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No. of bitstreams: 1 TeseAMF.pdf: 4750654 bytes, checksum: f48dd93469cee1d3b92bc7a4295b054d (MD5) Previous issue date: 2016-11-07 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Cancer is a leading cause of death, according to World Health Organization, preceding diseases as diabetes and tuberculosis, conditions as malnutrition and even though interpersonal violence. Many types of cancer are also correlated to major risk factors associated to mortality and morbidity, such as obesity, alcohol abuse and smoking. In 2012 14.1 millions of new cases of cancer arisen with 8.2 million of deaths worldwide. Ginger rhizome (Zingiber officinale Roscoe) is word know use as spice on cooking and widely use as medicinal herb in preparations. Several studies describe its anti-inflammatory, antimicrobial, antioxidant and anti-emetic activities. Much of the ginger bioactivity is due to its phenolic compounds [6], [8], and [10]- gingerol, which have anti-proliferative and antiangiogenic action on tumor cells, as demonstrated by several in vivo and in vitro studies. Several studies have revealed advantages of three-dimensional culture techniques (3D) over traditional two-dimensional monolayer cultures (2D). 3D cultures better mimic the tumor microenvironment found in vivo, as well as the interaction of cells with the extracellular matrix (ECM). Three-dimensional culture has produced different responses compared to those found in 2D cultures, such as increased resistance of tumor cells to various drugs, and increased selective sensitivity to tumor cells in relation to normal cells. On this work, techniques for three-dimensional culture were used to test the effects of [10]-gingerol on breast tumor cells. The aim of this study was to evaluate the effects of [10]-gingerol in different hallmarks of malignancy correlated with metastatic process, such as adhesion, proliferation, migration, invasion and also its effects on apoptosis, both in 2D and 3D. Results demonstrated that [10]-gingerol changes the morphology of MDA-MB-231 malignant cells in lower concentrations and shorter times when compared to nonmalignant MCF-10A cells, suggesting an specific and concentration-dependent action for [10]-gingerol on malignant cells. [10]-gingerol was also able to inhibit migration and invasion of MDA-MB- 231 cells at low concentrations and to induce apoptosis at higher concentrations. We observed that [10]-gingerol presented higher IC50 in proliferation assays with MCF-10A non tumor cells compared to tumor cells. The compound also inhibited migration of non-tumor cell lines at higher concentrations compared to tumor cells. Moreover, [10]-gingerol inhibited MDA-MB- 231 cell adhesion to different ECM components, such as laminin, fibronectin and vitronectin, even at low concentrations. Western blotting and real time quantitative PCR assays suggested that [10]-gingerol was able to act by the intrinsic pathway of apoptosis, increasing Bax/Bcl-2 ratio and caspase-9 and caspase-3 mRNA and protein levels in MDA-MB-231 cells. On 3D assays the results showed selectivity of [10]-gingerol against the malignant T4-2 lineage. The compound was also able to revert the malignant phenotype and to induce apoptosis in this cell line. These results suggest that [10]-gingerol has potential to be a new anticancer drug in the future. / O câncer é uma das principais causas de morte no mundo, segundo dados da Organização Mundial da Saúde, estando à frente de doenças como o diabetes e a tuberculose, de condições como a desnutrição e até mesmo da violência interpessoal. Diversos tipos de câncer estão ainda relacionados aos principais fatores de risco associados à mortalidade e morbidade no mundo, tais como a obesidade, o consumo excessivo de álcool e o tabagismo. Em 2012 foram 14,1 milhões de casos novos de câncer com 8,2 milhões de mortes no mundo. O rizoma do gengibre (Zingiber officinale Roscoe) é utilizado no mundo todo como especiaria culinária e como erva medicinal em diversas preparações. Existem muitas pesquisas descrevendo suas propriedades antieméticas, antimicrobianas, anti-inflamatórias, antioxidantes e antitumorais. Os compostos farmacologicamente ativos do gengibre são os compostos fenólicos pungentes [6]-gingerol, [8]- gingerol e [10]-gingerol, os principais componentes do seu extrato bruto. Tais compostos são os responsáveis pela bioatividade do gengibre e por seus efeitos sobre células tumorais, demonstrados em diversos modelos de câncer, em estudos in vivo e in vitro. Muitas pesquisas revelam vantagens das técnicas de cultura tridimensional (3D) sobre as culturas tradicionais em monocamada. Culturas em 3D mimetizam melhor o microambiente tumoral encontrado in vivo, bem como a interação das células com a matriz extracelular (MEC). Em culturas 3D já foram observadas respostas diferentes das encontradas em culturas bidimensionais (2D), como a maior resistência de células tumorais a diversas drogas, além de sensibilidade seletiva aumentada para células tumorais em relação às células normais. Neste trabalho, técnicas de cultura tridimensional foram aplicadas em testes utilizando a molécula de [10]-gingerol em células tumorais de mama. O objetivo deste estudo foi avaliar os efeitos do [10]-gingerol em diferentes marcadores de malignidade, importantes no processo metastático, como adesão, proliferação, migração, invasão, bem como seus efeitos sobre a apoptose, tanto em cultura 2D, quanto 3D. Os resultados mostraram que o [10]-gingerol altera a morfologia de células tumorais da linhagem MDA-MB-231 em concentrações e tempos menores que a de células não tumorais de mama da linhagem MCF-10A, o que sugere uma ação seletiva do [10]-gingerol dependente de concentração e tempo de tratamento. O [10]-gingerol também inibiu a migração e a invasão das células da linhagem MDA-MB-231 em baixas concentrações e induziu apoptose em concentrações mais altas. Observamos que o [10]-gingerol apresentou, em ensaios de proliferação com células não tumorais da linhagem MCF-10A, um IC50 maior comparado com as células tumorais. O composto também inibiu a migração das células não tumorais em concentrações maiores comparado às células tumorais. Além disso, o [10]-gingerol diminuiu a capacidade de adesão das células MDA-MB-231 a diferentes componentes da MEC, como laminina, fibronectina e vitronectina mesmo em baixas concentrações. Ainda, confirmamos sua capacidade de causar danos ao DNA e induzir apoptose. Ensaios de western blotting e qPCR sugerem que o [10]-gingerol atua pela via intrínseca da apoptose, aumentando a razão Bax/Bcl- 2 e ativando as caspases-9 e -3. Com relação aos ensaios em 3D, os resultados demonstraram que o composto [10]-gingerol age seletivamente sobre células tumorais, sendo capaz de reverter o fenótipo maligno e induzir apoptose nas células da linhagem T4-2. Estes resultados sugerem que o [10]-gingerol tem potencial para futuramente se tornar uma nova droga antitumoral. / FAPESP: 2012/18908-6 / FAPESP: 2015/08146-0

Master of Science / Department of Animal Sciences and Industry / Duane L. Davis / The three transcription factors, Nanog, Oct-4 and Sox-2, are central regulators of pluripotency in embryonic stem cells. Porcine umbilical cord (PUC) matrix stem cells also express these transcription factors. Wharton’s jelly is composed of an extracellular matrix high in hyaluronic acid and various collagens and serves as a reservoir for several growth factors and cytokines. We expect that Wharton’s jelly includes a stem cell niche that provides a microenvironment that maintains and supports the stem-cell characteristics of PUCs. The mechanisms by which the PUCs remain primitive within the Wharton’s jelly are unknown. We developed methods for producing an extracellular matrix product extracted from porcine Wharton’s jelly that we named Pormatrix (PMX). When PMX is incubated at 37[degrees]C, it becomes a matrical gel that provides a matrix allowing PUC attachment and growth. Concentrating the protein in PMX by filtration provides a low molecular weight by-product which we refer to as flow through (FT). In Experiment 1, PUCs were seeded on Pormatrix, Matrigel or plastic substrates in the presence or absence of FT. PUCs cultured on Matrigel, Matrigel+FT, Plastic+FT and PMX had higher expression of Nanog compared to PUCs cultured on PMX+FT (P-value <0.05). In Experiment 2, the PMX and Matrigel were diluted to low protein concentrations (1.2-1.5 mg/ml protein) so that gelling did not occur. Adding FT to PMX, Matrigel and plastic increased gene expression of Nanog 2.78 fold compared to treatments without FT (P =0.10). Sox-2 expression was increased by adding FT to Matrigel but adding FT to the other matrix proteins had no effect resulting in a tendency for a matrix*FT interaction(P=0.10). The transcription factor Oct-4 remained unchanged regardless of treatment. To evaluate the effects of in vitro maintenance on Nanog, Oct-4 and Sox-2 we measured the relative gene expression in PUCs over the first six passages in vitro. Nanog, Oct-4 and Sox-2 did not differ over these passages. This may indicate that during the first six passages in vitro, PUCs remain relatively primitive. In summary, we prepared an extract from Wharton’s jelly from porcine umbilical cords. The extract supported PUC attachment and growth and appeared to regulate gene expression. Perhaps with further investigation the interactions of PUCs with their in vivo environment can be elucidated.

Submitted in fulfillment of the requirements for the Degree of Master of Technology: Biotechnology, Department of Biotechnology and Food Technology, Faculty of Applied Sciences, Durban University of Technology, South Africa,2009. / Nearly half of all individuals living with HIV worldwide at present are woman and the best current strategy to prevent sexually transmitted HIV is antiretrovirals (ARVs). Microbicides are ARV’s which directly target viral entry and avert infection at mucosal surfaces. However, most promising ARV entry inhibitors are biologicals which are costly to manufacture and deliver to resource-poor areas. Microbicides formulated as simple gels, which are currently not commonly used in ARV therapy, show immense potential for use in prevention and treatment of multidrug-resistant viral infections in developing countries. Among the most potent HIV entry inhibitory molecules are lectins, which target the high mannose N-linked glycans which are displayed on the surface of HIV envelope glycoproteins. Of the microbicides, the red algal protein griffithsin (GRFT) has potent anti-HIV inhibitory activity and is active by targeting the terminal mannose residues on high mannose oligosaccharides. It has a total of 6 carbohydrate binding sites per homodimer, which likely accounts for its unparalleled potency. The antiviral potency of GRFT, coupled with its lack of cellular toxicity and exceptional environmental stability make it an ideal active ingredient of a topical HIV microbicide. v Scytovirin (SVN) is an equally potent anti-HIV protein, isolated from aqueous extracts of the cyanbacterium, Scytonema varium. Low, nanomolar concentrations of SVN have been reported to inactivate laboratory strains and primary isolates of HIV- 1. The inhibition of HIV by SVN involves interactions between the protein and HIV-1 envelope glycoproteins gp120, gp160 and gp41. Current recombinant production methods for GRFT and SVN molecules are unfortunately hampered by inadequate production capacities. This project therefore aimed to determine if these molecules can be produced in plant cell culture systems. The transgenic tobacco cell culture system was evaluated to determine if it can be an alternative, cost effective production system for these molecules. Results of the study show that the microbicide genes can be cloned into plant transformation vectors, used to successfully transform SR1 tobacco cell lines and adequately produce 3.38ng and 10.5ng of GRFT and SVN protein respectively, per gram of SR1 tobacco callus fresh weight. The promising results attained in this study form the basis for further work in optimising plant cell based production systems for producing valuable anti-HIV microbicides, a possible means to curbing the elevated HIV infection rates worldwide. / CSIR

We have studied the effects of exogenous salt on whole plant and suspension culture cells of the halophytic tomato Lycopersicon pennellii. Under low salt conditions (2.9 dS/M) plants showed enhanced (halophytic) growth (107% of control). At moderate (7.5 dS/M) and high (18.5 dS/M) salt levels, salt stress reduced growth to about 78% and 40% of control respectively. Salt-induced changes in root mRNAs were analyzed via two-dimensional PAGE of cell free translation (CFT) products. We have identified 14 proteins whose levels were enhanced by exogenous salt. One of these proteins was unique to low salt induced halophytic growth. This system allowed for discrimination between proteins up-regulated at all salt levels and those up-regulated only during salt stress induced growth reduction. Ten proteins were identified whose levels were reduced by exogenous salt. Once again, one could identify a subset of proteins whose levels were reduced only under salt stressed conditions. Proteins identified in this study are candidates for roles in growth maintaining stress adaptive metabolism in L.pennellii. These data underscore the complexity of the genetic control of salt metabolism in higher plants. The effects of exogenous salt on protein synthesis and accumulation were studied in suspension cultures of L.pennellii. Two salt levels were applied to the cells. Under low salt conditions (LS, 10 mM), L.pennellii cells showed enhanced (halophytic) growth. Under high salt conditions (HS, 50 mM), the cells showed reduced (salt-stressed) growth. Changes in proteins with time were analyzed by a combination of cell free translation, in vivo labeling and total accumulated protein. In vivo labeling studies showed that the pattern of steady state protein synthesis was disrupted shortly after addition of salt. High salt induced greater disruption in the pattern. Over time, the steady state levels of most proteins shifted back towards those of the unstressed-control. However, the level of several proteins remained altered. Analysis of proteins whose levels increased with exogenous salt showed differences in the response patterns that may allow for discrimination between proteins involved in growth maintaining and stress shock responses.

Despite intensive research, the cellular and molecular mechanisms involved in human cellular susceptibility to prion infection remain poorly defined, in part due to the continuing lack of cultured human cells that are susceptible to infection with human prions. Such culture models would present distinct advantages including speed and expense compared with animal models, and would provide systems in which to investigate the interaction between PrPC and PrPSc, the basis of cellular susceptibility, the nature of the species barrier and the mechanism of prion propagation in situ. This study sought to examine whether non-neuronal cells might provide opportunities to establish human cell lines replicating human prions. A human follicular dendritic cell-like cell line (termed HK) was obtained, further characterised and then tested for its ability to support human prion replication. The mechanisms of internalisation, intracellular trafficking and the eventual fate of exogenous PrPSc taken up by these cells were also examined. This thesis similarly examined the cellular response of human embryonic stem cells (hESC) to acute exposure to human and animal prions. PrPC was found to be abundantly expressed by HK cells and HK cell extracts were found to support conversion to PrPSc in a cell-free conversion assay. However, HK cells exposed to infectious brain homogenates failed to accumulate PrPSc or become infected in vitro. Exposed HK and hESC did display a readily detectable, time dependent uptake of PrPSc from medium spiked with prion-infected brain homogenates that was independent of the species, disease phenotype and PRNP codon 129 genotype of the human source and the recipient cells. The exposed cells showed intensely labelled intracellular accumulations of PrPSc with coarse granular morphology, largely in the juxtanuclear region of cytoplasm. However, when the brain-spiked medium was withdrawn and cells were given control medium, the intensity and extent of PrPSc immunostaining rapidly diminished. Co-localisation studies implicated caveolae-mediated endocytic uptake of exogenous PrPSc, apparently preceding uptake via clathrin coated pits in HK cells. Evidence suggesting that the endosomal recycling compartment and lysosomes are involved in intracellular trafficking and degradation of exogenous PrPSc was also found. Understanding the cell biology of these processes may help to explain why the majority of cultured cells are refractory to prion infection in vitro. Internalization of misfolded PrP and its subsequent degradation in the lysosomal compartment might function as a self-protective cellular mechanism, serving to eliminate non-native, presumably dysfunctional and potentially dangerous PrP conformers, whether generated endogenously or acquired through exposure to exogenous prion infectivity.

B virus is an alphaherpesvirus, endemic to macaque monkeys, capable of deadly human zoonosis with an 80% mortality rate in untreated cases. The macaque monkey is widely used in biomedical research and the threat of B virus poses an occupational hazard to researchers, veterinarians, and animal handlers. B virus establishes a life-long latent infection in sensory neurons of the peripheral nervous system (PNS) in the natural host. In human infections, B virus readily transits to the central nervous system (CNS) and destroys brain tissues. Identifying immune correlates of B virus infection in the PNS of the natural host is critical in understanding viral lethality in the human host. The lack of an accurate animal model and restrictions on handling potentially infected nervous tissue previously limited studies of B virus infection in macaque ganglia. To address this barrier, a long-lived mixed neuron/glia cell culture model was established from macaque DRG explants using a novel methodology that relied on cellular migration from whole tissues. Utilizing this model, the hypothesis tested was that acute B virus infection of macaque ganglia triggers cellular defense networks to promote leukocyte recruitment and impact leukocyte activation. Chemokines were upregulated in B virus-infected cultures and infected cell media induced leukocyte chemotaxis. Leukocytes were less effectively activated by media from infected cells when compared to media from mock-infected cells. To identify factors responsible for this, focused microarrays were performed and cytokine profiles were quantified from B virus and mock-infected culture supernatants. IL-6 protein levels were significantly reduced in B virus infected cultures. This observation led to the hypothesis that IL-6 downregulation impairs leukocyte activation and, indeed, when IL-6 was added to B virus-infected culture supernatants to control levels, these cultures were far more effective at eliciting leukocyte activation when compared with mock-infected cultures. Collectively, these data support the hypothesis that acute B virus infection of macaque ganglia triggers cellular defense networks to promote leukocyte recruitment and impact leukocyte activation and identifies a potential viral mechanism to impair leukocyte functionality. Additionally, this work presents a novel methodology for establishing long-lived mixed neuron/glia cultures from postnatal/adult macaque DRGs.

Thesis (PhD (Genetics))--University of Stellenbosch, 2010. / Includes bibliography. / ENGLISH ABSTRACT: The slow growth rate of Haliotis midae impedes the optimal commercial production of this most profitable South African aquaculture species. To date, no comprehensive effort has been made to identify genes associated with growth variation in farmed H. midae. The aim of this study was therefore to investigate growth variation in H. midae and to identify and quantify the expression of selected growth-related genes. Towards this aim, molecular methodologies and cell cultures were combined as a time-efficient and economical way of studying abalone transcriptomics and cell biology. Modern Illumina sequencing-by-synthesis technology and subsequent sequence annotation were used to elucidate differential gene expression between two sibling groups of abalone demonstrating significant growth variation. Following transcriptome sequencing, genes involved in growth and metabolism, previously unknown in H. midae, were identified. The expression of selected target genes involved in growth was subsequently analyzed by quantitative real-time PCR (qPCR). The feasibility of primary cell cultures for H. midae was furthermore investigated by targeting embryo, larval and haemolymph tissues for the initiation of primary cell culture. Larval cells and haemocytes could be successfully maintained in vitro for limited periods. Primary haemocyte cultures demonstrated to be a suitable in vitro system for studying gene expression and were subsequently used for RNA extraction and qPCR, to evaluate differential growth induced by bovine insulin and epidermal growth factor (EGF). Gene expression was thus quantified in fast and slow growing abalone and in in vitro primary haemocyte cultures treated with different growth stimulating factors. The results obtained from transcriptome analysis and qPCR revealed significant differences in gene expression between large and small abalone, and between treated and untreated haemocyte cell cultures. Throughout in vivo and in vitro qPCR experiments, the up-regulation of genes involved in the insulin signaling pathway provides evidence for the involvement of insulin in enhanced growth rate for various H. midae tissues. Besides the regulation of target genes, valuable knowledge was also gained in terms of reference genes, during qPCR experimentation. By quantifying the stable expression of two genes (8629, ribosomal protein S9 and 12621, ornithine decarboxylase) in various tissues and under various conditions, suitable reference genes, that can also be used in future H. midae qPCR studies, were identified. By providing evidence at the transcriptional level for the involvement of insulin, insulin-like growth factors (IGFs) and insulin-like growth factor binding proteins (IGFBPs) in improved growth rate of H. midae, the relevance of investigating ways to stimulate insulin/IGF release in aquaculture species was again emphasized. As nutritional administration remains the most probable route of introducing agents that can stimulate the release of insulin-related peptides, continuous endeavours to stimulate abalone growth through a nutritional approach is encouraged. This is the first time next generation sequencing is used towards the large scale transcriptome sequencing of any haliotid species and also the first time a comprehensive investigation is launched towards the establishment of primary cell cultures for H. midae. A considerable amount of sequence data was furthermore annotated for the first time in H. midae. The results obtained here provide a foundation for future genetic studies exploring ways to optimise the commercial production of H. midae. / AFRIKAANSE OPSOMMING: Die stadige groeitempo van Haliotis midae belemmer die optimale kommersiele produksie van hierdie mees winsgewende Suid-Afrikaanse akwakultuur spesie. Tot op hede is geen omvattende poging aangewend om gene verwant aan groeivariasie in H. midae te identifiseer nie. Die doel van hierdie studie was dus om groeivariasie in H. midae te ondersoek en om spesifieke groei-gekoppelde gene te identifiseer en hul uitdrukking te kwantifiseer. Ter bereiking van hierdie doel is molekulêre metodes en selkulture gekombineer as 'n en tydsbesparende en ekonomiese manier om perlemoen transkriptomika en selbiologie te bestudeer. Moderne Illumina volgordebepaling-deur-sintese tegnologie en daaropvolgende annotasie is gebruik om verskille in geenuitdrukking tussen naby-verwante groepe perlemoen, wat noemenswaardige groeivariasie vertoon, toe te lig. Na afloop van die transkriptoom volgordebepaling is gene betrokke by groei en metabolisme, vantevore onbekend in H. midae, geïdentifiseer. Die uitdrukking van uitgesoekte teikengene betrokke by groei is vervolgens ge-analiseer deur kwantitatiewe "real-time PCR" (qPCR). die lewensvatbaarheid van 'n primêre selkulture vir H. midae is ook ondersoek deur embrio, larwe en hemolimf weefsels te teiken vir die daarstelling van primêre selkulture. Larweselle en hemosiete kon in vitro suksesvol onderhou word vir beperkte periodes. Primêre hemosietkulture het geblyk 'n gepaste in vitro sisteem te wees om geenuitdrukking te bestudeer en dit is vervolgens gebruik vir RNS ekstraksie en qPCR, om differensiële groei, geïnduseer deur insulien en epidermale groeifaktor (EGF), te evalueer. Geenuitdrukking is dus gekwantifiseer in vinnig- en stadiggroeiende perlemoen en in in vitro primêre hemosiet selkulture wat behandel is met verskillende groei stimulante. Die resultate wat verkry is van transkriptoomanalise en qPCR het noemenswaardige verskille in geenuitdrukking tussen groot en klein perlemoen, en tussen behandelde en onbehandelde hemosiet selkulture uitgelig. Die op-regulering van gene betrokke by die insulien sein-padweg, tydens in vivo en in vitro qPCR eksperimente, bied getuienis vir die betrokkenheid van insulien in die verhoogde groeitempo van verskeie H. midae weefsels. Benewens die regulering van teikengene is waardevolle kennis ook ingewin in terme van verwysingsgene tydens qPCR eksperimentering. Deur die stabiele uitdrukking van twee gene (8629, ribosomale proteien S9 en 12621, ornitien dekarboksilase) te kwantifiseer in verskeie weefsels en onder verskeie kondisies is gepaste verwysingsgene, wat ook in toekomstige H. midae qPCR eksperimente aangewend kan word, geïdentifiseer. Deur getuienis vir die betrokkenheid van insulien, insuliensoortige groeifaktor en insuliensoortige groeifaktor-bindingsproteïene by verbeterde groei van H. midae op transkripsievlak te bied, is die toepaslikheid van bestudering van maniere om insulienvrystelling in akwakultuurspesies te stimuleer, beklemtoon. Aangesien voeding die mees waarskynlike roete is om middele wat insuliensoortige peptiedvrystelling stimuleer daar te stel, word vogehoue pogings om perlemoengroei deur die regte voeding te stimuleer, aangemoedig. Hierdie is die eerste studie wat volgende generasie volgordebepaling (“next generation sequencing”) gebruik vir die grootskaalse transkriptoom volgordebepaling van enige haliotied spesie. Dit is ook die eerste keer dat ‘n omvattende ondersoek geloods word na die daarstelling van primêre selkulture vir H.midae. ‘n Aansienlike hoeveelheid volgorde data is ook vir die eerste keer geannoteer in H. midae. Die resultate wat hier verkry is bied ‘n basis vir toekomstige genetiese studies wat maniere ondersoek om die kommersiële produksie van perlemoen te optimiseer.

Studies investigating the effect of 5-HT receptors mediating a response in the neonatal intestine have been limited. There are evidences that the development of new neurones continues past postnatal term and this suggests that receptors expression may differ during maturation. Thus, 'in vitro' experiments were carried out to investigate the effects of ACh, atropine, 5-HT and its related drugs on intact intestinal segments taken from the ileum of adult and neonate rats. The application of ACh (3nM-1mM) and 5-HT (3nM-1mM) induced contractions in a concentration dependent manner in all tissues examined. The 5-HT induced contractions were only sensitive to antagonism by atropine (1μM) in segments taken from the neonates but not adults. The pre-treatment with methysergide (5-HT1/2/5-7 receptor antagonist), ritanserin (5-HT2 receptor antagonist), granisetron (5-HT3 receptor antagonist) and RS 23597 (5-HT4 receptor antagonist) at 1μM or a combination of ritanserin, granisetron, plus RS 23597 at 1μM significantly reduced or abolished contractile responses induced by 5-HT. SB 269970A (5-HT7 receptor antagonist) and WAY 100635 (5-HT1A receptor antagonist) at 1μM failed to influence contractile responses induced by 5-HT or the challenges to 5-HT receptor agonists, 5-CT (5-HT1A/7 receptor agonist) and 8-OH-DPAT (5-HT1A receptor agonist) at a concentration range of 10nM-0.1mM, indicating the unlikely involvement of 5-HT1A and 5-HT7 receptors in the mediation of contractile responses in the neonatal rat ileum. Results indicate differences in cholinergic receptor involvement during postnatal maturation and suggest the involvement of 5-HT2, 5-HT3 and 5-HT4 receptors in the mediation of contractile responses to 5-HT in the neonatal rat ileum. There is a growing need to decrease animal usage in pharmacological experiments. This may be achieved by the development of 'in vitro' cell culture models. Thus attempts were also made to develop a cell culture model of neonatal intestine to further investigate the action of pharmacologically active agents. The isolation of individual cell populations from segments taken from the intestine of rat neonates were achieved by ligation of both ends of the intestine prior to incubation in trypsin so that a gradual dissociation could be monitored. This was supported by histological procedures, determining the time required to extract large numbers of cells from different intestinal layers. Differential adhesion and selective cytotoxicity techniques were used for further purification of intestinal smooth muscle cells (ISMC), neuronal cells, and a coculture of ISMC and neuronal cells, and these were characterised through immunostaining with antibodies to α-smooth muscle actin, α-actinin and the 5-HT3 receptor. A protocol for cryopreservation of ISMC was designed in order to protect cells against genetic instability, enhance cell availability and reduce animal usage. Results showed that cells extracted from the intestine are viable for up to 4-months. ISMC functionality was analysed via the application of known pharmacologically active drugs on ISMC, which were plated onto glass and silicone elastomer substrate. The cultured ISMC responded to the application of drugs such as potassium chloride (KCl), carbachol, 5-HT and noradrenaline (NA). Large population of cocultures seeded onto silicone elastomers or cholesteric liquid crystal substrates (LC) were assessed for their ability to produce a collective response to KCl application. Attempts were made to detect any deformations of the substrate surface due to the exposure to KCl and NA. Cholesteric LC substrates seemed to be the most suitable material for investigating the cellular tensions. The availability of cell cultures allowed the development of an intestinal model of inflammation. This was achieved through the use of lipopolysaccharide (LPS)-induced inflammation and was confirmed by assessing the levels pro-inflammatory mediators interleukin (IL-8) and nitric oxide (NO), which were significantly elevated. Reduction of IL-8 ad NO was also examined using granisetron and L-NAME and Chaga mushroom extract. Granisetron and L-NAME reduced the NO production during short incubation times. However, an elevated level of NO was observed when longer treatment times were examined. The Chaga mushroom extract caused a significant reduction in NO production in the model of inflammation. This indicates that this model may be a valuable tool for the investigation of other pro-inflammatory mediators and may contribute for the investigation of more selective drugs in the management of intestinal inflammation in neonates.

published_or_final_version / abstract / Zoology / Master / Master of Philosophy

Male fertility is largely controlled by the hypothalamic-pituitary axis, a careful balance between stimulating and suppressing gene expression and the secretion of hormones. The critical factors for male fertility have in the past been thought to be limited to testosterone and the gonadotropins. Estrogen has only recently been demonstrated to be both a crucial requirement for fertility and a cause of infertility. Reports in the early 1990s demonstrated a decrease in mean sperm counts over the last 50 years. A hypothesis for this observation is the increase of xenoestrogens in the environment that are able to mimic and potential disrupt the natural estrogens involvement in fertility. Although the mechanisms of estrogens involvement are not yet defined, the Sertoli cells are a potential sites of action as they possess receptors for the hormone and are able to locally produce it. Sertoli cells both act to protect and provide for the male germ cells and the developing spermatozoa. Pyrethroids are common synthetic insecticides of which some have previously shown estrogenic activity. Therefore this investigation examined the effects of pyrethoids, whose estrogenicity was confirmed via the yeast assay, on the estrogen receptor expression in mouse Sertoli cells as a model for general effects of estrogenic chemicals on male fertility. The results first confirmed the estrogenicity of some pyrethroids and these pyrethroids when exposed to mouse Sertoli cells effected estrogen receptor mRNA expression however in a different way to the natural ligand 17β-estradiol.