Thesis (MS)--Montana State University--Bozeman, 2008. / Typescript. Chairperson, Graduate Committee: David L. Dickensheets. Includes bibliographical references (leaves 69-70).

Thesis (Ph. D.)--University of Texas at Austin, 2004. / Supervisor: Rebecca Richards-Kortum. Vita. Includes bibliographical references. Also available from UMI.

Thesis (Ph. D.)--University of Texas at Austin, 2006. / Vita. Includes bibliographical references.

Septic arthritis has the potential to be a highly destructive joint disease. Although numerous bacterial species are capable of inducing septic arthritis, Staphylococcus aureus is most commonly implicated, accounting for up to 65% of cases. Whilst this organism is known to produce a diverse array of potential virulence factors, studies investigating a variety of S. aureus-related infections have implicated alpha(Hla)-, beta(Hlb)- and gamma(Hlg)-haemolysins as key damaging toxins, with the ‘pore-forming’ Hla considered to be the most potent. The work presented in this study focused on gaining further insight into the interaction between S. aureus toxins and in situ chondrocytes during an episode of septic arthritis. An in vitro bovine osteochondral explant model of S. aureus-induced septic arthritis was developed in this study. Utilising fluorescence-mode confocal laser scanning microscopy (CLSM), the model, which avoided the complexities of a host immune response, permitted an assessment of the following: (1) the spatial and temporal quantification of in situ chondrocyte viability following exposure to both a laboratory ‘wild-type’ (S. aureus 8325-4) and clinical strains of S. aureus; (2) the influence of Hla, Hlb and Hlg on in situ chondrocyte viability through the use of specific ‘haemolysin-knockout’ mutant strains; (3) the influence of altered culture medium osmolarity and extracellular Ca2+ on Hla-induced in situ chondrocyte death; and (4) dynamic changes in intracellular Ca2+ within in situ chondrocytes following Hla exposure. S. aureus 8325-4 and S. aureus clinical strains rapidly reduced in situ chondrocyte viability ( > 45% chondrocyte death at 40hrs). The increased acidity, observed during bacterial culture, had a minimal effect on chondrocyte viability. Chondrocyte death commenced within the superficial zone (SZ) of cartilage and rapidly progressed to the deep zone (DZ). Simultaneous exposure of SZ and DZ chondrocytes to S. aureus 8325-4 toxins (achieved with the use of subchondral bone-free explants) demonstrated that SZ chondrocytes were more susceptible to the toxins than DZ chondrocytes. When explants were cultured in the presence of a selection of isogenic S. aureus mutants, with varying Hla, Hlb and Hlg production capabilities (all originating from S. aureus 8325-4), Hla-producing mutants induced significant in situ chondrocyte death compared to toxin deficient controls (Hla-Hlb-Hlg-). In contrast, mutants producing Hlb and Hlg in the absence of Hla were unable to induce significant chondrocyte death. Hla alone was therefore identified as the key damaging toxin to in situ chondrocyte viability. Raised culture medium osmolarity had no influence on Hla-induced in situ chondrocyte death. In the absence of Hla, a high extracellular Ca2+ concentration (20mM) had no influence on chondrocyte viability during the experimental period. Hla-induced chondrocyte death increased in the presence of raised extracellular Ca2+ concentrations thereby confirming a role of Ca2+ in the chondrocyte death pathway. There was no significant difference between S. aureus growth in high and low Ca2+ culture media. Finally, when live osteochondral explants stained with the Ca2+-sensitive fluorophore Fluo-4 were cultured with an Hla-containing S. aureus supernatant (S. aureus 8325-4 (Hla+Hlb+Hlg+)) there was a significant rise in intracellular Ca2+ in comparison to those explants exposed to a non-Hla-containing supernatant (S. aureus DU5938 (Hla- Hlb-Hlg-)). The Hla-induced Ca2+ transients were always followed by chondrocyte death. Thus, it is likely that Hla-induced chondrocyte death was associated with a rise in intracellular Ca2+. These findings are of translational relevance. Firstly, toxins released by S. aureus have a rapid and fatal action on in situ chondrocytes, thereby advocating the prompt and thorough removal of bacteria and their toxins during the treatment of septic arthritis. Secondly, the identification of Hla alone as the key damaging toxin to in situ chondrocyte viability, with its destructive action being associated with a rise in intracellular Ca2+, may enable the development of future targeted therapeutic strategies in order to reduce the extent of cartilage destruction during and after an episode of septic arthritis.

Orientador: Altair Antoninha Del Bel Cury / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-11T05:18:05Z (GMT). No. of bitstreams: 1 Pereira-Cenci_Tatiana_D.pdf: 1303169 bytes, checksum: c75a9e7acaf1e652f846b5eded8376b5 (MD5) Previous issue date: 2008 / Resumo: A candidose é a infecção oral fúngica mais comum diagnosticada em humanos, com prevalência de até 77,5% em usuários de próteses removíveis. Embora tenha sido inicialmente associada apenas à Candida albicans, outras espécies de Candida podem ser responsáveis por mais de 50% dos casos de infecção. Ainda, fatores como presença de saliva, bactérias e características de materiais utilizados para confecção de próteses removíveis parecem desempenhar importante papel na adesão, colonização e formação de biofilme por Candida. Assim, este trabalho objetivou (i) discutir os fatores que controlam a adesão inicial, colonização e formação de biofilme de Candida em um artigo de revisão, no intuito de apontar diretrizes para estudos futuros e ainda, mostrar de que forma estes fatores podem ser controlados, ajudando na prevenção da doença; (ii) verificar a influência in vitro de alguns dos fatores supracitados na formação de biofilme de C. albicans sobre a superfície de hidroxiapatita, resina acrílica e reembasador temporário e; (iii) avaliar in situ a formação de biofilme sobre espécimes de resina acrílica e reembasadores de próteses inseridos nas próteses totais de 21 voluntários. Para avaliação da formação de biofilme de C. OBS.: O resumo na integra poderá ser visualizado no link ou texto completo da tese digital. / Abstract: Candida-associated stomatitis is the most common fungal oral infection in humans, with a prevalence reported in up to 77.5% of a population wearing dentures. Disease-associated Candida species have shifted from C. albicans to non-albicans species, these latter being responsible for more than 50% of the infections. Additionally, several factors as the presence of saliva, bacteria and dental prostheses materials¿ characteristics seem to be related to the adhesion, colonization and biofilm formation of Candida. This study aimed (i) to discuss the factors that govern initial adherence, colonization and biofilm formation of Candida by means of a review article, in order to suggest future research and show how these factors may be controlled, therefore helping to prevent the disease; (ii) to verify the influence of several of these factors in the biofilm formation of C. albicans in vitro, on hydroxyapatite, acrylic resin and soft denture liner; (iii) to evaluate in situ biofilm formed on acrylic resin and denture liner specimens inserted in the lower dentures of 21 volunteers. For C. Note: the complete abstract is avaiable with the link or full eletronic digital theses or dissertations. / Doutorado / Protese Dental / Doutor em Clínica Odontológica

The accurate detection and quantification of human diabetic somatic polyneuropathy (DSPN) are important to define at risk patients, anticipate deterioration, and assess new therapies. Current methods lack sensitivity, require expert assessment and have major shortcomings when employed to define therapeutic efficacy. In recent years, in vivo corneal confocal microscopy (IVCCM) has shown potential as a surrogate endpoint for DSPN.This study aims to investigate fundamental aspects of IVCCM such as repeatability and optimal scanning methodology and establish changes in corneal nerve morphology in relation to the severity of DSPN and regeneration in response to normalisation of hyperglycaemia. Furthermore, it aims to provide a novel fully automated image analysis algorithm for the quantification of corneal nerve morphology and establish the diagnostic ability of CCM.IVCCM shows high repeatability which is enhanced with more experienced observers. Central corneal innervation is comparable to adjacent peripheral innervation in mild diabetic neuropathy but the central cornea may be more sensitive to change. Corneal nerve loss is symmetrical and progressive with increasing neuropathic severity and corneal nerves show significant regenerative capacity following rapid normalisation of glycaemic control after simultaneous pancreas and kidney transplantation. The novel image analysis algorithm strongly correlates with human expert annotation and therefore represents a rapid, objective and repeatable means of assessing corneal nerve morphology. Automated image quantification may replace human manual assessment with high diagnostic validity for DSPN.

Rotavirus (RV) is a viral pathogen that causes severe, watery diarrhea and vomiting in the young of humans and other animals. RV infections result in over 200,000 pediatric deaths around the world each year, especially in developing nations. Within the infected host cell, RV forms inclusion bodies, called viroplasms, where many stages of viral replication occur. The RV polymerase, known as VP1, must localize to viroplasms during infection where it replicates the virus' RNA genome. The work described in this dissertation focused on identifying region(s) of VP1 essential for its viroplasmic localization and its function as a polymerase. We found that a single amino acid change in a region of the polymerase called the N-terminal domain negatively impacted its capacity to localize to viroplasms during infection as well as its enzymatic activity in a test tube. Follow up studies using VP1 proteins from divergent strains and a mutant containing only the N-terminal domain of VP1 provided more insight into polymerase localization determinants. In total, our work suggests that the VP1 N-terminal domain plays an important role in localizing the polymerase to viroplasms via interactions with other viral proteins and supporting its function as a polymerase. / Ph. D. / Rotavirus (RV) is a viral pathogen that causes severe, watery diarrhea and vomiting in the young of humans and other animals. RV infections result in over 200,000 pediatric deaths around the world each year, especially in developing nations. Within the infected host cell, RV forms inclusion bodies, called viroplasms, where many stages of viral replication occur. The RV polymerase, known as VP1, must localize to viroplasms during infection where it replicates the virus’ RNA genome. The work described in this dissertation focused on identifying region(s) of VP1 essential for its viroplasmic localization and its function as a polymerase. We found that a single amino acid change in a region of the polymerase called the N-terminal domain negatively impacted its capacity to localize to viroplasms during infection as well as its enzymatic activity in a test tube. Follow up studies using VP1 proteins from divergent strains and a mutant containing only the N-terminal domain of VP1 provided more insight into polymerase localization determinants. In total, our work suggests that the VP1 N-terminal domain plays an important role in localizing the polymerase to viroplasms via interactions with other viral proteins and supporting its function as a polymerase.

Confocal microscopes utilize point illumination and pinhole detection to reject out-of-focus light. Because of the point illumination and detection pinhole, confocal microscopes typically utilize point scanning for imaging, which limits the overall acquisition speed. Due to the excellent optical sectioning capabilities of confocal microscopes, they are excellent tools for the study of three-dimensional objects at the microscopic scale. Fluorescence confocal microscopy is especially useful in biomedical imaging due to its high sensitivity and specificity. However, all designs for confocal microscopes must balance tradeoffs between the numerical aperture (NA), field of view (FOV), acquisition speed, and cost during the design process. In this dissertation, two different designs for an array confocal microscope are proposed to significantly increase the acquisition speed of confocal microscopes. An array confocal microscope scans an array of beams in the object plane to parallelize the confocal microscope to significantly reduce the acquisition time. If N beams are used in the array confocal microscope, the acquisition time is reduced by a factor of N. The first design scans an array of miniature objectives over the object plane to overcome the trade-off between FOV and NA. The array of objectives is laterally translated and each objective scans a small portion of the total FOV. Therefore, the number of objectives used in the array limits the FOV, and the FOV is increased without sacrificing NA. The second design utilizes a single objective with a high NA, large FOV, and large working distance designed specifically for whole brain imaging. This array confocal microscope is designed to speed up the acquisition time required for whole brain imaging. Utilizing an objective with a large FOV and scanning using multiple beams in the array significantly reduces the time required to image large three-dimensional volumes. Both array confocal microscope designs use beam-splitting gratings to efficiently split one laser beam into a number of equal energy outgoing beams, so this dissertation explores design methods and analyses of beam-splitting gratings to fabrication errors. In this dissertation, an optimization method to design single layer beam-splitting gratings with reduced sensitivity to fabrication errors is proposed. Beam-spitting gratings are typically only designed for a single wavelength, so achromatic beam-splitting grating doublets are also analyzed for possible use in array confocal microscopes with multiple excitation wavelengths. An analysis of the lateral shift between grating layers in the achromatic grating doublet proves grating profiles with constant first spatial derivatives are significantly less sensitive than continuous phase profiles. These achromatic grating doublets have designed performance at two wavelengths, but the diffraction angles at the two wavelengths differ. To overcome that limitation, scale-invariant achromatic gratings are designed, which not only provide designed performance at two wavelengths, but also equal diffraction angles at two wavelengths.

The aim of this study was to investigate in vitro transdermal diffusion of a small peptide namely arginine vasopressin (AVP) with the aid of the novel PheroidTM drug delivery system. Generally, peptides seem unfit for transdermal permeation, but it was thought prudent to explore the suitability of this lipid-based system after success was achieved with entrapment of tuberculostatics, bacteria and viruses. Bestatin (a selective aminopeptidase inhibitor) was employed to circumvent any skin-related degradation of the active. Therefore, the effect of bestatin on the preservation of AVP during diffusion was investigated. Vertical Franz cell diffusion studies were conducted with female abdominal skin, with AVP at a concentration of 150 pglml in the donor phase and Hepes buffer as the receptor phase over a twelve-hour period. To prove entrapment of AVP within the lipid structures of the PheroidsTM, fluorescentlylabelled samples were monitored by means of confocal laser scanning microscopy (CLSM), which revealed definite entrapment. In vitro permeation profiles for AVP exhibited a biphasic character, with the majority of permeation occurring during the first two hours. The PheroidTM delivery system proved to be advantageous when applied as delivery medium. The inclusion of bestatin has an enhancing effect on permeation probably due to its protection of AVP. / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2007.

Drosophila melanogaster and many other insects harbor intracellular bacterial symbionts that are transmitted vertically from infected host mothers to their offspring. Many of these bacteria alter host reproductive developmental processes in order to increase their transmission success. For example, Spiroplasma, a spirochete that naturally infects D. melanogaster, selectively kills males during mid-embryogenesis while sparing females. Previous studies suggested that Spiroplasma interacts genetically with the male-specific dosage compensation pathway, which causes ~2-fold up-regulation of most genes located on the male’s single X chromosome so that their expression matches the levels found in females who have two Xs. To further test this idea, I used confocal microscopy to visualize dosage compensation complex (DCC) localization and activity in infected as well as uninfected embryos. In the presence of Spiroplasma, the DCC became abnormally mis-localized across the nucleus. This pattern was accompanied by abnormal acetylation of histone H4K16, a mark induced by DCC activity and needed for proper X chromatin remodeling. My results imply that Spiroplasma directly targets the DCC by misdirecting it to uncompensated regions of the genome, an effect that leads to abnormal gene mis-regulation and consequent lethality (work from other members in our group). To further investigate this interaction, we transgenically expressed low levels of MSL-2 in both Spiroplasma infected and uninfected embryos in order to cause ectopic formation of the DCC in the female sex. I found that when infected, female embryos expressing the DCC showed significantly reduced viability in comparison to uninfected transgenic females. This result supports the notion that Spiroplasma uses the DCC in a dominant gain-of-function manner to kill embryos.