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<p>Basella alba (BA) and Hibiscus macranthus (HM) are used by traditional healers in Cameroon to treat male sexual fertility problems. Previous studies showed that in vivo administration of the leaf extracts of both plants caused a significant increase in rat seminal vesicle weight and spermatozoa numbers was accompanied by a significant increase in serum testosterone. The aim of this study was to establish the effects of BA and HM extracts on Sertoli cell functions. TM4 cell line was used in this study as it exhibited properties similar to the Sertoli cells (Mather, 1982). Sertoli cell play a key role in spermatogenesis by regulating and supporting germ cell development. Therefore, any alterations in Sertoli cell physiology or structure may lead to impaired spermatogenesis, germ cell loss and male infertility. Developing germ cells in the seminiferous tubule require a constant supply of lactate and pyruvate (Jutte et al, 1981 / 1982) and toxicant induced alterations in these nutrients have been shown to induce germ cell necrosis (Monsees et al., 2000). TM4 Sertoli cells were cultured in DMEM/Ham F-12 (M) for one day and exposed to<br /> 0.01, 0.1, 1, 10, 100 &mu / g/ml of BA and HM extracts, respectively, for four further days. The extracts were dissolved in 0.5 % DMSO in M, while 0.5 % and 2% DMSO in M were used as negative or positive controls, respectively, and 100mM ethanol as positive control where indicated. Results obtained from the Sertoli cells exposed to BA extracts, showed that the plant extract had no significant effect on the cell viability but induced a significant concentration-dependent increase in lactate (19-67%) and pyruvate levels (39-102%) and a concentration-dependent decrease in the protein content (9-42%). The H&amp / E histological study confirmed that the BA extract had no cytotoxic effect, as there were no changes in the morphology of the cell. Likewise, apoptotic study using DAPI showed no alteration in the nucleus when compared to the negative control. The HM plant extract significantly enhanced mitochondrial dehydrogenase activity (7fold) in the Sertoli cells but caused only slight alterations in the lactate and pyruvate levels. There was no effect seen in the protein content of the Sertoli cells. H&amp / E and DAPI staining revealed that there were neither changes in the morphology of the cells nor any alteration regarding the mitotic and apoptotic indices. Thus, the HM extract did not have a cytotoxic effect on the cells. This study demonstrated that the Basella alba methanol extract may enhance spermatogenesis as it stimulated the source of energy required for the development of germ cells without exerting a cytotoxic effect. The Hibiscus macranthus extract stimulated mitochondrial dehydrogenase activities and may thus trigger changes in Sertoli cell physiology. In summary, both plant extracts enhanced certain Sertoli cell<br /> functions and thus might explain the positive in vivo effects of the combined plant extracts on rat spermatogenesis observed by Moundipa et al. (1999).</p>

Biochemical and ultrastructural changes associated with chilling injury (CI) in soybean seeds imbibed at 5°C and 25°C were investigated. Soybean seed germination appeared to be affected by chilling temperatures and initial seed moisture content. Seeds with higher moisture contents exhibited 85% germination, while low moisture content seeds had a 32% germination. Leakage rates were greater in chilled seeds, indicating that membrane integrity in the tissues was impaired at chilling. The low rates of potassium ion leakage between 6 and 24 hours of imbibition compared to the high peroxide levels observed during this period led to the suggestion that lipid peroxidation was a better marker of CI than leakage. Transient changes in lipid hydroperoxide levels were observed in chilled and non-chilled seeds and axes. However, in axes, the increase in lipid hydroperoxides after 12 hours of imbibition at chilling temperatures was associated with an 18% decline in linoleic acid levels of total lipid fraction. Similarly, a 10% decline was observed in the polar lipid fraction. These results suggest that the capacity of seeds to control lipid peroxidation may be an important component in CI and that a consequence of peroxidation is likely to be a loss of fatty acid unsaturation. Sugar levels were not affected by chilling and non-chilling temperatures and no relationship could be established with CI. Antioxidant defense enzymes (catalase and superoxide dismutase) were expressed at chilling and non-chilling temperatures and increases were observed after 24 hours of imbibition which showed an apparent correlation with increases to lipid hydroperoxide levels. Enzyme levels decreased after 48 hours of imbibition at a time which coincided with the decline observed in the peroxide levels. Overall, no marked differences were observed in chilled and non-chilled cells at the ultrastructural level, except that vacuolar reserve mobilization was markedly impeded. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 1996.

The putative peroxisome proliferator-activated receptor (PPAR) α ligand, conjugated linoleic acid (CLA) induced cytoplasmic lipid droplet (LD) formation in H4IIE rat hepatoma cells. Currently, the mechanism(s) by which CLA isomers affects hepatic LD formation is unclear. We have investigated the role of PPARα and fatty acid (FA) activation in the regulation of hepatic LD formation induced by CLA isomers [cis-9,trans-11 (c9,t11), trans-10,cis-12 (t10,c12)] and linoleic acid (LA) in an in vitro model of lipid accumulation. Dose response of c9,t11 and t10,c12 CLA isomers as well as LA in quiescent H4IIE cells was assessed by Oil Red O staining and subsequent quantification after 24 hours. LD formation was induced by the CLA isomers similar to LA in a dose-dependent manner. However, treatment with the acyl CoA synthetase (ACS) inhibitor, triacsin C, resulted in significantly reduced LD formation. A similar reduction in lipid accumulation was observed with the PPARα activator, Wy14643. Furthermore, CLA isomers promoted H4IIE viability at 60 µM but decreased viability at a higher dose of 180 µM. To further understand the role of PPARα in hepatic steatosis, we studied the level and phosphorylation of PPARα in livers of male lean and fa/fa Zucker rats fed either a control diet or fa/fa Zucker rats fed a CLA isomer (0.4% wt/wt c9,t11 or 0.4% wt/wt t10,c12) diet for 8 weeks. Immunoblotting results showed that only the t10,c12 CLA isomer significantly reduced phospho-PPARα S21 compared to the lean control (ln Ctl) and it was associated with a significant increase in the phosphorylation of p38 mitogen activated protein kinase (MAPK).These changes were not observed with the c9,t11 CLA isomer. Taken together, we have shown that CLA isomers directly induce LD formation in quiescent H4IIEs by activation of the lipid storage pathway which was significantly reduced by triacsin C or Wy14643. Also, we demonstrate for the first time that only the t10,c12 CLA isomer significantly reduced PPARα phosphorylation while it increased p38 MAPK phosphorylation. These results indicate that the anti-steatotic effects of the t10,c12 CLA isomer is associated with changes in PPARα phosphorylation and thereby its activity in a MAPK-independent manner.

Conservation frequently requires immediate responses to prevent further declines of imperilled populations, often in the absence of detailed information. Consequently, population distribution patterns are often used to guide conservation decisions. However, distribution patterns may be misleading if threats have restricted species to low quality habitat. This issue means it is not always apparent where management efforts should be concentrated for maximum conservation gain. My aim was to improve the effectiveness of threatened species conservation by investigating this issue in whio (blue duck - Hymenolaimus malacorhynchos), a New Zealand riverine duck that has undergone serious declines. I used population and spatial modelling to answer three questions: (1) what are the threats to whio, (2) how can these threats be managed, and (3) managing which whio habitats will give the greatest conservation gain? A spatial analysis of contemporary whio habitat using boosted regression trees revealed whio are only secure in 1 % of their historical range, with predation likely causing significantly greater range contraction (83 %) than habitat modification (29 %). In that analysis, I identified 39,000 km of occupiable whio habitat, providing extensive opportunities to expand their contemporary range through management. Intensive monitoring identified stoats (Mustela erminea) as the primary cause of whio population declines, with stoat predation severely reducing whio nest survival (10 % and 54 % in the absence and presence of stoat control, respectively). Population viability analyses indicated whio populations in the absence of stoat control were at high risk of extinction (λ = 0.74) but large-scale, low-intensity predator control was useful for short-term whio conservation. However, whio populations with stoat control still had a declining population growth rate (λ = 0.95) and further intervention may be required to prevent whio extinctions. Such management needs to target high quality habitat to ensure the greatest conservation value. Analyses of habitat quality revealed whio fitness was highest in warm, low gradient rivers, although fitness gradients differed between North and South Islands. Comparisons of fitness relationships with spatial model predictions showed that South Island whio occurred more frequently in poorer habitat, indicating they may occupy a relict distribution. Limited resources for conservation mean identifying effective management techniques is critical for species persistence. My modelling approach enabled the effectiveness of whio management to be assessed and areas of high quality habitat where such management should provide the greatest benefit to be identified. These tools are directly applicable to the conservation management of many threatened species by quickly informing managers in situations where distributions may not follow habitat quality.

Poly-γ-glutamic acid (γ-PGA) is a naturally occurring biopolymer made up of repeating units of glutamic acid and can be potentially used for multiple applications. This study compared the production of γ-PGA by eight bacteria (B. subtilis 23856, B. subtilis 23857, B. subtilis 23858 B. subtilis 23859, B. subtilis natto, B. licheniformis 1525, B. licheniformis 6816 and B. licheniformis 9945a) in GS and E media. B. subtilis natto and B. licheniformis 9945a have been investigated extensively for γ-PGA production, however, the remaining six have not previously been used. Using the eight bacteria, yields of up to 22.3 g/l were achieved in shake flasks. On characterization, it was observed that γ-PGA with different properties (crystallinity, acid/salt form and molecular weights ranging from 3,000 Da to 871,000 Da) was produced. Production of γ-PGA by B. subtilis natto in GS medium was scaled up using a fermenter and was tested for novel probiotic applications. The survival of probiotics during freeze drying, storage and ingestion was improved by combining them with a γ-PGA matrix. For L. paracasei, 10% γ-PGA protected the cells significantly better (P < 0.05) than 10% sucrose during freeze drying, whereas for B. longum and B. breve, it showed comparable cryoprotectant activity (P > 0.05) to 10% sucrose. This study also demonstrated the potential use of a non-dairy foodstuff (orange juice) for delivery of probiotics. Two Bifidobacteria strains protected with γ-PGA survived significantly better (P < 0.05) in orange juice for 39 days, with a log reduction in viability of less than 2.99 CFU/ml, when compared to unprotected cells, which showed complete loss in viability by day 20. In addition, γ-PGA protection improved survival of Bifidobacteria in a solution mimicking the environment of the stomach. γ-PGA-protected Bifidobacteria showed little (< 0.47 log CFU/ml) or no loss in viability when stored in simulated gastric juice (pH 2.0) for four hours, whereas unprotected cells died within two hours.

Distraction osteogenesis (DO) is a surgical procedure in which applied strain stimulates new bone growth; however, the underlying mechanisms by which bone cells respond to load are still uncertain. An organ culture model of DO was developed and validated by using linear distraction on the femoral shafts of 5 day old Wistar rats. Two loading regimes were utilized: distracting the bones for 2 hrs on day 1 (GRP I); distracting the bones for 2 hrs on days 1, 3, and 5 (GRP II). After 1 week in culture, the bones were compared to unloaded contralateral controls and assessed for changes. Structural, dimensional, massing, micro-CT, areal, and viability properties were obtained from testing. Relative to paired controls, distracted bones demonstrated an increase in failure load (9.15% GRP I, 18.85% GRP II), increase in stiffness (31.28% GRP I, 53.21% GRP II), increases in areal and polar moments of inertia, and viability (6.21% GRP I, 13.02% GRP II). Our results suggest that DO can be modeled successfully with an organ culture, and continued use of this system will help to gain insight into the mechanisms and pathways by which distraction osteogenesis occurs.

The Southern Ground Hornbill (SGH) (Bucorvus leadbeateri) has recently been listed globally by the IUCN Red list as Endangered or Critically Endangered. In South Africa, the declining population of the SGH has led it to be listed nationally as Vulnerable and falls under the “Protected Species” legislation. Research into the habitat preferences and identification of suitable reintroduction areas have been few since the initiation of reintroduction attempts, therefore resulting in mixed outcomes of previously-reintroduced SGH. The aim of this study was to identify and verify optimal reintroduction sites for the SGH, in the Musina area in the Limpopo Province. Five main objectives were determined, namely: to identify optimal reintroduction areas; to develop a habitat profile of the designated area; to determine the availability of prey items; to determine the availability of large trees needed for nesting and roosting; to evaluate pre-release sites by involving a number of stakeholders. Three study sites were selected on the farm Greyghost Safaris (Ludwigslust), each made up of a different plant community. Sampling was carried out a total of four times during the year: one sampling bout in each season. A niche-based modelling technique was used to describe the suitability of a particular habitat (“ecological space”) then used to project it onto another geographical space. Floristic and faunal surveys were conducted to determine the species composition of prey items and composition and frequency of the herbaceous and woody layer. High-resolution, natural-colour aerial imagery was used in order to determine the availability of trees for nesting and roosting. Pre-release site evaluations and stakeholder engagements were conducted by means of interviews with landowners on and around the study sites. The Limpopo River Valley and across to the KNP on the eastern border of the Limpopo Province, is suitable re-introduction sites for the SGH. This study site provided a good opportunity to explore what an optimal site for reintroduction should resemble. / Thesis (MSc (Zoology))--North-West University, Potchefstroom Campus, 2012.

The Southern Ground Hornbill (SGH) (Bucorvus leadbeateri) has recently been listed globally by the IUCN Red list as Endangered or Critically Endangered. In South Africa, the declining population of the SGH has led it to be listed nationally as Vulnerable and falls under the “Protected Species” legislation. Research into the habitat preferences and identification of suitable reintroduction areas have been few since the initiation of reintroduction attempts, therefore resulting in mixed outcomes of previously-reintroduced SGH. The aim of this study was to identify and verify optimal reintroduction sites for the SGH, in the Musina area in the Limpopo Province. Five main objectives were determined, namely: to identify optimal reintroduction areas; to develop a habitat profile of the designated area; to determine the availability of prey items; to determine the availability of large trees needed for nesting and roosting; to evaluate pre-release sites by involving a number of stakeholders. Three study sites were selected on the farm Greyghost Safaris (Ludwigslust), each made up of a different plant community. Sampling was carried out a total of four times during the year: one sampling bout in each season. A niche-based modelling technique was used to describe the suitability of a particular habitat (“ecological space”) then used to project it onto another geographical space. Floristic and faunal surveys were conducted to determine the species composition of prey items and composition and frequency of the herbaceous and woody layer. High-resolution, natural-colour aerial imagery was used in order to determine the availability of trees for nesting and roosting. Pre-release site evaluations and stakeholder engagements were conducted by means of interviews with landowners on and around the study sites. The Limpopo River Valley and across to the KNP on the eastern border of the Limpopo Province, is suitable re-introduction sites for the SGH. This study site provided a good opportunity to explore what an optimal site for reintroduction should resemble. / Thesis (MSc (Zoology))--North-West University, Potchefstroom Campus, 2012.

Humans can be exposed to waterborne bacterial pathogens and numerous outbreaks have been reported involving these microorganisms around the world. Many different enteric pathogens can be found in source waters used for drinking water. Assessing these pathogens and their possible threat to public health has always been important. Waterborne pathogens can be difficult to detect, and despite a large variety of recognized microbial detection techniques, the cause of many outbreaks has not been unidentified. Effective and rapid pathogen detection techniques are required to achieve reliable data for microbial source water quality, outbreak investigations, and for drinking water treatment efficacy monitoring. Bacteria have long been detected using classical culture-based methods, with the rationale that living cells are able to grow/replicate. However, many pathogenic bacteria in source waters may turn into viable but not culturable (VBNC) cells and are thus undetectable by growth-based methodologies. Alternatively, PCR-based techniques have been developed to detect both non-culturable and culturable bacteria. Yet with these techniques, post-death DNA persistency can inaccurately overestimate the number of viable cells. This problem may be circumvented by an alteration to the PCR procedure that is reported to be able to block PCR amplification of DNA that originates from dead cells. This alteration involves a chemical pre-treatment step prior to PCR using a photoreactive intercalating dye, propidium monoazide (PMA). In this research, a successful modification was made to the PMA-PCR method that can result in substantial suppression of the PCR signal from dead cells, and provide results that can more accurately measure bacterial pathogen viability. PMA-PCR was applied to high concentrations (1 × 107 cells mL-1) of heat-killed cells of Salmonella enterica and Campylobacter jejuni. Using PMA-PCR in combination with primers that amplified a relatively short fragment of the S. enterica invA gene (119 bp), only a 3-log reduction of the dead cell PCR signal was obtained. Similarly, for C. jejuni using PCR primers that amplified a relatively short fragment of DNA (174 bp of cpn60 gene), only a 1-log reduction of the PCR signal was observed for dead cells. Therefore, PMA treatment followed by PCR amplification of short DNA fragments resulted in incomplete signal inhibition of heat killed Salmonella and Campylobacter. To further investigate how PCR conditions can affect the ability of PMA to inhibit PCR amplification, primers were then used that could amplify a larger fragment of DNA. PCR amplification of a longer DNA fragment (1614 bp of invA gene for S. enterica and 1512 bp of cpn60 gene for C. jejuni) strongly suppressed the signal (7 log reduction) for both heat-killed Salmonella and Campylobacter. For UV-treated S. enterica and C. jejuni, short amplicon PMA-PCR showed no or very low PCR signal reduction, in part due to intact membranes directly after UV irradiation. Long amplicon qPCR, however, resulted in dead cell signal removal and PMA pretreatment had no effect on PCR signal suppression. This study used quantitative PCR and the PMA-PCR viability assays to evaluate the levels and occurrences of four groups of pathogenic bacteria in surface water samples from two locations on the Grand River, Ontario, Canada, to demonstrate the reliability of the PMA-PCR technique for the enumeration of viable cells. The bacterial groups investigated included S. enterica, thermophilic Campylobacter, Escherichia coli O157:H7, and Arcobacter butzleri. Small numbers of dead cells (not more than 0.5 log 100 mL-1) were present, detected as the difference between PMA-PCR and PCR without PMA treatment. In this particular river, pathogen enumeration by PCR was only slightly influenced by false positive signal detection due to the presence of dead cells or extracellular DNA and reliable bacterial pathogen detection could be attained by PCR without PMA pretreatment. Viable A. butzleri were detected at elevated concentrations (up to 4.8 log cells per 100 mL) in the Grand River. Arcobacter has not been previously studied in the Grand River and this is one of the few studies that have quantitatively assessed Arcobacter in the environment. This suggests that additional research is required on the pathogenicity of this organism and its occurrence in water. In the next stage of this research, both the improved viability assay (long amplicon PMA-PCR) and conventional quantitative PCR were applied to investigate the survival trends of selected enteric bacterial pathogens including Yersinia enterocolitica, S. enterica, C. jejuni, and A. butzleri. The target bacteria were inoculated into sterile or non-sterile river water to study the impact of background microbiota on cell survival. These experiments were perfomed at 3 different temperatures (5, 15, and 25°C) and at high/low dissolved oxygen (DO) concentrations (for C. jejuni, and A. butzleri only) to evaluate the effect of these potential environmental stresses on bacterial survival trends. The results indicated that the autochthonous microbiota in river water had a significant effect on the bacterial die-off. Although lower temperatures enhanced bacterial survival in non-sterile river water, it was found that PCR may overestimate the effect of temperature on survival and that the PCR viability assays (PMA-PCR) could more accurately measure the impact of temperature. The survival of viable C. jejuni was adversely affected by high DO levels only at a low temperature (5°C) and this effect was observed only when the PMA-PCR viability assay was applied. A. butzleri survival was not affected by water DO levels. This research provides an improved understanding of viable/active enteric waterborne bacteria and their survival in the aquatic microcosms as well as reliable data to better elucidate the effect of environmental factors on the occurrence of pathogenic bacteria. It can also offer valuable information for microbial risk assessments used by regulators and decision makers.