A DNA probe was developed for the identification of the periodontal pathogen, Actinobacillus actinomycetemcomitans. Chromosomal DNA was extracted from A. actinomycetemcomitans, digested with a restriction enzyme, Sau3A, ligated to plasmid DNA (pUC18) and transformed into JM109 cells to give a partial A. actinomycetemcomitans library. The library was screened using Southern blot analysis. Out of the nine inserts tested, one was found to be species specific as it did not cross-hybridise to Haemophilus aphrophilus, a closely related organism which occurs in the normal oral microflora, nor did it cross-hybridise with 7 species of Bacteroides tested. A level of detection of 104 cells or 50ng of A. actinomycetemcomitans was obtained. The probe has a length of 779bp and out of 30 restriction enzymes tested, only SspI was found to have a restriction site in the insert. The probe was tested on clinical specimens obtained from five different periodontitis patient groups and was shown to correlate with culture results in eighteen out of twenty-two cases in detecting A. actinomycetemcomitans.

Os objetivos deste trabalho foram detectar e identificar os fungos presentes em amostras de sementes de ipê-amarelo (Tabebuia serratifolia), ipê-roxo (Tabebuia impetiginosa), aroeira-pimenteira (Schinus terebinthifolius) e aroeira-salsa (Schinus molle), coletadas em diferentes localidades (Piracicaba-SP, Mogi-Guaçu-SP, Rio das Pedras-SP, Ijaci-MG, Itumirim-MG e Lavras-MG) uma parte das amostras foram submetidas à assepsia e a outra não; verificar o efeito desses fungos na germinação dessas sementes; avaliar a transmissão de fungos das sementes para as plântulas; avaliar a patogenicidade de Pestalotiopsis sp. em mudas de aroeira-pimenteira e comparar o efeito de diferentes fungicidas (captam, carbendazim+tiram e carboxim+tiram) na incidência dos fungos e na germinação. Constatou-se que os fungos mais frequentes e em maior incidência encontrados associados às sementes, tanto de ipê-amarelo quanto de ipê-roxo, foram Cladosporium sp., Alternaria alternata, Epicoccum sp., Phoma sp., Geotrichum sp., Penicillium sp., Trichothecium sp., Phomopsis sp., Drechslera sp., Aspergillus spp., Curvularia sp. e Fusarium spp. De maneira geral, a assepsia proporcionou uma redução drástica de todos os fungos, em ambas espécies. Não houve diferença significativa na porcentagem de plântulas normais, entre as amostras, porém o tratamento com hipoclorito de sódio, reduziu a germinação em 64%. Na transmissão observou-se, em média, 17% e 10% de plântulas com sintomas, nas amostras sem assepsia e com assepsia, respectivamente. Os fungos mais freqüentes transmitidos pelas sementes de ipê foram: Alternaria alternata., Fusarium spp., Aspergillus spp., Phoma sp. e Phomopsis sp. Em relação às espécies de aroeira, os fungos quantificados foram Cladosporium sp., Alternaria alternata, Aspergillus spp., Pestalotiopsis sp., Penicillium sp., Fusarium spp., Epicoccum sp., Nigrospora sp., Curvularia sp., Drechslera sp., Trichoderma sp., Myrothecium sp. e Phoma sp. A assepsia reduziu ou manteve a incidência dos fungos, exceto para Pestalotiopsis sp. e Aspergillus spp. que aumentaram em algumas amostras de sementes de aroeira-pimenteira. Não houve diferença estatística da germinação das com e sem assepsia e entre amostras; para aroeira-salsa não verificou-se germinação. Foi confirmada a transmissão, principalmente dos fungos Cladosporium sp., Aspergillus spp. e Pestalotiopsis sp. Em mudas de aroeira-pimenteira verificou-se a patogenicidade de Pestalotiopsis sp. No tratamento de sementes com fungicidas, todos, de uma maneira geral, mostram resultados satisfatórios no controle de todos os fungos detectados, tanto para ipê quanto para aroeira. Captam apresentou resultado satisfatório principalmente em sementes de ipê, onde não foi verificado efeito fitotóxico em relação aos demais fungicidas comparados. Porém, o uso de copolímero de poliéter e silicone, um espalhante adesivo utilizado para facilitar a distribuição do produto, interferiu de forma negativa nos resultados de germinação de sementes de ipê; para aroeira não foi verificado este efeito. Torna-se necessário comparar diferentes doses e produtos para não só controlar fungos associados às sementes mas evitar problemas durante a germinação das mesmas e garantir a produção de mudas sadias e vigorosas em viveiros. / The objectives of this work were to detect and to identify the fungi present in samples of native forest seeds of ipê-amarelo (Tabebuia serratifolia), ipê-roxo (Tabebuia impetiginosa), aroeira-pimenteira (Schinus terebinthifolius) and aroeira-salsa (Schinus molle), collected at different places (Piracicaba-SP, Mogí-Guaçu-SP, Rio das Pedras-SP, Ijaci-MG, Itunirim-MG and Lavras-MG), submiting some parts of the asepsis and the other no; to verify the effect of these fungi in the germination of these seeds; to evaluate the transmission of seed fungi associated to seedlings; to evaluate the patogenicity of Pestalotiopsis sp. in aroeira-pimenteira seedlings and to compare the effect of different fungicides (captan, carbendaxim+tiram and carboxim+tiram) in the fungi incidence and germination. The results showed that the most incident and frequent fungi found associated to the seeds of ipê-amarelo and ipê-roxo were Cladosporium sp., Alternaria alternata, Epicoccum sp., Phoma sp., Geotrichum sp., Penicillium sp., Trichothecium sp., Phomopsis sp., Drechslera sp., Aspergillus spp., Curvularia sp. and Fusarium spp. In general, the asepsis provided a drastic reduction of all fungi, in both species. There was no significantive difference in the normal seedling percentage, independent of the sample, however the treatment with sodium hipoclorite, in the asepsis, reduced the germination in 64%. In the transmission was observed, on average, 17% and 10% of seedlings with symptoms, in the samples without asepsis and with asepsis, respectively. The most frequent fungi transmitted by ipê seeds were: Alternaria alternata., Fusarium spp., Aspergillus spp., Phoma sp. e Phomopsis sp.. In relation to aroeira species, the quantified fungi were: Cladosporium sp., Alternaria alternata, Aspergillus spp., Pestalotiopsis sp., Penicillium sp., Fusarium spp., Epicoccum sp., Nigrospora sp., Curvularia sp., Drechslera sp., Trichoderma sp., Myrothecium sp. e Phoma sp.. The The asepsis reduced or maintained the fungi incidence, except for Pestalotiopsis sp. and Aspergillus spp. That increased in some aroeira-pimenteira seeds. The germination results showed that there was no statistical difference in relation to seeds with and without asepsis and among samples. The transmission was confirmed, mainly of the fungi Cladosporium sp., Aspergillus spp. e Pestalotiopsis sp.. In aroeira-pimenteira seedlings the patogenicity of Pestalotiospsis sp. was verified. In the seeds treatment with fungicides, all, in a general way, showed satisfactory results in the control of all detected fungi, as much for ipê as for aroeira. Captan presented satisfactory result principally in ipê seeds, where fitotoxic effect had not been verified in relation to the other compared fungicides. However, the use of copolímero of polyeter and silicon, an adhesive dispersed, used to facilitate the product distribution, negatively interfered on the germination results; for aroeira this effect had not been verified in the germination. To compare different doses, products not only to control fungi associated to the seeds but to avoid problems during the germination of the same ones and to guarantee the production of healthy seedlings and vigorous in nurseries becomes necessary.

人類乳頭瘤病毒 (HPV) 是引起宮頸癌的必要條件。在高危型HPV中，以HPV16在癌症樣本中最為常見，其全球盛行率達50%以上。近年來，用以辨認HPV16變異子譜系的序列特徵已經建立。雖然這個系統建基於全球的HPV16變異株，但是它只包含了四個亞洲地區。為了改善這個系統於亞洲樣本的準確性，是次研究收集了更多亞洲地區的序列。 / 是次研究提供了在香港和韓國收集的HPV16樣本的系統發生史及序列變異 (LCR、E6 和 E7)。此外，是次研究也檢測了HPV16變異株的在兩地的分佈和致癌風險。 / 是次研究從香港和韓國收集了329個HPV16呈陽性的宮頸樣本。利用LCR、E6、E7 和整合的LCR-E6基因序列以極大似然法來構建HPV16變異株的系統發生樹。序列變異會按照系統發生樹之拓撲結構來分類並詳細描述。卡方檢驗或費雪精確性檢定用於分析HPV16變異株在兩地的分佈和致癌風險。 / 是次研究結果顯示用以辨認HPV16變異子譜系的序列特徵需加以改善。我們建議採用A7287C/T作為亞洲子譜系的序列特徵，以替代原有的A7287C。有關HPV16變異株的地理分佈，亞洲和歐洲的變異株在香港 (亞洲變異株: 70%，歐洲變異株: 25.3%) 和韓國 (亞洲變異株: 61.2%，歐洲變異株: 20.2%) 均十分普遍。另外，1和2型亞美變異株在香港和韓國的分佈有著明顯差別 (1型亞美變異株: 2% 與12.4%，P < 0.001; 2型亞美變異株: 0% 與2.8%，P = 0.04)。 / 另外，是次研究發現亞洲子譜系於韓國民族中呈較高致癌風險 [比值比 (95% 置信區間) 2.02 (1.03-3.99)]。在進化支中，E6的第五進化支[2.44 (1.27-4.74)]和E7的第三進化支[2.02 (1.03-3.99)]也於韓國民族中呈較高致癌風險。在SNP中，E6 T178G [2.17 (1.11-4.23)]、兩個E7的SNPs (A647G [1.73 (0.88-3.42)]、T846C [2.27 (1.16-4.49)]) 和9個LCR SNPs (A7175C, T7177C, T7201C, C7270T, A7730C, G7842A [2.02 (1.03-3.99)], A7289C [2.04 (1.05-3.96)], T7781C [2.07 (1.02-4.22)] 和 C24T [2.36 (1.20-4.66)])於韓國民族中也呈較高致癌風險。這些進化支和SNPs都與亞洲子譜系有關聯。在香港方面，兩個LCR SNPs (A7289C [1.89 (0.92-3.87)] 和 T7781C [2.07 (0.92-4.71)])呈較高致癌風險。 / 是次研究發現的高危SNPs和進化支需要進一步的大型流行病學研究和生物化學實驗來核實。這些序列特徵可作為生物標誌物以檢測出與HPV有關的早期宮頸病變。 / Human papillomavirus (HPV) is necessary for the development of cervical cancer. Of those high-risk HPV types, HPV16 is the most common type detected in cervical cancer and accounts for a prevalence of greater than 50% worldwide. Recently, a sequence signature-based system for identifying the sub-lineages of HPV16 variants has been established. Although this system was developed from HPV16 variants collected worldwide, only four Asian regions were included. To improve the accuracy of this sub-lineage classification system for Asian samples, more sequence data from Asian regions were included in the current study. / The current study provided data on the phylogeny and the sequence variation of Long control region (LCR), E6 and E7 open reading frames (ORFs) of HPV16 isolates collected in Hong Kong and Korea. The distribution of HPV16 variants between two regions and the risk association of HPV16 variants with cervical cancer development were also examined. / A total of 329 HPV16-positive cervical samples were collected from Hong Kong and Korea. The phylogenetic trees were constructed for the LCR, E6, E7 and concatenated LCR-E6 sequences using the maximum likelihood method. The sequence variation of each region was delineated and grouped according to the tree topology. The distribution and risk association of HPV16 variants were examined using the chi-square test or Fisher’s exact test as appropriate. / The results showed that the previously described sequence signatures for classifying sub-lineages of HPV16 variants required further improvement, especially for the Asian sub-lineage. We proposed A7287C/T as a signature SNP of the Asian sub-lineage rather than A7287C as suggested by Cornet et al. In regard to the distribution of HPV16 variants, the Asian (As) and European (Eur) variants were commonly found in Hong Kong (As: 70%, Eur: 25.3%) and Korea (As: 61.2%, Eur: 20.2%). Furthermore, Asian American-1 and 2 (AA1 and AA2) variants were found to distribute significantly different between Hong Kong and Korea (AA1: 2% versus 12.4%, P < 0.001; AA2: 0% versus 2.8%, P = 0.04). / A key finding was that variants of the Asian sub-lineage carried a higher oncogenicity among Korean population [odds ratio (95% confidence interval) = 2.02 (1.03-3.99)]. In clade level, E6 clade 5 [2.44 (1.27-4.74)] and E7 clade 3 [2.02 (1.03-3.99)] were found to carry a higher oncogenicity among Korean population. In SNP level, E6 T178G [2.17 (1.11-4.23)], two SNPs of E7 ORF (A647G [1.73 (0.88-3.42)] and T846C [2.27 (1.16-4.49)]) and nine SNPs of LCR (A7175C, T7177C, T7201C, C7270T, A7730C, G7842A [2.02 (1.03-3.99)], A7289C [2.04 (1.05-3.96)], T7781C [2.07 (1.02-4.22)] and C24T [2.36 (1.20-4.66)]) were also found to carry a higher oncogenicity among Korean population. Those clades and SNPs were linked to the Asian sub-lineage. In contrast, only two SNPs of LCR (A7289C [1.89 (0.92-3.87)] and T7781C [2.07 (0.92-4.71)]) were found to associate with a higher oncogenicity among Hong Kong population. / The risk associations of SNPs, clades of the HPV16 Asian sub-lineage revealed by the current study should be verified by large-scale epidemiological studies and biochemical experiments. These signatures may serve as biomarkers for early detection of HPV-related cervical neoplasia. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Ma, Tsz Ue. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 148-156). / Abstracts also in Chinese. / Abstract of Thesis --- p.I / 論文摘要 --- p.V / Acknowledgements --- p.VIII / Contents --- p.X / Figures --- p.XIII / Tables --- p.XIV / Abbreviations --- p.XVI / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- History of Human Papillomavirus --- p.2 / Chapter 1.2 --- Biology of Human Papillomavirus --- p.4 / Chapter 1.2.1 --- Genome Organization and Protein Functions --- p.4 / Chapter 1.2.1.1 --- E5 Protein --- p.7 / Chapter 1.2.1.2 --- E6 Protein --- p.8 / Chapter 1.2.1.3 --- E7 Protein --- p.9 / Chapter 1.2.2 --- Life Cycle of Human Papillomavirus --- p.10 / Chapter 1.2.3 --- Taxonomy of Human Papillomavirus --- p.12 / Chapter 1.3 --- Cervical Cancer --- p.16 / Chapter 1.3.1 --- Natural History --- p.16 / Chapter 1.3.2 --- Risk Factors --- p.17 / Chapter 1.4 --- Epidemiology of Cervical Cancer --- p.19 / Chapter 1.4.1 --- Global Disease Burden --- p.19 / Chapter 1.4.2 --- Disease Burden in Hong Kong --- p.21 / Chapter 1.4.3 --- Disease Burden in South Korea --- p.22 / Chapter 1.5 --- Human Papillomavirus Type 16 --- p.23 / Chapter 1.6 --- Background and Objectives --- p.27 / Chapter Chapter 2 --- Materials and Methods --- p.30 / Chapter 2.1 --- Study Design --- p.31 / Chapter 2.2 --- Study Samples --- p.35 / Chapter 2.2.1 --- HPV16-Positive Samples --- p.35 / Chapter 2.2.2 --- Samples with Unknown HPV Status --- p.36 / Chapter 2.3 --- Laboratory Methods --- p.39 / Chapter 2.3.1 --- DNA Extraction --- p.39 / Chapter 2.3.2 --- Polymerase Chain Reaction --- p.40 / Chapter 2.3.2.1 --- PGMY09/11 PCR --- p.40 / Chapter 2.3.2.2 --- HPV16-Specific PCR --- p.42 / Chapter 2.3.3 --- Genotyping of HPV --- p.48 / Chapter 2.3.4 --- Purification of PCR Products --- p.51 / Chapter 2.3.5 --- Sequencing Reaction --- p.52 / Chapter 2.4 --- Data Analysis --- p.54 / Chapter 2.4.1 --- Sequence Edit and Alignment --- p.54 / Chapter 2.4.2 --- Sequence Variation of HPV16 Variants --- p.56 / Chapter 2.4.3 --- Construction of Phylogenetic Tree --- p.56 / Chapter 2.4.4 --- Distribution and Comparison of HPV16 Variants in Hong Kong and Korea --- p.57 / Chapter 2.4.5 --- Distribution of HPV16 Variants in Normal and Cancer Samples and Risk Association Study --- p.58 / Chapter Chapter 3 --- Results --- p.59 / Chapter 3.1 --- Study Samples --- p.60 / Chapter 3.1.1 --- HPV16-Positive Samples --- p.60 / Chapter 3.1.2 --- Samples with Unknown HPV Status --- p.61 / Chapter 3.2 --- Sub-lineage Identification of HPV16 Variants --- p.63 / Chapter 3.2.1 --- Based on the Phylogenetic Analysis in the Current Study --- p.63 / Chapter 3.2.1.1 --- Concatenated LCR-E6 Phylogenetic Tree --- p.63 / Chapter 3.2.1.2 --- LCR Phylogenetic Tree --- p.66 / Chapter 3.2.1.3 --- E6 Phylogenetic Tree --- p.69 / Chapter 3.2.2 --- Based on the Single Nucleotide Polymorphisms Proposed by Cornet et al. --- p.74 / Chapter 3.2.2.1 --- Single Nucleotide Polymorphisms of LCR Sequence --- p.74 / Chapter 3.2.2.2 --- Single Nucleotide Polymorphisms of E6 Open Reading Frame --- p.78 / Chapter 3.3 --- Sequence Variation of HPV16 Variants --- p.82 / Chapter 3.3.1 --- LCR Sequence --- p.82 / Chapter 3.3.2 --- E6 Open Reading Frame --- p.91 / Chapter 3.3.3 --- E7 Open Reading Frame --- p.95 / Chapter 3.4 --- Distribution of HPV16 Variants in Hong Kong and Korea --- p.100 / Chapter 3.4.1 --- Sub-lineage Level --- p.100 / Chapter 3.4.2 --- Clade Level of E6 Open Reading Frame --- p.101 / Chapter 3.4.3 --- Clade Level of E7 Open Reading Frame --- p.102 / Chapter 3.4.4 --- Single Nucleotide Polymorphisms Level --- p.105 / Chapter 3.4.4.1 --- LCR Sequence --- p.105 / Chapter 3.4.4.2 --- E6 Open Reading Frame --- p.107 / Chapter 3.4.4.3 --- E7 Open Reading Frame --- p.108 / Chapter 3.5 --- Risk Association and distribution of HPV16 Variants in normal and Cancer samples --- p.112 / Chapter 3.5.1 --- Sub-lineage Level --- p.112 / Chapter 3.5.2 --- Clade Level of E6 Open Reading Frame --- p.114 / Chapter 3.5.3 --- Clade Level of E7 Open Reading Frame --- p.115 / Chapter 3.5.4 --- Single Nucleotide Polymorphisms Level --- p.122 / Chapter 3.5.4.1 --- LCR Sequence --- p.122 / Chapter 3.5.4.2 --- E6 Open Reading Frame --- p.125 / Chapter 3.5.4.3 --- E7 Open Reading Frame --- p.126 / Chapter Chapter 4 --- Discussion --- p.132 / Chapter 4.1 --- HPV16 Variant Sub-lineages --- p.133 / Chapter 4.2 --- Comparison of HPV16 variants between Hong Kong and Korea --- p.137 / Chapter 4.3 --- Risk Association of HPV16 Variants --- p.138 / Chapter 4.4 --- Strength and Weakness --- p.144 / Chapter 4.5 --- Implications for Future Work --- p.146 / References --- p.148

Li Jun. / "March 2002." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (p. 189-216). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Char Tsui Shan. / Thesis submitted in: October 2000. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 92-114). / Abstracts in English and Chinese. / Abstract (in English) --- p.i / Abstract (in Chinese) --- p.iv / Acknowledgements --- p.vi / Table of Contents --- p.vii / Contents of Chapter 1: Introduction --- p.viii / Contents of Chapter 2: Materials & Methods --- p.viii / Contents of Chapter 3: Results --- p.x / Lists of Tables --- p.xi / List of Figures --- p.xii / Chapter Chapter 1: --- Introduction --- p.1-24 / Chapter Chapter 2: --- Material & Methods --- p.25 -41 / Chapter Chapter 3: --- Results --- p.42 -76 / Chapter Chapter 4: --- Discussion --- p.77 -89 / Chapter Chapter 5: --- Future Work --- p.90 -91 / References --- p.92 -107 / Chapter Chapter 1: --- Introduction / Chapter 1.1. --- Taxonomy of Enterococci --- p.1 / Chapter 1.2. --- Natural Habitat of Enterococci --- p.2 / Chapter 1.3. --- Characteristics of Enterococci --- p.2 / Chapter 1.4. --- Identification to Species Level of Enterococci --- p.2 / Chapter 1.5. --- Clinical Significance of Enterococci --- p.5 / Chapter 1.6 --- Clinical Management of Enterococcal Infections --- p.8 / Chapter 1.7. --- Antimicrobial Susceptibilities of Enterococci --- p.8 / Chapter 1.8. --- Antimicrobial Resistance of Enterococci --- p.11 / Chapter 1.9. --- Molecular typing of enterococci --- p.16 / Chapter 1.10. --- Epidemiological Studies of Enterococci --- p.19 / Chapter 1.11. --- Objectives --- p.21 / Chapter Chapter 2. --- Materials & Methods / Chapter 2.1. --- Collection of strains --- p.23 / Chapter 2.2. --- Quality control strains --- p.23 / Chapter 2.3. --- Identification of strains to genus level --- p.23 / Chapter 2.4. --- Identification of strains to species level --- p.24 -27 / Chapter 2.4.1. --- By commercially available biochemical method / Chapter 2.4.2. --- By molecular method 226}0ؤ multiplex PCR / Chapter 2.4.2.1. --- Source of primers / Chapter 2.4.2.2. --- Extraction of bacterial DNA / Chapter 2.4.2.3. --- Multiplex PCR / Chapter 2.4.2.4. --- Analysis of PCR products agarose gel electrophoresis / Chapter 2.4.3. --- By conventional biochemical methods --- p.27 / Chapter 2.5. --- Antimicrobial susceptibility testing --- p.27 -30 / Chapter 2.5.1. --- Preparation of antimicrobial agent stock solution / Chapter 2.5.2. --- Preparation of agar medium / Chapter 2.5.3. --- Preparation of inoculum and inoculation of plates / Chapter 2.5.4. --- Incubation and reading of plates / Chapter 2.6. --- Mechanisms of antibiotic resistance --- p.30 -35 / Chapter 2.6.1. --- Amplification of vancomycin resistance genes in enterococci by multiplex PCR / Chapter 2.6.1.1. --- Isolates / Chapter 2.6.1.2. --- Primers / Chapter 2.6.1.3. --- Extraction of bacterial DNA and PCR / Chapter 2.6.2. --- "Detection of gene coding for aminoglycoside-modifying enzyme (AAC6'-APH2"") by PCR" / Chapter 2.6.2.1. --- Isolates / Chapter 2.6.2.2. --- Primers / Chapter 2.6.2.3. --- Extraction of bacterial DNA and PCR / Chapter 2.6.3. --- Detection of β-lactamase in enterococci / Chapter 2.7. --- Molecular typing of enterococci by pulsed-field gel electrophoresis --- p.35 -37 / Chapter 2.7.1. --- Incorporation of chromosomal DNA into agarose plugs / Chapter 2.7.2. --- Digestion of chromosomal DNA in agarose plugs with restriction enzyme SmaI / Chapter 2.7.3. --- Pulsed-field gel electrophoresis / Chapter 2.7.4. --- Cluster analysis / Chapter 2.7.5. --- Data analysis / Chapter 2.8. --- Research plan --- p.37 / Chapter Chapter 3. --- Results / Chapter 3.1. --- Identification of enterococci by API 20 Strep and PCR --- p.39-46 / Chapter 3.2. --- Antimicrobial susceptibilities of enterococci --- p.47 -54 / Chapter 3.3. --- Detection of vancomycin resistance genes in enterococci by PCR --- p.55 -57 / Chapter 3.4. --- "Detection of gene coding for aminoglycoside-modifying enzyme (AAC6'- APH2"") by PCR" --- p.58 -60 / Chapter 3.5. --- Detection of β-lactamase in enterococci --- p.61 / Chapter 3.6. --- Resistance pattern of enterococci --- p.62 -64 / Chapter 3.7. --- Multiresistant enterococci investigated by pulsed-field gel electrophoresis

Os objetivos deste trabalho foram detectar e identificar os fungos presentes em amostras de sementes de ipê-amarelo (Tabebuia serratifolia), ipê-roxo (Tabebuia impetiginosa), aroeira-pimenteira (Schinus terebinthifolius) e aroeira-salsa (Schinus molle), coletadas em diferentes localidades (Piracicaba-SP, Mogi-Guaçu-SP, Rio das Pedras-SP, Ijaci-MG, Itumirim-MG e Lavras-MG) uma parte das amostras foram submetidas à assepsia e a outra não; verificar o efeito desses fungos na germinação dessas sementes; avaliar a transmissão de fungos das sementes para as plântulas; avaliar a patogenicidade de Pestalotiopsis sp. em mudas de aroeira-pimenteira e comparar o efeito de diferentes fungicidas (captam, carbendazim+tiram e carboxim+tiram) na incidência dos fungos e na germinação. Constatou-se que os fungos mais frequentes e em maior incidência encontrados associados às sementes, tanto de ipê-amarelo quanto de ipê-roxo, foram Cladosporium sp., Alternaria alternata, Epicoccum sp., Phoma sp., Geotrichum sp., Penicillium sp., Trichothecium sp., Phomopsis sp., Drechslera sp., Aspergillus spp., Curvularia sp. e Fusarium spp. De maneira geral, a assepsia proporcionou uma redução drástica de todos os fungos, em ambas espécies. Não houve diferença significativa na porcentagem de plântulas normais, entre as amostras, porém o tratamento com hipoclorito de sódio, reduziu a germinação em 64%. Na transmissão observou-se, em média, 17% e 10% de plântulas com sintomas, nas amostras sem assepsia e com assepsia, respectivamente. Os fungos mais freqüentes transmitidos pelas sementes de ipê foram: Alternaria alternata., Fusarium spp., Aspergillus spp., Phoma sp. e Phomopsis sp. Em relação às espécies de aroeira, os fungos quantificados foram Cladosporium sp., Alternaria alternata, Aspergillus spp., Pestalotiopsis sp., Penicillium sp., Fusarium spp., Epicoccum sp., Nigrospora sp., Curvularia sp., Drechslera sp., Trichoderma sp., Myrothecium sp. e Phoma sp. A assepsia reduziu ou manteve a incidência dos fungos, exceto para Pestalotiopsis sp. e Aspergillus spp. que aumentaram em algumas amostras de sementes de aroeira-pimenteira. Não houve diferença estatística da germinação das com e sem assepsia e entre amostras; para aroeira-salsa não verificou-se germinação. Foi confirmada a transmissão, principalmente dos fungos Cladosporium sp., Aspergillus spp. e Pestalotiopsis sp. Em mudas de aroeira-pimenteira verificou-se a patogenicidade de Pestalotiopsis sp. No tratamento de sementes com fungicidas, todos, de uma maneira geral, mostram resultados satisfatórios no controle de todos os fungos detectados, tanto para ipê quanto para aroeira. Captam apresentou resultado satisfatório principalmente em sementes de ipê, onde não foi verificado efeito fitotóxico em relação aos demais fungicidas comparados. Porém, o uso de copolímero de poliéter e silicone, um espalhante adesivo utilizado para facilitar a distribuição do produto, interferiu de forma negativa nos resultados de germinação de sementes de ipê; para aroeira não foi verificado este efeito. Torna-se necessário comparar diferentes doses e produtos para não só controlar fungos associados às sementes mas evitar problemas durante a germinação das mesmas e garantir a produção de mudas sadias e vigorosas em viveiros. / The objectives of this work were to detect and to identify the fungi present in samples of native forest seeds of ipê-amarelo (Tabebuia serratifolia), ipê-roxo (Tabebuia impetiginosa), aroeira-pimenteira (Schinus terebinthifolius) and aroeira-salsa (Schinus molle), collected at different places (Piracicaba-SP, Mogí-Guaçu-SP, Rio das Pedras-SP, Ijaci-MG, Itunirim-MG and Lavras-MG), submiting some parts of the asepsis and the other no; to verify the effect of these fungi in the germination of these seeds; to evaluate the transmission of seed fungi associated to seedlings; to evaluate the patogenicity of Pestalotiopsis sp. in aroeira-pimenteira seedlings and to compare the effect of different fungicides (captan, carbendaxim+tiram and carboxim+tiram) in the fungi incidence and germination. The results showed that the most incident and frequent fungi found associated to the seeds of ipê-amarelo and ipê-roxo were Cladosporium sp., Alternaria alternata, Epicoccum sp., Phoma sp., Geotrichum sp., Penicillium sp., Trichothecium sp., Phomopsis sp., Drechslera sp., Aspergillus spp., Curvularia sp. and Fusarium spp. In general, the asepsis provided a drastic reduction of all fungi, in both species. There was no significantive difference in the normal seedling percentage, independent of the sample, however the treatment with sodium hipoclorite, in the asepsis, reduced the germination in 64%. In the transmission was observed, on average, 17% and 10% of seedlings with symptoms, in the samples without asepsis and with asepsis, respectively. The most frequent fungi transmitted by ipê seeds were: Alternaria alternata., Fusarium spp., Aspergillus spp., Phoma sp. e Phomopsis sp.. In relation to aroeira species, the quantified fungi were: Cladosporium sp., Alternaria alternata, Aspergillus spp., Pestalotiopsis sp., Penicillium sp., Fusarium spp., Epicoccum sp., Nigrospora sp., Curvularia sp., Drechslera sp., Trichoderma sp., Myrothecium sp. e Phoma sp.. The The asepsis reduced or maintained the fungi incidence, except for Pestalotiopsis sp. and Aspergillus spp. That increased in some aroeira-pimenteira seeds. The germination results showed that there was no statistical difference in relation to seeds with and without asepsis and among samples. The transmission was confirmed, mainly of the fungi Cladosporium sp., Aspergillus spp. e Pestalotiopsis sp.. In aroeira-pimenteira seedlings the patogenicity of Pestalotiospsis sp. was verified. In the seeds treatment with fungicides, all, in a general way, showed satisfactory results in the control of all detected fungi, as much for ipê as for aroeira. Captan presented satisfactory result principally in ipê seeds, where fitotoxic effect had not been verified in relation to the other compared fungicides. However, the use of copolímero of polyeter and silicon, an adhesive dispersed, used to facilitate the product distribution, negatively interfered on the germination results; for aroeira this effect had not been verified in the germination. To compare different doses, products not only to control fungi associated to the seeds but to avoid problems during the germination of the same ones and to guarantee the production of healthy seedlings and vigorous in nurseries becomes necessary.

<p>While the many completely sequenced genomes of bacterial pathogens contain all the determinants of the host-pathogen interaction, and also every possible drug target and recombinant vaccine candidate, computational tools for selecting suitable candidates for further experimental analyses are limited to date. The overall objective of my PhD project was to attempt to design reusable systems that employ the two most important features of bacterial evolution, horizontal gene transfer and adaptive mutation, for the identification of potentially novel virulence-associated factors and possible drug targets. In this dissertation, I report the development of two novel technologies that uncover novel virulence-associated factors and mechanisms employed by bacterial pathogens to effectively inhabit the host niche. More importantly, I illustrate that these technologies may present a reliable starting point for the development of screens for novel drug targets and vaccine candidates, significantly reducing the time for the development of novel therapeutic strategies. Our initial analyses of proteins predicted from the preliminary genomic sequences released by the Sanger Center indicated that a significant number appeared to be more similar to eukaryotic proteins than to their bacterial orthologs. In order determine whether acquisition of genetic material from eukaryotes has played a role in the evolution of pathogenic bacteria, we developed a system that detects genes in a bacterial genome that have been acquired by interkingdom horizontal gene transfer.. Initially, 19 eukaryotic genes were identified in the genome of Mycobacterium tuberculosis of which 2 were later found in the genome of Pseudomonas aeruginosa, along with two novel eukaryotic genes.</p> <p>Surprisingly, six of the M. tuberculosis genes and all four eukaryotic genes in P. aeruginosa may be involved in modulating the host immune response through altering the steroid balance and the production of pro-inflammatory lipids. We also compared the genome of the H37Rv M. tuberculosis strain to that of the CDC- 1551 strain that was sequenced by TIGR and found that the organisms were virtually identical with respect to their gene content, and hypothesized that the differences in virulence may be due to evolved differences in shared genes, rather than the absence/presence of unique genes. Using this observation as rationale, we developed a system that compares the orthologous gene complements of two strains of a bacterial species and mines for genes that have undergone adaptive evolution as a means to identify possibly novel virulence &ndash / associated genes. By applying this system to the genome sequences of two strains of Helicobacter pylori and Neisseria meningitidis, we identified 41 and 44 genes that are under positive selection in these organisms, respectively. As approximately 50% of the genes encode known or potential virulence factors, the remaining genes may also be implicated in virulence or pathoadaptation. Furthermore, 21 H. pylori genes, none of which are classic virulence factors or associated with a pathogenicity island, were tested for a role in colonization by gene knockout experiments. Of these, 61% were found to be either essential, or involved in effective stomach colonization in a mouse infection model. A significant amount of strong circumstantial and empirical evidence is thus presented that finding genes under positive selection is a reliable method of identifying novel virulence-associated genes and promising leads for drug targets.</p>

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Wenjun Ma / Influenza A virus (IAV) is a negative sense, single-stranded, segmented RNA virus with eight gene segments. It is an important respiratory pathogen which causes annual epidemics and occasional pandemics worldwide in humans and leads to considerable economic problems for the livestock industry. To control and prevent this significant disease, understanding the pathogenesis of IAVs is critical. Although some molecular mechanisms regarding virulence have been determined, IAV pathogenesis is not completely understood and is difficult to predict. The eight viral gene segments of IAV were thought to encode for 10 viral proteins. Since 2001, eight additional viral proteins have been identified, including PB1-F2, PB1-N40, PA-X, NS3, PA-N155, PA-N182, M42, and PB2-S1. However, the functions of these novel proteins in influenza virus replication as well as pathogenesis have not been fully elucidated. Although PB1-F2 protein is an important virulence factor of IAV, the effects of this protein on viral pathogenicity of swine influenza virus (SIV) remain unclear. In Chapter 2, we investigated the contribution of the PB1-F2 protein to viral pathogenicity of a virulent triple-reassortant (TR) H1N1 SIV in different hosts, pigs and mice. Our data indicate that PB1-F2 expression in virulent TR H1N1 SIV modulates virus replication and pathogenicity in the natural host, pigs, but not in mice. In addition, single amino acid (aa) substitution at position 66 (N/S) in the PB1-F2 has a critical role in virulence in mice but no effect was found in pigs. A novel IAV protein, PA-X consists of the N-terminal 191aa of PA protein and a unique C-terminal 41 (truncated form) or 61 (full-length form) aa residues encoded by +1 ribosomal frameshifting. Although several studies have demonstrated the PA-X protein as an important immune modulator and virulence factor, the impact of different expressions of PA-X protein including full-length, truncated or PA-X deficient forms on viral pathogenicity and host response remains unclear. In Chapter 3, we showed that expression of either truncated or full-length PA-X protein in 2009 human pandemic H1N1 (pH1N1) viruses suppresses host antiviral response by host shutoff activity which promotes viral growth and virulence in mice when compared to loss of PA-X expression. Furthermore, full-length PA-X expression displayed stronger impact on viral pathogenicity and host immune response compared to truncated PA-X expression. Taken together, our results provide new insights into the impact of PB1-F2 and PA-X proteins on virus replication, pathogenicity and modulation of host immune responses. This knowledge is important for better understanding of IAV pathogenesis.

Acquisition of a superantigen pathogenicity island (SaPI) significantly increases virulence in Staphylococcus aureus. Horizontal transfer of SaPIs occurs at high frequency and depends upon a helper bacteriophage, either through direct infection or SOS-mediated induction of a lysogen. SaPIs hijack the packaging machinery of the helper phage, leading to the formation of SaPI-containing transducing particles that can introduce the pathogenicity island into neighboring SaPI-negative cells. All SaPIs contain a conserved core of genes, some of which are co-transcribed as an operon and encode functions involved in helper exploitation. The goal of this study was to more fully understand the intricate relationships between the SaPI elements and their helper bacteriophages, specifically any regulatory crosstalk that might occur between them. We demonstrated phage-host interactions in 80 and 80α, and SaPI1 and SaPIbov1-mediated crosstalk with helper phage 80α. The phage Sri protein was shown to be a bi-functional protein that both derepresses SaPI1 and interferes with host chromosome replication. Incoming SaPI1 experiments showed that SaPI1 modulates the levels of the N-terminal part of orf14 mRNA. Induction experiments using the 80α ΔrinA phage as a genetic tool, reveal several new phage genes that SaPI1 targets for expression modulation. Finally, a novel SaPI1 interference mechanism was identified. In an 80α ΔrinA mutant, which cannot activate its late operon, SaPI1 can directly turn on expression of the packaging and structural genes in a noncanonical manner, initiating from the 2nd gene in the operon, the large terminase subunit.

De 50 à 100 millions de cas de maladie de dengue sont recensés chaque année dans le monde. Le virus de la dengue présente aujourd’hui un problème de santé publique avec son émergence en Europe et particulièrement en France. L’infection par le virus peut être asymptomatique ou être responsable de trois pathologies: l’une avec des symptômes grippaux (DF), une autre avec des hémorragies modérées (DHF) et une dernière avec des hémorragies sévères entraînant un syndrome de choc (DSS).Les facteurs de l’hôte jouent un rôle important dans le développement de formes sévères mais les facteurs viraux impliqués restent peu décrits. Le but de ce travail de thèse était de mieux comprendre ces facteurs viraux au travers de l’étude des dynamiques de circulation de souches de dengue 3 génotype III en Afrique et de la caractérisation de trois souches de dengue de sérotype I du Cambodge. Ce travail nous a permis de mettre en évidence la circulation de variants pendant les épidémies, permettant de supposer que la présence de variants permet une meilleure dissémination, ainsi que des caractéristiques génotypiques et phénotypiques particulières in vitro aux souches associées aux formes hémorragiques et aux formes avec syndrome de choc chez l’homme. Ces travaux ont été complétés par le développement d’un système original de détection du virus de la dengue et des autres virus du genre Flavivirus. Ce travail de thèse a permis d’identifier de potentiels facteurs de virulence propres au virus, ouvrant la voie à la recherche sur le rôle de certaines protéines virales dans la pathogénicité. / From 50 to 100 million cases of dengue illness occurred every year in the world. Today, dengue virus is a public health problem with its emergence in Europe, particularly in France. DENV infection can be asymptomatic or be responsible for three distinct pathologies: one with flu-like symptoms (DF), another with moderate hemorrhage (DHF) and the last one with severe bleeding leading to shock syndrome (DSS). Host factors have an important role in the development of severe forms but implicated viral virulence factors stay not well described. The aim of this research work was to better understand these viral factors through study of dengue serotype 3 genotype III dynamics of circulation in Africa and through the characterization of three dengue serotype 1 strains in Cambodia. This work highlighted the circulation of variants during epidemics, allowing us to suppose that the presence of variants permits a better dissemination, as well as specific phenotypic and genotypic characteristics in vitro of strains associated with hemorrhagic forms or forms with shock syndrome in humans. These works were completed by the development of an original system of detection of dengue virus and other viruses of genus Flavivirus. This research work allowed identifying potential virulence factor specific to virus, opening the way for research on the role of certain viral proteins in pathogenicity.