This thesis is focused on the ARP2/3 complex, which is a de novo actin cytoskeleton nucleator. This highly conserved complex is composed of seven subunits and regulates branching of actin filaments at a constant angle of 70 degrees. In plant and animal cells ARP2/3 is involved in various processes, which are connected with the initiation of actin polymerization; for example it participates in determining the direction and speed of cell growth and the movement of vesicles and organelles within the cell. The mutation of individual subunits is lethal for animal cells, but in plants, these mutants have only mild symptoms such as distorted trichomes or changes in epidermal cells. The aim of the presented work was to study the function of the ARP2/3 complex by the method of partial silencing of subunits using RNA interference. Specifically, it was the ARPC1 subunit of Arabidopsis thaliana and the ARPC2 subunit studied on the cellular model, the tobacco BY-2 cell line. Experimental work involved the creation of DNA constructs for induction of silencing, transformation of plant material, silencing rate analysis, and phenotype tracking in selected lines. Although lines with reduced transcript levels of the given ARP2/3 complex subunit were found, no phenotypic changes were observed in these lines. Key words...

The Ca2+ -signaling pathway is an important mechanism of cell signaling. Ca2+ /Cal- modulin (CaM)-dependent protein kinases (CaMKs) are members of Ser/Thr protein kinase family. CaMKs are regulated by Ca2+ /CaM binding in response to increase in intracellular level of Ca2+ . An important member of this protein family is Ca2+ /CaM- dependent protein kinase kinase (CaMKK), which is an upstream activator of CaMKI and CaMKIV. There are two isoforms of CaMKK, CaMKK1 and CaMKK2. CaMKK1 is regulated not only by Ca2+ /CaM-binding, but also by phosphorylation by cAMP-dependent protein kinase A (PKA). PKA phosphorylation induces inter- action with the 14-3-3 proteins. Previous studies of interaction between CaMKK1 and 14-3-3 proteins suggested, that the interaction with 14-3-3 proteins keeps CaMKK1 in the PKA-induced inhibited state and blocks its active site. However, the exact mecha- nism of this inhibition is still unclear mainly due to the absence of structural data. Main aim of this diploma thesis was to characterize the protein complexes between CaMKK1, Ca2+ /CaM and 14-3-3γ using analytical ultracentrifugation, small angle X-ray scattering, and chemical cross-linking coupled to mass spectrometry. Analytical ultracentrifugation revealed concentration-dependent dimerization of CaMKK1, which is...

It is estimated that free-roaming dogs comprise on average 75% of dog populations. Interactions between free-roaming dogs and wildlife occur across the globe but little is known about these interactions in the Lowveld region of South Africa, where wildlife areas are fenced off from surrounding communities. Even with extensive fences dogs are still entering the reserves. The study site, a private reserve in eastern Mpumalanga Province on the western boundary of the Greater Limpopo Transfrontier Park, has a high density of human settlements on its boundary. These communities own dogs, many of which are free-roaming. Between January 2009 and March 2014, 170 stray dogs were destroyed inside the reserve and 65.3% of the samples returned a positive result for rabies. Dogs are not limited to the reserve edges and have been documented several kilometres into the reserve. Eleven geographical factors were used in the spatial analysis: (i) camps or lodges in the reserve, (ii) fence line of the reserve, (iii) water points within the reserve, both natural and man-made (excluding rivers but including pans, dams and waterholes which hold water for most of the year), (iv) access roads from gates to camps and lodges, (v) access gates into the reserve, (vi) pickets (field ranger accommodation) and general staff accommodation, (vii) rivers in stream order from 1 to 6, (viii) vulnerable points for erosion along the fence line, and (ix) villages bordering the reserve. GPS locations of dogs shot were used to obtain nearest distance to each factor. Generalized linear models (GLM) were then used to analyse the spatial data of distance of dogs shot to the nearest factor. Dogs were significantly more likely to be shot further away from pickets and closer to minor rivers. There was a significant interaction between these two factors (p < 0.0001). Dogs that were shot further from villages (odds ratio 1.42, 95% confidence intervals 1.18 1.71, p = 0.0002) and closer to water (odds ratio 0.41, 95% confidence intervals 0.21 0.81, p = 0.009) were more likely to test positive for rabies. A univariate GLM, with distance to fence as the only explanatory variable, showed a significant association between this and rabies test result: for every 1 km further away from the fence the odds of a dog testing rabies positive increases by 1.68 (95% confidence intervals 1.20 2.36, p = 0.002). However the fence is likely not to be the influencing factor but rather other factors close to the fence. To remove the effect of the fence an analysis of the subset of dogs found further than 200m into the reserve found a positive association between distance from a village and a positive rabies test result (odds ratio 1.58, 95% confidence intervals 1.18-2.32, p = 0.007). Temporal analysis of the data shows a higher average monthly number of dogs shot during the wet season (Oct-Mar) as well as a higher variance although the cycle is not strictly seasonal. An upgrade of the reserve fence for security reasons coincided with a decrease in number of dogs destroyed in the reserve in 2012-1014. Home range analysis of most susceptible predators was collected from daily sightings data and overlaid with rabies hotspots, which gives management an indication of the need to vaccinate predators with home ranges in close proximity to the fence. The spatial results gives management an indication to increase efforts to destroy free-roaming domestic dogs further into the reserve as the likelihood of a positive rabies result is greater. Ultimately efforts should be focused outside the reserve in the communities to eradicate rabies from the host (domestic dogs) before there is a significant transfer of rabies to wildlife. / Dissertation (MSc)--University of Pretoria, 2015. / tm2016 / Veterinary Tropical Diseases / MSc

Q fever is a zoonotic disease caused by Coxiella burnetii which infects humans and a wide range of hosts including birds, mammals, ticks, fish and reptiles. Rift Valley fever (RVF) virus (RVFV) is a mosquito-borne pathogen of livestock, wildlife and humans and is found throughout much of sub-Saharan Africa, occasionally causing large scale abortions and mortality. There is little detailed knowledge of the distribution and level of occurrence of these two pathogens in South Africa. The aim of this study was to estimate the prevalence of antibodies against C. burnetii and RVFV in goats in Moretele municipality, and to identify factors associated with seropositivity to the two zoonotic pathogens. Multi-stage random sampling was conducted and sera were collected from 216 goats in 32 goat herds. A questionnaire was completed for each participating goat owner to collect information on potential animal, management and environmental risk factors, as well as potential animal health consequences of the two infections. Rift Valley fever virus antibody testing was done by ID Screen® Rift Valley Fever Competition ELISA test kit (IDVet, Grabels, France) and C. burnetii antibody testing was done by LSIVETTM Ruminant Q Fever - Serum/Milk ELISA test kit (Life Technologies, Carlsbad, California, USA) with confirmation using a virus neutralisation test (VNT). 32/216 goats tested positive for C. burnetii antibodies and the overall seroprevalence, adjusted for clustering and sampling weights was 16% (95% CI: 10.6-23.5%). The intraclass correlation co-efficient (ICC) was 0.06, indicating low to moderate clustering within herds. Multiple logistic regression revealed age as the only factor that was significantly associated with seropositivity to C. burnetii, with a higher seroprevalence in animals ≥2 years of age (26%) than in animals ≤6 months of age (6%) (odds ratio (OR) = 6.6; 95% CI: 1.6-26.7; P = 0.010). Regarding potential consequences of infection, females with a history of abortion were more likely to be seropositive (OR = 4.6; 95% CI: 1.1-20.2; P = 0.043). Goats in herds that reported >2 abortions in the 12 months prior to sampling tended to have a higher odds of seropositivity than animals in herds with no reported abortions (OR = 2.5; 95% CI: 0.9-6.8; P = 0.071). 1/216 goats tested ELISA-positive for RVF virus antibodies and 3 samples were “doubtful”; however, they all tested VNT-negative. The estimated seroprevalence of RVFV was therefore 0% (95% CI: 0.0-1.4%). In this study, the herd seroprevalence of C. burnetii was high at 51% and there was possibility that C. burnetii is a likely cause of abortions in goats in Moretele municipality of South Africa. Seropositivity to RVFV could not be demonstrated in this study; but if present, the virus is likely to be circulating at very low levels. / Dissertation (MMedVet)--University of Pretoria, 2019. / Production Animal Studies / MMedVet / Unrestricted

The aim of this study was to investigate the prevalence and epidemiological significance of tuberculosis (TB) in bovine and humans living at a wildlife/livestock/human interface, as well as the risk factors associated with TB transmission at that interface. The Mnisi community was chosen as it is located at the western border of the Kruger National park (KNP) and enables research at the wildlife/livestock/human interface. The first objective of the study entailed investigating the presence of bovine tuberculosis (BTB) in 10% of Mnisi?s livestock, using the comparative intradermal skin test. A low individual prevalence of 0.33 % (95% CI.0.14 ? -0.79) was detected. Further investigations into the causative agent in livestock, using genotyping techniques identified the KNP parental strain, M. bovis KNP VNTR -1 strain.1 Supporting records from the provisional Mpumalanga Veterinary Services and the physical location of dip-tanks where BTB was detected, it was established the infection was a result of spillback infection from wildlife in the neighbouring KNP. The epidemiological significance of BTB in human TB was investigated through the isolation and genetic characterisation of the Mycobacterium tuberculosis complex (MTBC) strain population in the Mnisi community. Mycobacterium bovis was not detected in the human population. However, a high genetic diversity of M. tuberculosis was observed among the 13 isolates obtained.The M. tuberculosis isolates were identified as the following eight families: T; Beijing; LAM 11_ZWE; EAI5; MANU1; X1; X2; and S families. The predominant lineage was as T family, sub-lineage ST53. Based on the high diversity (8 clusters/13isolates) and the predominance of the T family, it was concluded that the TB population structure within the Mnisi community was largely impacted by human migration from urban towns and neighbouring Mozambique. A questionnaire was administered to investigate BTB transmission risk factors at the livestock/human interface. It was established that there were low risk levels of BTB transmission at the human/livestock interface mainly based on the fact that the majority of the households in the community obtained pasteurised milk commercially, and although undercooked/raw meat and organs were preferred, the majority of respondents reported that they discarded the meat if changes in meat quality were observed. / Thesis (PhD)--University of Pretoria, 2016. / tm2016 / Veterinary Tropical Diseases / PhD

No description available.

Our understanding of non-histone acetylation as a means of cellular regulation is in its infancy. Using a mass spectrometry approach we identified acetylated lysine residues and monitored acetylation changes across the proteome as a consequence of metabolic stress (hypoxia). We observed changes in acetylation status of non-histone lysines in tumor cells. Through the use of small molecule inhibitors of histone deacetylase enzymes (HDACs) and siRNA screening identified HDAC6 as a pro-survival regulator of lysine acetylation during hypoxia. The phospho-binding protein 14-3-3ζ acts as a signaling hub controlling a network of interacting partners and oncogenic pathways. We show here that lysines within the 14-3-3ζ binding pocket and protein-protein interface can be modified by acetylation. The positive charge on two of these lysines, K49 and K120, is critical for coordinating 14-3-3ζ-phosphoprotein interactions. Through screening, we identified HDAC6 as the K49/K120 deacetylase. Inhibition of HDAC6 blocks 14-3-3ζ interactions with two well-described interacting partners, Bad and AS160, which triggers their dephosphorylation at S112 and T642, respectively. Expression of an acetylation-refractory K49R/K120R mutant of 14-3-3ζ rescues both the HDAC6 inhibitor-induced loss of interaction and S112/T642 phosphorylation. Furthermore, expression of the K49R/K120R mutant of 14-3-3ζ inhibits the cytotoxicity of HDAC6 inhibition. These data demonstrate a novel role for HDAC6 in controlling 14-3-3ζ binding activity.

3'-Carboxymethyl-3'-deoxyadenosine derivatives were prepared from 2'-O-TBDMS-3'-deoxy-3'-[(ethoxycarbonyl)methyl]adenosine (1) via simple and efficient procedures. Conversion of 1 to 5'-azido-2'-O-TBDMS-3', 5'-dideoxy -3'-[(ethoxycarbonyl) methyl] adenosine (4) was accomplished via a novel one-pot method employing 5'-activation (TosCl) followed by efficient nucleophilic displacement with tetramethylguanidinium azide. Compound 4 was converted to a 5'-[(N-methylcarbamoyl)amino] derivative (5) via one-pot reduction/acylation employing H2/Pd-C followed by treatment with p-nitrophenyl N-methylcarbamate. The latter step of this two-step process required an efficient source of p-nitrophenyl N-methylcarbamate, thus a highly efficient new method for preparing p-nitrophenyl N-alkylcarbamate was developed. N6-phenylcarbamoyl groups were introduced by treatment with phenylisocyanate, and an efficient new method for lactonization of 2'-O-TBDMS-3'-deoxy-3'-[(ethoxycarbonyl)methyl]adenosines to give corresponding 2', 3'-lactones was also developed. Target compounds were evaluated for anti-HIV and anti-HIV integrase activities, but were not active at the concentrations tested.

No description available.

For the past 40 years, acoustic sensing has been a major avenue for the growth of interfero- metric fiber-optic sensors. Fiber-optic acoustic sensors have found uses in military, commer- cial, and medical applications. An interferometric fiber-optic acoustic sensor is presented utilizing the Michelson interferometer configuration with Faraday mirrors to eliminate po- larization fading. A 3 × 3 coupler is used as the beamsplitting component, and a symmetric demodulation algorithm is applied to recover the phase signal. This sensor has a theoretical resolution of 5.5 pico-strains and room to improve. Such improvements are discussed in the conclusion. / Master of Science