Thesis (M.S.)--University of Alabama at Birmingham, 2006. / Description based on contents viewed Feb. 12, 2009; title from PDF t.p. Includes bibliographical references (p. 40-41).

Typescript. Includes vita and abstract. Bibliography: Includes bibliographical references (leaves 151-160). Description: xiii, 160 leaves : ill. ; 29 cm.

The increasing incidence of disseminated (invasive) candidiasis is probably attributable to iatrogenic factors and to improved pre and postmortem evaluation. Premortem diagnosis of such infections have seldom been made early enough for successful treatment. In order to increase the likelihood of successful antifungal chemotherapy, rapid diagnosis of such infections is vital. However, present diagnostic procedures for invasive candidiasis are insensitive and often do not reliably differentiate superficial from invasive infections. This study was undertaken to produce DNA probes and to optimize conditions for rapid and efficient detection of Candida DNA. Seven random Candida albicans DNA fragments (2-7 kbp) were cloned into plasmid pACYC 184. These recombinant plasmids were labeled with either ³²p or biotin and used as probes. Two of the four recombinant plasmids tested were genus specific. The other two were slightly cross reactive with other yeasts (Saccharomyces cerevisiae and Hansenula anomala). Probes labeled with ³²p were twice as sensitive as the biotin probes. One ³²p labelled recombinant (#66) detected 7 Pg of target DNA , which corresponds to approximately 2 X 10⁵ C.albicans cells. With refined simple DNA extraction procedures for C.albicans (in serum), these recombinant probes could possibly be suitable for clinical application. / Medicine, Faculty of / Pathology and Laboratory Medicine, Department of / Graduate

Traditionally, bacterial culture has been used for bacterial detection, allowing study of living microorganisms. Molecular methods are rapid and allow simultaneous identification of numerous species and uncultivated phylotypes. The objective of this doctoral thesis was to investigate the role of the oral microbiota, including poorly characterized and uncultivated bacteria, in dental caries and periodontitis, by comprehensive molecular, clinical, and statistical methods. The microbiota of 275 pre-school children (75 with caries and 200 caries-free) was examined by whole genomic DNA probes, 16S rDNA cloning and sequencing, and PCR. Streptococcus mutans, exhibiting a combined association with Streptococcus sobrinus, was significantly associated with Early Childhood Caries (ECC). Plaque from children with Severe Early Childhood Caries (S-ECC) was diverse with 138 identified and 107 unidentified taxa, which possibly included novel phylotypes. Other species/phylotypes associated with childhood caries included Lactobacillus gasseri (p<0.01), Lactobacillus fermentum, Actinomyces israelii, and Actinomyces odontolyticus (all p<0.05, ECC), Veillonella parvula (p<0.01), Veillonella atypica (p<0.05), and Veillonella sp. HOT-780 (p<0.01, S-ECC). Lactobacillus acidophilus and Lactobacillus reuteri, both used as probiotic therapy species, were detected more frequently in caries-free children than those with ECC. Fastidious periodontal species, including Parvimonas micra, Aggregatibacter actinomycetemcomitans, Eubacterium brachy, Filifactor alocis (all p <0.05), and Porphyromonas gingivalis (p<0.01), were also more frequently detected in children with dental caries than in caries-free children. Other variables associated with ECC were race, dental visit, snacking (all p<0.05), and visible dental plaque (p<0.01). The oral microbiota of early periodontitis in young adults (N=141) was analyzed by whole genomic and oligonucleotide DNA probes, and PCR. Species detected more frequently in early periodontitis than periodontal health included Treponema denticola, F. alocis, Porphyromonas endodontalis, Bacteroidetes sp. HOT-274 (oral clone AU126), and A. odontolyticus (p<0.01) by oligonucleotide DNA probes, and P. gingivalis (p<0.001) and T. forsythia (p=0.03) by PCR. Subgingival samples exhibited a higher prevalence of periodontitis-associated species than samples from tongue surface, including A. actinomycetemcomitans, T. denticola, T. forsythia (all p<0.05), and uncultivated TM7, Treponema, and Actinobaculum clones (all p<0.05). P. gingivalis (p<0.01) by PCR was associated with periodontal disease progression. Early periodontitis was associated with older age (p=0.01), male gender (p=0.04), and cigarette smoking (p=0.05). The role of bacterial subgroups in periodontitis was examined by studying the serotypeability of 313 genotyped clinical A. actinomycetemcomitans isolates (189 subjects). A total of 95 strains (30 subjects) remained non-serotypeable, although PCR revealed presence of the serotype- specific genes. The absence of the immunodominant serotype-specific antigen was confirmed by immunoblot assays. No major DNA rearrangement in the studied serotype-specific gene clusters was found. In summary, detection of previously cultured species and uncultivated phylotypes revealed the diversity of the oral microbiota in dental diseases and health already early in life. Bacterial species have insufficiently characterized subgroups that may have attributes to evade the host response. Molecular approaches used in this study enable comprehensive, culture-independent characterization of the oral microbiome that may in the future lead to identification of diagnostic bacterial profiles for dental diseases.

Rotaviruses are one of the most significant causes of diarrheal disease in the world. Their presence in groundwater and drinking water supplies constitutes a health risk to the population. The study of rotaviruses in the environment has been hampered by the lack of accessible and consistent detection methodologies. Gene probes and other molecular techniques are a novel approach for the detection of these viruses in water. The feasibility of these new techniques for the detection and study of rotaviruses in the environment has been assessed using the simian SA-11 and the culturable human Wa rotavirus strains as models. Two general approaches have been undertaken consisting of hybridization of probes with genomic RNA and hybridization with mRNA produced by the virion-incorporated transcriptase. Hybridization of gene probes with genomic dsRNA of rotaviruses in environmental concentrates resulted in the detection of 10 4 immunofoci of Wa rotavirus. In vitro transcription serves as an amplification method with sensitivity 100- to 1000-fold greater than when probing for genomic RNA. The sensitivity obtained in Wa-seeded distilled water and environmental concentrates after in vitro transcription is 2 and 20 immunofoci, respectively. Proteins in environmental concentrates decrease the efficiency of probe hybridization by 10-100 fold. Also, transcriptase-inhibiting factors found in environmental samples decrease the production of mRNA. Both proteins and transcriptase-inhibiting factors can be reduced significantly with Sephadex G-200 columns. Passage of environmental concentrate through Sephadex G-200 spun columns, followed by in vitro transcription, was used to detect rotaviruses in environmental samples. Rotaviruses were detected by this combination of techniques in eight of 20 sewage samples, one of 16 tap water samples, five of 32 ground water samples, and two of nine surface water samples. Only one of 17 samples which tested positive with Wa cDNA 4 was positive for non-specific probe binding. The probing of rotavirus mRNA, amplified by the virion-incorporated transcriptase, is a practical and feasible method for monitoring these viruses in the environment.

O método de fixação do arco ortodôntico aos bráquetes cerâmicos é principalmente realizado por meio de ligaduras elastoméricas estéticas e têm sido alvo de atenção, pois, apesar da praticidade, acumulam grande quantidade de biofilme que causa degradação de cor, comprometendo permanentemente a estética. O objetivo desta pesquisa foi avaliar, in vivo, a contaminação microbiana da saliva e de 4 diferentes marcas comerciais de ligaduras estéticas: 1- Unistick Pearl American Orthodontics, Sheboygan, WI, USA; 2- Power Sticks Pearl Ortho Technology, Tampa, Flórida, EUA; 3- Ease to tie, Obscure - 3M Unitek, Monrovia, CA, USA; 4 - Ligadura modular marfimMorelli Ortodontia-Brasil, por meio da técnica Checkerboard DNA-DNA Hybridization. Foram selecionados 23 pacientes (entre 11 e 25 anos) de ambos os sexos, em tratamento ortodôntico corretivo. Foi coletado 1 ml de saliva não estimulada no momento da inserção e trinta dias após a colocação das ligaduras estéticas. Decorridos 30 dias da colocação, as ligaduras elastoméricas foram removidas, processadas individualmente e estocadas a -20°C, até o momento da análise microbiológica. Os dados foram analisados utilizando o software SAS software for Windows versão 9.1.3. Na saliva, não foram encontradas alterações estatisticamente significantes, exceto para o grupo de bactérias do complexo laranja (p=0,0493). Os resultados encontrados nas ligaduras elastoméricas estéticas mostraram acúmulo significativo nas bactérias do complexo laranja (p=0,0167), enquanto que nos outros complexos e nas bactérias cariogênicas não observamos diferenças estatisticamente relevantes. Evidenciou-se que a ligadura estética da marca comercial Morelli, apresentou menores níveis de contaminação bacteriana (p=0,0108) nos meninos. Assim, conclui-se que as ligaduras elastoméricas estéticas, no período de 30 dias, foram multicolonizadas por várias espécies de micro-organismos, especialmente as do complexo laranja, reforçando a necessidade de uma higiene bucal rigorosa em pacientes ortodônticos. / The attachment for orthodontic archwires to ceramic brackets are mainly done by means of aesthetic elastomeric ligatures, therefore, they have been subject of attention because they accumulate a great amount of biofilm, causing color degradation then compromising their aesthetic visual. The objective of this research was to analyze, in vivo, the microbial contamination of saliva and 4 different brands of aesthetic ligatures: 1- Unistick Pearl - American Orthodontics, Sheboygan, WI, USA; 2- Power Sticks -Pearl Ortho Technology, Tampa, Florida, EUA; 3- Ease to tie, Obscure - 3M Unitek, Monrovia, CA, USA; 4 - Ligadura modular marfimMorelli Ortodontia-Brasil, through the Checkerboard DNA-DNA Hybridization technique. Twenty-three patients (between 11 and 25 years old) of both genders, under orthodontic treatment were selected. One milliliter of non-stimulated saliva, at time of inserting the ligatures and 30 days after its insertion, was collected. Thirty days after the placement the elastomeric ligatures were removed, individually processed and stored at -20°C, until the microbiological analysis. The data were analyzed using the SAS software for Windows version 9.1.3. No statistically significant changes were found in the saliva values, except for the bacterias of the orange complex (p = 0.0493). The obtained results in the aesthetic elastomeric ligatures showed a significant increase in the orange bacterial complex (p=0,0057), while in the others complexes no statistically differences were observed. The Morelli commercial brand of aesthetic ligature showed lower bacterial contamination levels (p=0,0108) in boys. Thus, it can be concluded that the aesthetic elastomeric ligatures, in a 30-day period, were multi-colonized by various species of microorganisms, especially from the orange complexes, reinforcing the need for a thorough oral health care in orthodontic patients.

Tin-hung Ho. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1990. / Bibliography: leaves 137-151. / ACKNOWLEDGEMENT --- p.1 / CONTENT --- p.3 / ABBREVIATIONS --- p.7 / ABSTRACT --- p.8 / Chapter CHAPTER 1 --- INTRODUCTION --- p.10 / Chapter CHAPTER 2 --- LITERATURE REVIEW / Chapter 2.1 --- The cervix and cervical cancer --- p.12 / Chapter 2.2 --- Human papillomaviruses --- p.23 / Chapter 2.3 --- Culture of cancer cells --- p.36 / Chapter 2.4 --- Methods for the detection of HPV infection --- p.40 / Chapter CHAPTER 3 --- MATERIALS AND METHODS / Chapter 3.1 --- Characterization of cervical carcinoma cell lines / Chapter 3.1.1 --- Materials and solutions --- p.47 / Chapter 3.1.2 --- Establishment of cervical carcinoma cell lines --- p.50 / Chapter 3.1.3 --- Morphological studies of cervical carcinoma cells --- p.52 / Chapter 3.1.4 --- Examination of cervical carcinoma cells cultured on collagen gel --- p.54 / Chapter 3.1.5 --- Growth kinetics study --- p.55 / Chapter 3.1.6 --- Plating efficiency test --- p.56 / Chapter 3.1.7 --- Spheroid formation assay --- p.56 / Chapter 3.1.8 --- Chromosome number study --- p.57 / Chapter 3.2 --- Immunocytochemical studies / Chapter 3.2.1 --- Materials and solutions --- p.58 / Chapter 3.2.2 --- Immunocytochemical test for keratin --- p.59 / Chapter 3.2.3 --- Test for HPV capsid antigens --- p.60 / Chapter 3.3 --- Molecular studies of HPV in cervical carcinoma cells / Chapter 3.3.1 --- Materials and solutions --- p.62 / Chapter 3.3.2 --- Preparation of HPV DNA probes --- p.66 / Chapter 3.3.3 --- DNA extraction from the cervical carcinoma cells --- p.74 / Chapter 3.3.4 --- Detection of HPV DNA sequences by the use of hybridization techniques --- p.76 / Chapter 3.3.5 --- Copy number and physical state studies of HPV --- p.81 / Chapter 3.3.6 --- Study of the transcriptional activity of HPV DNA in cultured cervical carcinoma cells --- p.83 / Chapter CHAPTER 4 --- RESULTS / Chapter 4.1 --- Characterization of cervical carcinoma cell lines / Chapter 4.1.1 --- Morphological studies --- p.89 / Chapter 4.1.2 --- Examination of cervical carcinoma cells cultured on collagen gel --- p.90 / Chapter 4.1.3 --- Growth kinetics study --- p.93 / Chapter 4.1.4 --- Plating efficiency test --- p.94 / Chapter 4.1.5 --- Spheroid formation assay --- p.95 / Chapter 4.1.6 --- Chromosome number study --- p.98 / Chapter 4.2 --- Immunocytochemical studies / Chapter 4.2.1 --- Immunocytochemical test for keratin --- p.99 / Chapter 4.2.2 --- Test for HPV capsid antigen --- p.99 / Chapter 4.3 --- Molecular studies of HPV in cervical carcinoma cell lines / Chapter 4.3.1 --- Preparation of HPV DNA probes --- p.101 / Chapter 4.3.2 --- Detection of HPV DNA by the use of hybridization techniques --- p.102 / Chapter 4.3.3 --- Copy number and physical state studies --- p.105 / Chapter 4.3.4 --- Analysis of the transcriptional activity --- p.108 / Chapter CHAPTER 5 --- DISCUSSIONS / Chapter 5.1 --- Characterization of cervical carcinoma cell lines / Chapter 5.1.1 --- Morphological features of two cervical carcinoma cell lines --- p.110 / Chapter 5.1.2 --- Other characteristics of the cell lines --- p.112 / Chapter 5.2 --- Immunocytochemical studies / Chapter 5.2.1 --- Test for keratin antigens --- p.117 / Chapter 5.2.2 --- Test for HPV capsid antigens --- p.117 / Chapter 5.3 --- Molecular studies of HPV in cervical carcinoma cell lines / Chapter 5.3.1 --- Establishment of methods --- p.121 / Chapter 5.3.2 --- Detection of HPV DNA sequences by nucleic acid hybridizations --- p.123 / Chapter 5.3.3 --- Copy number and physical state studies --- p.128 / Chapter 5.3.4 --- Transcriptional analysis of HPV DNA in cervical carcinoma cell lines --- p.132 / CONCLUSION --- p.134 / REFERENCES --- p.137 / ILLUSTRATIONS --- p.152

O método de fixação do arco ortodôntico aos bráquetes cerâmicos é principalmente realizado por meio de ligaduras elastoméricas estéticas e têm sido alvo de atenção, pois, apesar da praticidade, acumulam grande quantidade de biofilme que causa degradação de cor, comprometendo permanentemente a estética. O objetivo desta pesquisa foi avaliar, in vivo, a contaminação microbiana da saliva e de 4 diferentes marcas comerciais de ligaduras estéticas: 1- Unistick Pearl American Orthodontics, Sheboygan, WI, USA; 2- Power Sticks Pearl Ortho Technology, Tampa, Flórida, EUA; 3- Ease to tie, Obscure - 3M Unitek, Monrovia, CA, USA; 4 - Ligadura modular marfimMorelli Ortodontia-Brasil, por meio da técnica Checkerboard DNA-DNA Hybridization. Foram selecionados 23 pacientes (entre 11 e 25 anos) de ambos os sexos, em tratamento ortodôntico corretivo. Foi coletado 1 ml de saliva não estimulada no momento da inserção e trinta dias após a colocação das ligaduras estéticas. Decorridos 30 dias da colocação, as ligaduras elastoméricas foram removidas, processadas individualmente e estocadas a -20°C, até o momento da análise microbiológica. Os dados foram analisados utilizando o software SAS software for Windows versão 9.1.3. Na saliva, não foram encontradas alterações estatisticamente significantes, exceto para o grupo de bactérias do complexo laranja (p=0,0493). Os resultados encontrados nas ligaduras elastoméricas estéticas mostraram acúmulo significativo nas bactérias do complexo laranja (p=0,0167), enquanto que nos outros complexos e nas bactérias cariogênicas não observamos diferenças estatisticamente relevantes. Evidenciou-se que a ligadura estética da marca comercial Morelli, apresentou menores níveis de contaminação bacteriana (p=0,0108) nos meninos. Assim, conclui-se que as ligaduras elastoméricas estéticas, no período de 30 dias, foram multicolonizadas por várias espécies de micro-organismos, especialmente as do complexo laranja, reforçando a necessidade de uma higiene bucal rigorosa em pacientes ortodônticos. / The attachment for orthodontic archwires to ceramic brackets are mainly done by means of aesthetic elastomeric ligatures, therefore, they have been subject of attention because they accumulate a great amount of biofilm, causing color degradation then compromising their aesthetic visual. The objective of this research was to analyze, in vivo, the microbial contamination of saliva and 4 different brands of aesthetic ligatures: 1- Unistick Pearl - American Orthodontics, Sheboygan, WI, USA; 2- Power Sticks -Pearl Ortho Technology, Tampa, Florida, EUA; 3- Ease to tie, Obscure - 3M Unitek, Monrovia, CA, USA; 4 - Ligadura modular marfimMorelli Ortodontia-Brasil, through the Checkerboard DNA-DNA Hybridization technique. Twenty-three patients (between 11 and 25 years old) of both genders, under orthodontic treatment were selected. One milliliter of non-stimulated saliva, at time of inserting the ligatures and 30 days after its insertion, was collected. Thirty days after the placement the elastomeric ligatures were removed, individually processed and stored at -20°C, until the microbiological analysis. The data were analyzed using the SAS software for Windows version 9.1.3. No statistically significant changes were found in the saliva values, except for the bacterias of the orange complex (p = 0.0493). The obtained results in the aesthetic elastomeric ligatures showed a significant increase in the orange bacterial complex (p=0,0057), while in the others complexes no statistically differences were observed. The Morelli commercial brand of aesthetic ligature showed lower bacterial contamination levels (p=0,0108) in boys. Thus, it can be concluded that the aesthetic elastomeric ligatures, in a 30-day period, were multi-colonized by various species of microorganisms, especially from the orange complexes, reinforcing the need for a thorough oral health care in orthodontic patients.

by Yam Hin Fai. / "June 1998." / Thesis (Ph.D.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (p. 118-151). / 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. / Abstract in Chinese.

The study of the DNA has found application beyond our understanding of its cellular function and into a variety of materials assembly and nucleic acid detection systems. The current research investigates double-stranded DNA probes in both a colloidal particle assembly and fluorescent assay format utilizing competitive hybridization events. In both contexts, the affinity of the dsProbes is tuned by the sequence design parameters of duplex length and complementarity. These systems were incubated with nucleic acid targets of interest and, based on the mechanism of competitive hybridization, were responsive to the presence of a high affinity competitive target. In the case of the particle assemblies, incubation with the competitive target resulted in observable disassembly of particle structures. In the case of fluorescently labeled dsProbes, incubation with competitive targets resulted in a quantifiable loss of fluorescence as determined by flow cytometry. Utilizing the fluorescently labeled dsProbe system, the kinetics of competitive hybridization was characterized for nucleic acid targets of varying specificity and strand context. The results indicate promise for the development of the competitive hybridization approach in nucleic acid detection systems providing advantages over current single-stranded probe designs. By utilizing a fluorescently labeled dsProbe approach, it is unnecessary to chemically modify the target of interest to impart a signaling mechanism. Additionally, as the process of competitive hybridization of dsProbes with targets of interest is an affinity driven process, discrimination of targets based on specificity is decoupled from standard measures such as elevated temperature protocols, an important step in translating nucleic acid technologies from the controlled laboratory environment to field applications.