Tuberculosis (TB), a severe disease caused by Mycobacteria tuberculosis (MTb), remains a globally severe health problem. In modern days, emergence of drug resistant TB is a new threat to public health since it can lead to treatment failure, increased transmission of TB to other hosts and further development of drug resistant complications. The traditional diagnostic method for TB by Acid Fast Bacilli (AFB) smear and Löwenstein–Jensen medium (LJ) culture are poor in sensitivity and time consuming respectively. There is a need for rapid diagnosis and identification of MTb. Nucleic acid amplification like PCR and real-time PCR are options for rapid diagnosis. However, such techniques require sophisticated technique and complex equipment. The high cost would constitute a barrier for countries with a high demand but only limited resources. Loop-mediated isothermal amplification (LAMP) assay is a novel technique, proven by many studies as to its high sensitivity and highly specificity to MTb. Most importantly, LAMP assay is economical and affordable by developing countries. The first objective of this study was to evaluate the analytical sensitivity and specificity of LAMP assay. The specificity of LAMP assay was determined by performing LAMP assay on 19 clinical isolates, which had already been identified previously. The clinical isolates included 14 mycobacteria tuberculosis complex (MTb), and five mycobacteria other than tuberculosis (MOTT) strains that were positive for AFB smear and LJ culture but negative for IS6110 single-tube nested real time PCR. The specificity was 100%. The analytical sensitivity as well as the limit of detection (LOD) were determined by testing on a duplicate set of serial DNA dilution, where each duplicate consisted of dilution of 100,000, 10,000, 1000, 300, 100, 10 and 1 colony forming unit/milliliter (CFU/ml). The LOD of LAMP assay was about 3 CFU per reaction. The second objective of this study evaluated the diagnostic performance of LAMP assay against AFB culture and IS6110 single tube nested real time PCR for identification of MTb in 200 respiratory specimens from 123 patients. All the specimens have already been tested for IS6110 single tube nested real time PCR, and culture results and AFB smears results have been obtained for all the specimens. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of three diagnostic methods (AFB smear microscopy, LAMP assay amplification and IS6110 single-tube nested real time PCR) were calculated with 95% confidence interval using LJ culture as gold standard. The LAMP assay had a sensitivity of 80%, specificity of 92.6%, PPV of 60% and NPV of 97.1%. However, MTb might fail to grow on the LJ culture medium for various reasons, for instance, MTb might already be dead after antibiotic treatment of the patient, or there might be poor laboratory practices during the processing of the specimens. Since the LJ culture method could produce false negatives in the situations described above, an alternative to the LJ culture method, ‘Hybrid Method’ was used as the gold standard. Under this method, a specimen was regarded as positive if the LJ culture result was positive. On the other hand, if a specimen generated a negative result using the LJ culture method, the results from the LAMP assay and IS6110 nested real time PCR would be considered, i.e., if both the LAMP assay and IS6110 nested real time PCR gave positive results while the results of LJ culture were negative, the specimen was referred to be positive in this case. In other words, a specimen would be regarded as negative if and only if the LJ culture result was negative and at least one of the LAMP assay or IS6110 was negative at the same time. Along the same line, the sensitivity, specificity, PPV and NPV of the three diagnostic methods (AFB smear microscopy, LAMP assay amplification and IS6110 single-tube nested real time PCR) were calculated with 95% confidence interval against the Hybrid Method. After resolution, the LAMP assay had a sensitivity of 87.0%, specificity of 100%, PPV of 100% and NPV of 97.1%. Our results showed that the LAMP assay has a great potential to be a new TB diagnostic test, especially in developing countries, with its lot of advantages like ease of use, cheap and fast. The LAMP assay in the study showed a high specificity, however, the sensitivity has to be improved before application in clinical use. For comparison of clinical performance, IS6110 single tube nested real time PCR had a higher sensitivity than that of LAMP assay (100% vs 80% using culture as gold standard; 100% vs 87% using ‘Hybrid Method’ as gold standard). However, LAMP assay had a higher specificity than that of IS6110 single tube nested real time PCR (92.6% vs 90.7% using culture as gold standard; 100% vs 98% using ‘Hybrid Method’ as gold standard). LAMP had been proven to be a potential and powerful tool in clinical diagnosis of MTb. Further improvement on its sensitivity is required to enable its extensive use in the clinic in the future. / published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

TSPYL2 (Testis-specific Y-encoded-like protein 2) is a member of the Nucleosome Assembly Protein (NAP) superfamily. It is a nuclear protein expressed in the cerebral cortex and the hippocampus. Our group has generated Tspyl2 knockout (Tspyl2m) mice, which are deficit in hippocampal long-term potentiation (LTP) with downregulation of Nr2a and Nr2b. Since Nr2a and Nr2b, subunits of the N-Methyl-D-Aspartate receptors, and hippocampal LTP are important in learning and memory, our Tspyl2m mice are likely to have behavioral deficits particularly in those related to memory. TSPYL2 could also affect LTP via CREB-dependent gene expression, since other NAP members have shown interaction with CBP/p300 - transcriptional co-activators of CREB which are well-known to be involved in memory formation. Furthermore, TSPYL2 may be linked to X-linked mental retardation (XLMR), since it is located at Xp11.2, a region with a high density of XLMR genes; and one of its interacting partners, CASK, is a XLMR gene. This thesis examines the three issues mentioned above. First, to characterize the behavior of our Tspyl2m mice, a behavioral test battery including open-field with amphetamine challenge, social interaction, prepulse inhibition and fear conditioning were conducted. Second, to examine the role of TSPYL2 on CREB-dependent gene expression, I first examined the subcellular localization of HA-TSPYL2 and endogenous CBP, p300 and pCREB in HEK293 cells. Then the interactions between TSPYL2 and CBP were tested by mammalian two-hybrid assay and co-immunoprecipitation. Thereafter, luciferase assay was used to measure CRE-luc activity in HEK293 and NG108-15 cells with overexpression and knockdown of TSPYL2. Third, to investigate the potential role of TSPYL2 on XLMR, a mutation analysis on the TSPYL2 gene was conducted with a cohort of 82 male patients with unexplained mental retardation. The analysis included examining the methylation on the TSPYL2 upstream sequence, DNA sequencing of the TSPYL2 exons, and in silico splice site analysis of the identified sequence variants. In the behavioral test battery, our Tspyl2m mice were normal in social ability, but showed enhanced hyperlocomotion after amphetamine injection, and deficit in prepulse inhibition and cued fear conditioning. When expressed in HEK293 cells, HA-TSPYL2 colocalized completely with endogenous CBP, but not with p300 and pCREB. In mammalian two-hybrid assay, pVP16(AD)-TSPYL2 interacted with GAL4(DBD)-CBP; however, HA-TSPYL2 did not immunoprecipitate with CBP. The luciferase assay data indicated that TSPYL2 suppressed the transcription of CREB-target genes. Lastly, no methylation was detected in the target sites in the TSPYL2 upstream sequence. Seven TSPYL2 sequence variations being identified were not deleterious as predicted by splice site analysis. To sum up, our Tspyl2m mice were deficit in cued fear memory, a form of associative memory. Moreover, they resembled the glutamatergic antagonist-induced schizophrenic rodent models in having enhanced hyperlocomotion after amphetamine injection, and deficit in prepulse inhibition. TSPYL2 interacted with CBP and suppressed the CRE-luc activity. The importance of TSPYL2 in XLMR has yet to be determined by larger studies. I propose that TSPYL2 represses CREB-dependent gene expression via sequestration of CBP as one of the possible mechanisms of how TSPYL2 causes various behavioral phenotypes. / published_or_final_version / Paediatrics and Adolescent Medicine / Master / Master of Philosophy

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Arabinogalactan proteins (AGPs) are highly glycosylated cell wall proteins specific to plants. AGPs have been implicated in almost all aspects of plant development and defense responses, nevertheless, most of such studies are correlative. To define the specific functions of individual AGPs, direct evidence from analyses of genetic knockout mutants of individual AGPs is required. Up to now, only a few AGPs have been demonstrated to have defined functions by mutant analyses. This dissertation identified a non-classical AGP (AGP31), described its expression and characterized the null mutant of AGP31 in Arabidopsis. In agp31 mutant, microarray analyses revealed that the expressions of genes encoding a subset of seed storage proteins (SSP): CRU3, CRA1 and OLEOSIN2 were induced. Further analysis showed that induction by agp31 knockout was specific to these three SSP genes, indicating a novel pathway to regulate the SSP gene expression. Comprehensive characterizations of AGP31 were carried out. Yariv reagent staining and monosaccharide analysis of purified AGP31 showed that AGP31 was a bona fide galactose-rich AGP. The cell wall localization of AGP31 was confirmed by expression of an AGP31::eGFP fusion protein. AGP31 promoter-GUS reporter gene analysis showed that AGP31 was expressed in the vascular bundle throughout the plant, except in the flower. In the flower, it was expressed throughout the pistil except in the stigma. Detailed analysis showed that GUS staining occurred in all cell types in the vascular bundle of roots, while GUS staining was restricted to phloem cells in the inflorescence stem. AGP31 mRNA was down-regulated by several stress treatments, including wounding, methyl jasmonic acid (MeJA) and abscisic acid (ABA). In response to MeJA treatment of whole seedlings, AGP31 mRNA level decreased to about 30% of its original level within 8 hr and almost returned to its original level after 24 hr. Nuclei run-on assay showed that the down-regulation of AGP31 mRNA upon MeJA treatment was due to reduced transcription. The strong preferential expression in vascular tissues and negative regulations by MeJA and ABA suggest that AGP31 may be involved in vascular tissue function both during development and the defense response.

Cholera is a life-threatening diarrhoeal disease, which mainly affects inhabitants of developing countries due to poor socio-economic conditions and lack of access to potable water and sanitation. Toxigenic Vibrio cholerae are the aetiological agents of cholera. These bacteria are autochthonous to aquatic environments, hence water plays a central role both in the epidemiology and transmission of cholera. The aim of this study was to determine the prevalence of V. cholerae from 32 sites of major rivers in Mpumalanga province of South Africa using a polyphasic approach. Water samples (594) collected over for 4 months were cultured on thiosulphate-citrate-bile salt-sucrose agar, and oxidase positive (88) isolates were subjected to biochemical tests and duplex polymerase chain reaction targeting the outer membrane protein (ompW) and cholera toxin (ctxAB) genes. All ompW PCR positive V. cholerae isolates were subjected to rfbO1 PCR. Fifteen isolates from Crocodile, Komati and Gutshwa rivers were assigned to V. cholerae by both biochemical tests and PCR, of which no isolates were positive for ctxAB and rfbO1 genes. The polyphasic approach was effective at revealing non-O1 and non-toxigenic V. cholerae in some rivers. Such information is important for raising awareness regarding the presence of V. cholerae so that precautionary measures are taken on time.

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published_or_final_version / abstract / toc / Paediatrics and Adolescent Medicine / Master / Master of Philosophy