The metabolic disorder diabetes; is a global epidemic affecting people in developed countries and increasingly in developing countries. In two decades time, 350 million people will be diabetic at the current rate of prevalence. In a preliminary study, insulin resistant rats were treated with Prunus Africana (plant A) for 28 days. Plasma samples obtained from P. africana treated rats had increased insulin levels compared to normal and untreated insulin resistant rats (Karachi, 2009). The treatment of insulin resistant rats with P. africana also showed increased glucose uptake in rat adipose tissue (Karachi, 2009), suggesting that P. africana had anti-diabetic properties. The aim of the study was to investigate the mechanism of the anti-diabetic properties of P africana extract. Increased insulin secretion was confirmed by the increased Cpeptide concentration in plasma samples of rats treated with P. africana. In order to explain the high insulin levels, several hypothesis’ were investigated: (1) P. africana may increase insulin secretion in β cells, hence the effect of P. africana on insulin secretion by INS-1 cells was investigated; (2) P. africana may increase insulin secretion by prolonging the half-life of glucagon like peptide-1 (GLP-1) by decreasing dipeptidyl peptidase IV (DPP IV) activity; the effect of P. africana on DPP IV activity was determined spectrophotometrically, (3) P. africana may increase the half-life of insulin in the plasma by decreasing the activity of insulin degrading enzyme (IDE); the effect of P. africana on IDE in rat muscle and spleen samples was investigated. To explain the increased glucose uptake in adipose tissue observed in the previous study two parameters were investigated: (1) increased GLUT4 expression in P. africana treated rats; the effect of P. africana treatment on the expression of glucose transporter 4 (GLUT4) was determined using real-time polymerase chain reaction (RT-PCR), (2) P. africana may increase glucose utilization; the effect of P. africana on glucose utilization was determined in 3T3-L1 cells. The plant extract did not significantly increase insulin secretion by INS-1 cells in the absence of glucose. P. africana decreased DPP IV activity in rat plasma when compared to the untreated insulin resistant rats and this could be a mechanism by which insulin secretion is increased during plant treatment. P. africana decreased IDE activity (however not significantly) when compared to the untreated insulin resistant The effects of a Kenyan antidiabetic plant on insulin homeostasis KY Suleiman VII rats. P. africana appeared to have no effect on GLUT4 expression. The plant appeared to increase glucose utilization in 3T3-L1 cells in the absence of insulin suggesting that P. africana may have insulin like activity. In summary, this study indicates that P. africana is indirectly involved in inhibiting DDPIV. This in turn can increase the half life of GLP-1, which in turn can enhance the secretion of insulin. P. africana increases glucose utilization although there was no evidence that the GLUT 4 transporter has a higher expression in the plant treated rats. Further studies should be conducted to investigate the expression of GLUT1 under the same conditons.

The role of plants in the treatment of disease and enhancement of production in animals in South African rural communities is poorly documented. Rapid Rural Appraisal (RRA) methods were employed to describe the use of ethnoveterinary medicinal plants in cattle by Setswana-speaking people in the Madikwe area of the North West Province of South Africa. Information was gathered from key spokespersons through individual interviews, group interviews, guided field walks and observations. Ethnoveterinary uses in cattle of 46 plant species representing 24 families were recorded. Plants were used in 84 % of the total number of ethnoveterinary remedies. These plants were used alone (64 %) or in mixtures (36 %) for 43 indications. The most important indications for the use of ethnoveterinary remedies were retained placenta, diarrhoea, gallsickness, fractures, eye inflammation, general ailments, fertility enhancement, general gastrointestinal problems, heartwater, internal parasites, coughing, redwater and the reduction of tick burdens. Plant materials were prepared in various ways including, infusion (36 %), decoction (33 %), infusion or decoction (13 %), ground fresh material (6 %), sap expressed from fresh material (3 %), charred (2 %) and dried (1 %). Unprocessed, fresh material was used in 6 % of remedies. The most common dosage form was a liquid for oral dosing (83 %). Other dosage forms included, drops, licks, ointments, lotions and powders. Liquid remedies for oral dosing were administered using a bottle. The study indicated that Setswana-speaking people in the North West Province have a rich heritage of ethnoveterinary knowledge, which includes all aspects of ethnoveterinary medicinal plant use. The impact of ethnoveterinary medicinal plant use on medicinal plant population densities was also assessed through a comparison of the medicinal plant densities inside and outside the Madikwe Game Reserve. Belt transects were used in a stratified trial design to record plant densities. No statistically significant differences in medicinal plant densities that could be attributed to medicinal plant use, were found. / Dissertation (MSc)--University of Pretoria, 2000. / Paraclinical Sciences / Unrestricted

Diabetes mellitus is becoming an increasing concern all over the world. Many people especially in poor communities have been using medicinal plants to treat diabetes and its complications. Much work has been done to find scientific evidence to support the use of medicinal plants in many cases with good evidence to support the traditional use. There has been an increase in research on the use of botanicals for either the treatment and/or management of diabetes in many parts of the world. To start this study an informal survey on plant species used to treat diabetes was carried out with local inhabitants and herbal traders in the Newcastle region (KwaZulu Natal). The plant species were chosen based on their wide use by traditional healers and local inhabitants. The efficacy of the selected plant (Senna alexandrina, Cymbopogon citrates, Cucurbita pepo, Nuxia floribunda, Hypoxis hemerocallidea and Cinnamomum cassia) used to treat diabetes mellitus by traditional healers in KwaZulu Natal province of South Africa was evaluated under controlled laboratory conditions. With the exception of Senna alexandrina and Nuxia floribunda, there has been some independent evidence of the efficacy of these plant species In this study three relevant in vitro and semi in-vivo assays were selected to test the efficacy of different extracts on alpha amylase (carbohydrate digestive enzyme) activity, alpha glucosidase (glucose absorption) activity and islets of Langerhans insulin secretory activity. Hexane, ethyl acetate, acetone and methanol extracts were examined and screened for their phytochemical properties and activity in the selected assays Alpha amylase inhibitory assay Not all extracts of the plant species had α-amylase enzyme inhibitory activity. The acetone extracts of C. pepo and H. hemerocallidea had enzyme inhibition less than that of acarbose positive control (EC50 = 1.82, 0.92 and 0.56 mg/ml respectively). The other plant species that had substantial α-amylase inhibitory activity was the methanol extracts of C. citratus and C. cassia (EC50 = 0.313 and 0.12 mg/ml respectively), ethyl acetate extracts of C. citratus and N. floribunda (EC50 = 1.20 and 1.60 mg/ml respectively). The hexane extracts of C. cassia (0.72 mg/ml), N. floribunda (0.88 mg/ml), C. pepo (0.70 mg/ml) and S. alexandrina (0.083 mg/ml) all had α-amylase inhibitory activity.The best activity was present in the intermediate polarity extracts. If these more apolar plant extracts are not toxic or do not have negative side effects they may be much more efficient than acarbose in managing α-amylase activity. Alpha glucosidase inhibitory assay In contrast to the alpha amylase activity, the inhibitory activity of the non-polar (hexane and ethyl acetate) plant extracts was in general higher than that of polar extracts. With the methanol and acetone extracts the inhibitory activity varied from no activity in the methanol extract of C. cassia to highly active methanol extract of C. pepo (70.3%) and acetone extract of H. hemerocallidea (84.35%). Among the plants studied C. cassia and N. floribunda (bark) had the highest inhibitory activity in the hexane and ethyl acetate extracts, the acetone extract of H. hemerocallidea had the highest inhibitory activity. The hexane crude extracts ofN. floribunda and C. citratus had very high inhibitory activity at the highest concentration tested (1 mg/ml). The ethyl acetate crude extracts of all the plant species used in this study had an inhibitory activity above 90% against α-glucosidase at 1 mg/ml. When compared to acarbose all the plant species used in this screening study had good activity against the α-glucosidase enzyme with the exception of the methanol extract of C. cassia. The inhibitory activity of hexane and ethyl acetate extracts was close to that of the positive control. If the more non-polar plant extracts are not toxic or do not have negative side effects (not tested) it appears that they may be more or less efficient than acarbose in managing α-glucosidase activity. Islets of Langerhans as a target site Only with the H. hemerocallidea acetone extract was there an increase in insulin secretion of 2.5 mIU/L (Table 8) at 8 ug/ml. With all the other extracts the insulin levels were less than 0.2 mIU/L. The positive controls of acarbose and glibenclamide at a concentration of 1 mg/ml stimulated insulin secretion to 11.5 and 19.8 mIU/L respectively. In comparison, the positive controls acarbose and glibenclamide control produce a 5-8 fold greater increase in insulin secretion although the exposure was at a 100-fold higher concentration. This would indicate that the H. hemerocallidea acetone crude extract contains a very potent secretogogue compound. It is possible that higher concentrations of the other plant extracts may also have led to stimulation of insulin production. If the more non-polar plant extracts are not toxic or do not have negative side effects and are biologically available, it appears that they may be much more efficient than acarbose and glibenclamide in managing insulin secretion. Conclusion The best overall activity was observed in the non-polar and intermediate solvents (hexane and ethyl acetate). Although the organic extracts had good activity, it does not explain the use of aqueous extracts by traditional healers because water extracts were not active in the assays. The activity of the C. pepo acetone leaf extract and N. floribunda ethyl acetate bark extract is the first reported evidence of activity with regard to diabetes mellitus. From the in vitro results, it can be concluded some extracts of all the traditionally used species have some merit in the management of diabetes mellitus type II, as suggested by the ethnomedicinal leads. In may be worthwhile following up on this work by isolating the compounds responsible for the biological activities. / Dissertation (MSc)--University of Pretoria, 2010. / Paraclinical Sciences / unrestricted

Please read the abstract in the dissertation Copyright 2002, University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria. Please cite as follows: Rakuambo, NC 2002, The traditional use of medicinal plants to treat erectile dysfunction and the isolation of their bioactive compounds, MSc dissertation, University of Pretoria, Pretoria, viewed yymmdd < http://upetd.up.ac.za/thesis/available/etd-03122012-172112 / > E12/4/140/gm / Dissertation (MSc)--University of Pretoria, 2002. / Plant Science / unrestricted

Apixaban is a novel oral anticoagulant that prevents clotting by directly inhibiting Factor Xa in the coagulation cascade. Due to its different pharmacokinetics, previous standards for testing anticoagulant concentrations are ineffective at measuring apixaban. In this study, Hyphen Biomed Biophen Direct Xa Inhibitor and Biophen Heparin chromogenic kits from Aniara Diagnostica were used along with a NanoDrop™ One/OneC Microvolume UV-Vis Spectrophotometer to see if either of these kits provide acceptable precision and accuracy for the quantification of apixaban in plasma samples, as well as if there is a significant difference in these two kits at varying concentrations of apixaban. Apixaban is a novel oral anticoagulant that prevents clotting by directly inhibiting Factor Xa in the coagulation cascade. Due to its different pharmacokinetics, previous standards for testing anticoagulant concentrations are ineffective at measuring apixaban. In this study, Hyphen Biomed Biophen Direct Xa Inhibitor and Biophen Heparin chromogenic kits from Aniara Diagnostica were used along withused witha NanoDrop™ One/OneC Microvolume UV-Vis Spectrophotometer to see if either of these kits provide acceptable precision and accuracy for the quantification of apixaban in plasma samples,as well as and to evaluate if there is a significant difference in these two kits at varying concentrations of apixaban.

Alzheimer's disease (AD) is commonly characterized by a loss of cognitive function due to the deterioration of neuronal synapses from the presence of senile amyloid beta-42 (Aß42) plaques. Evaluating cognitive deficits caused by Aß42 using human cortical neurons poses a challenge due to sourcing difficulties, and the use of animal models to assess drug efficacy creates biological hurdles from lack of species translatability. Recent advances in induced-pluripotent stem cell technology have enabled the development of mature, human-based cortical neuron models. The development of an hiPSC-cortical neuron differentiation protocol facilitates the exploration of disease onset and functional analysis from a patient-derived cell source, and further investigation of potential therapeutic treatments, while eliminating biological efficacy concerns. Long-term potentiation (LTP) was utilized as an in vitro correlate for memory and learning to quantify cognitive deficits in sporadic AD (SAD) and familial AD (FAD) systems and assess drug treatments for the prevention of Aß42-induced neurotoxicity. Synaptic connectivity and LTP induction through high-frequency stimulation was simulated through cortical neurons cultured on microelectrode arrays (MEAs), such that the functional activity of the neuronal population could be assessed overtime. AD therapeutic treatments were shown to block the Aß42-induced neurotoxic loss of synaptic plasticity and maintain persistent LTP in a model for SAD. Subsequently, FAD was assessed through the differentiation of patient-derived AD iPSCs, where LTP proficiency could be evaluated to relate to clinical cognitive evaluations. This study established a serum-free, in vitro human-derived iPSC-cortical neuron protocol that could be adapted to validate disease mechanisms and drug efficacy in patient-derived neural networks as a potential platform for precision medicine.

Extracts from medicinal mushrooms were prepared and tested for anti-microbial activity against food pathogens and food spoilage microorganisms. The inhibitory activity was measured using a disk diffusion assay and with optical density (OD). For OD, 7 fractions were collected using HPLC for 4 (A. blazei Murrill, G. lucidum, G. frondosa, I. obiquus) medicinal mushrooms and 6 fractions from L. edodes and 8 fractions from P. linteus. The results from disk diffusion assay showed that most mushrooms displayed significant inhibition compared to the ethanol. The exceptions were: A. blazei Murrill, I. obliquus, and L. edodes against E. coli O157:H7; I. obliquus against L. monocytogenes V7; I. obliquus against S. cerevisiae Y99; L. edodes against Z. bailii Y03; and I. obliquus against Z. bailii/bisporus Y108. Inhibition was more effective in yeasts than bacteria. The result from Bioscreen C showed that against L. monocytogenes V7, fraction 7 in A. blazei Murrill; fraction 1, 4 and 5 in G. lucidum; fraction 4 in G. frondosa; and fraction 4 and 5 in I. obliquus significantly inhibited the growth compared to ethanol. Against Z. bailii Y03, fraction 7 in A. blazei Murrill; all fractions from G. lucidum, G. frondosa, and P. linteus; fraction 1, 2, 3, and 6 from I. obliquus; and fraction 4 and 6 from L. edodes significantly inhibited growth compare to ethanol. The results indicated that there is significant antimicrobial activity against food pathogens and spoilage organisms in the medicinal mushrooms studied. / Ph. D.

The proteasome is a large protein complex which is responsible for the majority of protein degradation in eukaryotes. Following FDA approval of the first proteasome inhibitor bortezomib for the treatment of multiple myeloma (MM) in 2003, there has been an increasing awareness of the significant therapeutic potential of proteasome inhibitors in the treatment of cancer. As of 2017, three proteasome inhibitors are approved for the treatment of MM but in clinical trials with patients bearing solid tumors these existing proteasome inhibitors have demonstrated poor results. Notably, all three FDA-approved proteasome inhibitors rely on the combination a peptide backbone and reactive electrophilic warhead to target the proteasome, and all three primarily target the catalytic subunits conferring the proteasome’s chymotrypsin-like (CT-L) activity. It is our hypothesis that compounds with non-peptidic structures, non-covalent and reversible modes of action, and unique selectivity profiles against the proteasome’s distinct catalytic subunits could have superior pharmacodynamic and pharmacokinetic properties and may bear improved activity against solid tumors relative to existing proteasome inhibitors. In an effort to discover such compounds we have employed an approach which combines computational drug screening methods with conventional screening and classic medicinal chemistry. Our efforts began with a computational screen performed in the lab of Dr. Chang-Guo Zhan. This virtual screen narrowed a library of over 300,000 drug-like compounds down to under 300 virtual hits which were then screened for proteasome inhibitory activity in an in vitro assay. Despite screening a relatively small pool of compounds, we were able to identify 18 active compounds. The majority of these hits were non-peptide in structure and lacked any hallmarks of covalent inhibition. The further development of one compound, a tri-substituted pyrazole, provided us with a proteasome inhibitor which demonstrated cytotoxic activity in a variety of human solid cancer cell lines as well as significant anti-tumor activity in a prostate cancer mouse xenograft model. We have also evaluated the in vitro pharmacokinetic properties of our lead compound and investigated its ability to evade cross-resistance phenomena in proteasome inhibitor-resistant cell lines. We believe that our lead compound as well as our drug discovery approach itself will be of interest and use to other researchers. We hope that this research effort may aid in the further development of reversible non-peptide proteasome inhibitors and may eventually deliver new therapeutic options for patients with difficult-to-treat solid tumors.

Schumanniophyton magnificum and Glypheae brevis are important medicinal plants growing wild in the West African rain forest. They are used in folkloric medicine in the treatment of epilepsy and convulsion as well as for some other diseases. The purpose of this work was to investigate the aspect of folkloric use in order to support folkloric claims and document the findings. The extracts were prepared from ground plant material by a continuous extraction method. Five hundred grams of ground plant material were continuously de-fatted with 2 L petroleum ether (60°- 80°) in a Soxhlet apparatus for about 5 h. The resulting marc was dried and the chemical constituents extracted hot in a Soxhlet apparatus for about 8 to 10 h with 2 L aqueous ethanol (70%). The efficacy of the extraction method was confirmed using standard bioassays and phytochemical analyses. The anti-convulsant activity of the crude extracts was evaluated in vivo against chemically induced convulsions using three different animal models, namely the strychnine, the picrotoxin and the pentylenetetrazole tests. The acute and delayed toxicity test results showed that in all the animal models investigated very high doses, about four times higher than the protective doses of the extracts, were required to kill 50% of the population of animal used. Phytochemical assays of the extracts indicated the presence of alkaloids only in S. magnificum root extract and glycosides in extracts from both species. The glycosides were positive to Baljet, Xanthydrol and Keller-Kiliani tests for cardiac glycosides. S. magnificum and G. brevis chemical constituents were initially isolated with a sequential fractionation method starting with a highly non-polar solvent and gradually increasing to a more polar solvent. The fractions were pooled on the basis of TLC similarity profiles when viewed under the UV light at 254 and 366 nm and were found to have two and four major UV absorbing fractions for S. magnificum and G. brevis respectively. Radio-receptor binding tests were used to assess the anti-convulsant activities of the hydro-alcoholic crude extracts, the organic and aqueous fractions of the crude extracts, partially purified components and pure components in in vitro tests against some standard GABA[A] receptor antagonists, muscimol and isoguvacine respectively. The anti-convulsant activities resided in the aqueous fractions of the hydro-alcoholic crude extracts of both plants. The purely organic fractions of G. brevis demonstrated no activity while all the fractions of the aqueous component demonstrated some degree of activity. The anti-convulsant activity of S. magnificum was found only in one fraction-Fraction 1. This Fraction was further investigated and one of the components appear to be responsible for the activity. The structure of the active constituent was 5,7dihdroxy-2 methylbenzopyran-4-one, a noreugenin. A second bioactive compound, schumanniofoside, was identified from Fraction M[5.2] from S. magnificum. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 2002.

Yip Pui Ying. / Thesis submitted in: November 2004. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 147-184). / Abstracts in English and Chinese. / Abstract --- p.i / 摘要 --- p.iii / Acknowledgment --- p.v / Abbreviations --- p.vii / Table of contents --- p.viii / List of Figures --- p.xii / List of Tables --- p.xvii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1. --- The importance of characterization of Chinese medicinal materials and the development of Chinese medicine in Hong Kong --- p.1 / Chapter 1.2. --- Methods for characterization of Chinese medicinal materials --- p.5 / Chapter 1.3. --- Molecular characterization of Chinese medicinal materials --- p.8 / Chapter 1.3.1. --- DNA sequencing --- p.9 / Chapter 1.3.2. --- DNA fingerprinting --- p.14 / Chapter 1.3.3. --- Nucleic acid hybridization --- p.19 / Chapter 1.4. --- Objectives --- p.20 / Chapter Chapter 2 --- Characterization of Plant and Fungal Materials by rDNA ITS Sequence Analysis --- p.22 / Chapter 2.1. --- Introduction --- p.22 / Chapter 2.2. --- Materials and Methods --- p.22 / Chapter 2.2.1. --- Chinese medicinal materials used in this study --- p.22 / Chapter 2.2.1.1. --- Plants and fungi for interspecific ITS study --- p.22 / Chapter 2.2.1.2. --- Plant for intraspecific ITS study and locality study --- p.33 / Chapter 2.2.2. --- Extraction of total DNA --- p.35 / Chapter 2.2.3. --- PCR amplification of ITS1 and ITS2 regions of rRNA gene --- p.35 / Chapter 2.2.4. --- Purification of PCR products --- p.38 / Chapter 2.2.5. --- Cloning using pCR-Script´ёØ Amp SK(+) Cloning Kit --- p.38 / Chapter 2.2.5.1. --- Polishing --- p.38 / Chapter 2.2.5.2. --- Ligation of inserts into pCR-Script´ёØ Amp SK(+) cloning vector --- p.38 / Chapter 2.2.5.3. --- Transformation --- p.40 / Chapter 2.2.5.4. --- PCR screening of white colonies --- p.40 / Chapter 2.2.5.5. --- Purification of PCR screening products --- p.41 / Chapter 2.2.6. --- Sequencing of ITS regions --- p.41 / Chapter 2.2.6.1. --- Cycle sequencing reaction --- p.41 / Chapter 2.2.6.2. --- Purification of sequencing extension products --- p.41 / Chapter 2.2.6.3. --- Electrophoresis by genetic analyzer --- p.42 / Chapter 2.2.7. --- Sequence analysis and alignment --- p.42 / Chapter 2.3. --- Results --- p.42 / Chapter 2.3.1. --- Extraction of total DNA --- p.42 / Chapter 2.3.2. --- PCR amplification of ITS1 and ITS2 regions of rRNA gene --- p.44 / Chapter 2.3.2.1. --- Interspecific ITS study --- p.44 / Chapter 2.3.2.2. --- Intraspecific ITS study --- p.46 / Chapter 2.3.3. --- Sequence analysis and alignment --- p.47 / Chapter 2.3.3.1. --- Interspecific ITS study --- p.47 / Chapter 2.3.3.2. --- Intraspecific ITS study --- p.56 / Chapter 2.4. --- Discussions --- p.60 / Chapter 2.4.1. --- rDNA regions used for studying Chinese medicinal materials --- p.60 / Chapter 2.4.2. --- The results agreed with previously published works --- p.60 / Chapter 2.4.3. --- Explanation of interspecific results within the Ganoderma genus --- p.60 / Chapter 2.4.4. --- Implications from interspecific comparisons --- p.60 / Chapter 2.4.5. --- Implications from intraspecific comparisons --- p.61 / Chapter Chapter 3 --- .Characterization of Astragalus membranaceus by DNA Fingerprinting / Chapter 3.1 --- Introduction --- p.62 / Chapter 3.2 --- Materials and Methods --- p.62 / Chapter 3.2.1 --- Extraction of total DNA --- p.62 / Chapter 3.2.2 --- Generation and detection of DNA fingerprints by AP-PCR --- p.63 / Chapter 3.2.3 --- Analysis of DNA fingerprints --- p.63 / Chapter 3.3 --- Results --- p.63 / Chapter 3.3.1 --- Generation of DNA fingerprints by AP-PCR --- p.63 / Chapter 3.3.2 --- Fingerprint analysis --- p.69 / Chapter 3.4 --- Discussion --- p.85 / Chapter 3.4.1 --- RP-PCR has been used on Chinese medicinal materials --- p.85 / Chapter 3.4.2 --- AP-PCR used instead of RAPD --- p.85 / Chapter 3.4.3 --- Reproducibility and amount of bands --- p.86 / Chapter 3.4.4 --- Alternatives of electrophoresis process --- p.88 / Chapter 3.4.5 --- Explanation of results --- p.88 / Chapter 3.4.6 --- Distinguishing Neimengu and Shanxi samples --- p.89 / Chapter 3.4.7 --- Further studies --- p.90 / Chapter Chapter 4 --- Characterization of Plant and Fungal Materials by DNA-DNA Hybridization on Microarrays --- p.91 / Chapter 4.1 --- Introduction --- p.91 / Chapter 4.2 --- Materials and Methods --- p.92 / Chapter 4.2.1 --- Samples for microarray study --- p.92 / Chapter 4.2.2 --- Extraction of total DNA --- p.95 / Chapter 4.2.3 --- Amplification and sequencing of ITS 1 region of rRNA gene --- p.95 / Chapter 4.2.4 --- Preparation of labeled probe --- p.95 / Chapter 4.2.5 --- Amplification of ITS1 fragments --- p.97 / Chapter 4.2.6 --- Preparation of slides --- p.103 / Chapter 4.2.7 --- Hybridization and washing --- p.104 / Chapter 4.2.8 --- Scanning and data analysis --- p.105 / Chapter 4.3 --- Results --- p.105 / Chapter 4.3.1 --- DNA extraction --- p.105 / Chapter 4.3.2 --- Amplification and sequencing of ITS1 region of rRNA gene --- p.107 / Chapter 4.3.3 --- Preparation of labeled probe and amplification of ITS1 fragments… --- p.112 / Chapter 4.3.4 --- Preparation of slides --- p.112 / Chapter 4.3.5 --- Scanning and data analysis --- p.116 / Chapter 4.4 --- Discussion --- p.134 / Chapter 4.4.1 --- Implications --- p.134 / Chapter 4.4.2 --- Applying the findings --- p.134 / Chapter 4.4.3 --- Ways to maximize specificity --- p.137 / Chapter 4.4.4 --- Optimisation --- p.138 / Chapter 4.4.5 --- Microarray may be more advantageous over sequencing --- p.138 / Chapter Chapter Five --- General Discussion and Summary --- p.140 / Chapter 5.1. --- Objectives of this study --- p.140 / Chapter 5.2. --- rDNA ITS sequencing --- p.140 / Chapter 5.2.1. --- Description of the approach and summary of the results --- p.140 / Chapter 5.2.2. --- Implications from the results --- p.140 / Chapter 5.2.3. --- Advantages and limitations of DNA sequencing --- p.141 / Chapter 5.3. --- AP-PCR fingerprinting --- p.141 / Chapter 5.3.1. --- Description of the approach and summary of the results --- p.141 / Chapter 5.3.2. --- Advantages and limitations of DNA fingerprinting --- p.142 / Chapter 5.4. --- DNA-DNA hybridization on microarrays --- p.143 / Chapter 5.4.1. --- Description of the approach and summary of the results --- p.143 / Chapter 5.4.2. --- Implications from the results --- p.143 / Chapter 5.4.3. --- Advantages and limitations of DNA hybridization on microarrays. --- p.144 / Chapter 5.5. --- Overall summary --- p.144 / Chapter 5.6. --- Future studies --- p.146 / References --- p.147 / Appendix --- p.185