O presente estudo trata das atitudes morais de atletas juvenis nos Jogos Coletivos de Invasão (JCI): futebol de campo, futsal, basquetebol e handebol. Um dos objetivos desta pesquisa é (a) identificar qual das dimensões atitudinais – empenho, convenção, antidesportivismo ou trapaça – representa as ações mais assumidas declaradamente pelos jovens atletas, de 13 a 16 anos, dos JCI. Os demais objetivos são os seguintes: (b) traduzir e adaptar para a língua portuguesa (Brasil) o Questionário de atitudes no esporte (QAE-23) de Gonçalves et al. (2006); (c) proceder à validade de conteúdo QAE-23; (d) apresentar a ordem hierárquica das dimensões atitudinais, através da análise intrassexo; (e) realizar um estudo comparativo intersexo. Para tanto, foi aplicado o QAE-23. As respostas aos itens do QAE-23 foram dadas conforme uma escala de tipo Likert, bidirecional, graduada em cinco pontos, partindo de “discordo firmemente da declaração” (1) a “concordo firmemente com a declaração” (5). A pesquisa contou com a participação de um grupo de 265 atletas. Para cumprir com o segundo objetivo foram realizadas duas traduções independentes por profissionais da área (inglêsportuguês e português-inglês), que asseguraram a melhor tradução e adaptação do QAE-23 para a língua portuguesa (Brasil). Em seguida, procederam-se os estudos sobre a pertinência e a clareza dos itens do QAE-23, a partir dos dados obtidos nas respostas dos juízesavaliadores. Os resultados dos Coeficientes de validade de conteúdo (CVC), tanto para a pertinência como para a clareza dos itens (entre 0,7 e 0,8 em quatro itens; e superior a 0,8 em 19 itens), asseguraram a validação do conteúdo do QAE-23. Após o cumprimento dessas etapas, o QAE-23 foi submetido às respostas dos 265 atletas para cumprir com os demais objetivos do estudo. As atitudes mais assumidas declaradamente pelos atletas, de maneira geral fazem parte da dimensão empenho, seguida, respectivamente, pela convenção (2º), antidesportivismo (3º), e trapaça (4º). Se considerarmos apenas as respostas dos atletas do sexo masculino, os resultados foram os seguintes: empenho ( X = 23,23); seguido por um par de dimensões indissociáveis estatisticamente, convenção (X = 19,89) e antidesportivismo ( X = 19,11); e trapaça (X = 16,96). Para as atletas do sexo feminino aparecem: convenção ( X = 23,21) e empenho ( X = 22,95), indissociáveis estatisticamente, seguidas pelo antidesportivismo ( X = 17,50) e trapaça ( X = 9,85). A comparação inter-sexo mostrou que a dimensão trapaça está mais evidente no sexo masculino, enquanto que a dimensão convenção está mais evidente no sexo feminino, conforme verificação do valor das médias nos dois grupos. Através dos resultados foi possível concluir que os atletas da referida faixa etária buscam se empenhar ao máximo para obter rendimento durante os treinamentos e competições. Além disso, destaca-se a forma evidente como se expressa a competição no sexo masculino, em razão do destaque dado à dimensão trapaça. Já para as atletas do sexo feminino, a sociabilidade parece ser um aspecto determinante, em razão do destaque dado para as atitudes relacionadas à dimensão convenção. Espera-se que esse estudo possa contribuir para um plano de atividades que priorize o desenvolvimento moral dos atletas. / The present study deals with the moral attitudes of the athletes of the Invasion Collectives’ Games (ICG): soccer, futsal, basketball and handball. One of the aims of the research is (a) to identify wich dimensions of attitude – commitment, convention, gamesmanship or cheating – represents the actions reportedly taken over by young athletes from ICG from 13 to 16 years. The others objectives are: (b) to translate and adapt to Portuguese language (Brasil) the Sports attitudes questionnaire (SAQ-23) from Gonçalves et al. (2006); (c) make the contend validity of QAE-23; (d) present the hierarchical order of the attitudinal dimensions, through the intrasex analysis; (e) realize a inter-sex comparative study. To this end, was applied the (SAQ-23). The answers to the items on QAE-23 were given as a Likert scale, bidirectional, graduated in five points, from “strongly disagree the statement” (1) to “strongly agree with the statement” (5). The research counted with the participation of a group of 265 athletes. To meet the second objective, two independent translations were done by professionals of the area (English/Portuguese and Portuguese/English) wich ensured the best translation and adaptation of QAE-23 for the Portuguese language (Brasil). Then were conducted the studies on pertinence and clarity of the items of QAE-23, from data obtained in the answers of judgesevaluators. The results of the Contend validity coefficients (CVC) for both, the pertinence as to the clarity of the items (between 0,7 and 0,8 on four items, and more than 0,8 in nineteen items), ensured the validation of content of QAE-23. After the accomplishment of these steps, the QAE-23 was submitted to the answers of 265 athletes to comply with the others objectives of the study. The attitudes more reportedly taken by athletes in general are part of the dimension commitment, followed respectively by the convention (2), gamemanship (3) and cheating (4). If we consider only the answers of male athletes, the results were as follows: commitment (23,23), followed by a couple of dimensions that are statistically indissociable, convention (19,89) and gamesmanship (19,11), and cheating (16,96). For female athletes appear: convention (23,21) and commitment (22,95), that are statistically indissociable, followed by gamesmanship (17,50) and cheating (9,85). The inter-sex comparison showed that the dimension cheating is most evident in males, whereas the dimension convention is most evident in females, as verification of the value of the averages in both groups. Through the results it was concluded that the athletes in this age group seek to engage for maximum performance during training and competitions. Furthermore, it highlight the latent form expressed by the competition among males, due to the emphasis on dimension cheating. As for the females athletes, sociability appears to be a crucial aspect, because of the emphasis given to the attitudes toward dimension convention. It is hoped that this study may contribute to an activity plan that give priority to the moral development of young athletes.

Anêmonas-do-mar do gênero Diadumene Stephenson, 1920 foram encontradas em grande abundância fixadas sobre substratos naturais e estruturas artificiais na Baía de Paranaguá. Em 1989, em trabalho realizado no mesmo local, a quantidade de anêmonas deste gênero, não identificadas em nível de espécie, era baixa. Sua grande abundância atual pode ser explicada por sua alta taxa de reprodução assexuada, por fissão longitudinal, um dos fatores que poderiam caracterizar essas anêmonas como sendo uma espécie exótica e invasora. A elucidação dos aspectos sistemáticos dessas anêmonas e a compreensão de alguns de suas características biológicas são essenciais para definir o status da espécie na costa brasileira. Para isso, foram realizadas cinco (5) coletas no Iate Clube de Paranaguá, entre agosto de 2009 e agosto de 2010. Em cada coleta, aproximadamente 200 espécimes foram coletados e levados ao laboratório para serem submetidos aos estudos taxonômicos ou aos experimentos de resposta a parâmetros ambientais. A definição da espécie foi realizada com base em ferramentas taxonômicas comumente utilizadas para a ordem Actiniaria (morfologia externa, interna, elementos musculares e cnidoma) e através do seqüenciamento do gene mitocondrial 16s rRNA. Além disso, foi testada a tolerância da espécie a diferentes salinidades, à dessecação e a preferência por locais iluminados, sendo que os dois primeiros testes revelam se existe uma adaptabilidade característica de espécies invasoras, e o último sua capacidade de colonizar um local de águas calmas, mas onde há grande interferência humana por ser um porto movimentado A análise morfológica mostrou que a espécie de Paranaguá apresentava diferenças, segundo a literatura, em relação a todas as demais espécies do gênero Diadumene, sendo os caracteres responsáveis pela diferença: presença de cínclides na coluna dispostas em fileiras longitudinais, presença de nematocistos do tipo macrobásico p-amastigóforo nos tentáculos e nematocistos do tipo microbásico p-amastigóforo nos acôncios. No entanto, a análise molecular mostrou que a população da Baía de Paranaguá provavelmente é oriunda de uma população da espécie Diadumene cincta Stephenson, 1925. Nos experimentos, notou-se uma grande tolerância da espécie aos diversos fatores ambientais, mostrando que a espécie pode apresentar a plasticidade fenotípica característica de espécies invasoras. D. cincta já apresenta registros na literatura que a caracterizam como espécie invasora nos mares europeus e na costa atlântica européia, e acredita-se que a sua introdução na Baía de Paranaguá tenha se dado através de incrustação em cascos de navios, já que a região onde a anêmona foi encontrada é próxima ao Porto de Paranaguá. O monitoramento das espécies exóticas encontradas na costa brasileira é de grande importância, já que estas podem vir a causar impactos no ambiente ou nas atividades socioeconômicas que afetam o ser humano. Além disso, é imprescindível que sejam realizados mais estudos sobre as características das populações das espécies marinhas introduzidas no local, inclusive D. cincta / A great number of sea anemones of the genus Diadumene Stephenson, 1920 were found fixated on natural and artificial substrates at the Paranaguá Bay. In 1989, in a study conducted at the same location, the number of anemones of this genus, not identified to a specific level, was much lower. The great abundance of anemones that has currently been observed may be explained by the population\'s high rate of asexual reproduction, by longitudinal fission, one of the factors that may characterize these anemones as an exotic and invasive species. The elucidation of the systematic aspects of these anemones and the understanding of some of their biological characteristics are essential for defining the status of this species along the Brazilian coast. For this purpose, approximately 200 specimens were collected in five different dates, between August 2009 and August 2010, at the Iate Club of Paranaguá. The specimens were taken to the laboratory and submitted to taxonomical studies and experiments of response to environmental parameters. The definition of the species was based on the taxonomical features usually used for the order Actiniaria (external and internal morphology, muscle elements and cnidoma) and on the sequencing of the mitochondrial 16S rRNA gene. The species\' tolerance to different salinities and to air exposure was tested, as was it\'s preference for illuminated places. The first two tests would reveal if the sea anemone has an adaptability that is characteristic of invasive species, while the latter would test its ability to colonize calm waters with great human interference (due to the proximity to the Paranaguá Port). The morphological analysis revealed that the species from Paranaguá differed from the other species of the genus, because of the presence of the following characteristics: cinclids on the capitulum in longitudinal rows, macrobasic p-amastigophores in the tentacles and microbasic amastigophores in the acontia. However, molecular analysis showed that population of Paranaguá was probably originated from a population of the species Diadumene cincta Stephenson, 1925. The experiments showed that the species has a high tolerance to the tested environmental factors, showing that it may present the phenotypic plasticity that is characteristic of invasive species. D. cincta is already registered as an invasive species in European seas and the Atlantic coast of Europe, and it is believed that its introduction in the Paranaguá Bay was by fouling on shiphulls, as the species was found in a location near the Port of Paranaguá. The monitoring of alien species found along the Brazilian coast is very important, since these species may cause impacts on the environment or on human socio-economic activities. Moreover, it is necessary to conduct more studies on the characteristics of populations of marine species introduced in the Paranaguá Bay, including D. cincta

Abstract Bladder cancer when superficial has a good prognosis but it has a high recurrence risk and about 10–15% of the superficial carcinomas will progress into muscle invasive or metastatic type. The most powerful factor for predicting the behavior of bladder carcinoma is the stage of the tumor. Invasion to the lamina propria increases the risk of recurrence and progress to muscle-invasive tumor. Also grade of the tumor and tumor multiplicity associates with high risk for recurrence. New markers are still needed to find those patients who need more and better treatments to avoid the recurrence and progress. The need for new non-invasive markers to diminish the need for frequent cystoscopy in follow-up is also obvious. Gelatinases MMP-2 and MMP-9 are known to associate to tumor invasion and progression. Also their tissue inhibitors TIMP-1 and TIMP-2 take part in these diversified processes and metastasis formation. In the present work the expression and clinical value of gelatinases MMP-2 and MMP-9 and their tissue inhibitors TIMP-1 and TIMP-2 were evaluated in bladder carcinoma. Primary tissue samples of 121 patients were analyzed for expression of MMP-2 and/or MMP-9 using immunohistochemistry. The serum samples of 87 patients who were treated in the Oncology Department of Oulu University Hospital were collected and studied with ELISA. The control group consisted of 44 healthy volunteers. Overexperssion of MMP-2 protein correlated significantly to disease-specific survival and showed an independent prognostic value as a biomarker. High MMP-9 expression instead correlated to favorable overall survival of bladder cancer patients. Circulating proMMP-2, TIMP-2 and MMP-2:TIMP-2 complex levels were lower in cancer patients than in healthy volunteers in control group. High levels of all these three markers correlated with better prognosis in bladder cancer patients.

Ovarian cancer (OC) accounts for 4% of all cancer cases and 4.2% of all cancer deaths worldwide. OC is the most lethal gynecological cancer because it lacks early disease symptoms and does not have a specific diagnostic marker. As a result, more than 70% of OC patients are diagnosed in later stages when the disease has already metastasized and the 5-year survival rate has decreased to less than 20% compared with approximately 90% survival for women diagnosed with early stage disease. Therefore, I initiated my studies with a computational analysis of the 27 most commonly reported literature-derived ovarian cancer (LDOC) protein biomarkers. I found that LDOC protein biomarkers share many biochemical features including a preponderance for a stable protein structure, the ability to be secreted, and functionality related to extracellular matrix (ECM) modification, immune response and/or energy production. Subsequently, I analyzed the human proteome to identify proteins that also share these biochemical features. Of the 70,616 proteins in the human proteome, 683 proteins were found to have similar biochemical features to the 27 LDOC proteins. I also identified a subset of 21 potential additional protein regulators of ovarian cancer (APROC) that interact with LDOCs. Three of the APROCs identified were amylase proteins AMY1A, AMY2A, and AMY2B which cleaves alpha 1, 4-glycosidic bonds in polysaccharides. Amylase is reportedly overexpressed in and secreted by ovarian tumors but its functional contribution to OC remains unknown[1]. In this thesis, I posit that amylase contributes to OC invasion. I initiated my studies by computational characterizing the different amylase isozymes to predict which amylase isozyme(s) is most likely overexpressed in and contributory to OC invasion. I found that AMY1 and AMY2B have unique regions of disorder and unique phosphorylation sites indicating that AMY1 and AMY2B would be more likely to interact with other proteins, and to be easily secreted. Using OC patient serum samples, I was able to validate AMY1 and AMY2B overexpression by western immunoblotting. I then developed an in vitro model system to study the molecular contribution of amylase to OC invasion using normal ovarian surface epithelial (IOSE) and OC cell lines. I showed that OC cells generally overexpress and secrete metabolically active amylase isozymes AMY1 and AMY2B. Abrogating amylase activity using siRNA silencing technology decreased the capacity of OC cells to invade collagen coated Boyden chambers and increased sulfated glycosaminoglycans (sGAG) production. Since a survey of OC cell lines indicated that cancer cells have a bulkier glycocalyx compared to IOSE cells and immunogold labeling studies indicated the presence of amylase within the immediate OC microenvironment, my data suggest that, by cleaving alpha 1, 4-glycosidic bonds in glycoconjugates present within ECM, amylase may remodel the ECM to promote an invasive cancer phenotype. Amylase is therefore a target for therapeutic intervention in OC patients with hyperamylasemia. I established Spirulina, a dietary supplement, as a novel transcriptional inhibitor of amylase. Spirulina inhibited amylase expression in OC cell lines at both the message and protein levels. Spirulina reduced OC cell invasion and migration in vitro, putatively by decreasing amylase expression.

Abstract Tumor invasion and metastasis are the major causes of treatment failure or death for carcinoma patients. Matrix metalloproteinases (MMPs) are zinc dependent endopeptidases implicated in tumor invasion and metastasis. The expression of MMP-2 has been previously linked to invasiveness of carcinoma cells. The MMP-2 immunoreactive protein was studied here in squamous cell carcinoma of the utrine cervix and in adenocarcinoma of the breast by using a specific monoclonal antibody in immunohistochemical stainings. Immunoreactive protein of latent MMP-2 was found to be an early event in neoplastic transformation of the cervix in 60 patients. All cases of early stage cervical carcinoma expressed the latent MMP-2 protein, suggesting that MMP-2 could be a prerequisite for invasive behavior. In early stage cervical carcinoma the high score of MMP-2 expression seemed to be associated with poor histological differentiation and lymph node metastases. The intensitivity (score) of the immunoreaction was not, however, associated with clinical behavior of this disease. New predictive markers would be useful in selecting breast carcinoma patients to different modalities of adjuvant therapy. The MMP-2 protein has been found in breast carcinoma tumor cells in immunohistochemical analyses. MMP-2 has been found to be expressed in breast carcinoma in some preliminary studies, but there are no reports so far that would show a correlation of MMP-2 to survival in breast carcinoma. In the current study comprising 373 patients the expression of MMP-2 protein was found immunohistochemically in primary breast carcinomas. It is shown here for the first time that immunoreactive protein of MMP-2 in primary breast carcinoma is associated with a shortened relapse-free survival (RFS) or relative overall survival (OS). MMP-2 correlated to the risk of failure during the anti-estrogen adjuvant therapy in postmenopausal breast carcinoma patients with axillary lymph node metastasis without a high tumor burden. It was also found here that premenopausal patients with a node positive breast carcinoma showing MMP-2 positivity relapsed early after the primary operation. Young patients (< 40 years) with MMP-2 positive tumors had a poor outcome when compared to other node-positive premenopausal breast carcinoma patients. A patient group with a high risk for an early relapse was identified from node-positive, premenopausal breast carcinoma patients. In conclusion, the present data show for the first time MMP-2 immunoreactive protein to be a prognostic factor in breast carcinoma, indicating further studies to explore the value of this enzyme in clinical decision making.

Abstract Traditional clinicopathological factors are not accurate enough to predict the behavior of head and neck squamous cell carcinoma (HNSCC). The most powerful indicator of prognosis is the stage of the disease. New prognostic markers have, however, been searched for in order to better identify patient groups in need of different treatments or follow-up. Gelatinases (MMP-2, -9) are endopeptidases associated with tumor invasion and angiogenesis, and their tissue inhibitors (TIMP-1, -2) are also linked to cancer cell invasion and metastasis formation. In some cancer types they are even prognostic and relate with a more aggressive clinical course of the disease. In the present work the expression and the clinical significance of tumor tissue and circulating immunoreactive proteins for MMP-2, -9, TIMP-1 and -2 were assessed in HNSCC. The study group included 74 patients with HNSCC and 44 healthy controls. The expression of immunoreactive proteins was examined in paraffin-embedded tumor sections by immunohistochemical staining using specific antibodies, and the pretreatment serum levels of those proteins were quantitatively measured by ELISA assay. Immunohistochemical overexpression of MMP-9 in tumor was for the first time found to predict the prognosis for shortened survival in HNSCC, the cause-specific survival rates being 45% and 92% and relapse-free survival being 42% and 79% in MMP-9 positive or negative cases, respectively. Additionally, tissue TIMP-1, MMP-2 and TIMP-2 positivity were all also linked with poorer survival of patients with HNSCC. However, these differences remained less distinct than with MMP-9. The expression of gelatinases and their inhibitors in tumor tissue was also an indicator for later lymph node or hematogenic relapses in HNSCC patients. Circulating MMP-9 and TIMP-1 levels were significantly higher in HNSCC patients than in healthy controls. Further, the cause-specific and relapse-free survival rates were lower among HNSCC patients with high MMP-9 and TIMP-1 serum levels compared to patients with low levels of circulating MMP-9 and TIMP-1. A significant correlation was shown between circulating MMP-9 and MMP-9 immunohistochemical staining in the corresponding tumors. No correlation was found between tissue or circulating levels of gelatinases or their inhibitors and the traditional clinical or histopathological factors, except for the association between tissue and circulating TIMP-1 and the size of the primary tumor. Taken together, these results suggest that tissue expression of gelatinases and their inhibitors as well as pretreatment circulating MMP-9 and TIMP-1 levels could be prognostic in estimation of the clinical course of HNSCC. The results indicate further studies are needed with larger patient materials.

The global movement of aquatic non-indigenous species can have severe ecological, environmental and economic impacts emphasizing the need to identify potential invaders and transport pathways. Initial transport is arguably the most important stage of the invasion process owing to its role in selectively determining potential invasion candidates. This study characterizes a well defined human-mediated dispersal mechanism, ballast water transport, as a vector for the introduction of non-indigenous zooplankton. Ballast water exchange in the open ocean is the most widely adopted practice for reducing the threat of aquatic invasions and is mandatory for most foreign vessels intending to release ballast in Canadian waters. Ships entering Canadian ports are categorized into the following three shipping classes based on current regulations: overseas vessels carrying exchanged ballast water, intra-coastal vessels carrying exchanged ballast water or intra-coastal vessels carrying un-exchanged ballast water. This study characterizes zooplankton communities associated with each of these shipping classes sampled from ports on Canada’s Pacific coast, Atlantic coast and the Great Lakes Basin. Ballast water samples were collected and analyzed from 77 vessels between 2006 - 2007. The ballast water environment was found to be diverse, with over 193 zooplankton taxa, 71 of which were non-indigenous to their receiving environments. Intracoastal vessels containing un-exchanged coastal water transported the greatest density of non-indigenous zooplankton into Canadian ports. Total zooplankton density was found to be negatively correlated with ballast water age The absence of mandatory ballast water exchange and the younger ballast water age of coastal un-exchanged vessels is likely responsible for the higher density of non-indigenous zooplankton in intracoastal un-exchanged vessels. Propagule pressure, invasion history and environmental suitability are all useful in evaluating invasion potential and all suggest that intracoastal un-exchanged vessels pose the greatest invasion threat to Canadian aquatic ecosystems. In conclusion, although the risk of primary introductions from overseas ports may have been reduced through open-ocean exchange of ballast water, secondary introductions from previously invaded ports in North America may be the primary threat to Canadian aquatic ecosystems via this transport vector. / Science, Faculty of / Earth, Ocean and Atmospheric Sciences, Department of / Graduate

Obese pregnant women have increased risk of a number of pregnancy complications, including poor maternal health, fetal growth restriction (FGR) and fetal demise. The success of pregnancy is dependent on precise regulation of the immune response within the utero-placental environment. Rats as a model for human related pregnancy complications are beginning to be widely used because of the similarities between these species in terms of trophoblast invasion and spiral artery remodeling. However our knowledge of immune cells and cytokine localization in the rat utero-placental tissue relating to these processes is limited. Therefore our first aim was to characterize the immune cell populations, such as uterine natural killer (uNK) cells, neutrophils and macrophages in the rat utero-placental unit at two crucial gestational ages relevant to trophoblast invasion and spiral artery remodeling, gestational day (GD) 15 and GD18. In addition, we characterized the cytokine distribution of TNFα, IFNγ and IL-10 in the utero-placental tissue at both above mentioned gestational ages. Our study has demonstrated co-localization of TNFα and IFNγ with uNK cells in the perivascular region of the spiral arteries in the rat mesometrial triangle. Neutrophils were localized at the maternal fetal interface and in the spiral artery lumen of the rat mesometrial triangle at both gestational ages. TNFα and IL-10 demonstrated a temporal change in the localization from GD15 to GD18 which coincides with the leading edge of trophoblast invasion into the mesometrial triangle. The results of the current study furthers our knowledge of the localization and temporal expression of uterine immune cells and relevant cytokines, and provides a base to research the function of these immune cells and cytokines during rat pregnancy as a model to study human pregnancy and complications related to immune functions. Since obesity is associated with a peripheral and systemic pro-inflammatory state in humans, our second objective was to investigate whether maternal obesity could alter the utero-placental and systemic immune response in the rats. To characterize maternal obesity induced changes in uterine immune state we used pregnant rats fed a control diet (normal weight; CD) or a high fat diet (obese; HFD) at GD15 and GD18. We performed immunohistochemistry to localize TNFα and IL-10, and quantified the levels of TNFα, IL-1β and IL-10 in the uterine tissue by immunoassay. To assess the systemic immune state, circulating levels of pro-inflammatory cytokine MCP-1 were assessed by immunoassay. We demonstrated an increased concentration of the pro-inflammatory marker TNFα and a reduced anti-inflammatory IL-10-positive cell distribution in the rat mesometrial triangle in response to a HFD. In addition increased circulating MCP-1 was observed in the HFD-fed dams at both gestation ages. HFD induced obesity in our rat model leads to an increase in uterine and systemic pro-inflammatory markers. These markers have demonstrated the potential to alter utero-placental development. Pregnancy complications such as FGR and fetal demise have been shown to be associated with impaired placental development as a result of altered trophoblast invasion and aberrant maternal spiral artery remodeling. Therefore, our third aim was to compare these parameters between the CD-fed rats and HFD-fed rats at GD15 and GD18. Early trophoblast invasion was increased by approximately 2-fold in HFD-fed dams with a concomitant increase in the expression of matrix metalloproteinase-9 protein, a mediator of tissue remodeling and invasion. By late gestation reduced trophoblast invasion was observed in HFD-fed dams. Furthermore, we also observed in late gestation significantly higher levels of smooth muscle actin surrounding the uterine spiral arteries of HFD-fed dams, suggesting impaired spiral artery remodeling. We also determined the impact of human serum from obese mothers on trophoblast invasion. We compared the invasion of HTR-8/SVneo cells treated with pooled first-trimester serum from obese women with or without fetal growth restriction vs. cells treated with serum from normal-weight women with or without fetal growth restriction. First-trimester serum from obese pregnant women reduced invasion of the trophoblast cell line HTR8/SVneo compared to serum from normal-weight pregnant women. Taken together, the results of this study suggest that maternal obesity can negatively influence crucial utero-placental development processes resulting in the poor pregnancy outcomes and increased fetal demise. To summarize, the HFD increased the pro-inflammatory marker TNFα which was associated with altered trophoblast invasion profiles and impaired vascular remodeling. These disturbances in utero-placental development were also associated with decreased birth weights (indication of FGR) and increased rates of stillbirths in our obese rat model. In conclusion, we have made progress in defining the influence of maternal obesity (HFD) on utero-placental development. The importance of these studies is evident since FGR represents a leading cause of perinatal morbidity and mortality. Furthermore, FGR fetuses have an increased risk of becoming obese in their lifetime as a result of fetal programming, therefore resulting in the propagation of a transgenerational obesity cycle. Therefore by understanding the mechanisms by which maternal obesity influences utero-placental development leading to FGR, we may be able to impact short term morbidity and prevent the programming of obesity in future generations. In addition, characterization of maternal obesity’s influence on utero-placental development will also help in the search for therapeutics or intervention strategies to help optimize fetal growth and improve pregnancy outcomes in obese women.

Invasive lobular carcinoma (ILC) is the second most common form of breast cancer. ILC presents at later stages with many challenges, therefore improved diagnostic and therapeutic targets are needed. A microRNA (miRNA) genome analysis identified miR-23c and miR-23b-3p as possible regulators of ILC invasion due to their significantly increased expression in invasive compared to minimally invasive ILC cell lines. By decreasing the levels of miR-23c and miR-23b-3p using hairpin inhibitors, the invasive MDA-MB-330 cell line had significantly reduced invasion, while overexpressing these miRNAs using mimics in the minimally invasive MDA-MB-134VI cell line increased invasion. During the course of this study, it became apparent that limited tools exist for studying invasive ILC. Therefore, two more invasive ILC cell line models were created by isolating and expanding MDA-MB-134VI cells that had invaded through Matrigel® coated invasion chambers. This thesis has thus created new models of invasive ILC as well as identified miR-23c and miR-23b-3p as regulators of MDA-MB-330 and MDA-MB-134VI cell line invasion.

Malaria is a global pandemic that affects millions of people each year. It is a parasitic infection caused by the Plasmodium family, with Plasmodium falciparum being the most virulent strain. Malaria is transmitted to humans by the female Anopheles mosquito. The parasite undergoes two different cycles of its life cycle within the human host: the liver and intraerythrocytic life cycle. The latter consists of an asexual and sexual cycle. The intraerythrocytic cycle is perhaps the most important stage of the parasite's life cycle as it promotes the spread of the disease within and between hosts. The focus of this investigation was aimed at the invasion process of the merozoites into the erythrocytes. The Plasmodium merozoite utilises a cascade of proteins during the erythrocyte invasion process, which is a swift action that takes place in approximately 30 seconds. A number of surface proteins are expressed during merozoite development and are distributed along the merozoite surfaces to assist with attachment and invasion, the most crucial being MSP-1, AMA-1 and RON-2. MSP-1 and AMA-1 are vital targets for the development of malaria vaccines. AMA-1 is the central target protein of this investigation as it plays an essential role in the invasion process. AMA-1 commits the merozoite to invade the erythrocyte, as it assists the RON proteins in the formation of an irreversible tight-junction with the membrane of the erythrocyte. Antibodies, specific to AMA-1, bind to the protein, which prevents the formation of the tight junction and inhibits the invasion of the merozoite into the erythrocyte, therefore preventing the spread of the disease. However, before invasion, AMA-1 undergoes a number of proteolytic processes. It is synthesized as an 83 kDa (AMA-183) precursor protein in the apical organelle of the merozoite. This is then cleaved at the N-terminus to give rise to a 66 kDa (AMA-166) fragment, which is secreted onto the surface of the merozoite. The AMA-166 fragment is then cleaved into either a 48 kDa (AMA-148) or 44 kDa (AMA-144) fragment. One of these three fragments is then used by the merozoite for erythrocyte invasion. The aim of this investigation was to isolate and characterise each of the fragments of the Plasmodium falciparum AMA-1 (PfAMA-1) protein using the 3D7 lab strain of P. falciparum and to visualise the merozoite-erythrocyte invasion process, to possibly identify which of the AMA-1 fragments are involved in the invasion process. In order to achieve this large clusters of merozoites from sorbitol-synchronised cultures were isolated. Schizonts were isolated from culture by magnetic separation and incubated with E64 to prevent the release of merozoites. Merozoites that were required for the isolation of PfAMA-1 were harvested from the schizonts by saponin lysis, then homogenised, separated by SDS-PAGE and digested for LC-MS/MS analysis. Merozoites that were required for the visualisation procedures were not incubated with E64, to allow natural egression from the erythrocyte. The transmission electron microscopy results produced clear images of the merozoiteerythrocyte invasion process and the positioning of PfAMA-1 on the merozoite, before and after schizont rupture, was visualised from results obtained from confocal microscopy. Then PfAMA-1 was identified in isolated merozoite samples by LC-MS/MS analysis. However, due to its low abundance, isolation of high enough concentrations of PfAMA-1 to characterise its different fragments was not achieved. Further investigation into the development of the culturing and isolating methods could help in future projects aimed at isolating higher concentrations of merozoite proteins from synchronised cultures with a lower merozoite egression window period, in order to accomplish detailed analysis on invading proteins for the future development of treatments against malaria. / Dissertation (MSc)--University of Pretoria, 2016. / Pharmacology / MSc / Unrestricted