 --- cDNA microarray analysis --- p.92 / Chapter 4.2.1 --- Generation of reliable data using cDNA microarray --- p.92 / Chapter 4.2.1.1 --- Reproducibility of signal and normalized ratio --- p.92 / Chapter 4.2.2 --- Comparison of data between multiple slides --- p.96 / Chapter 4.2.2.1 --- Assession of data quality and statistical significance --- p.96 / Chapter 4.2.2.2 --- Interpretation of gene expression data from single and multiple hybridizarion --- p.97 / Chapter 4.3 --- Candidate genes differentially expressed in HCC and its surrounding normal counterpart --- p.99 / Chapter 4.3.1 --- Protein up-regulated in HCC --- p.99 / Chapter 4.3.1.1 --- Extracellular matrix protein --- p.99 / Chapter 4.3.1.2 --- Protein involved in other metabolism --- p.100 / Chapter 4.3.1.3 --- Protein involved in transcription and translation --- p.100 / Chapter 4.3.2 --- Protein down-regulated in HCC --- p.101 / Chapter 4.3.2.1 --- Membrane associated protein --- p.101 / Chapter 4.3.2.2 --- Protein involved in other metabolism --- p.102 / Chapter 4.3.2.2 --- Secretory protein --- p.104 / Chapter 4.3.3 --- Novel protein differentially expressed in HCC --- p.107 / Chapter 4.4 --- "TBC1 domain containing protein, T2L522" --- p.108 / Chapter 4.4.1 --- Possible involvement of T2L522 gene in HCC --- p.109 / Chapter 4.4.2 --- Tissue distribution and expression pattern of T2L522 --- p.110 / Chapter 4.4.3 --- Potential interacting partner of T2L522 --- p.110 / Chapter 4.4.4 --- Subcellular localization of T2L522 --- p.112 / Chapter 4.5 --- Summary --- p.113 / Appendix --- p.114 / References --- p.141

by Chan Ping Lung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 108-118). / Abstracts in English and Chinese. / Abstract (in Chinese) --- p.ii / Abstract (in English) --- p.iv / Acknowledgements --- p.vii / Table of Contents --- p.viii / List of Tables --- p.xiii / List of Figures --- p.xiv / List of Appendices --- p.xv / List of Abbreviations --- p.xvi / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Epidemiology of catheter associated infections --- p.1 / Chapter 1.1.1 --- Catheter associated infections --- p.1 / Chapter 1.1.2 --- Risk and mortality of CAI --- p.2 / Chapter 1.1.3 --- Etiology of CAI --- p.3 / Chapter 1.1.3.1 --- Venous catheters --- p.4 / Chapter 1.1.3.2 --- Urinary catheters --- p.4 / Chapter 1.2 --- Pathogenesis of CAI --- p.5 / Chapter 1.2.1 --- Central venous catheters (CVC) --- p.6 / Chapter 1.2.2 --- Urinary catheters --- p.7 / Chapter 1.3 --- Adhesion mechanisms --- p.7 / Chapter 1.3.1 --- Definition of adhesion --- p.7 / Chapter 1.3.2 --- Adhesion mechanism --- p.8 / Chapter 1.3.2.1 --- The phase one --- p.8 / Chapter 1.3.2.2 --- The phase two --- p.10 / Chapter 1.4 --- Catheters --- p.10 / Chapter 1.5 --- Biology of Candida albicans --- p.11 / Chapter 1.5.1 --- Taxonomy of Candida albicans --- p.11 / Chapter 1.5.2 --- Morphology --- p.12 / Chapter 1.5.3 --- Genome --- p.13 / Chapter 1.5.4 --- Biology of Candida albicans cell wall --- p.14 / Chapter 1.5.4.1 --- Constituting molecules of Candida albicans cell wall --- p.14 / Chapter 1.5.4.2 --- Organization of Candida albicans cell wall --- p.15 / Chapter 1.6 --- Agglutinin like sequence gene family --- p.16 / Chapter 1.6.1 --- Gene structure of agglutinin like sequence genes --- p.16 / Chapter 1.6.2 --- Sequence similarity --- p.17 / Chapter 1.6.3 --- Sequence variability --- p.18 / Chapter 1.6.4 --- Expression of ALS genes --- p.19 / Chapter 1.6.5 --- The Als proteins --- p.20 / Chapter 1.6.6 --- Functions of Als proteins --- p.21 / Chapter 1.7 --- Adhesion assay --- p.23 / Chapter 1.7.1 --- Adhesion model --- p.24 / Chapter 1.7.2 --- Factors affecting static adhesion model --- p.25 / Chapter 1.7.3 --- Quantitation methods of adherent cells --- p.27 / Chapter 1.7.3.1 --- Sonication --- p.27 / Chapter 1.7.3.2 --- Staining methods --- p.28 / Chapter 1.7.3.3 --- ATP bioluminescence --- p.28 / Chapter 1.8 --- Research model --- p.29 / Chapter Chapter 2 --- Aim of study --- p.31 / Chapter Chapter 3 --- Materials and Methods --- p.32 / Chapter 3.1 --- Preparation of bacteriological reagents --- p.32 / Chapter 3.2 --- Confirmation of identity of Candida albicans and of Saccharomyces cerevisiae --- p.33 / Chapter 3.3 --- Cell culture of fibroblasts --- p.36 / Chapter 3.3.1 --- Preparation of cell culture reagents --- p.36 / Chapter 3.3.2 --- Recovery of freezing fibroblasts --- p.37 / Chapter 3.3.3 --- Establishment of cell line --- p.37 / Chapter 3.4 --- Preliminary study of adherence of Candida albicans to fibroblasts and to catheters --- p.38 / Chapter 3.4.1 --- Adherence to fibroblasts --- p.38 / Chapter 3.4.1.1 --- Preparation of fibroblasts --- p.38 / Chapter 3.4.1.2 --- Preparation of culture of Candida albicans and of Saccharomyces cerevisiae --- p.39 / Chapter 3.4.1.3 --- Adhesion assay --- p.41 / Chapter 3.4.2 --- Adherence to catheters --- p.42 / Chapter 3.4.2.1 --- Preparation of catheters --- p.42 / Chapter 3.4.2.2 --- Adhesion assay --- p.42 / Chapter 3.5 --- "Confirmation of expression of ALS1, ALS5 smaller allele, and ALS6 of Candida albicans in YPD broth" --- p.44 / Chapter 3.5.1 --- RNA extraction of Candida albicans --- p.45 / Chapter 3.5.2 --- "RT-PCR of ALS1, ALS5 smaller allele, and ALS6" --- p.46 / Chapter 3.5.2.1 --- Primers --- p.46 / Chapter 3.5.2.2 --- RT-PCR --- p.47 / Chapter 3.6 --- Establishment of quantitation system of adhesion assay --- p.49 / Chapter 3.6.1 --- Absorbance measurement of Candida albicans stained with safranin --- p.49 / Chapter 3.6.1.1 --- Preparation of Candida albicans culture --- p.49 / Chapter 3.6.1.2 --- Staining of Candida albicans --- p.50 / Chapter 3.6.1.3 --- Viable count of Candida albicans adhered on the 6-well plate --- p.51 / Chapter 3.6.2 --- ATP bioluminescence --- p.52 / Chapter 3.7 --- Effect of inoculum size on adhesion to catheters --- p.53 / Chapter 3.7.1 --- Preparation of adhesion chambers --- p.53 / Chapter 3.7.2 --- Preparation of catheters --- p.54 / Chapter 3.7.3 --- Preparation of Candida albicans culture --- p.54 / Chapter 3.7.4 --- Adhesion assay --- p.55 / Chapter 3.8 --- "Transformation of Saccharomyces cerevisiae with ALS1, ALS5 smaller allele, and ALS6" --- p.57 / Chapter 3.8.1 --- DNA extraction of Candida albicans --- p.58 / Chapter 3.8.2 --- "PCR of ALS1, ALS5 smaller allele, and ALS6" --- p.59 / Chapter 3.8.3 --- Gel extraction --- p.60 / Chapter 3.8.4 --- Restriction digestion of PCR products of ALS genes and cloning plasmids --- p.61 / Chapter 3.8.5 --- "Ligation of ALS1, ALS5 smaller allele, ALS6 with pYES6CT cloning plasmids" --- p.62 / Chapter 3.8.6 --- Transformation of ligated plasmid into Escherichia coli --- p.63 / Chapter 3.8.7 --- Miniprep of plasmids --- p.64 / Chapter 3.8.8 --- DNA sequencing --- p.65 / Chapter 3.8.9 --- Transformation of Saccharomyces cerevisiae --- p.66 / Chapter 3.8.10 --- "Detection of Alsl，Als5, and Als6 protiens expression" --- p.68 / Chapter 3.8.10.1 --- Preparation of cultures in SC synthetic medium --- p.68 / Chapter 3.8.10.2 --- Protein extraction --- p.69 / Chapter 3.8.10.3 --- Dot blot of cell wall lysates --- p.69 / Chapter 3.9 --- Adhesion of transformed Saccharomyces cerevisiae to fibroblasts --- p.71 / Chapter 3.9.1 --- Preparation of fibroblasts and of Saccharomyces cerevisiae cultures --- p.71 / Chapter 3.9.2 --- Adhesion assay --- p.72 / Chapter 3.10 --- "Adhesion of transformed Saccharomyces cerevisiae to FEP, polyurethane, and silicone catheters" --- p.72 / Chapter 3.10.1 --- "Preparation of catheters, adhesion chambers and transformed Saccharomyces cerevisiae cultures" --- p.73 / Chapter 3.10.2 --- Adhesion to catheter fragments --- p.73 / Chapter 3.11 --- Statistical analysis --- p.74 / Chapter Chapter 4 --- Results --- p.75 / Chapter 4.1 --- Confirmation of identity of Candida albicans and of Saccharomyces cerevisiae --- p.75 / Chapter 4.1.1 --- Candida albicans --- p.75 / Chapter 4.1.2 --- Saccharomyces cerevisiae --- p.75 / Chapter 4.2 --- Cell culture of fibroblasts --- p.76 / Chapter 4.3 --- "Preliminary studies of adherence of Candida albicans to fibroblasts and to FEP, polyurethane, and silicone catheters" --- p.76 / Chapter 4.3.1 --- Adherence to fibroblasts --- p.76 / Chapter 4.3.2 --- Adherence to catheters --- p.77 / Chapter 4.4 --- "Confirmation of expression of ALSl, ALS5 smaller allele, and ALS6 of Candida albicans in YPD broth" --- p.78 / Chapter 4.5 --- Establishment of quantitation system of adhesion assay --- p.79 / Chapter 4.5.1 --- Absorbance measurement of Candida albicans stained with safranin --- p.79 / Chapter 4.5.2 --- ATP bioluminescence --- p.79 / Chapter 4.6 --- Effect of inoculum size on adhesion to catheters --- p.80 / Chapter 4.7 --- "Transformation of Saccharomyces cerevisiae with ALS1, ALS5 smaller allele, and ALS6" --- p.81 / Chapter 4.7.1 --- "PCR of ALSl, ALS5 smaller allele, and ALS6" --- p.81 / Chapter 4.7.2 --- Ligation of PCR products with pYES6CT plasmids --- p.82 / Chapter 4.7.3 --- "DNA sequencing results of ALS1, ALS5 smaller allele, and ALS6 ligated plasmids" --- p.83 / Chapter 4.7.4 --- "Detection of Alsl, Als5, and Als6 proteins expression" --- p.84 / Chapter 4.8 --- Adhesion of transformed Saccharomyces cerevisiae to fibroblasts --- p.84 / Chapter 4.9 --- "Adhesion of transformed Saccharomyces cerevisiae to FEP, polyurethane and silicone catheters" --- p.85 / Chapter Chapter 5 --- Discussion --- p.89 / Chapter 5.1 --- Limitations of static adhesion assay model --- p.89 / Chapter 5.2 --- Quantitation System --- p.90 / Chapter 5.2.1 --- Staining method --- p.90 / Chapter 5.2.2 --- ATP bioluminescence assay --- p.91 / Chapter 5.3 --- "Preliminary studies of adherence of Candida albicans to fibroblasts and to FEP, polyurethane, and silicone catheters" --- p.93 / Chapter 5.4 --- Effect of inoculum size on adhesion to catheters --- p.94 / Chapter 5.5 --- Selection of ALS genes --- p.96 / Chapter 5.6 --- Adhesion assay of transformed Saccharomyces cerevisiae to fibroblasts --- p.97 / Chapter 5.7 --- Adhesion assay of transformed Saccharomyces cerevisiae to catheters --- p.99 / Chapter 5.8 --- Alternative research model --- p.101 / Chapter 5.9 --- Implications and future work --- p.102 / Chapter Chapter 6 --- Conclusion --- p.107 / References --- p.108 / Tables --- p.119 / Figures --- p.123 / Appendices --- p.136

No description available.

A bactéria Escherichia coli é responsável por perdas econômicas significativas mundialmente, incluindo-se aquelas que ocorrem na produção avícola. O controle e a prevenção da colibacilose aviária são complexos, pois envolve a distinção de isolados que comumente habitam o trato gastrointestinal das aves daquelas consideradas patogênicas. Embora tenha sido assumido que a maioria dos isolados não possui potencial zoonótico, estudos recentes têm sugerido que isolados isoladas de humanos e de aves poderiam compartilhar o maquinário genético necessário para causar a doença no hospedeiro. Desta forma, os animais de produção poderiam atuar como reservatórios de estirpes potencialmente patogênicas para humanos. O objetivo deste trabalho foi realizar a caracterização molecular em grupos filogenéticos de E. coli isoladas de aves (APEC) e de humanos (UPEC) e propor um futuro acompanhamento da flutuação da patogenicidade dos isolados APEC em planteis avícolas. Foram selecionadas 450 isolados UPEC e 460 APEC para classificação em quatro grupos filogenéticos (A, B1, B2 e D) através de um protocolo de multiplex-PCR. Estes resultados foram comparados com a presença ou ausência de 38 genes associados à virulência e com o índice de patogenicidade in vivo estabelecido para cada isolado em estudo anterior. Em relação aos isolados APEC, 31,1% foram classificadas no grupo D, 25,2% no grupo B2, 24,1% no grupo B1 e 19,6% no grupo A. Entre os isolados UPEC, 53,6% das foram classificadas no grupo B2, 25,3% no grupo D, 15,1% no grupo A e apenas 6,0% no grupo B1. Os isolados virulentos geralmente classificam-se no grupo B2, porém algumas podem ser classificadas no grupo D. Enquanto que os isolados comensais em geral pertencem aos grupos A e B1. Observou-se associação entre determinados genes e os grupos filogenéticos, tanto para isolados APEC quanto UPEC. Observou-se diferença significativa entre os índices de patogenicidade conforme a fonte de isolamento, sendo que os isolados de lesões apresentaram os maiores índices. Também foi observada uma associação direta entre os índices de patogenicidade obtidos in vivo e os grupos filogenéticos. Os isolados do grupo B2 e D apresentaram maiores índices em relação aos isolados B1 e A. Uma vez que a distribuição dos isolados APEC nos grupos filogenéticos apresentou associação significativa com a patogenicidade, o multiplex-PCR torna-se uma importante ferramenta disponível para o screening da patogenicidade das amostras isoladas na cadeia avícola. / Escherichia coli is responsible for significant economic losses, including those occurring in poultry production. The control and prevention of avian colibacillosis are complex because it involves the distinction of pathogenic strains and those that are commonly found in the gastrointestinal tract flora of health birds. Although it has been assumed that most strains do not have zoonotic potential, recent studies have suggested that strains isolated from humans and poultry could share the genetic machinery needed to cause the disease in the host. Therefore, production animals could act as reservoirs of strains potentially pathogenic to humans. The aim of this study was to carry out the molecular characterization in phylogenetic groups of strains of E. coli isolated from poultry (APEC) and humans (UPEC), and to propose a future monitoring of the pathogenicity of APEC strains in poultry farms. A total of 450 UPEC and 460 APEC strains were selected for classification into four phylogenetic groups (A, B1, B2 and D) using a multiplex-PCR protocol. These results were compared with the presence or absence of 38 virulence-associated genes and the in vivo pathogenicity index established for each strain in a previous study. Regarding the APEC strains, 31.1% were classified in group D, 25.2% in group B2, 24.1% in group B1 and 19.6% in group A. Among the UPEC strains, 53.6% were classified in group B2, 25.3% in group D, 15.1% in group A and only 6.0% in group B1. Virulent strains are generally classified in group B2, but some may be classified in group D. While commensal isolates generally belong to groups A or B1. It was observed an association between certain genes and phylogenetic groups, both for APEC and UPEC strains. A significant difference was observed among pathogenicity indices according to the source of isolation, and the strains isolated from lesions presented the highest indices. A direct association between pathogenicity indices obtained in vivo and phylogenetic groups was also observed. Strains of groups B2 and D showed higher indices compared to strains from B1 and A. Since the distribution of APEC strains in phylogenetic groups showed a significant association with pathogenicity, multiplex-PCR becomes an important tool available for screening pathogenicity of the isolated samples in the poultry chain.

Yeung Sze-wan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references. / Abstracts in English and Chinese. / ACKNOWLEDGEMENT --- p.i / ABSTRACT --- p.ii / ABBREVIATIONS --- p.vii / TABLE OF CONTENTS --- p.ix / Chapter CHAPTER 1 --- INTRODUCTION --- p.1-1 / Chapter 1.1 --- Anatomy of the Cervix --- p.1-1 / Chapter 1.2 --- Histology --- p.1-1 / Chapter 1.2.1 --- Squamous Epithelium --- p.1-1 / Chapter 1.2.2 --- The Endocervical Epithelium --- p.1-3 / Chapter 1.2.3 --- The Squamo-columnar Junction --- p.1-4 / Chapter 1.2.3.1 --- The Embryology --- p.1-4 / Chapter 1.2.3.2 --- Definition --- p.1-4 / Chapter 1.3 --- Human Papillomaviruses (HPVs) --- p.1-6 / Chapter 1.3.1 --- Structure of the Viruses --- p.1-6 / Chapter 1.3.2 --- The Nomenclature --- p.1-7 / Chapter 1.3.3 --- HPVs Genomic Structure and Properties of Gene Products --- p.1-7 / Chapter 1.3.4 --- Target Tissues --- p.1-8 / Chapter 1.3.5 --- Role of HPVs in the Carcinogenesis of Lesions --- p.1-9 / Chapter 1.3.6 --- Risk Groups of HPVs --- p.1-10 / Chapter 1.4 --- Pathology --- p.1-11 / Chapter 1.4.1 --- Macroscopic Features --- p.1-11 / Chapter 1.4.2 --- Symptoms and Diagnosis --- p.1-12 / Chapter 1.4.3 --- Histopathology --- p.1-13 / Chapter 1.4.3.1 --- Histopathological Grading of Cervical Intraepithelial Neoplasia --- p.1-19 / Chapter 1.4.3.2 --- Staging of Cervical Cancer --- p.1-24 / Chapter 1.5 --- Epidemiology of Cervical Intraepithelial Neoplasia and Cervical Cancer --- p.1-27 / Chapter 1.5.1 --- Descriptive Epidemiology --- p.1-28 / Chapter 1.5.2 --- Risk Factors --- p.1-30 / Chapter 1.6 --- Human Papillomavirus Type 16 --- p.1-42 / Chapter 1.6.1 --- Role of HPV16 in CIN and Cervical Carcinoma --- p.1-42 / Chapter 1.6.2 --- Viral Load of HPV 16 in CIN --- p.1-43 / Chapter 1.6.3 --- HPV 16 Viral Load as a Screening Tool --- p.1-46 / Chapter 1.7 --- Quantitation of HPV 16 --- p.1-48 / Chapter 1.7.1 --- Methods in Viral Quantification --- p.1-48 / Chapter 1.7.2 --- Selection of Methodology --- p.1-51 / Chapter 1.7.3 --- Correlation of HPV 16 Viral Loading with Severity of Cervical Lesions --- p.1-54 / Chapter CHAPTER 2 --- AIMS OF STUDY --- p.2-1 / Chapter CHAPTER 3 --- MATERIALS AND METHODS --- p.3-1 / Chapter 3.1 --- Materials --- p.3-1 / Chapter 3.1.1 --- Patients and Specimens --- p.3-1 / Chapter 3.2 --- Methods --- p.3-3 / Chapter 3.2.1 --- DNA Extraction --- p.3-3 / Chapter 3.2.2 --- Polymerase Chain Reaction --- p.3-7 / Chapter 3.2.3 --- Gel Electrophoresis --- p.3-8 / Chapter 3.2.4 --- Real-time Quantitation Polymerase Chain Reaction --- p.3-11 / Chapter 3.2.5 --- Statistical Analysis --- p.3-15 / Chapter CHAPTER 4 --- RESULTS --- p.4-1 / Chapter 4.1 --- Grading of Cervical Smears --- p.4-1 / Chapter 4.2 --- Incidence of HPV 16 Detected in Cervical Smears --- p.4-2 / Chapter 4.2.1 --- Detection of HPV 16 in Women for Routine Pap Smear --- p.4-2 / Chapter 4.2.2 --- Detection of HPV 16 in Women for Colposcopic Examination --- p.4-5 / Chapter 4.3 --- Quantification of HPV 16 by Real-time PCR --- p.4-5 / Chapter 4.3.1 --- Range of Detection --- p.4-10 / Chapter 4.3.2 --- Standard Curve --- p.4-12 / Chapter 4.3.3 --- Reproducibility of Quantitative Real-time PCR --- p.4-17 / Chapter 4.3.4 --- Sensitivity of Quantitative Real-time PCR --- p.4-17 / Chapter 4.3.5 --- Detection and Quantification of HPV 16 E6/7 Genes in HPV16 Positive Cervical Scrapes --- p.4-21 / Chapter 4.4 --- Comparison of HPV 16 Copy Number Detected among Three Lesion Groups --- p.4-22 / Chapter 4.5 --- Clinical Analysis --- p.4-27 / Chapter 4.6 --- HPV 16 DNA Copy Number in Lesion Groups --- p.4-28 / Chapter CHAPTER 5 --- DISCUSSION --- p.5-1 / Chapter 5.1 --- Selection of Material (Scrapes) --- p.5-1 / Chapter 5.2 --- Detection of HPV 16 in Cervical Scrapes --- p.5-3 / Chapter 5.2.1 --- Selection of HPV Type --- p.5-3 / Chapter 5.2.2 --- Techniques in Detecting HPV Viral Load --- p.5-3 / Chapter 5.2.2.1 --- Advantages of Quantitative Real-time PCR --- p.5-6 / Chapter 5.2.2.2 --- Parameters Affecting the Performance of Real-time PCR --- p.5-8 / Chapter 5.2.3 --- Selection of Detection Sites --- p.5-9 / Chapter 5.2.4 --- Standard Curve Establishment --- p.5-10 / Chapter 5.3 --- Comparison between Real-time PCR and Traditional PCR --- p.5-12 / Chapter 5.4 --- Role of HPV Viral Load in Cervical Neoplasm --- p.5-13 / Chapter 5.5 --- HPV Infection in Hong Kong Chinese Women --- p.5-17 / Chapter 5.6 --- Clinical Significance of HPV 16 Viral Load Detected in Cervical Neoplasm --- p.5-18 / Chapter 5.7 --- Future Prospect --- p.5-20 / Chapter CHAPTER 6 --- CONCLUSION --- p.6-1 / REFERENCES --- p.R-I