A peptide nucleic acid (PNA) encoded 1296 member peptide library was synthesised and incubated with a variety of cell types. Library members entering cells were extracted, hybridised onto DNA microarrays and the peptide identity was determined via deconvolution. Global consensus analysis highlighted the tetrapepide, Glu-Llp- Glu-Glu (Llp is 6-hexamine-N-aminoacetic acid), a surprise in view of the basic residues typically observed in cell penetrating peptides. When evaluated, Glu-Llp- Glu-Glu revealed cellular uptake comparable to a known basic peptide (tetraLlp). In depth delineation via clustering analysis allowed assessment of differential cellular uptake, with the identified peptides showing clear cellular specificity. This was verified by peptide synthesis and cellular uptake analysis by flow-cytometry, and in all cases an endocytic uptake mechanism was confirmed. This approach establishes a strategy for the identification of short peptides as tools for selective delivery into specific cell types. The incubation of a 10,000 member PNA-encoded peptide library with D54 and HEK293T transfected with CCR6 cells followed by microarray analysis allowed detailed information on the interaction between peptide-ligands and cell surface receptors to be extracted. This allowed the identification of new ligands for integrins and G-protein coupled receptors and offers a novel approach to ligand discovery allowing the comparative analysis of different cell types for the identification of differences in surface-receptor ligands and/or receptor expression between various cell types. In addition, this work included the development of a novel method for the indirect amplification of a PNA library by amplification of a complementary DNA library hybridised to the PNA. The generation of 10,000 defined pieces of DNA would have a myriad of applications, not least in the area of defined or directed sequencing and synthetic biology, but also in applications associated with encoding and tagging. By this approach DNA microarrays were used to allow the linear amplification of immobilised DNA sequences on an array followed by PCR amplification. Arrays of increasing sophistication (1; 10; 3875; 10,000 defined oligonucleotides) were used to validate the process, with sequences verified by selective hybridisation to a complementary DNA microarray with DNA sequencing demonstrating error rates of ca ≈ 0.2%. This technique offers an economical and efficient way of producing hundreds to thousands of specific DNA primers, while the DNA-arrays can be used as “factories” allowing specific DNA oligonucleotide pools to be generated with or without masking. This study also demonstrated a significant variance observed between the sequence frequencies found via Solexa sequencing compared to microarray analysis.

Thesis (MScAgric (Soil Science))--University of Stellenbosch, 2006. / High potassium content in grape juice and wine are associated with low quality red wine in warm wine producing countries. In an attempt to reduce the potassium content of juice, must and wine, a field experiment was laid out on the farms Meerlus and Kersfontein in the Paardeberg area near Wellington in 1998 on granite derived soils to investigate the effect of canopy management and fertiliser applications on berry K accumulation and wine quality. Four fertiliser applications, three canopy treatments and a MgSO4 foliar spray were studied. The three fertiliser treatments being: none (control), CaSO4, Ca(OH)2, and MgSO4 applications. The canopy treatments were: thin to two shoots per bearer, tip, vertical shoot positioning (VSP) and the removal of yellow leaves and lateral shoots (canopy 1), thin to three shoots per bearer, top after véraison and VSP (canopy 2) and VSP with top after véraison (canopy 3/control). Magnesium sulfate sprays were applied at véraison for two seasons (1999/00 and 2000/01). Seasonal effects produced the most significant differences in this experiment. Canopy treatments did not affect juice K concentration at harvest. Canopy 1 and 2 produced significantly lower wine pH values at Kersfontein. Fertiliser treatments had no effect on juice K concentration nor did it affect wine quality. Magnesium sulphate foliar sprays did not affect juice K concentration at harvest but significantly lowered juice and wine pH, improved wine colour density and total phenolic content. It appears for this experiment that soil K content before véraison, shoot growth at and after véraison and water stress after véraison were the main factors determining juice K concentration at harvest.

No description available.

The Human Immunodeficiency Virus and Acquired Immune Deficiency Syndrome (HIV/AIDS) continues to have enormous implications on the health, economic and psychosocial well-being of individuals, family structures, and communities. Sub-Saharan Africa is the region most affected by the HIV/AIDS. The purpose of this study is to investigate the social and cognitive factors associated with HIV test uptake in the general population of Kenya. Data from the 2009/2010 Kenya Demographic Health Survey were utilized. Univariate, bivariate and multivariate analyses were conducted using STATA/SE software. Results showed that there were statistically significant differences between men and women in previous HIV testing status and HIV test uptake. Over 90% of participants reported knowing a place to seek testing. The mean HIV related knowledge was higher in men than women (x =0.1; 95% CI 0.04-0.16) than women (x = 0.04; 95% CI [0.01- 0.1]). Differences were found in expressed HIV stigmatizing attitudes, with women reporting more stigmatizing attitudes than men. For example 9.9% of women compared to 4.7% of men reported very high HIV stigmatizing attitudes. Weighted multinomial regression analyses were conducted with individuals who had not been previously tested and unwilling to be tested utilized as the reference group. Among the factors found to be significantly associated with HIV uptake include: HIV related knowledge- higher levels of HIV related knowledge were associated with increased HIV test uptake for men and women, HIV related stigma- lower levels of HIV related stigma were significantly associated with HIV test uptake for women but not men, acceptance to teach condoms to children and knowledge of someone infected with HIV/AIDS was positively associated with HIV test uptake, gender- compared to men, women were significantly less likely to agree to be take the HIV/AIDS test if not previously tested (OR 0.79; 95% CI [0.64, 0.97]) but significantly more likely to accept the HIV/AIDS test when offered (OR 1.341; 95% CI [1.02, 1.76]). Other significant associations included: Age, education attainment, sex of head of household, and wanting to keep a family member's tuberculosis infection a secret.

No description available.

No description available.

Diagnosis of diabetes mellitus, presence of hyperglycemia, and/or insulin resistance confer cardiovascular risk, particularly for diastolic dysfunction. Diabetes is associated with elevated myocardial norepinephrine (NE) content, enhanced sympathetic nervous system (SNS) activity, altered resting heart rate, and depressed heart rate variability. Positron emission tomography (PET) using the NE analogue [11C]meta-hydroxyephedrine ([11C]HED) provides an index of myocardial sympathetic neuronal integrity at the NE reuptake transporter (NET). The hypothesis of this project is that (i) hyperglycemia imparts heightened sympathetic tone and NE release, leading to abnormal sympathetic neuronal function in the hearts of diabetic rats, and (ii) these abnormalities may be reversed or prevented by treatments to normalize glycemia. Sprague Dawley rats were rendered insulin resistant by high fat feeding and diabetic by a single dose of streptozotocin (STZ). Diabetic rats were treated for 8 weeks with insulin, metformin or rosiglitazone, starting from either 1 week (prevention) or 8 weeks (reversal) after STZ administration. Sympathetic neuronal integrity was evaluated longitudinally by [11C]HED PET. Echocardiography measures of systolic and diastolic function were completed at serial timepoints. Plasma NE levels were evaluated serially and expression of NET and β-adrenoceptors were tested at the terminal endpoints. Diabetic rats exhibited a 52-57% reduction of [11C]HED standardized uptake value (SUV) at 8 weeks after STZ, with a parallel 2.5-fold elevation of plasma NE and a 17-20% reduction in cardiac NET expression. These findings were confirmed by ex vivo biodistribution studies. Transmitral pulse wave Doppler echocardiography established an extension of mitral valve deceleration time and elevated early to atrial velocity ratio, suggesting diastolic dysfunction. Subsequent treatment with insulin but not metformin restored glycemia, reduced plasma NE by 50%, normalized NET expression, and recovered [11C]HED SUV towards non-diabetic age-matched control. Diastolic dysfunction in these rats persisted. By contrast, early treatment with insulin, metformin, or rosiglitazone delayed the progression of diastolic dysfunction, but had no effect on elevated NE and reduced [11C]HED SUV in diabetic rats, potentially owing to a latent decrease in blood glucose. In conclusion, diabetes is associated with heightened circulating and tissue NE levels which can be effectively reversed by lowering glycemia with insulin. Noninvasive interrogation of sympathetic neuronal integrity using [11C]HED PET may have added value in the stratification of cardiovascular risk among diabetic patients and in determining the myocardial effects of glycemic therapy.

The purpose of this study was to assess the changes in maximal oxygen consumption and cardiorespiratory responses to maximal treadmill exercise of men, who engaged in intense physical training for more than 35-years. Thirteen men were first studied in 1969 and were re-examined in 1992 as a 25-year follow-up. The men were divided into two groups, group 1 (G1) and group 2 (G2). G1 (current age = 54.6 years) consisted of seven men who were Division I cross country runners in the late 1960's, and G2 (current age = 67.5 years) were highly active at the initial testing and were 14 years older than the men of G1 at all test dates. A maximal exercise test revealed a significant decrease in absolute and realtiveVO2ma, over the 35-years for both G1 and G2. G2 experienced a significant decline in VO2max when expressed in relative and absolute terms after the age of 56.5 years which occurred after the 25-year test. Maximal heat rate decreased over the 35-year period for both G1 (187.7 to 165.8 bpm) and G2 (181 to 164.7bpm), however only GI revealed a trend between the 25-and 35-year tests. O2pulse significantly decreased from the initial testing to the 35-year period in G2 (23.1 to 19.5 ml/beat). Body composition changes were evident with aging in that both GI and G2 had a significant increase in percent body fat over the 35-years however, only Gl had a significant increase in body weight at both the 25-and 35-year follow-up.These data suggest that aging and a reduced training regimen may have a significant effect upon VO2m and cardiorespiratory and body composition measures. It appears that after the age of 56 years, a greater reduction in VO2max occurs, which is accompanied by greater changes in stroke volume. Although the aerobic capacity of these men declined over the 35-year period, the values obtained by all men far exceeded the values reported for sedentary and less active men in other studies. Therefore, as previously reported, aging is associated with a reduction in aerobic capacity, however continued endurance training can reduce the rate at which aerobic capacity declines. / Fisher Institute for Wellness and Gerontology

The wide use of nanoscale materials in food and health care products raises the concern of their possible uptake across the gastrointestinal tract, but very limited data are available on their uptake kinetics, and the potential hazards for humans. In this study, the uptake mechanism of titanium dioxide (TiO2) across the isolated perfused fish intestine and human intestinal Caco-2 cells were evaluated. The in vitro preparation of the whole gut sac and the isolated perfused intestine of rainbow trout were performed using both bulk and nano TiO2 in a concentration of 1 mg l-1 for up to 4 h. The results showed that the Ti from both bulk and TiO2 NPs were mainly accumulated in the mid and hind intestine, with 80% or more of the accumulation in the mucosa rather than the underlying muscularis. Perfused intestines showed a saturable, time-dependent accumulation of the Ti from TiO2 and the uptake of Ti from exposure to NPs was faster than that of the bulk form. The uptake of Ti from exposure to TiO2 NPs increases 10 fold when the CO2 in the gas mixture was lowered to 0.5%. Subsequently, further investigation on the mechanisms of uptake of TiO2 was applied using different kinds of inhibitors. Adding 10 mmol l-1 cyanide did not stop Ti uptake from TiO2 exposures, and 100 µmol l-1 vanadate (ATPase inhibitor) caused a 2.8 fold reduction in the net uptake rate of Ti for the TiO2 NP exposure. Luminal additions of 120 IU ml-1 nystatin (endocytosis inhibitor) blocked the uptake of Ti from both bulk and TiO2 NPs treatments. The results indicate that Ti accumulation from TiO2 exposures was sensitive to both nystatin and vanadate; the former suggesting that there is an endocytosis involvement in the uptake of TiO2 across the intestinal epithelium. Human intestinal Caco-2 cell showed a steady, saturable and time-dependent accumulation of Ti over 24 h exposures to 1 mg l-1 TiO2 (for all forms of TiO2). A scanning electron microscope study indicated the appearance of the particles underneath the cells, increasing the evidence of the Ti uptake from different forms of TiO2 by Caco-2 cells. Both nystatin and vanadate increase the accumulation of TiO2 which suggests interference of these drugs with endocytic pathways. All the data in the thesis demonstrates Ti uptake across the intestinal epithelium from TiO2 exposures involving CO2-dependent and nystatin-sensitive mechanisms. The results in this thesis have contributed to some understanding on the behaviour, uptake and effects of the TiO2 NPs across the intestine; and highlight the possible dietary hazard of the NPs to human health.

The World Health Organization (WHO) states that 30,000 children under the age of five die each day worldwide. Around a quarter of these die from diarrheal disease caused by microbial infection. In addition to this high mortality rate, there are data emerging on the morbidity effects of diarrheal disease, for example a few episodes of diarrhea in the first two years of life can remove 10 IQ points and lead to growth deficiency. Vibrio cholerae, the causative agent of the diarrheal disease cholera, is a serious problem in third world countries, where sanitary and hygiene infrastructure is very poor, and claims several thousand lives every year. In order to better understand the pathogenicity regulation in V. cholerae, structural and functional investigations of a hypothetical protein family present in pathogenicity islands and a transcriptional regulator protein for DNA-binding were investigated. Two adjacent genes, vc1804 and vc1805, encode hypothetical proteins within the Vibrio pathogenicity island-2 (VPI-2) of Vibrio cholerae, and are part of a cluster of genes only present in pathogenic strains of the bacterium. Paralogous adjacent genes, vc0508 and vc0509, are also present within a second pathogenicity island, the Vibrio seventh pandemic island-2 (VSP-2), of V. cholerae O1 El Tor and O139 serogroup isolates. Sequence similarity suggests that the VC0508, VC0509, VC1804 and VC1805 proteins will share a similar fold. The crystal structures of VC0508, VC0509 and VC1805 have been determined to a resolution of 1.9, 2.4 and 2.1 Å, respectively. Several recombinant constructs of vc1804 were made, but no soluble proteins were expressed. This hypothetical protein family reveals structural homology to human mitochondrial protein p32. Human p32 is a promiscuous protein known to bind to a variety of partners including the globular head component of C1q. We have shown that VC1805 binds to C1q. One possibility is that VC1805 is involved in adherence of the bacterium to membrane-bound C1q in the gut. To explore the roles of VC0508, VC0509, VC1804 and VC1805 in vivo, gene knockout and animal model studies of those proteins are underway. The ferric uptake regulator (Fur), a metal-dependent DNA-binding protein, acts as both a repressor and activator of numerous genes involved in maintaining iron homeostasis in bacteria. It has also been demonstrated in Vibrio cholerae that Fur plays an additional role in pathogenesis, and this opens up the potential of Fur as a drug target for cholera. The first crystal structure of a Fur protein, from Pseudomonas aeruginosa, revealed a dimeric molecule with each monomer containing a dimerization domain, a helical DNA-binding domain and two metal binding sites: Zn1 is proposed to be a regulatory Fe-binding site, and Zn2 is proposed to be a structural Zn-binding site. Here we present the crystal structure of V. cholerae Fur (VcFur) that reveals a very different orientation of the DNA-binding domains. Accompanying these structural changes are alterations in the amino acids coordinating the zinc at the Zn2 site, and this lends support to this being the site regulated by iron. There is no evidence of metal binding to the cysteines that are conserved in many Fur homologues, including the much-studied E. coli Fur. An analysis of the metal binding properties shows that like other Fur proteins, VcFur can be activated by a range of divalent metals. EPR spectroscopy measurements of the movements of the DNA-binding domain, in the presence of DNA and different metals, are underway.