Asynchronous education activities have grown rapidly through popular distance education delivery techniques. This rapid growth has precluded science, technology, and engineering. Practice oriented disciplines have considered laboratories as key components of the curriculum. The laboratory is the difficulty of teaching such subjects through distance education. Studies have indicated that independent asynchronous study is not suitable for everyone. A qualitative study investigating two cases and utilizing a cross case analysis was performed with the goal of establishing some characteristics found in individuals who are successful, and those who are challenged by asynchronous laboratory study. Also considered were key factors which could aid or impede asynchronous laboratory studies. Case One involved a course on agricultural mechanical systems taught at a Texas four-year university with 13 participants. Case Two involved a course on electrical controls taught at the technical center of a Texas community college with 18 participants. Data were collected from observation - journaling, performance scores, and a questionnaire – interview process; then analyzed using the constant comparative method. To insure trustworthiness; credibility, transferability, and dependability were addressed. The cross-case analysis found no conflicts and reinforced the findings. The findings yielded a list of characteristics of individuals who were successful using asynchronous laboratory studies. Successes represented an elite student profile and support the suggestions of Lemckert and Florance (2002). Students were more likely to be successful when they (a) were autonomous self directed learners; (b) had a prerequisite knowledge framework; (c); had prerequisite technical skills; (d) had high reading and comprehension skills; (e) held intrinsic value for the educational experience; and (f) sought and used instructional resources. The factors discovered which aid or impede asynchronous laboratories are course design and curriculum issues. Asynchronous laboratory studies are more likely to be successful when they (a) provide a responsive system of feedback; (b) introduce study as small, step-wise experiences; (c) do not introduce independent complex concepts; (d) provide sufficient instructor time; (e) standardize computer software and applications; and (f) pilot-test and field-test laboratory equipment and activities. Conclusions drawn indicate limited applications of asynchronous laboratories for select prepared individuals with a critically designed curriculum.

Thesis (M.S.)--University of Florida, 2003. / Title from title page of source document. Includes vita. Includes bibliographical references.

This thesis is a study of the small animal colonies operated by the animal nutrition laboratory in the Department of Animal Husbandry. The four animal units studied are the albino rat, albino mouse, guinea pig and rabbit. The management practices used for each species are described fully. They include the housing, feeding, breeding and control of disease methods utilized. The factors discussed in the housing of the animals are the space utilization per animal and the type of cages. In addition, scale drawings and illustrations of the cages and cage racks are included. The method of feeding and the formula of each ration for each species is reported. The system of breeding used in each of the colonies is described. Control of disease is discussed with reference to the sanitation procedures practised. Growth and production data are reported for each species and a comparison made with the data published in the literature for other colonies. The number of animals involved in the study are 1,700 rats, 258 mice, 73 guinea pigs and 85 rabbits. The growth data includes size of litter, birth weights and weekly weights there after until weaning age. The production data comprises the percentage fertility and percentage weaned. In addition the results of a cost survey is reported. The cost per animal for each species includes the cost of housing, feeding and labour. The results reported here are comparable to those reported elsewhere. / Land and Food Systems, Faculty of / Graduate

RNA viruses are among the most virulent microorganisms that threaten the health of humans and livestock. Among the most socio-economically important of the known RNA viruses are those found in the family Orthomyxovirus. In this era of rapid low-cost genome sequencing and advancements in computational biology techniques, many previously difficult research questions relating to the molecular epidemiology and evolutionary dynamics of these viruses can now be answered with ease. Using sequence data together with associated meta-data, in chapter two of this dissertation I tested the hypothesis that the Influenza A/H1N1 2009 pandemic virus was introduced multiple times into Africa, and subsequently dispersed heterogeneously across the continent. I further tested to what degree factors such as road distances and air travel distances impacted the observed pattern of spread of this virus in Africa using a generalised linear modelbased approach. The results suggested that their were multiple simultaneous introductions of 2009 pandemic A/H1N1 into Africa, and geographical distance and human mobility through air travel played an important role towards dissemination. In chapter three, I set out to test two hypotheses: (1) that there is no difference in the frequency of reassortments among the segments that constitute influenza virus genomes; and (2) that there is epochal temporal reassortment among influenza viruses and that all geographical regions are equally likely sources of epidemiologically important influenza virus reassortant lineages. The findings suggested that surface segments are more frequently exchanges than internal genes and that North America/Asia, Oceania, and Asia could be the most likely source locations for reassortant Influenza A, B and C virus lineages respectively. In chapter four of this thesis, I explored the formation of RNA secondary structures within the genomes of orthomyxoviruses belonging to five genera: Influenza A, B and C, Infectious Salmon Anaemia Virus and Thogotovirus using in silico RNA folding predictions and additional molecular evolution and phylogenetic tests to show that structured regions may be biologically functional. The presence of some conserved structures across the five genera is likely a reflection of the biological importance of these structures, warranting further investigation regarding their role in the evolution and possible development of antiviral resistance. The studies herein demonstrate that pathogen genomics-based analytical approaches are useful both for understanding the mechanisms that drive the evolution and spread of rapidly evolving viral pathogens such as orthomyxoviruses, and for illuminating how these approaches could be leveraged to improve the management of these pathogens.

Background Outbreaks of community-acquired Pseudomonas aeruginosa are typically small and localized. We investigated an increase in P. aeruginosa clinical isolates in Cape Town, South Africa during a severe drought. Methods Cases were defined as P. aeruginosa isolated from any clinical sample, and “wild-type” as susceptibility to all antibiotics tested. Residential addresses of community-acquired wild-type cases were mapped. Whole genome sequencing and multi-locus sequence typing were used to determine clonality and identify virulence genes. A modified case-control study in a subset of patients with bloodstream infection compared demographic and clinical characteristics between sequence types. Results The outbreak lasted 10 months from December, 2016 to September, 2017 with 3,321 documented cases. At the peak, cases reached 2.3-fold baseline and the city's dams reached a nadir of 19% capacity. Cases were distributed widely across the city. Multi-locus sequence type (ST) 303 was found in 27 of 42 (64%) blood culture isolates of P. aeruginosa during the outbreak, one of 19 (5%) before, and none of 11 after. ST303 infection was independently associated with younger age, but not with co-morbidities nor increased mortality. Fifty-one virulence genes were differentially present in ST303 compared with other sequence types, including genes involved in biofilm formation, iron uptake, and gut penetration. Conclusion The investigation confirmed a citywide outbreak of P. aeruginosa coinciding with and potentially related to a severe drought. We identified a predominant outbreak-associated clone, ST303, which harboured genes that could contribute to virulence and survival in drought-related conditions. Enhanced surveillance for P. aeruginosa during periods of drought is recommended.

Background Bacillus Calmette-Guerin (BCG) remains the only licensed vaccine for use against tuberculosis (TB), however, it is poorly efficacious against pulmonary TB in adults. The poor efficacy has been attributed in part to coinfections with many other unrelated pathogens that overlap geographically with Mycobacterium tuberculosis (M. tuberculosis). In this study, we used a murine model to investigate the effect of Plasmodium species virulence and the timing of infection on BCG-induced immune responses and efficacy against M. tuberculosis both in vivo by aerosol challenge and ex vivo by Mycobacterial Growth Inhibition Assay (MGIA). Methods and Results To assess the impact of malaria parasite virulence on BCG, wild-type C57BL/6 mice were vaccinated intraperitoneally with BCG Pasteur and 6 weeks later, they were infected with either virulent Plasmodium berghei (P. berghei) or less-virulent Plasmodium chabaudi chabaudi AS (P. chabaudi) infected red blood cells with appropriate controls. The mice were euthanased at 7, 10, 17, 26 days after malaria infection for multiparameter flow cytometry analysis. We compared P. berghei and P. chabaudi, their effects on B cells, effector and memory T cells and the outcome on BCG-induced protective efficacy against M. tuberculosis H37Rv infection by subsequent aerosol challenge. P. berghei induced a significant decrease in the frequency of central memory T cell (CD44hiCD62Lhi), marginal zone B cell (B220+AA4.1-CD1dlo), and follicular B cell (B220+AA4.1-CD1dhi ) populations. In contrast, infection with the less virulent P. chabaudi induced depletion in the marginal zone B cells but not the follicular B cells or the central memory T cells. A strong effector T cell/effector memory T cell (CD44hiCD62Lo) response enhanced by BCG vaccination was observed in both species. The reduction in the central memory T cells observed during P. berghei infection was attributed to T cell apoptosis. It should be noted that the observed changes in T cell and B cell populations described above are relative proportions of these cells among splenocytes. Surprisingly, BCG-mediated protection against M. tuberculosis H37Rv by aerosol challenge was retained in both the virulent P. berghei and less virulent P. chabaudi species despite modulations in the immune responses. We also investigated the effect of malaria infection post BCG vaccination and malaria infection prior to BCG vaccination on the cytokine responses and the efficacy of BCG vaccine both in vivo and ex vivo. Splenocytes from wild type C57BL/6 mice infected with P. yoelii 17XNL 4 weeks post BCG vaccination were analysed 13 days after P. yoelii 17XNL infection. We compared the cytokine responses and growth inhibition by MGIA in four groups of 6 mice each: naïve, BCG, BCG-malaria, and malaria control. BCG Pasteur Aeras was used as a surrogate for M. tuberculosis in the MGIA. Restimulation with PPD-T induced a significant increase in both the proportion and absolute cell numbers of CD4+ IFNγ+ and CD4+ TNF+ T cells in the BCG vaccinated compared to the naïve control. No significant differences in the proportion and cell numbers of CD4+ IFNγ+ T cells were observed between the BCG and BCGmalaria groups restimulated with PPD-T. This indicates that the presence of malaria did not significantly hamper the IFNγ response to BCG. In contrast, a significant (p<0.05) reduction was detected in the proportion of CD4+TNF+ T cells in the BCG-malaria group compared to the BCG group following restimulation with PPD-T. P. yoelii 17XNL infection also led to significant production of IL-4 by CD4+ T cells in the groups that were infected with malaria with or without BCG vaccination when restimulated with PPD-T or P. yoelii 17XNL peptide pool. Minimal production of CD4+ IL-17 T cells and CD8+ T cells were observed when restimulated with PPD-T. MGIA revealed a significant decrease in net bacterial growth (0.2 log 10) in the BCG and BCG-malaria splenocyte cultures compared to the naïve and malaria control splenocyte cultures, indicating that BCG immunized mice were able to control the growth of mycobacteria in the presence of malaria. For P. yoelii 17XNL infection prior to BCG vaccination, wild type C57BL/6 mice (n=6-11) were infected with either P. yoelii 17XNL or P. chabaudi and after 13 (acute malaria) and 21 (cleared malaria) days or 7 (acute malaria) and 28 (cleared malaria) days respectively, mice were vaccinated with BCG. They were either sacrificed 6 weeks after BCG vaccination for cytokine analysis and MGIA or challenged with M. tuberculosis H37Rv by aerosol for bacterial growth determination. BCG vaccination caused a significant increase in the proportion of CD4+ IFN-γ + and CD4+TNF+ T cells compared to the naïve control when restimulated with PPD-T. However, we observed a significant reduction in the proportion of CD4+TNF+ but not CD4+ IFN-γ + T cell responses in the acute malaria-BCG group compared to the BCG vaccinated mice following in vitro restimulation with PPD-T. Interestingly, these cytokine levels were significantly elevated upon clearance of the P. yoelii 17XNL parasites. Therefore, acute malaria infection at the time of BCG vaccination induces a transient decrease in Th1 response which is restored once the parasite is cleared. An ex vivo MGIA assay on splenocytes showed a significant decrease (0.2 log reduction) in net bacterial growth in all BCG vaccinated groups except the acute malaria-BCG group, compared to the unvaccinated groups with or without malaria. Interestingly, a significant increase in net bacterial growth was observed in the acute malaria group compared to the BCG vaccinated group, indicating that acute P. yoelii 17XNL infection prior to BCG vaccination decreases the protective efficacy of BCG in the MGIA. In an in vivo aerosol challenge with M. tuberculosis, a significant decrease was observed in bacterial burden in the spleen (0.6 log reduction) and lungs (1 log reduction) of mice that were vaccinated during acute P. chabaudi infection, vaccinated when malaria had cleared or vaccinated in the absence of malarial infection. This was further confirmed by higher numbers of acid-fast bacilli observed in all unvaccinated groups compared to all vaccinated groups with or without malaria, implying BCG vaccine efficacy is maintained in an acute or cleared P. chabaudi infection prior to BCG vaccination. This discrepancy between the in vivo aerosol challenge and ex vivo growth inhibition assay may be attributed to the different Plasmodium species with different clinical characteristics used in the study or the different kinetics in the two assays used. Conclusions Therefore, malaria parasite virulence does not inhibit the ability of BCG to control the growth of M. tuberculosis in vivo despite alterations in the immune responses. Additionally, neither Plasmodium infection prior to BCG vaccination nor Plasmodium infection post BCG vaccination abolishes the efficacy of BCG against M. tuberculosis although a decrease in CD4+ Th1 cytokine responses and concomitant increase in bacilli burdens are observed in the group of acute Plasmodium infection prior to BCG vaccination.

Aim: The purpose of this study is to verify the performance of the Roche Elecsys ® antinucleocapsid (qualitative) and anti-spike (quantitative) SARS-CoV-2 immunoassays to determine whether the performance of the assays is acceptable for diagnostic use in the Groote Schuur Hospital virology/chemistry laboratory, as well as other National Health Laboratory Service (NHLS) laboratories in South Africa. Methods: We performed a verification study using de-identified remnant serum or plasma samples. Standard verification experiments including sensitivity, specificity and precision were performed. Pre-pandemic samples were used to assess specificity. Samples with a linked positive SARS-CoV-2 polymerase chain reaction (PCR) result on a respiratory sample >10 days before the serum/plasma collection date were used to assess sensitivity. Additionally, postvaccine humoral response and other parameters was assessed in a cohort of laboratory staff. Results: For the anti-nucleocapsid antibody assay, specificity was 99.7% based on 316 samples and sensitivity 91.3% based on 404 samples. For the anti-spike antibody assay, the specificity based on 194 samples was 100%, and the sensitivity based on 384 samples was 93.8%. Both assays demonstrated acceptable precision. Furthermore, the anti-spike antibody assay sensitivity was >92% during the first three waves in South Africa, dominated by different SARS-CoV-2 variants. Post-vaccine seroconversion in 115 staff with no evidence of prior natural infection was 99% and hybrid immunity produced higher anti-spike antibody titres compared to vaccine-only participants. Conclusion: Both immunoassays met our acceptance criteria. Both assays can be used for seroprevalence studies. The anti-nucleocapsid immunoassay assay is valuable in confirming past natural infection in patients with previous asymptomatic infection, previous symptomatic infection where no PCR was done or PCR-negative patients who present to hospital with COVID-19 during the second week of illness or later. Most importantly, the antispike immunoassay can be used as a reliable, cheap, and easily accessible surrogate marker of post-vaccine humoral immune response and we recommend using this to confirm and monitor humoral immune response in patients with risk factors for non-seroconversion following vaccination and increased risk for morbidity and mortality following infection with SARS-CoV-2.

A biological method for peptide synthesis provides increased production capacity of inexpensive peptide pharmaceuticals with environmentally safe procedures relative to current chemical peptide synthesis. Most precursor peptides are readily produced from yeast and bacterial systems using recombinant DNA technologies but require C-terminal amidation for maximum biological activity. Peptidylglycine α-amidating monooxygenase (PAM) is the only enzyme that catalyses the C-terminal amidation of peptides in vivo through its two catalytic cores, peptidylglycine α-hydroxylating monooxygenase (PHM) and peptidylglycine αamidating lyase (PAL). The cost and limited quantities of the commercial PAM variants available have necessitated research into low cost, scalable quantities of PAM and peptide amidation to enable inexpensive biological peptide production. In the present study, an assay for measuring the product of PAM activity, glyoxylate, was developed based on a 2-aminobenzaldehyde-glycine-glyoxylate (AGG) absorbance assay. The AGG chromophore synthesised was identified with ultra-performance liquid chromatography mass-spectroscopy (UPLC-MS). PAM activity was measurable with glyoxylate between 25 µM and 1600 µM with the AGG assay. Furthermore, the activity of PHM alone was measured by the inclusion of an alkaline hydrolysis step to lyse glyoxylate as a substitute for PAL catalytic activity. Multiple candidate proteins and DNA sequences for PAM were identified by genetic sequence searches and a novel fungal PHM modelled in silico. Fourteen PAM, PHM, PAL and truncated constructs were expressed in the non-conventional yeast host, Yarrowia lipolytica. The novel fungal PHM's nutrient, temperature, and pH conditions were optimised to maximise protein expression. Enzyme purification was optimised with scalable industrial appropriate methods to purify milligram amounts of fungal PHM. The AGG assay was validated with a commercially obtained PAM, demonstrating a simple medium-throughput method to measure PAM activity. The novel fungal PHM was characterised with a pH optimum of 4.0 and maximum enzymatic activity at 45°C. Deglycosylation of fungal PHM enhanced enzyme activity by 1.83 fold, but lowered the temperature optimum to 37°C. The novel PHM and alkaline hydrolysis catalysed the conversion of the peptide pharmaceutical precursor for exenatide into its final bioactive form.

Thesis PlanB (M.S.)--University of Wisconsin--Stout, 2009. / Includes bibliographical references.

The increasing demand for the chemical monitoring of water qualities emphasises the importance of an efficient and workable laboratory management system to remain profitable and competitive in a fast growing industry. The management of information is therefore becoming increasingly important as the effectiveness and profitability of the water laboratory is largely measured against its management systems and continual improvement programmes. Effective information management forms an important part of laboratory management to ensure that data are updated and remain current. One way of proving its effectiveness, the laboratory must provide proof of a controlled and procedurised documentation system and the availability of updated data and information. The effective control of data and information in the water laboratory by using some kind of information management system is therefore essential. Laboratory managers are becoming aware of the need for an effective, computerised laboratory data and information management system as the entry of data and results into a manual system has several disadvantages. The laboratory manager is increasingly seeking for ways to improve the efficiency of his laboratory and more time must therefore be spent on managing the laboratory, rather than to facilitate the distribution and control of information.