Submitted in fulfillment of the requirements for the Degree of Master of Technology: Biological sciences at the ML Sultan Technikon, 2000. / Plant medicine is an important area of commercial activity in South Africa. This is a rapidly expanding market, thus we are evaluating natural and stressinduced compounds (phytoalexins) from plants as agents that may be able to control mycotoxins. Natural compounds from Bridelia micrantha, Warburgia salutaris, Lippia javanica and Scenecio serratuloides and stress-induced compounds (phytoalexins) from Citrus sinensis cv Valencia were screened for antitunqal and antimycotoxic activity by bioautography against a test organism (Cladosporium cladosporoides) and mycotoxin producing fungi (Fusarium moniliforme and Aspergillus flavus). / M

Pseudomonas aeruginosa is a dreaded opportunistic pathogen that causes severe and often intractable infections in immunocompromised and critically ill patients. This bacterium is also the primary cause of fatal lung infections in patients with cystic fibrosis and a leading nosocomial pathogen responsible for nearly 10% of all hospital-acquired infections. P. aeruginosa is intrinsically recalcitrant to most classes of antibiotics and has the ability to acquire additional resistance during treatment. In particular, resistance to the widely used β-lactam antibiotics is frequently mediated by the expression of AmpC, a chromosomally encoded β-lactamase that is ubiquitously found in P. aeruginosa strains. This dissertation delved into the role of a recently reported chromosomal β-lactamase in P. aeruginosa called PoxB. To date, no detailed studies have addressed the regulation of poxB expression and its contribution to β-lactam resistance in P. aeruginosa. In an effort to better understand the role of this β-lactamase, poxB was deleted from the chromosome and expressed in trans from an IPTG-inducible promoter. The loss of poxB did not affect susceptibility. However, expression in trans in the absence of ampC rendered strains more resistant to the carbapenem β-lactams. The carbapenem-hydrolyzing phenotype was enhanced, reaching intermediate and resistant clinical breakpoints, in the absence of the carbapenem-specific outer membrane porin OprD. As observed for most class D β-lactamases, PoxB was only weakly inhibited by the currently available β-lactamase inhibitors. Moreover, poxB was shown to form an operon with the upstream located poxA, whose expression in trans decreased pox promoter (Ppox) activity suggesting autoregulation. The transcriptional regulator AmpR negatively controlled Ppox activity, however no direct interaction could be demonstrated. A mariner transposon library identified genes involved in the transport of polyamines as potential regulators of pox expression. Unexpectedly, polyamines themselves were able induce resistance to carbapenems. In summary, P. aeruginosa carries a chromosomal-encoded β-lactamase PoxB that can provide resistance against the clinically relevant carbapenems despite its narrow spectrum of hydrolysis and whose activity in vivo may be regulated by polyamines.

M.Sc. / Polygalacturonase-inhibiting proteins (PGIPs) are cell wall-associated plant proteins that inhibit endopolygalacturonases from phytopathogenic fungi. It has been proposed that pgip encoding genes could be utilised for engineering increased resistance in transgenic crops against important fungal pathogens such as Botrytis cinerea. During this study a pgip gene from Malus domestica cv Granny Smith apple fruit was cloned by the degenerate and inverse polymerase chain reaction (PCR) techniques. An alignment of the pear and bean PGIP sequences was used to design degenerate PCR primers in highly conserved regions. Degenerate PCR allowed the amplification of a 351bp internal fragment of the pgip gene, termed ipgip. The DNA sequence of ipgip was used to design inverse PCR primers. A Southern blot of apple genomic DNA probed with the ipgip fragment was used to identify restriction enzyme sites for inverse PCR. Inverse PCR enabled cloning of the remainder of the gene, from which a composite pgip gene sequence was constructed. The composite apple pgip gene comprised an open reading frame of 990bp that is predicted to encode a 330 amino acid polypeptide. The polypeptide contains a putative 24 amino acid N-terminal leader sequence that may function as a signal peptide for secretion. The deduced apple PGIP contains nine cysteine residues and seven potential N-linked glycosylation sites. Ten loosely conserved leucine-rich repeat motifs characteristic of PG1Ps were identified in the apple PGIP sequence. The apple PGIP showed 97% and 55% amino acid identity to the pear and bean PGIPs, respectively. The full-length apple pgip gene was re-isolated from genomic DNA by PCR using primers designed to the 5' and 3' ends of the composite pgip gene. The apple pgip gene was cloned into a plant transformation vector and transformed into tobacco by Agrobacterium-mediated transformation. Phenotypically normal transgenic tobacco plants were produced. Stable transgene insertion into the transgenic tobacco genomes was verified by PCR and Southern blot analyses. Sequence analysis of the pgip construct used for transformation revealed two potential mutations in the deduced amino acid sequence. The substitutions of Asp residues with Asn and Tyr at positions 43 and 196, respectively, could interfere with the secondary structure of the expressed transgene protein. To test whether the apple PGIP was effective against Botrytis cinerea, protein extracts were prepared from apple fruit and transgenic tobacco and tested for inhibitory activity against B. cinerea polygalacturonases. Biochemical assays showed that a heat-denaturable PGIP extract prepared from apple fruit inhibited the polygalacturonases produced by a virulent isolate of Botrytis cinerea grown on pectin and apple cell walls. Protein extracts prepared from transgenic tobacco did not show any inhibitory activity towards Botrytis polygalacturonases. This suggests the absence of active PGIP in the extracts possibly due to inefficient transcription of the transgene or due to the introduced mutations.

One of the factors that maintains fruit quality is its microbial flora. Fruit holds a natural non-pathogenic epiphytic microflora but can become contaminated with pathogenic microorganisms during export, causing either postharvest decay or possibly resulting in a food safety risk. In order to study microbial dynamics on fruit surfaces and the environment fruit moves through in the export chain, fruit washings were made, surfaces were sampled and total populations and diversities determined per cm2. Hygiene and safety levels for fruit export environments were hereby determined by sampling various points along the apple export chain, which included two farms and a harbour in South Africa and two harbours, two repacking facilities and two retail centres in Europe. In this first study of its kind, all the surfaces that were sampled exceeded the international standard for cleaning efficacy of food-processing equipment that is <5 cfu/cm2, while several areas exceeded the maximum acceptable index level of microbial air contamination of 22 cfu/h in food industries. Washing of containers on a harbour in South Africa did not have a significant impact on microbial populations. Regarding fruit quality, it was determined that apple microflora fluctuate throughout the export process and that postharvest pathogens that are known to cause great economic losses in the apple industry, proved to be of little significance in this investigation. The presence of six foodborne pathogens i.e. Shigella sonnei, Salmonella muenchen, Escherichia coli, Listeria monocytogenes, Staphylococcus aureus and Staphylococcus epidermidi/s was monitored throughout the chain. Of these, only S. aureus and E. coli were recorded, although pathogenicity was not confirmed for the latter. Staphylococcus aureus was found in containers and at a retail centre in Europe, and S. aureus and S. epidermidis were recorded on apple surfaces for the first time. Escherichia coli was present in great numbers in fruit washing water on a farm in South Africa. Since the standard for food premises is very stringent and perhaps inapplicable for fresh fruit handling and holding facilities, future research should include development of a more realistic hygiene standard for fresh fruit environments. / Dissertation (MSc(Agric) : Plant Pathology)--University of Pretoria, 2006. / Microbiology and Plant Pathology / unrestricted

Bacterial aspartate transcarbamoylases (ATCase's) are divided into three classes that correspond to taxonomic relationships within the bacteria. The opportunistic pathogen Moraxeila catarrhalis has undergone several reclassifications based on traditional microbiological criteria. The previously uncharacterized ATCase from M. catarrhalis was purified to homogeneity and its chemical properties characterized. The ATCase from M. catarrhalis is a class C ATCase with an apparent molecular mass of 480-520 kDa. The M. catarrhalis ATCase is a dodecomer composed of six 35 kDa polypeptides and six 45 kDa polypeptides. The enzyme has an unusually high pH optimum of greater than pH 10. The enzyme exhibited hyperbolic kinetic with a Km for aspartate of 2 mM. A single, separate 78 kDa dihydroorotase from M. catarrhalis was identified and it was not associated with ATCase. These data support the reclassification of M. catarrhalis out of the Neisseriaceae family.

Plant fungal pathogens are a major threat to food security worldwide. The most important method of protecting plants against fungal attack is the use of fungicides, but the development of resistance towards synthetic fungicides is of great concern. Moreover, the health risks associated with the use of chemical fungicides increase the need to search for safe, efficacious and environmentally friendly fungicides. Plants produce antifungal agents by secondary metabolism to protect themselves from fungal attack, and therefore many plant species have substantial antifungal activity. The use of plant extracts could enable the development of inexpensive and environmentally acceptable fungicides based on locally available natural products. This study was undertaken to investigate weedy and invasive plant species for antifungal activity against plant pathogens in order to develop a useful product using a widely available resource. Acetone leaf extracts of seven invasive species (Chromoleana odorata, Ipomoea alba, Tecoma stans, Passiflora suberosa, Passiflora subpeltata, Aristolochia sp, Solanum seaforthianum) were screened against eight plant fungal pathogens viz Rhizoctonia solani, Fusarium oxysporium, Penicillum janthinellum, Penicillum expansum, Aspergillus parasiticus, Aspergillus niger, Pythium ultimum and Phytophthora nicotiana, using microdilution assay and bioautography. The acetone extract of Tecoma stans had reasonable antifungal activity with an average minimal inhibitory concentration (MIC) value against all the fungi of 550 ìg/ml and clear zones on bioautograms indicating inhibition of fungal growth of a compounds with an Rf of 0.082 in BEA against several of the fungal pathogens. Due to the clear compound on bioautography and availability of Tecoma stans, this species was selected for further work. Bioassay-guided fractionation of the leaves of the Tecoma stans dichloromethane (DCM) extract obtained from solvent-solvent fractionation resulted in one major compound, oleanolic acid. The isolated compound had antifungal activity with an average MIC value of 130 ìg/ml against the 10 plant pathogenic fungi and clear bands with an Rf value of 0.082 on bioautograms, indicating fungal growth inhibition. It was surprising that the MIC value of the crude DCM extract was as high as that of the only compound with antifungal activity based on bioautography. These results clearly indicated the possibility of synergisms especially since the average total activity of the extract was nearly 6.5 times higher than that of oleanolic acid with total activity values of 60154 ml for the extract and 9262 ml for oleanolic acid. Cellular cytotoxicity of DCM extract and oleanolic acid was investigated using tetrazoliumbased colorimetric assay (MTT) on Vero monkey kidney cells. The toxicity of the extract and oleanolic acid was determined by LC50 values. The DCM extract and oleanolic acid were toxic with and LC50 of 0.413 mg/ml and 0.129 mg/ml respectively, lower than that of berberine the toxic compound used as control. However therapeutic index which can be defined here as the LC50 in (ìg/ml)/MIC in (ìg/ml), indicated that though the extract and oleanolic acid were toxic, they could be used under controlled conditions against infections of certain of the fungal pathogens. The crude extract had a high therapeutic index value of 21 against microorganisms T. harzianum, R. solani, F. oxysporium and P. expansum; and oleanolic acid had high therapeutic index values of 16 and 64 of against T. harzianum and R.solani respectively. This high therapeutic index value of crude extract and oleanolic acid means that, crude extract and oleanolic acid may be used for treatment of infections by these tested fungi with very little toxicity under controlled conditions. Oleanolic acid had very low antibacterial activity (MIC >250 ìg/ml). against two Grampositive (Staphylococcus aureus, ATCC 29213 and Enterococcus faecalis, ATCC 29212) and two Gram-negative bacteria (Escherichia coli, ATCC 27853 and Pseudomonas aeruginosa, ATCC 25922). Animal pathogenic fungi were more resistant than the plant fungal pathogens. Based on the good activity of the DCM crude extract, the surprising selectivity in activity against different fungi coupled with reasonably good therapeutic indexes and the wide availability of T stans leaves opens up the possibility that a commercial product to protect plants against certain pathogens may be developed from T. stans leaves. Copyright / Dissertation (MSc (Veterinary Science))--University of Pretoria, 2008. / Paraclinical Sciences / unrestricted

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第21311号 / 農博第2296号 / 新制||農||1064(附属図書館) / 学位論文||H30||N5145(農学部図書室) / 京都大学大学院農学研究科応用生物科学専攻 / (主査)教授 髙野 義孝, 教授 田中 千尋, 教授 寺内 良平 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

The ability to sense and respond to environmental stimuli is essential for the survival of all living things. As a result, nature has evolved an uncountable number of ways to detect environmental signals. At the cellular level, G protein-coupled receptors (GPCRs) are used by eukaryotes, including fungi and humans, to convert extracellular molecular binding events into intracellular responses. Recently, synthetic biologists have shown that biological sensing systems can be repurposed to suit human needs, developing tools such as diagnostic devices and drug screening platforms. In this thesis, I present work exploring the potential of fungal GPCRs to be used as sensing elements in yeast-based biosensors. Chapter 1 gives background information related to synthetic biology, biosensors, and yeast signaling pathways. Chapter 2 describes the development of the baker's yeast Saccharomyces cerevisiae into a diagnostic device for detection of fungal pathogens, using fungal GPCRs. In Chapter 3 I demonstrate that the substrate specificity of fungal GPCRs can be altered using directed evolution. Chapter 4 describes experiments further probing the native binding abilities of fungal GPCRs, specifically examining protein ligands. Finally, in Chapter 5 we move beyond fungal GPCRs and engineer yeast to detect other stimuli, in the context of an engineered living material.

Denna studie är en kvantitativ undersökning med syftet att undersöka hur synen på hälsa skiljer sig mellan yrkesverksamma idrottslärare i landet. En jämförelse gjordes också på basis mellan kön, ålder och examinationsår. Enkäten utformades utifrån Antonovskys salutogena perspektiv, teorin KASAM och dess tre komponenter begriplighet, hanterbarhet och meningsfullhet och besvarades utav 237 respondenter från Facebook grupperna Idrottslärare och Sveriges Idrottslärare. De överblickande resultaten visade på att idrottslärare överlag svarade positivt på de allmänna perspektivet och de tre KASAM-komponenterna. De enda statistiskt säkerställda skillnaderna var mellan män och kvinnor där kvinnor svarade mer positivt än män vad gäller både salutogenitet och hanterbarhet. Dock kan man inte säga att detta är något som utspelar sig i praktiken då man som idrottslärare kan tycka att något är viktigt men sedan inte praktiskt utöva det. Det vi ser i denna undersökning är idrottslärarnas upplevelser utav lektionerna och inte vad som verkligen händer på lektionerna. Sedan kan ju en idrottslärares upplevelser skilja sig mot vad eleverna upplever. / This study is a quantitative study aimed at examining how the perception of health differs between professional sports teachers in the country. A comparison was also made on the basis of gender, age and examination year. The questionnaire was designed from Antonovsky's salutogenic perspective, the KASAM theory and its three components comprehensibility, manageability and meaningfulness and was answered by 237 respondents from the Facebook groups Idrottslärare and Sveriges Idrottslärare. The overall results showed that sports teachers generally responded positively to the general perspective and the three KASAM components. The only statistically significant differences were between men and women, where women responded more positively than men in terms of both salutogenicity and manageability. However, you cannot say that this is something that takes place in practice as you as a sports teacher may think that something is important but then not practically practice it. What we see in this survey is the sports teachers' experiences of the lessons and not what really happens at the lessons. Then, after all, a sports teacher's experiences can differ from what the students experience.

Fish of the genus Cottus were found infected with heterophyid metacercariae. Laboratory animals were infected with the metacercariae, and adult heterophid trematodes were recovered. These flukes were found to represent an undescribed species of the genus Euryhelmis. Various streams in the Willamette Valley and coastal areas were sampled for infected Cottus sp. Snails of the genera Oxytrema and Fluminicola were collected. Several possible definitive hosts were examined. A partial review of the subfamily Apophallinae and a complete review of the genus Euryhelmis are presented. Euryhelmis cotti n. sp. is placed in the subfamily Apophallinae, and its life cycle is partially described.