Submitted by Claudinei Pracidelli (cpracide@ipen.br) on 2016-06-22T11:39:54Z No. of bitstreams: 0 / Made available in DSpace on 2016-06-22T11:39:54Z (GMT). No. of bitstreams: 0 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Tese (Doutorado em Tecnologia Nuclear) / IPEN/T / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP / FAPESP:11/21708-6

Submitted by Claudinei Pracidelli (cpracide@ipen.br) on 2016-06-22T11:39:54Z No. of bitstreams: 0 / Made available in DSpace on 2016-06-22T11:39:54Z (GMT). No. of bitstreams: 0 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A deficiência de hormônio de crescimento (DGH) é a deficiência mais comum entre os hormônios pituitários. A terapia utilizada atualmente consiste de injeções diárias de hormônio de crescimento humano recombinante (r-hGH), entretanto esta terapia apresenta alguns inconvenientes, como a necessidade de frequentes injeções de r-hGH durante um longo período de vida, dependendo da severidade da deficiência, e o alto custo do hormônio, em razão dos dispendiosos processos de purificação. Uma alternativa ao tratamento padrão seria aquele no qual fossem evitados estes tipos de inconvenientes e o processo de liberação da proteína fosse sustentável, por um longo período e promovesse níveis normais e sustentáveis do fator de crescimento semelhante à insulina I (IGF-I), o principal mediador dos efeitos do GH. Uma alternativa é a terapia gênica in vivo, baseada na administração de DNA plasmidial em diversos órgãos/tecidos, seguida de eletroporação. É considerada uma metodologia bastante promissora e que tem sido alvo de vários estudos para diversos tipos de deficiências sistêmicas. Neste trabalho foram realizadas diversas administrações de um plasmídeo contendo o gene do hormônio de crescimento humano, nos músculos quadríceps exposto ou tibial anterior sem exposição, seguidas de eletroporação, em camundongos anões e imunodeficientes (lit/scid) com 40-80 dias de idade, na tentativa de obter uma correção fenotípica do nanismo, mediante a avaliação de parâmetros de crescimento. A administração deste plasmídeo no músculo tibial anterior, em camundongos com a idade inicial de 40 dias, foi capaz de proporcionar uma normalização dos níveis de mIGF-I, quando comparados aos dos camundongos não-deficientes de GH. Além disso, foram obtidos valores de catch-up dos parâmetros de crescimento longitudinal de 36-77%. Visando uma maior eficiência na expressão de GH, foram construídos plasmídeos parentais, e a partir destes, foram produzidos minicírculos de DNA com os promotores do CMV e Ubiquitina C e com os cDNAs de hGH e mGH. Estes minicírculos de DNA foram transfectados em células HEK 293 e foram até 2 vezes mais eficientes em relação aos plasmídeos convencionais com o promotor do CMV. Estes dados são bastantes promissores e abrem caminho para ensaios mais eficientes, utilizando este tipo de protocolo de terapia gênica para a DGH, visando uma normalização de todos os parâmetros de crescimento. / Tese (Doutorado em Tecnologia Nuclear) / IPEN/T / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP / FAPESP:11/21708-6

Thesis (MSc)--University of Stellenbosch, 2005. / ENGLISH ABSTRACT: Water quality analysis forms the basis in assessing and monitoring catchments. As urban development continuously increase, pollution sources increase in either point source (wastewater treatment works, industrial effluents) and/or non-point source origin (storm water discharge, domestic pollutants), accumulating pollutants in the environment. It was only recently discovered that certain pollutants have subtle disrupting effects on the endocrine system resulting in health related problems associated with the reproductive system and thyroid system (growth and development) of animals and potentially humans. Natural water resource management proves to include limited biological assays measuring endpoints for cytoxicity, inflammatory activity and endocrine disruption. The broad objective of this study was therefore to include several bioassays, not normally used in municipal (City of Cape Town) monitoring programmes, along with water quality data collected by the City of Cape Town. The Eerste/Kuils River catchment system, Western Cape, under the auspices of the City of Cape Town was chosen, and although this catchment does not contribute to drinking water resources, is subjected to a range of anthropogenic influences (industrial effluents, household wastewater, agricultural runoff). Within the short time-frame available for this study (six months) two months, July (following a dry summer and autumn season) and October (following a wet winter and early spring season) were selected for water quality monitoring. Spatial variation (with relevance to specific point and non-point contamination) among sampling sites were also obtained by choosing several (n=10) along the catchment. Specific aim of the study therefore included: Firstly (Chapter 2), the use of in vitro bioassays, lactate-dehydrolises assay (LDH) for cytotoxic activity, pro-inflammatory hormone Interleukin-6 (IL-6) secretion by human blood cells and a specific Salmonella ELISA for faecal contamination, in conjunction with routine chemical and biological (mostly microbiological) monitoring activities. The study indicated significant variation among sites in all microbiological measures as well in IL-6 secretion and Salmonella presence. Between months, variations were also evident in certain variables. Secondly (Chapter 3), two bioassays using the yolk precursor protein, vitellogenin (Vtg) as endpoint was implemented in a) an in vitro Xenopus laevis liver slice assay (five day exposure) and b) an in vivo Zebrafish (Danio rerio) bioassay (seven day exposure) assessing estrogenic activity in the Eerste/Kuils River catchment. Although estrogen spiked positive control water samples stimulated Vtg production in vitro as well as in vivo, no dramatic estrogenic activity was measured at any of the selected sites. Thirdly (Chapter 4), a bioassay using the thyroid controlled metamorphosis in Xenopus laevis tadpoles to assess effects on the thyroid hormonal system was implemented. Thyroid stimulatory activity, compared with a negative control sample, was measured at two sites along the catchment. Although the practical implementation of the tadpole semi-static exposure protocol (water replacement) proved to be labour intensive, all the added bioassays proved to be valuable tools to add valuable information regarding water quality. It is clear that more research related to anthropogenic influences along the Eerste/Kuils River catchment system are needed, specifically in monitoring monthly variations to better understand annual variation in several of the endpoints studied. / AFRIKAANSE OPSOMMING: Waterkwaliteit vorm die basis vir die evaluering en monitering van opvangsgebiede. Voortdurende stedelike ontwikkeling gee aanleiding tot ‘n toename in die voorkoms van besoedelstowwe in die natuurlike omgewing deur gelokaliseerde (punt) bronne (rioolwerke/industriële uitvloeisel) en/of nie gelokaliseerde (nie punt) bronne (vloed uitlaat/huishoudelike uitvloeisels) van besoedeling. Dit het onlangs aan die lig gekom dat van hierdie chemise besoedelstowwe subtiel die endokriene sisteem versteur en so aanleiding gee tot gesondheidsprobleme in terme van die voortplantingsisteem en tiroied sisteem (groei en ontwikkeling) by diere en moontlik ook die mens. Daar is beperkte gebruik van biologiese toetse wat inligting verskaf oor sitotoksiteit, inflammatoriese aktiwiteit en endokriene versteuring. Die doel van hierdie studie was dus om van hierdie biologiese toetse, wat normaalweg nie deel uitmaak van die roetine munisipale (Stad van Kaapstad) opvangsgebied monitering nie, gebruik te maak. Die Eerste-, Kuilsrivier, Wes Kaap, onder beheer van Stad Kaapstad is gekies en alhoewel die opvangsgebied nie water bydra tot drinkwaterbronne nie, word die opvangsgebied beïnvloed deur verskeie mensgemaakte bronne van besoedeling (afloop vanuit omliggende lanbougebiede). Binne die kort tydsraamwerk van die projek (ses maande) is besluit om twee maande, Julie (volg ‘n droë somer en herfs seisoen) en Oktober (volg ‘n nat winter en vroeë lente seisoen) vir water kwaliteit monitering te kies. Ruimtelike variasie langs die loop van die opvangsgebied is ingesluit deur moniteringspunte (n=10), met in ag name van die potensiële besoedelingsbronne. Spesifieke doelwitte van die projek sluit in: Eerstens (Hoofstuk 2), om die in vitro biotoetse, laktaat hidrolise (LDH) vir sitotoksisiteit, proinflammatoriese hormoon Interleuikin-6 (IL-6) vir inflammatoriese aktiwiteit, vrygestel deur menslike bloedselle en ‘n Salmonella ELISA vir ontlasting besoedeling saam met bestaande chemise en biologiese (hoofsaaklik migrobiologiese) veranderlikes te gebruik. Die studie het getoon dat beduidende variasie in alle mikrobiologiese toetse asook IL-6 vrystelling en Salmonella voorkoms bestaan het tussen versamelpunte. Maandelikse variasie in sekere van die veranderlikes het ook voorgekom. Tweedens (Hoofstuk 3), is twee biotoetse wat die dooiervoorloperproteïen, vitellogeen (Vtg) as eindpunt gebruik geimplimenteer in a) ‘n in vitro Xenopus laevis lewersnit biotoets (vyf dag blootstelling) en b) ‘n in vivo Zebravis (Danio rerio) biotoets (sewe dag blootstelling) om estrogenisiteit in die Eerste-, Kuilsrivier opvangsgebied te evalueer. Alhoewel, die estrogeen behandelde positiewe kontrole water monsters Vtg produksie veroorsaak het in beide die in vitro lewer-kulture en in vivo vistoets, is geen dramatiess estrogeniese aktiwiteit by enige van die moniteringspunte gevind nie. Derdens (Hoofstuk 4), is ‘n biotoets wat die tiroïedbeheerde metamorfose in Xenopus laevis paddavisse gebruik om effekte op die tiroïedsisteem te evalueer. Die differensiële stimulering (versnelling), in vergelyking met ‘n negatiewe kontrole watermonster, van die tiroied sisteem is by twee moniteringspunte in die opvangsgebied waargeneem. Alhoewel die praktiese implementering van die paddavis semi-statiese (water word gereeld vervang) biotoets arbeidsintensief is, het alle bykomstige biotoetse waardevolle bykomstige inligting oor water kwaliteit verskaf wat ‘n belangrike bydrae tot ingeligte bestuursbesluite kan verleen. Dit is duidelik dat meer navorsing int verband met die menslike invloed langs die Eerste-, Kuilsrivier opvangsgebied nodig is, veral met maandlikse monitering vir seisoenale veranderinge.

Indiana University-Purdue University Indianapolis (IUPUI) / Type I diabetes is an autoimmune disorder characterized by the loss of insulin producing islet cell mass. While daily insulin injection provides an easy means of glycemic control, it does not prevent long-term complications associated with diabetes. Islet transplantation has been suggested as a permanent cure for type 1 diabetes. However, the recurrence of host immunity and shortage of donor islets hinder the prevalence of islet transplantation. Biomaterial strategies provide an alternative route to solving the problems associated with host immune response and shortage of donor islets. One highly recognized platform for achieving these goals are hydrogels, which are hydrophilic crosslinked polymers with tissue-like elasticity and high permeability. Hydrogels prepared from poly(ethylene glycol) (PEG) derivatives are increasingly used for a variety of tissue engineering applications, including encapsulation of pancreatic islets and serving as a material platform for pseudo-islet differentiation. PEG hydrogels formed by mild and rapid thiol-ene photo-click reactions are particularly useful for studying cell behaviors in three-dimension (3D). Thiol-ene PEG-based hydrogels can be rendered biodegradable if appropriate macromer and cross-linker chemistry is employed. However, the influence of hydrogel matrix properties on the survival, growth, and morphogenesis of cells in 3D has not been fully evaluated. This thesis aims at using norbornene-functionalized PEG macromers to prepare thiol-ene hydrogels with various stiffness and degradability, from which to study the influence of hydrogel properties on pancreatic cell fate processes in 3D. Toward establishing an adaptable hydrogel platform for pancreatic tissue engineering, this thesis systematically studies the influence of hydrogel properties on encapsulated endocrine cells (e.g., MIN6 beta-cells) and exocrine cells (PANC-1 cells), as well as human mesenchymal stem cells (hMSC). It was found that thiol-ene photo-click hydrogels provide a cytocompatible environment for 3D culture of these cells. However, cell viability was negatively affected in hydrogels with higher cross-linking density. In contrast to a monolayer when cultured on a 2D surface, cells with epithelial characteristic formed clusters and cells with mesenchymal features retained single cell morphology in 3D. Although cells survived in all hydrogel formulations studied, the degree of proliferation, and the size and morphology of cell clusters formed in 3D were significantly influenced by hydrogel matrix compositions. For example: encapsulating cells in hydrogels formed by hydrolytically degradable macromer positively influenced cell survival indicated by increased proliferation. In addition, when cells were encapsulated in thiol-ene gels lacking cell-adhesive motifs, hydrolytic gel degradation promoted their survival and proliferation. Further, adjusting peptide crosslinker type and immobilized ECM-mimetic bioactive cues provide control over cell fate by determining whether observed cellular morphogenesis is cell-mediated or matrix-controlled. These fundamental studies have established PEG-peptide hydrogels formed by thiol-ene photo-click reaction as a suitable platform for pancreatic tissue engineering