Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2006. / Härtill 3 uppsatser.

Thesis (Ph.D.). / Written for the Dept. of Experimental Medicine. Title from title page of PDF (viewed 2008/01/12). Includes bibliographical references.

Thesis (MSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: The use of toxicity tests to evaluate the quality of streams affected by mixtures such as acid mine drainage (AMD), adds value to assessments whereby site-specific toxicological data may identify toxicants that pose a threat to humans. To successfully evaluate the risk of combined mixtures, an improved understanding of the individual components, their uptake, metabolism, excretion and mode of action is required. This study aimed to identify the extent of AMD toxicity in a dose dependant manner on the MCF-7 cell line. The first study site associated with gold mining was chosen as the Tweelopies Stream situated in the Gauteng province of South Africa. The AMD effluent (un-neutralised) contaminating the Tweelopies Stream had undergone pH-neutralisation using a reactor-bed limestone technology incorporating the use of both calcium carbonate (CaCO3) powder and limestone beds. The second study site, the Kromdraai River, is situated in the eMalahleni region of South Africa where a predominance of coal mining exists. The pH -neutralisation of the AMD (un-neutralised) contaminated Kromdraai River was performed using a caustic soda (NaOH) precipitation technique. This study demonstrated the rapid and effective application of the comet assay as a screening tool for AMD-associated DNA breakages in the human cell line, MCF-7. Moreover, the study analysed parameters of cellular survival, DNA fragmentation and variations in morphologies indicative of cellular death. Collectively, the cyto-genetic aberrations observed in the MCF-7 cells as a result of exposure to gold and coal mining associated AMD, confirms the urgency of incorporating high-throughput screening in ecological toxicity assessment to evaluate cellular damage at genetic levels in low dose exposures where detection might be missed. / AFRIKAANSE OPSOMMING: Die gebruik van toksisiteitstoetse om die gehalte van strome te evalueer wat geraak word deur mengsels soos suur mynwater (SM), gee waarde aan spesifieke toksikologiese data van gifstowwe wat 'n bedreiging vir die mens kan identifiseer. Om die risiko van gekombineerde mengsels en hul individuele komponente beter te begrip en suksesvol evalueer, is hul opname, metabolisme, uitskeiding en modus van aksie nodig. Hierdie studie het gepoog om die omvang van SM-toksisiteit in 'n dosis afhanklike wyse op die MCF-7-sellyn te identifiseer. Die eerste studie-area wat gekies is, hou verband met goudmyn-ontginning, en is die Tweelopiesspruit, geleë in die Gauteng-provinsie van Suid-Afrika. Die SM-uitvloeisel (on-geneutraliseerde) wat die Tweelopiesspruit besoedel, het pH-neutralisasie ondergaan met behulp van die integrasie van 'n reaktor-bed kalksorpsietegnologie wat gebruik maak van beide kalsiumkarbonaat (CaCO3) poeier en kalksteenbeddens. Die tweede studie-area, is die Kromdraairivier geleë in die eMalahleni-streek van Suid-Afrika, waar steenkoolontginning die oorheersende aktiwiteit is. Die pH-neutralisasie van die SM (on-geneutraliseerde) in die geval van die Kromdraairivier word met behulp van 'n bytsoda (NaOH) neerslag tegniek, uitgevoer. Hierdie studie het die komeet-toets getoon as 'n vinnige en doeltreffende toepassing vir SM-geassosieerde DNA-breekskade in die menslike sel lyn, MCF-7. Verder het die studie parameters van sellulêre oorlewing, DNA-fragmentasie en variasies in sel morfologieë wat ‘n aanduiding van sellulêre dood is, ontleed. Gesamentlik dui die resultate daarop dat die sitogenetiese afwykings wat in die MCF-7-selle waargeneem is, as 'n gevolg van blootstelling aan goud- en steenkool-geassosieerde SM is. Die studie het verder die dringendheid van die integrasie van hoë-deurset tegnologieë in ekologiese toksisiteitstoetse in selle wat genetiese skade mag ondergaan, na 'n lae dosis blootstelling waar opsporing dalk gemis word, ondersteun.

Phosphatidylinositol 5-phosphate (PtdIns5P) is the least well-characterised member of the phosphoinositide family of essential regulatory phospholipids. PtdIns5P levels are altered within cells in response to a number of stimuli and evidence is accumulating to suggest that it possesses important functions in cellular signalling. However, the physiological role of this lipid remains imperfectly understood. Previous studies have shown that PtdIns5P is elevated in adipocytes in response to insulin, and microinjection of PtdIns5P into these cells promotes plasma membrane insertion of the insulin-regulated glucose transporter GLUT4 (Sbrissa et al., 2004). This finding suggests a potential role of PtdIns5P as a mediator in insulin-stimulated glucose uptake, a process essential for efficient glucose homeostasis. As approximately 75% of postprandial glucose disposal is carried out by skeletal muscle, it is important to investigate the role of PtdIns5P in the response of this tissue to insulin. Therefore, this work has used differentiated myotubes of the rat muscle cell line, L6, to explore the effects of altered PtdIns5P levels on insulin-stimulated glucose uptake. This cell model had not been previously used in the laboratory so it first required characterisation. Here insulin is shown to stimulate a transient increase of PtdIns5P in L6 myotubes, indicative of a signalling role in response to insulin. This project developed several tools to further investigate this potential role for PtdIns5P in the insulin response of myotubes. One such development was the successful overexpression of the PtdIns5P 4-kinase PIP4KIIalpha in these cells, which was able to abolish the insulin-stimulated PtdIns5P rise. This correlated with a loss of insulin-stimulated glucose uptake (upon PIP4KIIalpha expression). Interestingly, artificial elevation of PtdIns5P in L6 myotubes increases glucose uptake in the absence of stimulation. This phenomenon appears to result from the activation of PI3-kinase signalling, as it is abolished by the PI3-kinase inhibitor wortmannin, and involves activation of the PI3-kinase effector Akt. These results are consistent with the idea that insulin-stimulated PtdIns5P production contributes to the robust PI3-kinase/Akt activation necessary for insulin-stimulated glucose uptake in muscle.

Colorectal cancer (CRC), which includes cancer of the large bowel and rectum is the third most common cancer in men and the second in women and there is a poorer survival rate in less developed regions of the world such as West Africa mainly due to the ‘out of reach’ costs of chemotherapy. Evidence suggests that aspirin, a non-steroidal anti-inflammatory drug (NSAID) has the potential to decrease incidence of, or mortality from, a number of cancers including CRC through several mechanisms of action. However, this evidence is dampened by aspirin’s gastrointestinal (GI) toxicity, which have been found to be mostly age-dependent. The search for potential aspirin-related compounds with the same or better cytotoxic effects against cancer cells accompanied by a safer toxicity profile has been ongoing over the years and led to us to synthesise a number of novel aspirin analogues. One of the mechanisms of action suggested for the anticancer property of aspirin is the COX-dependent pathway. In this thesis SW480 cell line, a CRC cell line that is COX-2 negative and mismatch repair (MMR) proficient was used to study the possible COX-independent mechanism of action for aspirin, its analogues and diflunisal at 0.5 mM. Diflunisal was included in this study because it is also a salicylate with reports of having cytotoxic effects. OE33 and FLO1 oesophageal cancer cells were also employed in the epidermal growth factor receptor (EGFR) and synergy experiments to show effects were not just specific to SW480 cells alone. These aspirin analogues were synthesised, identified using nuclear magnetic resonance (NMR) and infra-red (IR) spectroscopy, and tested for purity using thin layer chromatography (TLC) and melting point. The findings of this study suggest that these compounds breakdown into salicylates and perturb epidermal growth factor (EGF) internalization with PN517 (fumaryldiaspirin) and PN590 (ortho-thioaspirin) also driving EGF co-localization with early-endosome antigen-1 (EEA1). The perturbation of the internalization of EGF by aspirin and PN517 was also observed by a time-lapse assay using live confocal imaging. These compounds also had specific effects on different tyrosine phosphorylation sites of the EGFR, with none but PN590 inhibiting 4 phosphorylation at Y1068, and all but PN502 (ortho-aspirin), PN548 (meta-aspirin) and PN549 (para-aspirin) inhibiting phosphorylation at Y1045 and Y1173. Given that the EGF internalization assay involved the cells being treated with compounds for 2 h, cells were also treated for this same time period and probed with pEGFR 1045, which resulted in the compounds having no significant effect on phosphorylation at that site which is responsible for the ubiquitination of the EGFR. Most of these compounds were apoptotic with some showing a combination of apoptosis and necrosis. Aspirin and its isomers drove apoptotic cell death in SW480 cells via the BCL2-BAX pathway while the thioaspirins appear to follow the p21 pathway by decreasing the expression of the protein. In addition, it was shown that PN502 (aspirin), PN517 and PN590 had synergistic effects when used in combination with oxaliplatin at ED50, ED75 and ED90 in SW480 CRC cells. The cytotoxicity of these compounds individually or in combination was determined using MTT assay followed by the use of the CompuSyn and CalcuSyn software to calculate combination index (CI), which indicated whether a drug combination was synergistic, antagonistic or additive. PN517 and PN524 were synergistic when used in combination with cisplatin in OE33 oesophageal cancer cells. Effect of these compounds on the EGFR indicates a delay or disruption of the signalling pathway involved in the proliferation of cancer cells, thus, translating into protection against tumour formation or progression while the synergistic effects of these compounds when used in combination with platinum compounds can provide patients with less toxic chemotherapeutic regimen especially in patients with CRC tumours that harbour mutant TP53 gene and normally resistant to oxaliplatin. It is therefore proposed that the perturbation of EGF internalization is a novel mechanism of action for aspirin and its analogues in cancer therapy. These positive findings shed light on the understanding of the possible mechanism of action for aspirins and gives hope for a more affordable, less toxic therapy for the prevention, treatment and management of cancer.

Embryonic haematopoiesis is a complex process under intensive research. Murine definitive Haematopoietic Stem Cells (HSCs) originates from the Aorta-Gonad-Mesonephros (AGM) region of E10.5 embryo. It is thought that definitive HSCs arise from endothelial lining of dorsal aorta. However, detail of HSC specification in the developing embryo remains elusive. One way to deciphering events occurred during HSC specification is to derive cell lines from the developing HSC niche. Previous work by Oostendorp et al. showed the AGM and fetal liver derived lines could maintain HSCs in vitro (Oostendorp, Harvey et al. 2002). In this study, I established a more robust immortalization system using normal SV40 large T antigen delivered via Neon™ electroporation system. The new immortalization system achieved direct immortalization without going through crisis. And it is compatible with small number of primary cells dissected from different haematopoietic niches. With my new system, multiple cell lines from different haematopoietic sites at different developmental points are derived. Moreover, some of these lines demonstrated ability to mature precursors from E9.5 embryo (pro-HSCs) to definitive HSC without help of growth factors. This result is better compared to OP9 stromal lines. Such data proved usefulness of using stromal cell lines to study haematopoietic specification.

Made available in DSpace on 2014-06-11T19:31:10Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-05-31Bitstream added on 2014-06-13T18:41:52Z : No. of bitstreams: 1 duarte_ra_dr_arafcf.pdf: 4649782 bytes, checksum: 99bc00374a88b89ba36ff755fc732fa3 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Universidade Estadual Paulista (UNESP) / O câncer de mama é a maior causa de morbidade e mortalidade entre as mulheres no mundo. Pesquisas revelam vários fatores prognósticos e preditivos para a identificação de pacientes com alto risco de agressividade, metastases e doença recorrente na condição de combater estas estatísticas. Por esta razão, é evidente a necessidade do desenvolvimento de estratégias de tratamento mais eficazes. Estudos prévios com Pterogyne nitens Tul. (Fabaceae-Caesalpinioideae), uma planta nativa do Brasil resultou o isolamento de dois alcalóides guanidínicos. Exibiram atividade seletiva direcionada a DNA deficiente de reparo, sugerindo potencial atividade anticâncer. Objetivo: O objetivo do presente estudo foi avaliar a citotoxicidade e apoptose induzidas pelos alcalóides pteroginina (PGN) and pteroginidina (PGD) em linhagem de adenocarcinoma (MCF-7) e carcinoma ductal invasivo (ZR-7531). Materiais e Métodos: As duas linhagens celulares foram tratadas pelos alcalóides em várias concentrações (0.25 – 10 mM) em dois tempos, t0 (24h) e t24 (24h seguido por 24h pós-tratamento). O ensaio de citotoxicidade foi determinado pelo teste de MTT; a morte celular (apoptose e necrose) foi analisada usando os métodos Hoechst 33342/iodeto propídio, Kit de Anexina V-FITC e atividade de Caspases 3/7. Resultados: Os tratamentos com os alcalóides demonstraram citotoxicidade concentração-resposta nas linhagens de câncer de mama. Para avaliação da apoptose foi observado um intense efeito concentraçãoresposta em apoptose tardia/necrose e discreto sinal para apoptose precoce em todas concentrações (p<0,01). No ensaio Hoechst/iodeto, observou diferença significante entre os estágios de apoptose precoce e tardia de ambas linhagens. A pteroginina no período t0 e t24, e pteroginidina no período t0 demonstraram possuir intenso efeito concentração... / Breast cancer is a major cause of morbidity and mortality among women worldwide. Research has elucidated several specific prognostic and predictive factors to identify patients at high risk of the aggressive disease, metastasis and recurrence of the disease in order to combat these statistics. For this reason, there is an obvious need to develop more efficacious treatment strategies. Previous studies on Pterogyne nitens Tul. (Fabaceae-Caesalpinioideae), a native plant of Brazil, resulted in the isolation of two guanidine alkaloids, exhibited selective activity towards a DNA repair-deficient, suggesting potential anti-cancer activity. Objective: The aim of the present study was to evaluate the citotoxicity and apoptosis induced by alkaloids pterogynine (PGN) and pterogynidine (PGD) in human adenocarcinoma cell line (MCF-7) and human invasive ductal carcinoma cell line(ZR-7531). Material and Methods: The two cell lines were treated by both alkaloids at several concentrations (0.25 – 10 mM) and two time points, 24h (t0) and 24h followed by 24h pos-treatment (t24). The cytotoxicity assay was determined by MTT assay; the cell death (apoptosis and necrosis) were analyzed using the dye Hoechst 33342/propidium iodide, Annexin V-FITC and Caspase 3/7 activity. Results: The treatments with the alkaloids demonstrated citotoxicity effect concentrationresponse in breast cell lines. Apoptosis evaluation, pterogynine and pterogynidine has an intense effect concentration-response of late apoptosis/necrosis and a discrete signal of early apoptosis in all of the concentrations (p<0.01). Hoechst/iodide assay, it was observe significant difference among the stages of early and late apoptosis in the both cells lines. Pterogynine for the period of t0 and t24, and pterogynidine for the period of t0 demonstrated to possess an intense concentrationresponse... (Complete abstract click electronic access below)

O núcleo é uma estrutura organizada e possui verdadeiras organelas nucleares. Entre elas, estão os nucléolos e os corpúsculos de Cajal (CBs). Estes compartimentos nucleares são estruturas dinâmicas, mantidos pela associação de macromoléculas envolvidas na expressão gênica que interagem entre si delimitando-as. A principal proteína encontrada nos CBs é a p-80-coilin e, portanto, o principal epítopo capaz de marcar essas estruturas. Suas funções específicas ainda tem sido alvo de estudo. Existe uma proteína em comum a ambas as estruturas, a fibrilarina que participa no processamento de rRNA. Estes corpúsculos já foram descritos na periferia dos nucléolos ou mesmo fisicamente ligados a ele. Acredita-se que os corpúsculos de Cajal participem da síntese de rRNA, maturação, transporte e associação das subunidades ribossômicas.Diante esta relação, este trabalho visa estudar a inter-relação entre estas estruturas em células normais e as respectivas linhagens de células tumorais em cultura antes e após tratamentos com actinomicina D. Esta droga se usada em baixas concentrações, bloqueia a transcrição dos genes que foram decodificados pela RNA polimerase I e II, e -amanitin, por sua vez bloqueia a transcrição de genes decodificado pela RNA polimerase II . Além disso, também visa investigar uma relação entre a proliferação das linhagens estudadas e freqüência dos corpúsculos de Cajal nas células controle e tratadas. O microscópio confocal de varredura a laser permitiu o estudo dessas estruturas em preparações imunofluorescência fornecendo uma análise tridimensional destas estruturas quando utilizados anticorpos específicos. Linhagens de células que apresentaram um crescimento mais lento foram aquelas que tinham uma maior freqüência de corpúsculos por núcleo. Por outro lado, aquelas que apresentaram um crescimento mais intenso, foram aquelas que apresentaram maior variação no número de corpúsculos por núcleo. Após o tratamento com inibidores de síntese de RNA, tanto os corpúsculos de Cajal quanto os nucléolos, apresentaram alterações morfológicas, às vezes apresentando um grande acúmulo na região dos corpúsculos ou desorganizando os nucléolos. Mudanças no tamanho e forma também puderam ser destacadas. / The nucleus is a structure that has sub-compartments which can be called nuclear organelles. Among them, may be cited the nucleoli and the Cajal bodies (CBs). These nuclear compartments are dynamic structures, maintained by association and stock of macromolecules involved in gene expression. The main protein found in the CB is a p-80-coilin and therefore the main epitope able to label these structures. Their functions are still to be clarified. There is a protein in common to the nucleolus and Cajal bodies, the fibrillarin that takes part in the processing of rRNA. The CBs can be found at the periphery of the nucleoli or even physically connected to them. It is believed that the CBs may have role in the synthesis of rRNA and maturation, transport and association of ribosome subunits. In view of this relationship between Cajal bodies and nucleoli, this work aims to study the interrelationship between these structures in normal cells and their respective tumor cell line in culture before and after treatments with actinomycin D, which in low concentrations, blocks the transcription of genes that were decoded by the RNA polymerase I and II and -amanitin, which is responsible for blocking the transcription of genes decoded by the RNA polymerase II and find out a relationship between cell proliferation and Cajal bodies frequencies in control and treated cells. The confocal microscope of laser scanning enabled the study of these structures in preparations immunofluorescent providing a three-dimensional analysis of these structures when used specific antibodies before and after treatment. Cell lines that shown low cell grow, appears to have lass CB/ nucleus in the other hand, cell lines that have fastest grow shown nuclei with more Cajal bodies frequencies and more variation in the number of Cajal/nucleus After treatment with inhibitors, both Cajal bodies as nucleoli, made quite clear morphological changes, sometimes giving large accumulation of proteins in organelles and sometimes appeared disorganized in the nucleoplasm. Changes in the size and shape were also highlighted. The tumor cell lines also showed changes compared to their normal cell type.

Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2017-04-03T14:55:33Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) dissertação final_bbc.pdf: 741010 bytes, checksum: 2a9032403833869b2c43a2062da8fcd6 (MD5) / Made available in DSpace on 2017-04-03T14:55:33Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) dissertação final_bbc.pdf: 741010 bytes, checksum: 2a9032403833869b2c43a2062da8fcd6 (MD5) Previous issue date: 2015-09-02 / FACEPE / A Influenza é uma doença infecciosa aguda, causada por um vírus pertencente à família Orthomyxoviridae. As drogas antivirais e a vacinação são importantes no controle da disseminação da doença, porém alguns vírus adquirem resistência a certas drogas, alertando a necessidade de novas drogas. Triagem de antivirais através de ensaios biológicos são laboriosos e demorados. Com intuito de facilitar a triagem de drogas foram desenvolvidas duas linhagens celulares repórteres distintas. A linhagem Vero-Gluc-NS-Neo é específica para o vírus da influenza, expressa o gene Gaussia luciferase na presença do vírus, e foi desenvolvida através da tranfecção de células Vero com o plasmídeo pGluc-NSNeo. A segunda linhagem, denominada de A549-ISRE-Luc-Hygro, foi desenvolvida a partir da transfecção de células A549 com o plasmídeo pISRE-Luc-Hygro, o qual expressa o gene repórter Firefly luciferase na presença do interferon do tipo (IFN-I). Seguida da transfecção, ambas linhagens foram selecionadas e submetidas a uma clonagem biológica por diluição limitante e os clones selecionados foram então caracterizados quanto à sua especificidade e sensibilidade no ensaio. Resultados importantes e promissores foram obtidos com a linhagem A549-ISRE-Luc-Hygro, a qual se mostrou eficiente para a triagem de antivirais para influenza e drogas indutoras do IFN-I. Em relação à linhagem Vero-Gluc-NS-Neo, apesar do plasmídeo construído se mostrar funcional e específico, não foi possível observar a expressão do gene repórter após a infecção viral, trazendo à tona questionamentos e mostrando ser necessária a realização de ensaios complementares / Influenza is an acute infectious disease caused by viruses belonging to the Orthomyxoviridae family. Antiviral drugs are vital in controlling the spread of the disease, but some viruses become resistant to certain drugs, prompting the need for new drugs. Antiviral screening through biological tests are laborious and time consuming. In order to facilitate the screening of drugs, it was developed two distinct cell lineages reporters. The Vero-Gluc-Neo-NS cells line is specific for the influenza virus expresses the Gaussia luciferase gene in the presence of the virus, and it was developed by transfection of Vero cells with pGluc-NS-Neo plasmid. The second cell line, A549-called ISRE-Luc-Hygro, was developed from the transfection of A549 cells with pISRE-Hygro-Luc plasmid, which expresses the Firefly luciferase reporter gene in the presence of type one interferon (IFN-I). Followed by transfection, both cell lines were selected and subjected to a biological cloning by limiting dilution and selected clones were then characterized for specificity and sensitivity in the assay. Important and promising results were obtained with A549-Hygro-ISRE-Luc cells, which proved to be efficient for screening of antiviral drugs for influenza and IFN-I inducing drugs. Regarding the Vero-Gluc-NS-Neo cell line, despite the plasmid constructed to show functional and specific, it was not possible to observe the reporter gene expression after viral infection, bringing up questions and shown to be necessary to carry out further testing.