With a population both increasing in number and age, comes a need for new diagnostic tools in the healthcare system, capable of diagnosing and monitoring multiple disorders in a cheap and effective way to provide personalised healthcare. Multiplex label-free biosensors have the potential to rejuvenate the current system. This thesis details the assessment of an ‘in house’ built labelfree array screening technology that has potential to be a point-of-care diagnostic for personalised medicine – the Array Reader. The performance of the Array Reader platform is considered in detail and optimised for both antibody and protein screening arrays. A Global Fit protocol is developed to extract kinetic constants for all protein-protein interactions, assuming a Langmuir adsorption binding model. Standard operating procedures are developed to provide optimised dynamic range, sensitivity, reproducibility and limit of detection of immuno-kinetic assay. A new antibody bio-stack signal amplification strategy is formed, improving the detection limit 60-fold. As a consequence, the bio-stack resulted in a novel method for determining the plasmon field penetration depth, defining the assay sensing volume at the nanoparticle surface. Antibody screening arrays were investigated with an IgG quantification assay to determine total IgG content from serum samples. It relied on the ability of protein A/G to bind antibodies via the Fc region. Specific antigens were used to measure the binding properties of the antibody Fab region. By characterising both regions, we have gained insight into the overall ability of an antibody to trigger an immune response. Protein screening assay were investigated targeting C-reactive protein (CRP), a marker of inflammation. The assays performance characteristics compared favourably with clinically used CRP assays. Finally, an antibody screening array was developed to assess the efficacy of a vaccine against Yersinia pestis in a non-human primate model. The vaccine screening array is an excellent example of the versatility of the platform and just one of many possible applications for the future.

Teladorsagia circumcincta, an economically-important abomasal nematode of small ruminants in temperate regions worldwide, is currently controlled with a combination of anthelmintics and pasture management. Anthelmintic resistance has emerged and vaccination is a potential alternative control strategy, as protective immunity in sheep can be acquired after repeated exposure to the parasite. Abomasal mucosal IgA responses in immune sheep have been correlated with delayed worm development and reduced faecal egg counts. However, recombinant vaccine development against parasitic nematodes has had limited success, and one of the reasons may be unsuitable expression systems for antigen production leading to incomplete or inadequate post-translational modifications such as glycosylation and tertiary protein folding, resulting in incorrect epitope structures for antibody binding. In this thesis, to address this issue, “native” infective larval (L3) antigen targets of protective immune responses and synthetic peptide sequences which mimic structural epitopes on these antigens were identified. Abomasal mucosal IgA was used as a probe to identify native immunogenic antigens from T. circumcincta L3. IgA was purified from abomasal mucus of animals rendered immune by repeated experimental infection and a custom antibody-affinity column was created and used to purify antigens from an L3 somatic PBS-soluble extract. Affinity purified L3-antigen-specific IgA levels in sheep with varying levels of immunity to T. circumcincta were positively correlated (rs = 0.853, P < 0.001) with both the total IgA concentration in efferent gastric lymph after parasite challenge, and with the percentage of inhibited fourth-stage (L4) larvae present in the gastric glands of the immune hosts (rs = 0.534, P = 0.007). In contrast, a negative correlation between the levels of affinity-purified L3 antigen-specific IgA and total T. circumcincta burden was observed (rs = -0.565, P = 0.004). Proteomic analysis of the IgA-affinity purified L3 extract identified a number of proteins which represent potential vaccine candidate molecules in other helminth species, including paramyosin, superoxide dismutase, galectin, activation-associated secreted proteins and fatty-acid retinol-binding proteins. As a first step towards the development of a novel vaccine based on IgA-binding peptide mimics of native structural epitopes, phage display libraries were used to screen antibodies, from sheep rendered immune to T. circumcincta by experimental infection. These antibodies were affinity-purified before use and specifically bound T. circumcincta L3 glycans or, alternatively, surface antigens on exsheathed T. circumcincta L3. Five peptide sequences which mimic L3 antigenic epitopes were identified and positive correlations existed between peptide-specific IgA levels and both the total IgA concentration in efferent gastric lymph after parasite challenge and the percentage of inhibited L4 present (rs > 0.621, P < 0.001 to P < 0.05). In contrast, negative correlations between the levels of peptide-specific IgA and the total nematode burden were observed (rs > -0.528, P < 0.01 to P < 0.05). In conclusion, the selected phage clones may therefore represent vaccine candidates if they could be presented to the ovine immune system in an appropriate fashion.

Following a spinal cord injury (SCI), participation in regular exercise can enhance physical capacity and performance in activities of daily living. With this in mind, the use of subjective ratings of perceived exertion (RPE) may provide an easy-to-administer alternative to traditional methods of regulating exercise intensity (e.g. heart rate and power output (PO)). A physically active lifestyle is also associated with a reduced risk of cardiovascular disease, in part because exercise exerts anti-inflammatory effects. Examining the plasma response of inflammation-mediating chemical messengers, known as cytokines, to traditional and novel exercise modalities may help maximise the anti-inflammatory potential of regular exercise. Participants with a cervical level SCI successfully self-regulated a 20 min bout of moderate intensity wheelchair propulsion (Chapter three). No differences in physiological or PO responses were observed during the imposed-intensity and self-regulated wheelchair propulsion in the trained population group. In a non-SCI group of novice wheelchair-users, a differentiated RPE specific to the exercising muscle mass (RPEP) was the dominant perceptual signal during submaximal wheelchair propulsion (Chapter four). The novice group successfully self-regulated a 12 min bout of moderate intensity wheelchair propulsion, comprising of a discontinuous 3 x 4 min protocol, using differentiated RPEP. In contrast, a more accurate self-regulation of light intensity wheelchair propulsion was observed when employing traditional overall RPE compared to RPEP. Following strenuous wheelchair propulsion, plasma concentrations of the inflammation-mediating cytokine interleukin-6 (IL-6) were significantly elevated in non-SCI and thoracic level SCI participants (Chapter five). Impaired sympathetic nervous system (SNS) function was associated with a reduced IL-6 response in participants with a cervical level SCI. The plasma IL-6 response to 30 min moderate intensity (60% VO2peak) arm-crank ergometry (ACE) was associated with an elevation in the anti-inflammatory cytokine IL-1 receptor antagonist (IL-1ra) independent of SNS activation (Chapter six). Light intensity ACE resulted in a small, significant plasma IL-6 response but no IL-1ra response. The addition of functional electrical stimulation-evoked lower-limb cycling to concurrent hand cycling, termed hybrid exercise, resulted in a greater plasma IL-6 response compared to moderate intensity hand cycling alone in participants with a thoracic level SCI (Chapter seven).

Thesis (PhD (Molecular Biology and Human Genetics))--University of Stellenbosch, 2005. / 157 leaves single sided printed, preliminary pages i-xvii and numbered pages 1-141. Includes bibliography, and abbreviations and a list of figures. / ENGLISH ABSTRACT: A gene coding for Arylaminie N-acetyltransferase (NAT) has been found in Mycobacterium tuberculosis, the casual agent of tuberculosis (TB). N-acetyltransferase acetylates and inactivates isoniazid (INH), which is a front line drug used in TB therapy. A guanine to adenine SNP at basepair 619 (G619A) has previously been identified in this gene, which results in a glycine to arginine change at amino acid 207 (G207R) (Upton et al. 2001). In this study the nat gene was further characterised. The frequency of the G619A SNP was analysed in 37 M tuberculosis strain families found in the Western Cape Province of South Africa, and it was found that the G619A SNP is conserved in two strain families (strain family 3 and strain family 28). Further sequence analysis identified a new thymine to cytosine SNP at base-pair 529 (T529C) resulting in a tyrosine to histidine change at amino acid 177 (Yl77H). This SNP was found only in isolates from strain family 3. These results imply that these SNPs may be used in epidemiology studies to classify isolates into these strain families. Using Real Time PCR, the expression of nat in M bovis BCG and M tuberculosis (reference strain H37Rv) was determined over a 7 and 28 day growth cycle, respectively. Using 16S rRNA as an endogenous control, the nat gene was shown to be expressed early during the growth curve and reach its maximum expression level at approximately mid-log phase. The expression of nat was induced in drug susceptible M tuberculosis isolates (reference strain H37Rv and isolate 1430 containing both SNPs) exposed to INH at a concentration of O.Oll-lg/ml, but minimal change in expression was observed in resistant isolates (isolate 816) exposed to INH at the same concentration. Mycobacterium bovis BCG cultures exposed to INH, at a final concentration of 0.28I-lg/ml, showed an increase in protein production. The increase of nat mRNA and NAT protein in M tuberculosis and M bovis BCG, respectively, implies that INH affects the expression of NAT. The NAT protein was localised to all fractions of the cell in Mycobacterium smegmatis, M bovis BCG and M tuberculosis, using the Western blot technique. However, protein fractions from the cell envelope region showed a protein (detected with specific NAT antibodies) that ran at a higher molecular weight (MW). This implies that the cytosolic hydrophilic NAT undergoes some type of post-translational process that may make it hydrophobic, and enable it to pass into the cell envelope region. These results show for the first time how nat is expressed during the entire growth cycle of M tuberculosis and M. bovis BeG. It was shown that nat is expressed early during the growth cycle of the bacterium reaching maximum expression levels at mid-log phase. These results are in concordance with those obtained using M. smegmatis nat mutants, which taken together, show that early expression of nat is important for early growth and development of mycobacteria. The results in this study also showed that NAT appeared to be translocated into the cell envelope of the bacterium, implying that NAT may be involved in one of the pathways needed for complete formation of the cell envelope. These results suggest that NAT may be an important target for drug development, as inhibitors of NAT could result in hindered growth and hence spread of the bacterium within its host. Inhibitors may also result in the incomplete development of the cell wall, enabling the host to combat the disease using its own immune system.

Thesis (PhD)--Stellenbosch University, 2012. / Please refer to full text for abstract.

published_or_final_version / abstract / Industrial and Manufacturing Systems Engineering / Doctoral / Doctor of Philosophy

published_or_final_version / Industrial and Manufacturing Systems Engineering / Master / Master of Philosophy

This work deals with the control of the human immune system. A standard immune system model is modified by introducing control signals corresponding to drug cocktail and immune suppressor treatments. The ultimate objective is to use these control signals to 'cure' a chronically-ill patient. Control is challenging for this system due to nonlinearities and time delays. In fact, it is shown that fundamental aspects of the system dynamics are lost when the system is linearised; hence, control approaches involving linearisation are fruitless. Feedback linearisation and some optimal control methods are also investigated and shown to be infeasible. However, it is shown that, for certain parameter values and initial conditions related to the virus and patient, a specific open-loop control scheme using only the drug cocktail achieves the objective. It is also proven that, unfortunately, this control scheme fails for other parameter values and initial conditions. A two-stage open-loop controller that uses both control inputs is then proposed. It is shown in simulation that the two-stage controller works over a larger set of parameters and initial conditions than the single-stage controller, but a rigorous analysis of the two-stage controller remains elusive.

Genome-scale ordered RNA structure (GORS) was first identified in 2004. It refers to the presence of secondary structure throughout the length of the RNA genomes of certain genera of RNA virus families, as predicted by bioinformatic analysis. It was also observed that the viruses containing GORS were able to establish persistent infections in their natural hosts, raising the possibility that the presence of GORS could play a role in viral avoidance of the innate immune system. This thesis describes the first study of GORS and its possible role in persistence. Two GORS viruses have been studied, equine rhinitis A virus (ERAV) and murine norovirus (MNV). A 55% seroprevalence of ERAV has been determined in a cohort of Scottish horses indicating a wide exposure to the virus. Equine faecal samples were screened for ERAV by PCR with the intention of identifying a virus, possibly from a persistently infected animal, which would not have undergone any cell culture adaptations as laboratory strains have. Newly identified viruses would then be sequenced, their secondary structures predicted and further studies carried out. Unfortunately, none of the 50 faecal samples screened were positive and clinical isolates of ERAV provided by the Animal Health Trust were sequenced but were identical to laboratory strains, so the study then focussed on MNV. Prevalence of MNV in laboratory mice was determined by PCR of faecal samples to be 67%. MNV was also discovered in the faeces of a pet shop mouse and a wild wood mouse (Apodemus sylvaticus). The complete genomes of 4 laboratory mouse MNVs, the pet shop mouse and wood mouse MNVs were sequenced. Phylogenetic analysis showed the wood mouse MNV had a p distance of 23% from other MNVs, although the laboratory mice and pet shop mouse were closely related to other MNVs. Structural analysis of the genomes of 6 sequenced MNVs, including the wood mouse virus, showed all were GORS viruses. A laboratory strain of MNV, MNV-3, was serially passaged in RAW 264.7 cells to test the hypothesis that in an animal with an intact immune system, there is a pressure for GORS viruses to maintain their genomic RNA structure as a means of immune avoidance, and that cell culture adaptation would attenuate the degree of secondary structure. The complete genome of passage 33 was sequenced, which revealed 7 base mutations, a mutation rate of 0.1 %, which was not considered significant enough to have affected the degree of secondary structure. In order to assess if structured and unstructured RNA behaved differently in cells, replication deficient RNA transcripts were made from the infectious clones of a panel of GORS and non-GORS viruses. These transcripts were electroporated into cells and their rate of decay measured, but there was no difference between the GORS and non-GORS transcripts. The full length and 4 kilobase transcripts were transfected into NIH3T3 cells and the degree of interferon-β induction measured by quantitative PCR and a luciferase reporter assay. The IFN-β response differed across the panel of viruses, and although none of the GORS viruses induced strongly, the non-GORS viruses were variable in their ability to induce an IFN-β response, some inducing strongly, other not at all. This result indicates that during exposure of viral genomes in the cytoplasm during infection, GORS-virus RNAs are unlikely to induce an interferon response, possibly contributing to their ability to persist. It is unclear why some non-GORS-viruses failed to induce IFN and there are likely to be other contributory factors.

Background: The prevalence of immunological disorders such as type 1 diabetes (T1D) is increasingly common amongst children, adolescents and young adults. There is also an increase in psychosomatic symptoms (depression, insomnia, anxiety, headaches and fatigue etc.) as well as a decrease in physical activity amongst young people, affecting the well-being and overall health of our younger population. It is therefore important to study the effects of psychological and physical stressors on the immune system, to evaluate their impact on juvenile health. Aim: This thesis explores the impact of psychological and physical stressors on the cellular immune system with special focus on diabetes-related immunity in a young population, using an interdisciplinary approach. Method: When exploring the impact of psychological and physical stressors such as psychological stress due to exposure to psychological stressful experiences or degree of physical activity/training on the cellular immune system in children, adolescents and young women, peripheral blood mononuclear cells (PBMC) were stimulated with antigens (tetanus toxoid (TT) and β-lactoglobulin (βLG)) as well as diabetes-related autoantigens (insulin, heat shock protein 60 (HSP60), tyrosine phosphatase-2 (IA-2) and glutamic acid decarboxylase 65 (GAD65)) and secreted cytokines and chemokines were measured by multiplex fluorochrome technique (Luminex). Populations of Thelper (Th) cells (CD4+), T-cytotoxic (Tc) cells (CD8+), B cells (CD19+), Natural Killer (NK) cells (CD56+CD16+) as well as regulatory T (Treg) cells (CD4+CD25+FoxP3+CD127-), and their expression of CD39 and CD45RA were studied by flow cytometry. Diabetes-related parameters (glucose, C-peptide,proinsulin, pancreatic polypeptide and peptide YY) were measured to studyβ-cell activity and appetite regulation and cortisol was used as a biological marker for psychological and physical stress. Results: Children in families exposed to psychological stress showed an imbalanced cellular immune response as well as an increased immune response towards diabetes-related autoantigens. Also, previous exposure to psychological stress as well as current exposure to psychological stress in young women showed an increased immune response towards diabetes-related autoantigens. Further, previous exposure to psychological stress in young women showed increased numbers of circulating CD56+CD16+ NK cells as wellas decreased numbers of circulating CD4+CD25+FoxP3+CD127- Treg cells. High physical activity in children showed decreased spontaneous immune response as well as a decreased immune response towards diabetes-related autoantigens, while low physical activity in children showed an increased immune response towards diabetes-related autoantigens. Further, endurance training in adolescents, especially in adolescent males and young adolescents, showed an increased immune response towards the diabetes-related autoantigen IA-2. Conclusion: It is evident that psychological and physical stressors such as exposure to psychological stress and degree of physical activity/training impact the cellular immune system. Experiences associated with psychological stress seem to have a negative effect on the cellular immune system in a young population, causing an imbalance in the immune system that could possibly induce diabetes-related immunity. High physical activity in children seems to have a protective effect against diabetes-related immunity. In contrast, low physical activity in children and endurance training in adolescents seems to induce diabetes-related immunity. It is very likely that psychological stressful experiences, low physical activity and intense training such as endurance training all play important roles in the immunological process leading to the development of type 1 diabetes.