A thesis submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Master of Science. Johannesburg, 2001 / Long distance running is characterised by a high injury rate (van Mechelen, 1995; Lysholm and Wiklander, 1987). It is an ever-growing sport, being tried by increasing numbers of people with varying degrees of athletic ability. Injuries are detrimental to training, increase the risk of sustaining another injury, and are expensive to treat placing demands on our already strained health care system. Runners are healthy people who would require less health care than most people if they could avoid injuries. Iliotibial band friction syndrome (ITBFS), is one of the most common running injuries experienced. Buttock and/or posterior thigh pain (BAOPTP) is another common condition, which is also resistant to treatment. It tends to become chronic and to result in ongoing morbidity. / IT2018

Background The optimal exercise load/intensity for exercise programs for individuals with metabolic syndrome (MetS) has not been investigated. One method of determining optimal exercise load is to measure the blood lactate transition threshold (BLTT), referred to as the anaerobic threshold (AT). The first part of this thesis (study 1) investigated the reproducibility of BLTT testing and the consequent determination of AT via the Mader method (Mader et al. 1986) and a modified form of the ADAPT method (Cheng et al. 1992) in patients with MetS. Furthermore, a comparison of the reproducibility of the AT determination using the Mader et al. (1986) method as opposed to the ADAPT method has not been investigated in MetS patients. The effect of specific exercise protocols on the different components of MetS has also not been investigated. Therefore, the second study in the thesis compared the effects on the components of the MetS of an exercise program that uses BLTT (specifically, the AT) to those of a comparable exercise program (not using AT) taken from the literature. The main aim of the study was to design an exercise program that optimized exercise responses and may thus improve metabolic characteristics in individuals with MetS. The third part of the thesis (study 3) focused on the relationship between cardiorespiratory fitness and the components of the metabolic syndrome. This study developed multiple regression models to find the principal variables that associated with peak vi oxygen consumption (VO2 peak) and AT in persons with MetS. Regression models were also developed to investigate whether these variables were associated with the individual metabolic and cardiovascular components of the metabolic syndrome. Methods In study 1, fifteen male patients diagnosed with MetS (age: 43.5 ± 7.52 years) and fifteen healthy, male participants (age: 44.1 ± 6.08 years) each performed a peak oxygen consumption and BLTT test simultaneously using an incremental protocol to exhaustion on a treadmill, at the same daily times, on three different days. Study 2 used three subject groups. One group consisted of ten participants (male, age: 48.3 ± 7.32 years) with MetS that exercised using the walking program of Leon et al. (1979) (MetSL). A second group consisted of ten participants (male, age: 40.8 ± 8.21 years) with MetS that exercised using velocity at AT to set training intensities (MetSV). A third group consisted of ten participants (male, age: 40.2 ± 7.90 years) without MetS that exercised using velocity at AT to set training intensities (Non-MetSV). Training durations and frequency varied from 20 – 90 minutes and 3 -5 days per week respectively. Height, body mass, waist circumference, blood pressure, fasting plasma triglyceride, total cholesterol, HDL-, LDL- cholesterol, insulin levels, VO2 peak and BLTT were measured in all groups before, during and after twenty weeks of exercise. In addition, oral glucose tolerance tests (OGTT) were administered to all participants. 0 min, 30 min and 2 hours plasma glucose and insulin levels were measured during the OGTT. HOMA-IR and insulinogenic indices were also calculated. Nutritional data were recorded at week 0, 8 and 20 of training. vii In study 3, thirty-one males diagnosed with MetS and twenty-four healthy male participants each performed a VO2 peak and a BLTT test. Height, mass, waist circumference, blood pressure, fasting plasma triglyceride, total cholesterol, HDLcholesterol and insulin levels were also measured. In addition, oral glucose tolerance tests (OGTT) were administered to all participants and HOMA indices were calculated. Results There was no significant difference in treadmill velocity at AT determined by the Mader method or the Modified ADAPT method within both groups of study 1 (p > 0.05). The mean treadmill velocity at AT was higher in the healthy compared to the MetS group using both the Mader and the ADAPT method. Regression analysis and ANCOVA in study 1 demonstrated that this difference was largely due to a higher VO2 peak in the healthy group. The study also found an association between VO2 peak and waist circumference. The coefficient of variation of repeat measurements for both the Mader method and the Adapt method was less than 4% indicating good reproducibility. This was confirmed by the typical error method of Hopkins (2000). Study 2 showed that body mass, BMI and waist circumference decreased significantly in all training groups with the training program using AT and the program not using AT showing similar outcomes in these variables among persons with MetS. Velocity at AT also improved in all training groups. While VO2 peak increased (p < 0.05) in both the MetS groups, it did not change significantly in the group without MetS. Similarly, the blood pressure response was favourable in the groups with MetS yet absent in the group viii without MetS. The training group with MetS that used AT was the only group to show significant, positive changes in any of the metabolic parameters (fasting insulin and HOMA). This group also showed the greatest change in the incidence of MetS. In study 3, presence of MetS, waist circumference and AT were found to associate with VO2 peak and VO2 peak was strongly correlated with AT. Age and body mass were found to correlate with fasting glucose, whilst only age correlated with HDL-cholesterol. Age and VO2 peak both correlated with systolic blood pressure but only VO2 peak had a significant association with diastolic blood pressure. Conclusions Study 1 demonstrated that BLTT tests are reproducible in persons with MetS. Study 2 demonstrated that an endurance exercise program using AT to set intensity is effective in eliciting favourable responses in individuals diagnosed with MetS. In addition, the training program using AT elicited the responses with a reduced exercise frequency and intensity. It also improved insulin sensitivity which was not affected by the walking program. The response to the exercise program that used AT was similar in persons with MetS and in persons without MetS, except in the central cardio-vascular adaptations of VO2 peak and in the metabolic parameters of fasting insulin and the HOMA index. Study 3 found that the lower VO2 peak of participants with MetS is associated with their higher waist circumference. The VO2 peak, in turn, was shown to correlate with anaerobic threshold. Therefore, reducing waist circumference in persons with MetS needs to be a focus of intervention programs for such a group. This study also found that both diastolic and systolic blood pressures were associated with cardio-respiratory fitness (VO2 peak). ix This further supports the benefit of increasing cardio-respiratory fitness in persons with MetS. The results of these studies showed that BLTT tests are simple, low-cost, reproducible ways of setting exercise intensity for persons with MetS that can be incorporated in the routine cardio-respiratory fitness assessment of an individual. Furthermore, the determination of AT from such tests can be used to design an individualized exercise program that can “reverse” the effects of MetS.

Trisomies 18 and 13 are rare genetic conditions (occurring around 1 in 6,000 and 10,000 newborns respectively) which are caused by an extra copy of either chromosome 18 or 13, similar to trisomy 21 (Down syndrome). The only known risk factor for these syndromes is maternal age, however previous cluster analyses have linked trisomy risk to a number of alternate factors, including radiation exposure and infection. Cases of trisomies 18 and 13 from the National Down Syndrome Cytogenetic Register (NDSCR) were scanned for temporal and spatial clusters throughout England and Wales between 2004 and 2010. No temporal clusters were detected, however there were multiple significant spatial clusters detected for both trisomies in London. These clusters were likely caused by advanced maternal age in the region, and it is also possible that regional differences in gestational age at the time of prenatal screening could have contributed to these clusters. In order to account for maternal age and gestational age at diagnosis, a novel method was developed in R to directly weight cases based on these factors. Applying weights to cases directly allowed both factors to be simultaneously accounted for by multiplying weights together. This method was evaluated using synthetic data and compared with an alternate method in the widely used program SaTScan. Both programs returned similar results when the weighting method was mild, but when extreme weights were applied at random significant clusters were observed in SaTScan but not in R. The NDSCR data was weighted and then rescanned for spatial clusters in both programs. No evidence of clustering was detected using the novel method, while SaTScan returned multiple highly significant clusters. These findings, combined with those obtained using the synthetic data, indicate that the novel method produces more reliable results than SaTScan when extreme adjustment is applied.

Patients with trisomy of chromosome 21, known as Down’s syndrome (DS), have a lower incidence of solid tumours than unaffected age-matched individuals. However, the cellular and molecular basis for this observation is not well understood. We hypothesised that a direct link between Down’s syndrome and angiogenesis exists, whereby the overexpression of human chromosome 21 (Hsa21) genes causes the repression of angiogenesis (gene dosage effects) resulting in the inhibition of solid tumour growth. In this project we investigated the angiogenic phenotype of an animal model of Down’s syndrome, the Tc1 mouse, which contains a large freely segregating fragment of Hsa21 containing over 200 human genes. We found that the growth of both B16F0 melanoma and Lewis Lung Carcinoma cells was impaired in Tc1 mice. Tumour vascularity also was reduced. This is supportive of the epidemiological data from the human DS population and supports the hypothesis that Hsa21 contains anti-angiogenic genes. Candidate genes were selected due to their endothelial specificity or likelihood to function in angiogenesis based on functional data or similarity to other proteins. Ex vivo RNAi assays were used to examine their roles in angiogenesis. We have found that reducing the expression of human Adamts1, Erg, Jamb or Pttg1ip in Tc1 tissue can restore its angiogenic potential, suggesting that the dosage of these genes (i.e. 3 copies instead of 2) can inhibit angiogenesis. Following from this study we also examined the role of selected adhesion related genes found on chromosome 21 in angiogenesis. Cldn14 encodes the tight junction molecule Claudin14 but its role in angiogenesis was unknown. We found that partial, but not complete, depletion of Cldn14 can increase the proportion of non-lumenated tumour blood vessels; decrease supporting cell association with tumour vessels; and increase endothelial cell proliferation in vivo, ex vivo and in vitro. Taken together this series of experiments has identified novel regulators of angiogenesis and has demonstrated the gene dosage effects of a subset of Hsa21 genes on angiogenic processes.

This thesis aimed to study the inter-relationship between the oral health status, aspects of the adaptive and innate immune response, and the oral microbiome of a well-defined cohort of Behçet’s syndrome (BS) patients. Methods Patients thought to have BS were referred to one of the two centres participating in this study (St Thomas’ Hospital and Royal London Hospital). Diagnosis was based on the International Study Group (ISG) criteria. Information about the frequency of the different symptoms of the disease along with the treatment protocol was collected on a data sheet and stored electronically in a database, in the period between January 2006 and August 2012. The data were then analysed at the end of the study period. The quality of life (QoL) and oral health status of BS patients in the UK was compared to those from Turkey. Furthermore, the oral health status of BS patients in the UK was compared to that of healthy control (HC) volunteers and recurrent aphthous stomatitis (RAS) patients. Saliva and oral swabs were cultured and purified to homogeneity and bacteria were identified by matrix-assisted laser desorption/ionisation-time of flight (MALDI-TOF). Salivary viral load and the serum immunoglobulin (Ig) G of the different herpes viruses were also examined. In order to determine whether there were differences in the innate response efficacy in BS patients, the expression of key molecular determinants of pathogen recognition was also analysed. Total ribonucleic acid (RNA) was purified from non-ulcerated buccal mucosal brush biopsies and analysed by real time polymerase chain reaction (qPCR) for the presence of toll-like receptor (TLR) 2 messenger RNA (mRNA) and TLR4 mRNA, and their splice variants. The functions of TLR2 and TLR4 were also investigated. Results In the investigated cohort of UK BS patients, all patients had a history of recurrent oral ulceration, 85.6% had dermatological lesions, 79.1% had rheumatological manifestations, 73.9% had genital ulceration, 68.6% had ocular involvement, 15% had neurological manifestations, and 10.5% had vascular involvement. The most frequent treatment was colchicine (54.2%), followed by topical corticosteroid therapy (53.6%) then azathioprine (43.8%). The QoL of BS patients from the UK and Turkey was affected to a similar extent. The Turkish BS patients had generally poorer oral health status in comparison to the UK BS patients. Nevertheless, the UK BS patients had also generally poorer oral health status in comparison to HC volunteers. The oral health status of the UK BS patients was comparable to those suffering from RAS. The oral mucosal and salivary microbial profile was variable between individuals in the same group and between individuals in different groups. The orally active BS patients’ oral mucosa showed the highest microbial community complexity and diversity compared to all the other investigated groups. Moreover, the BS patients had statistically higher salivary Epstein-Barr virus (EBV) shedding compared to HC volunteers, but not to RAS patients. Relapsed BS patients’ oral mucosa expressed higher levels of TLR2 and TLR4 mRNA. Investigation of the known splice variants of both receptors revealed that TLR2 mRNA variants b, d and e, and TLR4 variants 3 and 4 are significantly elevated in relapsed BS patients. A significant defect in the response to cognate agonists of TLR1/2 heterodimer and TLR4 was also observed in the whole BS patient cohort. Furthermore, BS patients expressed a lower cytomegalovirus (CMV) IgG level in comparison to all the investigated groups. Conclusion In conclusion, BS patients show higher levels of expression of some of the unusual splice variants of TLR2 and TLR4 mRNA, which might contribute to the observed functional defect in TLR1/2 heterodimer and TLR4. This defect in the key molecular determinants of pathogen recognition can lead to a failure in the adaptive immune responses’ modulation, resulting in the observed decrease in the expression of CMV IgG in BS patients and indeed the increase in susceptibility to oral infections. Furthermore, it is envisaged that the reported discrepancy in the oral microbiome of BS patients can be targeted in the future by probiotics to restore the balance of the oral microbial community, leading to better oral health which in turn will enable a better control of the BS immune response.

Thesis (M.D.)--Boston University School of Medicine

Down's syndrome (DS) is a congenital disorder caused by trisomy of chromosome 21, giving rise to symptoms including intellectual disability, poor muscle tone and characteristic facial features. Located on chromosome 21 is a gene encoding ubiquitin specific protease 25 (USP25), a member of the deubiquitinating family of enzymes. Studies of partial trisomies have revealed that although the USP25 gene is situated outside the critical region of chromosome 21 required to be triplicated to induce full DS symptoms, it is in a region linked to mild mental retardation and muscle hypotonia. Previous data has shown that, when overexpressed, USP25 forms novel rod shaped structures approximately 3-5 μm in length and 0.3-0.6 μm wide, and with a copy number, on average, of no more than 1-2 per cell. These structures do not associate with any known cellular organelle or with any component of the cytoskeleton. In this work, endogenous USP25 rods were detected in cultures of primary human foetal astrocytes, and a comparison of healthy and DS human primary foetal astrocytes revealed that in the DS culture a higher percentage of astrocytes contain rods. The domains of USP25 required for rod formation were identified using a series of GFP-tagged deletion constructs. USP25 rods could be purified from HEK293 cells using subcellular fractionation techniques and were assayed for deubiquitinating activity; however, none was detected suggesting that rods may be catalytically inactive. Interacting partners of USP25 were identified using mass spectrometry, but none were found to localise in rods. Additionally, a USP25 null human embryonic stem cell line was generated in order to interrogate the function of USP25 in astrocytes, and was found to be deficient in maintaining the integrity of an epithelial cell layer in an in vitro model of the blood brain barrier.

The presence of El Niño Southern Oscillation (ENSO) implies the presence of fluctuating rains in coastal areas and these changes influence the occurrence of febrile syndromes outbreaks. In Peru, Aedes aegypti is the vector responsible for various viruses such as the dengue, Zika, chikungunya, which is distributed in 18 Peruvian departments. These viruses cause similar clinical characteristics in the host and for this reason rapid, sensitive and specific diagnostic tests are needed so that the patient can receive timely treatment. / Revisión por pares

Abstract Fetal alcohol syndrome (FAS) is a common cause of mental retardation and is attributable to the teratogenic effects of alcohol exposure in utero in individuals with genetic susceptibility. The Coloured communities from the Western and Northern Cape regions have some of the highest recorded incidence rates (~70 affected children per 1000 live births) in the world. The candidate genes selected for this study belong to the family of alcohol dehydrogenase genes that code for enzymes which metabolise alcohol. The ADH1B and ADH1C genes have previously been examined in the Western Cape Coloured community and the enzyme encoded by the allele ADH1B*2 was significantly associated with protection against the development of FAS. ADH4, a new candidate gene, was selected due to its role in both the alcohol and retinol metabolic pathways. A case-control genetic association study was performed to examine the potential roles of the ADH1B, ADH1C and ADH4 genes in the etiology of FAS in two Coloured populations from the Northern and Western Cape. Single nucleotide polymorphisms found within the candidate genes were typed by PCR-based methods in samples from the FAS children, their mothers and controls. Significant associations were observed in the Western Cape cohort but were not replicated in the Northern Cape. Allelic association tests revealed that ADH1B*2 may be a protective marker as it occurred more commonly in the controls than the mothers (p= 0.038). The alleles of the polymorphic variant, ADH4.8, have been shown to influence the promoter activity of ADH4 (the ‘A’ allele has been shown to increase the activity of the promoter when compared to the ‘C’ allele as the same position). The alleles of this polymorphic marker were significantly associated with the risk for FAS. The ‘A’ allele was shown to occur more commonly in the mothers and FAS-affected children (p= 0.002 and 0.035 respectively) when compared to the controls, suggesting a role in disease susceptibility while the ‘C’ allele was shown to occur more commonly in the controls. Itwas also observed that ADH1B and ADH4.8 when examined together in a haplotype demonstrated an association with susceptibility to the disease. While the 2-C haplotype (ADH1B-ADH4.8) was shown to be associated with protection against the development of FAS, the 1-A haplotype was associated with increased susceptibility. The results suggest that mothers with the common ADH1B*1 allele and presumably a normal ADH1B function but an increased level of ADH4 (allele ‘A’) as a result of the promoter mutation, will, when the blood alcohol concentration is high, have an increased risk of having a child with FAS. Conversely when the mothers have a faster alcohol metabolising rate due to the allele ADH1B*2 and normal levels of ADH4 protein (allele ‘C’), the circulating alcohol in the blood is removed efficiently resulting in maternal protection against developing the disease. This study has also highlighted the genetic diversity within individuals of the South African Coloured population. Haplotype analysis and logistic regression revealed that the Western and Northern Cape Coloured communities are genetically different and as a result, the samples could not be pooled for analysis. Although the two groups of controls were genetically diverse, haplotype analysis revealed that the sample of mothers and FAS-affected children were not statistically different between the provinces thus possibly suggesting a similar genetic etiology for the disease. The results from this study suggest that the ADH genes do play a role in the pathogenesis of FAS.

No description available.