M. Tech. Biomedical Technology. / In most rural areas of developing countries, people use untreated water sources for consumption and sanitation purposes. The current health-related water quality testing techniques only determine the presence of indicator organisms and may not adequately predict the potential of water to result in adverse physiological effects after ingestion. The possibility of using an accurate and reliable in vitro bioassay that could directly predict the potential of water to cause an infection after ingestion was investigated in this study.

Prof. Paul Jagals Dr. Hafsatou Ndama Traoré

Water is essential to all life but many freshwater resources are polluted through human activities. Humans and wildlife are exposed to a wide range of contaminants through their water, many of which pose a risk to health. Some of the contaminants released into the environment have been reported to have the capability to disrupt the endocrine functions in humans and wildlife and they can mimic or antagonise the action of estrogenic. These endocrine disrupting chemicals (EDCs) interact with physiological systems and cause alterations in development, growth and reproduction in wildlife and humans. To achieve some measure of assessing the potential harm that the contaminants pose, we need to know the environmental concentration of the chemical concerned and to monitor their effect on the organisms. The water supply sector need to include EDCs in standard systems of routine water source monitoring which include indicator bacteria and nutrient species but before the system can be incorporated, methods to measure the occurrence of EDCs in aquatic environment need to be developed and validated and a reliable guidelines data need to be in place. The aim of this study was to develop an enzyme-linked immunosorbent assay (ELISA) to quantify vitellogenin (VTG) in Oreochromis mossambicus (Mozambique tilapia) VTG has been used successfully as a biomarker for estrogenic contamination in different studies. For this study, VTG was isolated and purified from plasma of 17β-estradiol exposed tilapia by gel filtration chromatography. The purity of the VTG isolate was confirmed by polyacrylamide gel electrophoreses (SDS-PAGE). Polyclonal antibodies against t-VTG were raised in rabbits and the specificity of the anti-t-VTG was confirmed by western blot. Using purified t-VTG as a standard and anti-t-VTG antibody, a homologous competitive ELISA was developed and validated. The standard curves of the ELISA, which were generated on different days, were identical which indicate that the assay is reliable, reproducible and repeatable. The intra-assay and inter-assay coefficient variation was 2.41 (n = 4) and 8.71 (n = 10) respectively. The serial dilution of plasma VTG from exposed tilapia showed a good parallelism with the standard t-VTG within the working range of the assay. The serial dilution of the reference fish did not cover the whole range of the t-VTG standard curve. By using the standard curve and the dilution of the exposed plasma, we were able to demonstrate that the ELISA was able to quantify VTG. With good laboratory practise, this ELISA can be use to quantify VTG in chemically exposed fish. It will also be ideal to continue analyzing the antibody to determine the appropriate dilutions necessary to ensure that the assay work its optimal capabilities. / Dr. I. Barnhoorn Prof. P. Jagals Prof. J.H.J. Van Vuren

Simple, reliable and cost-effective methods for detecting pathogens are a vital part of diagnostics inside and outside the clinic, in particular in the developing world. Paper based colorimetric techniques are a promising approach for biosensors and bioassays as they can be used at the point of sampling and require little equipment. This study reports on the development of a colorimetric cellulose bioassay that can detect pathogen DNA with covalently attached single-stranded DNA probes. Chemical activation of cellulose via tosylation and oxidation was investigated. The successful activation of cellulose was characterised by Fourier transform infrared spectroscopy, scanning electron microscopy and elemental analysis. Sulfhydryl and amine functionalised oligonucleotide probes complementary to a segment of IS6110 element in Mycobacterium tuberculosis genome were covalently immobilised on the cellulose strips for recognition of target nucleic acid. The detection of biotinylated target oligonucleotides was achieved with horseradish peroxidase (HRP) linked to streptavidin that binds biotin with high affinity. HRP catalysed the oxdidation of tetramethylbenzidine by hydrogen peroxide. The successful assay was confirmed by the appearance of blue coloured spots on cellulose strips incubated with biotinylated target oligonucleotides complementary to the surface attached probe. The study also showed that tosylated cellulose is more reliable for the detection of targets. Initial experiments have shown sensitivity upto 0.1 µM and considerable specificity. High probe immobilization efficiencies (>90%) have been observed. The assay was also effectively demonstrated with mycobacterial DNA. Additionally, the development of a label free assay based on a dual-probe approach was investigated, but did not yield conclusive results. The developed assay has the potential for use as a simple test for the detection of pathogen DNA in clinical samples since it requires minimal equipment and is cost effective. In addition, it also shows the potential use of tosylated cellulose as a prospective surface for attaching other types of biomolecules in an active conformation.

<p>Halten näringsämnen i livsmedel varierar bland annat med tillagningsmetod, lagringstid och lagringsförhållanden, som exponering för ljus och syre. Oklarheter finns för mikrovågors inverkan på näringsämnen och framförallt på vitamin B₁₂. Eftersom många äldre drabbas av brist på vitamin B₁₂, är detta av intresse eftersom många hemmaboende äldre människor får hemleverans av färdiga matportioner som är avsedda att värmas, ofta i mikrovågsugn. Syftet med studien var att kunna ge en indikation på om uppvärmning genom mikrovågor påverkar halten vitamin B₁₂ i färdiga kylda måltider. Detta gjordes genom litteratursammanställning om vitamin B₁₂ och mikrovågar samt genom bioassay med <em>Lactobacillus delbrueckii</em>. Vitamin B₁₂ i ouppvärmd lax, lax värmd i mikrovågsugn samt lax värmd i konventionell ugn analyserades. Tillväxten av bakterier uppskattades genom mätning av turbiditet. Högst vitamin B₁₂-halt sågs i mikrovågsvärmd lax, därefter ouppvärmd lax och ugnsvärmd lax. Statistiskt signifikanta skillnader sågs för analyserade vitamin B₁₂-halter mellan ouppvärmt- och ugnsvärmt prov respektive mellan mikrovågsvärmt- och ugnsvärmt prov. De varierande vitaminhalterna kan bero på metodfel, otillräcklig vitaminextraktion eller att bakterierna inte konsumerat allt tillgängligt vitamin vid turbiditetsmätning. Slutsatsen av studien är att halten vitamin B₁₂ i livsmedel inte verkar påverkas negativt av mikrovågor, däremot finns tendenser att halten minskar beroende av temperatur och tillagningstid.</p>

No description available.

The prevailing use of chemical pesticides has generated several problems including insecticide resistance, outbreak of secondary pests, safety risks for humans and domestic animals, contamination of ground water and decrease in biodiversity among other environmental concerns (Webster, 1982). These problems and the nonsustainability of control programs based mainly on conventional insecticides have stimulated increased interest in the development and implementation of costeffective, environmentally safe alternatives to chemical pesticides for insect pest control. One of the most promising strategies to help minimize dependence on chemical pesticides has been the recent application of entomopathogenic nematodes (EPNs) as biocontrol agents. EPNs in the families Steinernematidae and Heterorhabdidae have been shown to have considerable potential as biological control agents. As a natural process, biological control has the potential to play an important role in the suppression of field crop pests in agriculture. EPNs as biocontrol agents have the following advantages: high virulence, safety to non target organisms, ability to search for hosts, high efficacy in favourable habitats, high reproductive potential, ease of mass production, ease of application (Ferron & Deguine, 1996). To isolate the EPNs in South African soil, 200 soil samples were randomly collected from 5 locations in the agricultural research council (ARC) Pretoria, Gauteng province in April 2006; and 5 locations in Brits, North West province in March, 2006. At the different collection sites, soil samples were obtained from soils associated with various types of vegetation. The nematodes were collected from sandy soil by the insect-baiting technique and maintained successfully in vivo for 12 months on Galleria mellonella (G. mellonella), 4 months on Tenebrio molitor (T.molitor); 2 months Pupae and in vitro (lipid agar) for 2 weeks in the laboratory. Out of a total of 200 soil samples that were baited, 2 were found to be positive for EPNs.EPNs. IV In addition to completing Koch’s postulates, the colour of cadavers infected by the putative EPNs were also used as a diagnostic characteristic for categorizing the nematode isolates. Characterization and identification of the EPN isolates were based on morphological characters, as well as on a molecular marker (18S rDNA). On the basis of the morphological and molecular data that was obtained both of the EPNs isolates were placed in the family Heterorhabdidae: Heterorhabditis bacteriophora (H. bacteriophora) and Heterorhabditis zealandica (H. zealandica). Also from the phylogenetic trees generated from the 18S rDNA sequence, the indigenous putative H. bacteriophora was shown to be closely related to H. bacteriophora (accession number EF690469) and indigenous putative H. zealandica to H. zealandica (accession number AY321481). The two EPNs were found associated with Gram negative rod-shaped bacteria. The bacterial symbionts of the two isolates were isolated and a region of the 16S rDNA gene was sequenced. National Center for Biotechnology Information (NCBI-BLAST) results of the 16S rDNA sequence obtained showed the endosybiotic bacteria to be Photorhabdus luminescens laumondii (P. laumondii) (H. bacteriophora) and Photorhabdus sp (H. zealandica). Results of the tree showed that isolates from H. bacteriophora appeared to be closely related to P. luminescens subsp laumondii strain TT01 Ay 278646. The isolates from H. zealandica appeared to be most closely related to Photorhabdus sp Accession number: Q 614 Ay 216500). Bioassays were used to determine the infectivity of the two EPNs. In this experiment different infective juvenile (IJs) concentrations (5, 10, 25, 50, 100,200 400 and 500) of the two EPNs were applied per G. mellonella; T. molitor larva and pupae. The bioassay was carried out in two parts. In the first part, mortality data was collected for H. bacteriophora and H. zealandica. The results showed that the degree of susceptibility of G. mellonella, T. molitor larvae and pupae to each nematode species was different. When 24 h post-exposure mortality data for larvae exposed to the IJs of H. bacteriophora and H. zealandica were analyzed, ANOVA showed no differences V in mortality between insects exposed to different H. bacteriophora IJ doses (Fig: 8.1 ABC). However, there were significant differences in mortality between insects exposed to different IJ doses of H. zealandica such as 5 and 500 IJs/insect (Fig: 8.2 ABC) Therefore, no differences were noted when mortality data was compared between IJ doses at both 72 h and 96 h following IJ application to the insects. The highest susceptibility was observed with G. mellonella followed by T. molitor pupae and then T. molitor larvae. According to Caroli et al., (1996), the total mortality of insect such as G. mellonella and other lepidopterans, was reached within 24-72 h of exposure to nematodes at concentrations such as those tested here. In this study similar results were observed with high concentration of nematodes (100, 200 and 500). In the second part of the dose response bioassay, the number of progeny IJs emerging from EPN-infected cadavers was determined for all two EPNs. The results indicate that IJ progeny production differed among the three insect hosts used, the IJ doses they were exposed to, as well as the EPN species (Figs 8.3 & 8.4). The highest number of emerged IJs of H. zealandica was produced by G. mellonella (mean ± SEM: 220500 ± 133933 IJs), followed by T. molitor larvae (mean ± SEM: 152133 ± 45466 IJs) and the lowest then T. molitor pupae (mean ± SEM: 103366 ± 56933 IJs).

Neste trabalho, foram desenvolvidos e validados métodos analítico e microbiológico, bem como estudo preliminar de estabilidade, cinética de degradação e citotoxicidade da Ceftarolina Fosamila em pó para solução injetável, um antibiótico da classe das cefalosporinas de quinta geração, indicado para pneumonias adquiridas na comunidade e infecções graves, de pele e tecidos moles. A validação do ensaio microbiológico pelo método de difusão em ágar cilindros em placa, delineamento 3x3, apresentou resultados satisfatórios, como especificidade, linearidade na faixa de 2,0 - 8,0 μg/mL, precisão (109,42 %), exatidão (102,3 %) e robustez. Soluções de Cefatarolina Fosamila do produto acabado expostas à radiação UVC (254 nm) e à degradação térmica a 60 °C foram utilizadas para avaliar a especificidade do bioensaio. A robustez foi avaliada através da alteração da concentração do meio inoculado (0,8 e 1,2 %). O desenvolvimento e validação de método por CLAE foi avaliado através da especificidade, linearidade, precisão, exatidão e robustez. No método cromatográfico foi utilizado cromatógrafo à liquido de alta eficiência SHIMADZU com coluna Agilent® C18, fase móvel (água com trietilamina 1,0% pH 5,0:acetonitrila 87:13 v/v). O método apresentou-se específico, linear, no intervalo de 5,0 - 60,0 μg/mL, preciso (110,0 %), exato (100,68 %) e robusto. Os métodos microbiológico e cromatográfico validados foram comparados estatisticamente e verificou-se não haver diferença significativa entre eles quando comparados através do teste “t” de Student. No estudo preliminar de estabilidade constatou-se ser estável em hidrólise ácida (0,1 M) e luz UVA no período avaliado, e instável frente à degradação térmica (40 e 60 °C), oxidativa com peróxido de hidrogênio, básica em NaOH (0,1 M e 0,01 M) e luz UVC. As cinéticas de degradação frente à luz UVC e degradação térmica 60 °C mostraram que as amostras possuem cinética de degradação de ordem zero e de segunda ordem, respectivamente. O ensaio de citotoxicidade demonstrou não haver diferença entre a condição normal e a amostra submetida à degradação forçada, sugerindo que os possíveis produtos de degradação formados não alteraram o resultado. / In this work, analytical and microbiological methods were developed and validated, as well as a preliminary study of the stability, degradation kinetics and cytotoxicity to Ceftaroline Fosamil powder for injectable solution, this is a fifth generation cephalosporin antibiotic indicated for community-acquired pneumonia and severe infections of the skin and soft tissues. The validation of the microbial assay by diffusion method in 3x3 cylinder agar delineated showed satisfactory results in specificity, linearity in the range of 2.0 - 8.0 μg / mL, precision (109.42 %), accuracy (102.3 %) and robustness. The development and validation of the method by HPLC was evaluated through specificity, linearity, precision, accuracy and robustness. In the chromatographic method was used high performance liquid chromatograph from SHIMADZU with Agilent® C18 column, mobile phase (water with triethylamine 1.0 % pH 5.0: acetonitrile 87:13 v/v). The method was linear, specific in the range of 5.0 - 60.0 μg/mL, accurate (110.0 %), exact (100.68 %) and robust. The validated microbiological and chromatographic methods were compared statistically and there was no significant difference between them when compared through Student's t-test. In the preliminary stability study, it was found stable in acid hydrolysis (0.1M) and UVA light in the period evaluated, and instable against thermal degradation (40 and 60 °C), oxidative with hydrogen peroxide, basic in NaOH (0.1 M and 0.0 1M) and UVC light. Samples exposed in UVC light an thermal degradation at 60°C showed degradation kinetics following zero order and second order, respectively. The cytotoxicity assay showed no difference between the normal condition and the sample submitted to forced degradation, suggesting that the possible degradation products formed did not change the result.

Medicinal plants have provided important advances in the treatment of numerous diseases and many plant-derived drugs are currently in use or under investigation for the treatment of many ailments including cancer.<p>A phytochemical analysis of the methanol extract from the seed of <i>Saponaria vaccaria</i> L. cultivated in Saskatchewan was performed which resulted in the detection of several bisdesmosidic saponins. A high-performance liquid chromatographic method using photodiode array and single quadrupole electrospray mass detection for analysis and profiling was developed. Due to their structural similarities, purification of bisdesmosidic saponins was challenging. However, monodesmosidic saponin Vaccaroside B and cyclopeptides Segetalin A, Segetalin B, and a new cyclopeptide, segetalin I [whose structure was proposed to be cyclo(Gly1-Pro2-Tyr3-Tyr4-Pro5-Phe6)], were purified employing various chromatographic techniques such as HPLC, VLC, PTLC). <p>Crude methanol extracts of <i>S. vaccaria</i> seed were evaluated for cytotoxic activity using the methyl-thiazol-tetrazolium non-radioactive cell proliferation assay (MTT assay). Various concentrations of the extract (2-50 ug/mL) were tested against a series of four human cancer cell lines (WiDr, colon; MDA-MB-231, breast; NCI-417, lung and PC-3, prostate). The human foreskin (BJ)-derived normal human fibroblast cell line CRL-2522 was included as a non-cancerous control. Results showed that cytotoxic activities from the seed extract were greater than commercially available Quillaja saponaria saponin. <p>The human cancer cell lines were also exposed to fractions containing high titers of saponins as well as semi-purified saponins (ca. 80%). All bisdesmosidic saponins and fractions thereof showed cytotoxicity against the cell lines studied. The effect was particularly strong in breast and prostate cancer cell lines with IC50 values in the range 14 ug/mL. Monodesmosidic saponins, phenolics and cyclopeptides did not show activity even at the highest concentration (50 ug/mL) tested in this study. Chemical modifications of the saponin structures resulted in an overall decrease in activity, but an increase in selectivity in comparison to CRL-2522. Time and concentration-dependent studies using the nuclear stains propidium iodide and Hoechst 33342, demonstrated that the stimulation of apoptosis was the mechanism of cytotoxic action. When breast and prostate cell lines were exposed to small amounts (4-7 mM) of bisdesmosidic saponins Segetalin H (MW 1448) and Segetalin I (MW 1464), it was observed that apoptosis was induced at an early incubation time (4-10 h). Activation of caspases and changes in membrane potential were determined by flow cytometry.<p>As a result of this study, we propose that triterpene bisdesmosidic saponins from the seed of <i>Saponaria vaccaria</i> L. represent a new type of drug having potential antitumor/anticancer activity due to their ability to induce apoptosis in vitro in human cancer cell lines at low concentrations. These compounds are extracted from a plant that can be easily cultivated using conventional agricultural equipment in Western Canada.

Medicinal plants have provided important advances in the treatment of numerous diseases and many plant-derived drugs are currently in use or under investigation for the treatment of many ailments including cancer.<p>A phytochemical analysis of the methanol extract from the seed of <i>Saponaria vaccaria</i> L. cultivated in Saskatchewan was performed which resulted in the detection of several bisdesmosidic saponins. A high-performance liquid chromatographic method using photodiode array and single quadrupole electrospray mass detection for analysis and profiling was developed. Due to their structural similarities, purification of bisdesmosidic saponins was challenging. However, monodesmosidic saponin Vaccaroside B and cyclopeptides Segetalin A, Segetalin B, and a new cyclopeptide, segetalin I [whose structure was proposed to be cyclo(Gly1-Pro2-Tyr3-Tyr4-Pro5-Phe6)], were purified employing various chromatographic techniques such as HPLC, VLC, PTLC). <p>Crude methanol extracts of <i>S. vaccaria</i> seed were evaluated for cytotoxic activity using the methyl-thiazol-tetrazolium non-radioactive cell proliferation assay (MTT assay). Various concentrations of the extract (2-50 ug/mL) were tested against a series of four human cancer cell lines (WiDr, colon; MDA-MB-231, breast; NCI-417, lung and PC-3, prostate). The human foreskin (BJ)-derived normal human fibroblast cell line CRL-2522 was included as a non-cancerous control. Results showed that cytotoxic activities from the seed extract were greater than commercially available Quillaja saponaria saponin. <p>The human cancer cell lines were also exposed to fractions containing high titers of saponins as well as semi-purified saponins (ca. 80%). All bisdesmosidic saponins and fractions thereof showed cytotoxicity against the cell lines studied. The effect was particularly strong in breast and prostate cancer cell lines with IC50 values in the range 14 ug/mL. Monodesmosidic saponins, phenolics and cyclopeptides did not show activity even at the highest concentration (50 ug/mL) tested in this study. Chemical modifications of the saponin structures resulted in an overall decrease in activity, but an increase in selectivity in comparison to CRL-2522. Time and concentration-dependent studies using the nuclear stains propidium iodide and Hoechst 33342, demonstrated that the stimulation of apoptosis was the mechanism of cytotoxic action. When breast and prostate cell lines were exposed to small amounts (4-7 mM) of bisdesmosidic saponins Segetalin H (MW 1448) and Segetalin I (MW 1464), it was observed that apoptosis was induced at an early incubation time (4-10 h). Activation of caspases and changes in membrane potential were determined by flow cytometry.<p>As a result of this study, we propose that triterpene bisdesmosidic saponins from the seed of <i>Saponaria vaccaria</i> L. represent a new type of drug having potential antitumor/anticancer activity due to their ability to induce apoptosis in vitro in human cancer cell lines at low concentrations. These compounds are extracted from a plant that can be easily cultivated using conventional agricultural equipment in Western Canada.