Several key flexure sites exist in the muscle crossbridge including the actomyosin binding site which play important roles in the actomyosin crossbridge cycle. To distinguish between these sources of flexibility, a new single molecule assay was developed to observe the swiveling of rod about a single myosin. Myosins attached through a single crossbridge displayed mostly similar torsional characteristics compared to myosins attached through two crossbridges, which indicates that most of the torsional flexibility resides in the myosin subfragment-2, and thus the hinge between subfragment-2 and light meromyosin should contribute the most to this flexibility. The comparison of torsional characteristics in the absence and presence of ADP demonstrated a small but significant increase in twist rates for the double-headed myosins but no increase for single-headed myosins, which indicates that the ADP-induced increase in flexibility arises due to changes in the myosin head and verifies that most flexibility resides in myosin subfragment-2.

Class of 2007 Abstract / Objectives: The objective of this study was to determine the cost-effectiveness of using the Invader® UGT1A1 Molecular Assay to identify patients at risk of experiencing neutropenia from irinotecan monotherapy and FOLFIRI. Methods: Publicly available search engines were used to search for literature reporting the rate, treatment, and cost of adverse events and the population rate of genotypes. Drug acquisition costs were determined using average wholesale prices. Additional costs were determined using the Physicians’ Fee and Coding Guide. Monte Carlo Simulations were performed to reveal average cost, average effectiveness, and 95% confidence intervals. Tornado diagrams were derived to identify variables most likely to affect the outcomes and sensitivity analyses. Results: Monte Carlo simulations of monotherapy revealed a mean cost of treatment with genotype assay information of $6,620 (SD: $3,235; 95% CI: $5,408 to $15, 258) and an effectiveness ratio of 0.877 (SD: 0.328; 95% CI: 0 to 1). Without the genotype assay information the mean cost of treatment was $7,358 (SD: $4,183; 95% CI: $5,083 to $14,883) and the effectiveness ratio was 0.764 (SD: 0.425; 95% CI: 0 to 1). Monte Carlo simulations of FOLFIRI data showed a mean cost of treatment with genotype assay information of $6,105 (SD: $3,438; 95% CI: $4,706 to $14,556) and an effectiveness ratio of 0.858 (SD: 0.349; 95% CI: 0 to 1). When genotype information was unknown, the mean cost of FOLFIRI was $6,833 (SD: $4,288; 95% CI: $4,331 to $14,181) and the effectiveness ratio was 0.746 (SD: 0.435; 95% CI: 0 to 1). Conclusions: Genotyping patients exposed to irinotecan monotherapy or FOLFIRI may be a cost-effective means of identifying at risk patients but the results from this study while favorable are not statistically significant.

Master of Science / Department of Diagnostic Medicine/Pathobiology / Melinda J. Wilkerson / Tick-borne bacteria, Ehrlichia canis, Anaplasma platys, and Ehrlichia chaffeensis are significant zoonotic pathogens of dogs and humans worldwide. In tropical regions such as Grenada, West Indies, dogs represent a major reservoir for E. canis and A. platys, and they are often co-infected. The purpose of this study was to develop a serologic, multiplex bead-based assay to detect species-specific exposures to E. canis, A. platys, and E. chaffeensis in dogs for purposes of surveillance and public health. Peptides from specific outer membrane proteins of P30 for E. canis, OMP1X of A. platys, and P28-19/P28-14 of E. chaffeensis were coupled to magnetic beads and assays were optimized using the multiplex Luminex xMAP® platform. In experimentally infected dogs, the multiplex assay successfully detected antibodies for E. canis and E. chaffeensis, but not A. platys. In the Grenadian population (n=104), the multiplex assay and the in-house ELISA, the SNAP® 4Dx®, detected A. platys antibodies as well as Ehrlichia spp.. Multiplex assay results were found to have “good” and “very good” agreement with the ELISA and IFA for E. canis antibody-positive dogs (K value of 0.73 and 0.84 respectively), while ELISA and IFA had “very good” agreement with each other (K value of 0.85). A. platys multiplex results had only “poor” agreement with ELISA and IFA (K value of -0.02 and 0.01, respectively), while the ELISA and IFA tests had “moderate” agreement with each other (K value of 0.5). These tests showed the prevalence of exposure to E. canis to be comparable with previous studies (38% in 2014), but a doubling of exposure to A. platys determined by IFA and 4Dx® from 9% in 2006, to 20% in 2014. Bayesian modeling (performed on E. canis data only) suggested conditional independence between the IFA, 4Dx®, and MAG tests using consensus priors calculated from literature, and that the bead-assay had comparable sensitivity and specificity to the IFA and ELISA tests. In conclusion, the multiplex peptide assay performed well in detecting the seropositive status of dogs to E. canis and had good agreement with commercial assays; however, more work needs to be done to assess performance in populations of dogs with exposures to multiple species of Ehrlichia. Further, the reasons for low seroreactivity to A. platys need to be further investigated.

MSc (Med), Faculty of Health Sciences, University of the Witwatersrand / The hepatitis B virus remains a major public health problem even after decades of its discovery. Horizontal transmission during early childhood is the predominant mode of transmission in highly endemic regions such as sub-Saharan Africa. Infection exhibits a wide spectrum of clinical manifestations, from an asymptomatic stage to severe liver disease which may result in hepatocellular carcinoma (HCC). The HBV X protein (HBx) has been implicated in carcinogenesis, which often has a poor prognosis, consequently the use of highly specific monoclonal antibodies (mAbs) directed against HBx in an enzyme-linked immunosorbent assay (ELISA) could lead to early identification of HBV carriers at risk of developing liver cancer. A variety of mixed hybridoma cell cultures secreting anti-HBx antibodies were cloned and sub-cloned by “limiting dilution”. Clonal supernatants were assessed for anti-HBx antibody production by Indirect ELISA and Western/Immunoblotting. Monoclonal antibodies were then characterized according to their relative binding affinity (Indirect ELISA) and relative epitope specificity (Competitive ELISA). One of our monoclonal antibodies was found to bind to the same epitope on HBx as the commercial anti-HBx antibody and with the same high affinity. In the developed Sandwich ELISA, our monoclonal antibody proved effective as the „detecting‟ antibody when the commercial anti-HBx antibody was deployed as the „capture‟ antibody. This Sandwich ELISA will be further developed in our laboratory with the object of applying it to patient sera.

A very important aspect for the functioning of an organism is that cells adapt their behaviour to external stimuli. They continuously interact with their environment, and biochemical and physical cues can activate cellular signalling, which leads to changes in cell behaviour such as proliferation and shape. Understanding cells' interactions with their environment is also important for understanding diseases. For example mechanosensing, which is the sensing of the cell's mechanical environment, has been associated with cancer development. In order for a cell to be able to sense its mechanical environment, it needs to form attachments to the environment. In my thesis, I have worked on three different tasks: the development of a new measurement technique and the study of initial cell adhesion and of cell spreading. When a cell from suspension first comes into contact with a substrate, it forms initial attachment bonds with proteins on the substrate surface. These bonds are mediated through integrins, which are transmembrane heterodimers, binding to the cell's environment on one side and to the cell's cytoskeleton on the other side. I study this initial cell attachment by measuring the force needed to detach cells, called cell adhesion strength. For these experiments I built a detachment device, which allows the detachment of cells from a substrate by vibrating the substrate in liquid. The device combines cell incubation, detachment and imaging. I measured the dependence of initial integrin bond formation on external factors such as incubation temperature and substrate stiffness. Once initial integrin bonds are formed, many different proteins are recruited to the adhesion site in order to form stronger adhesions. Amongst these proteins are signalling proteins, which direct the behaviour of the cell as a whole. One of the first cellular reactions to a substrate after initial integrin binding is cell spreading. This can be seen by the cell changing its shape from spherical to dome-like on the substrate. Because cell spreading is a very early response of a cell to a substrate, the onset time of spreading can be used as a quantitative measure for the time it takes the cell to sense a substrate and signal shape change. In my work, I look at the distribution of the time of initial cell spreading in a population of cells. I measure this distribution under different growth conditions such as pH, change of incubation medium from DMEM to PBS, substrate stiffness and incubation temperature. In my detachment experiments, I observe that vibration accelerates cell spreading in those cells which remain on the substrate. This is a connection between the detachment experiments and the cell spreading experiments and it shows how cells react to external forces. By changing the medium temperature in the cell detachment and cell spreading experiments, I am able to analyse the kinetics of these two processes. I use a signalling network model to analyse the internal cellular signalling path that leads from a spherical to a spread cell.

by Michael Yiu-kwong Leung. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (p. 158-168). / 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.

Kwok Leung Wong. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (leaves 149-170). / Acknowledgments --- p.i / Abstract --- p.ii -v / Abbreviations --- p.vi-vii / Chapter Chapter 1 : --- General Introduction --- p.1 / Chapter Chapter 2 : --- Development of Polyclonal Antibodies Against 6-B-hydroxycortisol （6-B-OHC) And Its Applications / Chapter 2.1 : --- Introduction --- p.40 / Chapter 2.2 : --- Materials and methods --- p.43 / Chapter 2.3: --- Results --- p.55 / Chapter 2.4: --- Discussion --- p.73 / Chapter Chapter 3 : --- Development of Monoclonal Antibody-Based ELISA Against 6-B-hydroxycortisol (6-B-OHC) And Its Applications / Chapter 3.1 : --- Introduction --- p.76 / Chapter 3.2 : --- Materials and methods --- p.89 / Chapter 3.3: --- Results --- p.108 / Chapter 3.4: --- Discussion --- p.135 / Chapter Chapter 4 --- : General Conclusion --- p.141 / References --- p.149

Pantothenate-p-nitroanilide has been synthesized for use as a substrate in a continuous spectrophotometric assay of pantetheinase activity monitoring absorbance at 41 0 nm. Pantothenate-p-nitroanilide is a crystalline compound with a molecular weight of 338.0 and a melting point of 146-149°C. Use of this substrate in the described assay is suitable for enzyme activity determination in high protein content media such as blood serum. Serum pantetheinase activity was determined for rats of varying pantothenate nutriture. Rats with mildly (but significantly, p

Abstract not available

Graduation date: 1996