Endometrial adenocarcinoma (EAC) is the most frequently diagnosed gynecological cancer of the female genital tract in the Western world. Research studies in EC is difficult to conduct on human tumor samples due to the complex nature of tumor arousal and genetic heterogeneousness in the human population. Therefore, inbred animal models can be promising tools to use in EC research due to similar histopathology and pathogenesis as humans. Studies performed on MAP2K1 and CCND1 has shown that their altered expression play a crucial role in carcinogenesis. CCND1 has been demonstrated to have oncogenic properties when overexpressed in human neoplasias. The aim of this study is to investigate gene expression levels of MAP2K1 and CCND1 in BDII rat model of endometrial adenocarcinoma cells. Quantitative real-time PCR was used to analyze expression levels of MAP2K1 and CCND1 genes in BDII/Han rat model of endometrial cancer cells using TaqMan approach. The differences in gene expression levels of MAP2K1 and CCND1 between pathologically EAC malignant and nonmalignant cells showed an upregulation of MAP2K1 and CCND1 in EAC malignant cells. The analyzed data presented observable mean differences between MAP2K1 and CCND1 in several endometrial cell lines that were examined. Although no statistical significance was reached, an alteration in gene expression levels in malignant and nonmalignant endometrial cells could be observed. Furthermore, this present study shows observable upregulation of MAP2K1 and CCND1 in endometrial carcinoma cells vs. nonmalignant endometrium cells and encourages further investigation of the role of CCND1 and MAP2K genes in endometrial carcinogenesis.

Copepods comprise an ecologically important role in freshwater and marine ecosystems, which is why they are often considered an important ecotoxicological model organism. The International Organization for Standardization’s (ISO) 14669 protocol is the only guideline for the determination of acute toxicity in three European marine copepod species: Acartia tonsa. The goal of this project was to assess the feasibility of establishing and maintaining cultures of Acartia tonsa, as well as to refine current culturing and egg separation methods. Initial culture methodology proved difficult for consistent production of eggs and collection of nauplii. The development of an airlift system for the separation of eggs from nauplii and adults, based on size, successfully increased the availability of eggs, nauplii and adults. The sensitivity and relative conditions of the copepod species was assessed by running a series of 48h acute toxicity tests with the reference toxicants 3,5-dichlorophenol, 4,4’-methylenebis(2,6-di-tert-butylphenol. The acute 48 hour median lethal dose concentration (LC50), the no observed effect concentration (NOEC), and the lowest observed effect concentration (LOEC) was analyzed for the three reference compounds for of A. tonsa.

A qualidade do sedimento no estado de São Paulo tem sido caracterizada com o uso, dentre outras linhas de evidência, dos resultados obtidos com ensaios ecotoxicológicos utilizando o anfípoda Hyalella azteca. Protocolos internacionais como os da USEPA, ASTM, OECD e ENVIRONMENT CANADA descrevem procedimentos para o cultivo e realização desses ensaios utilizando também outros organismos bentônicos, como as larvas de inseto do gênero Chironomus sp (Insecta, Diptera). No Brasil, diversos estudos realizados com a espécie Chironomus sancticaroli não contam com metodologia nacional normatizada. Os protocolos descrevem metodologias para ensaios de sensibilidade à substância de referência como garantia da qualidade dos organismos antes de sua utilização, não estabelecendo outras metodologias para o monitoramento de variáveis biológicas em cultivo de organismos-teste ou ainda, critérios de aceitabilidade. Com o intuito de garantir a qualidade desses organismos cultivados em laboratório e adaptar uma metodologia para ensaios com sedimento, foram realizados ensaios para o monitoramento de variáveis biológicas em cultivo, determinação de critérios de aceitabilidade para essas variáveis e ensaios com amostras de rios e reservatórios do estado de São Paulo durante os anos 2006 a 2009. As variáveis sobrevivência, deformidade do mento em organismos cultivados e submetidos ao controle do ensaio, fecundidade, fertilidade e taxa de eclosão, foram monitoradas e o critério de aceitabilidade para cada uma foi estabelecido com base no cálculo do percentil. As variações encontradas nesses resultados sugerem a existência de possíveis interferentes como a qualidade da água de cultivo utilizada e a consangüinidade. Os critérios de aceitabilidade, determinados com base em percentis, servirão de ferramenta para o controle da qualidade dos organismos cultivados sob condições específicas para cada laboratório. A metodologia de ensaio adaptada mostrou bom desempenho, superando o critério de aceitabilidade estabelecido para a sobrevivência dos organismos do controle do ensaio. O quironomídeo apresentou maior sensibilidade quando comparado ao anfípoda comumente utilizado em ensaios com sedimento, enfatizando a existência de diferentes respostas às características físico-químicas do sedimento, associada à biologia de cada organismo, permitindo-se recomendar a utilização conjunta desses organismos na avaliação da qualidade do sedimento. / The sediment of Sao Paulo state has been evaluated by using, among other lines of evidence, results obtained from ecotoxicological tests using the amphipod Hyalella azteca. International protocols such as the USEPA, ASTM, OECD and Environment Canada describe procedures for culturing and tests with other benthic organisms such as insect larvae of the genus Chironomus sp (Insecta, Diptera). In Brazil, several studies were conducted with the species Chironomus sancticaroli without the use of a national standardized methodology. The protocols describe methods for testing the sensitivity to the reference toxicant as quality assurance and do not describe other methodologies for monitoring the biological variables in culturing of test organisms, or establish acceptability criteria. In order to ensure the quality of the organisms cultured in the laboratory and adapt a methodology for sediment, tests were conducted to establish the variation of biological variables in culture and determine an acceptability criteria for these variables. Tests with sediment samples from rivers and reservoirs from Sao Paulo state were performed during the years 2006 to 2009. Variables such as survival, menthum deformity, fecundity, fertility and hatching rate were monitored and the acceptability criteria for each one was established using percentile calculations. Variations in the monitoring of variables results suggest the existence of possible interferences such as water quality and inbreeding in culturing. The acceptability criteria determined, based on the percentile, will serve as a tool for quality control of culturing organisms under specific conditions to each laboratory. The adapted test methodology showed good performance, exceeding the acceptability criteria established to organism survival in the control test. The high sensitivity of chironomids when compared to amphipods emphasizing the existence of different responses to physicochemical and chemical characteristics of the sediment, associated with the biology of each organism, indicating that the combined use of these organisms in sediment quality assessment can be recommended.

A qualidade do sedimento no estado de São Paulo tem sido caracterizada com o uso, dentre outras linhas de evidência, dos resultados obtidos com ensaios ecotoxicológicos utilizando o anfípoda Hyalella azteca. Protocolos internacionais como os da USEPA, ASTM, OECD e ENVIRONMENT CANADA descrevem procedimentos para o cultivo e realização desses ensaios utilizando também outros organismos bentônicos, como as larvas de inseto do gênero Chironomus sp (Insecta, Diptera). No Brasil, diversos estudos realizados com a espécie Chironomus sancticaroli não contam com metodologia nacional normatizada. Os protocolos descrevem metodologias para ensaios de sensibilidade à substância de referência como garantia da qualidade dos organismos antes de sua utilização, não estabelecendo outras metodologias para o monitoramento de variáveis biológicas em cultivo de organismos-teste ou ainda, critérios de aceitabilidade. Com o intuito de garantir a qualidade desses organismos cultivados em laboratório e adaptar uma metodologia para ensaios com sedimento, foram realizados ensaios para o monitoramento de variáveis biológicas em cultivo, determinação de critérios de aceitabilidade para essas variáveis e ensaios com amostras de rios e reservatórios do estado de São Paulo durante os anos 2006 a 2009. As variáveis sobrevivência, deformidade do mento em organismos cultivados e submetidos ao controle do ensaio, fecundidade, fertilidade e taxa de eclosão, foram monitoradas e o critério de aceitabilidade para cada uma foi estabelecido com base no cálculo do percentil. As variações encontradas nesses resultados sugerem a existência de possíveis interferentes como a qualidade da água de cultivo utilizada e a consangüinidade. Os critérios de aceitabilidade, determinados com base em percentis, servirão de ferramenta para o controle da qualidade dos organismos cultivados sob condições específicas para cada laboratório. A metodologia de ensaio adaptada mostrou bom desempenho, superando o critério de aceitabilidade estabelecido para a sobrevivência dos organismos do controle do ensaio. O quironomídeo apresentou maior sensibilidade quando comparado ao anfípoda comumente utilizado em ensaios com sedimento, enfatizando a existência de diferentes respostas às características físico-químicas do sedimento, associada à biologia de cada organismo, permitindo-se recomendar a utilização conjunta desses organismos na avaliação da qualidade do sedimento. / The sediment of Sao Paulo state has been evaluated by using, among other lines of evidence, results obtained from ecotoxicological tests using the amphipod Hyalella azteca. International protocols such as the USEPA, ASTM, OECD and Environment Canada describe procedures for culturing and tests with other benthic organisms such as insect larvae of the genus Chironomus sp (Insecta, Diptera). In Brazil, several studies were conducted with the species Chironomus sancticaroli without the use of a national standardized methodology. The protocols describe methods for testing the sensitivity to the reference toxicant as quality assurance and do not describe other methodologies for monitoring the biological variables in culturing of test organisms, or establish acceptability criteria. In order to ensure the quality of the organisms cultured in the laboratory and adapt a methodology for sediment, tests were conducted to establish the variation of biological variables in culture and determine an acceptability criteria for these variables. Tests with sediment samples from rivers and reservoirs from Sao Paulo state were performed during the years 2006 to 2009. Variables such as survival, menthum deformity, fecundity, fertility and hatching rate were monitored and the acceptability criteria for each one was established using percentile calculations. Variations in the monitoring of variables results suggest the existence of possible interferences such as water quality and inbreeding in culturing. The acceptability criteria determined, based on the percentile, will serve as a tool for quality control of culturing organisms under specific conditions to each laboratory. The adapted test methodology showed good performance, exceeding the acceptability criteria established to organism survival in the control test. The high sensitivity of chironomids when compared to amphipods emphasizing the existence of different responses to physicochemical and chemical characteristics of the sediment, associated with the biology of each organism, indicating that the combined use of these organisms in sediment quality assessment can be recommended.

Symbiotic dinoflagellates in the genus Symbiodinium are critical to the success of scleractinian reef corals in shallow tropical seas. These symbionts are commonly isolated from hosts and cultured separately in liquid media (f/2 or ASP8a), but initial isolations can be prone to abundant contaminants that can persist long-term in culture. To help remove these contaminants, I developed a solid growth substrate composed of 1% f/2 medium in agar, supplemented with a variety of antibiotics, to help isolate individual clones and establish new ?axenic? cultures. I found that an antibiotic cocktail of kanamycin (50 µg/mL), ampicillin (100 µg/mL) and streptomycin (50 µg/mL) was the most effective at eliminating visual signs of contamination without apparent harm to a variety of Symbiodinium in culture. Photophysiological measurements of Symbiodinium grown on f/2 agar plates, taken using an Imaging Pulse Amplitude Modulated (I-PAM) fluorometer, were comparable with those grown in liquid f/2, both with and without antibiotics. Eight types of Symbiodinium in clades A-D grown on f/2 agar plates at low irradiance (19-46 µmol photons m-2 s-1) were exposed to higher irradiance conditions (50-90 µmol photons m-2s-1) for 13 days and their photosynthetic efficiencies (Fv/Fm) were compared using the I-PAM. There were significant differences among and within clades, except for two types in clade C (C2 and C3) which did not perform differently from eachother. All types showed reduced Fv/Fm after 12 days in higher light. Type D1a showed high mortality after 13 days of higher light stress. Finally, preliminary work to fluorescently label Symbiodinium determined that available vital stains were not generally practical for symbiosis studies. Attempts to transform Symbiodinium with a variety of plasmids containing fluorescent reporters and/or genes for antibiotic resistance were not successful, but did provide a baseline for future work. In summary, Symbiodinium cultures grown on solid substrates supplemented with antibiotics are useful for: (1) isolating individual cells or clones for subsequent applications and establishing and maintaining ?axenic? cultures that are free of observable contaminants; (2) directly comparing the photophysiology of different cultures using an I-PAM fluorometer; (3) quantifying cells on agar plates using the I-PAM and (4) selecting possible transgenic symbionts for symbiosis studies.

This research examines the understandings and practices of inclusive minded principals toward facilitating the development of inclusive school environments for intermediate level (Grade 7 and 8) students who are experiencing behavioural problems in their schools. Qualitative interviews with 16 principals across 4 school districts were conducted to explore how these inclusive minded principals conceptualize and understand the needs of this particular group of students, and what they consider to be their roles and responsibilities as principals in meeting these needs. The data suggest that despite the number of barriers that serve to hamper principals’ efforts to develop the ideal inclusive school, there are a great many strategies principals intentionally use to facilitate change toward more inclusive school cultures and pedagogy. These strategies emanate from, and are reflective of, an inclusive philosophy that is common among participants. Principals’ individual philosophies and ideologies serve as a compass in guiding decision-making and actions that affect staff, students, and the wider school community. In an inclusive school, these ideologies are reflective of the principles of inclusion, such as the need to create a culture of care wherein all students feel valued, supported, and experience a sense of belonging and individual self worth. The implications of this research toward improving the schooling experiences of students with behavioural problems as well as other marginalized groups of learners are discussed in the context of the call for a re-culturing of schools toward more inclusive environments.

This research examines the understandings and practices of inclusive minded principals toward facilitating the development of inclusive school environments for intermediate level (Grade 7 and 8) students who are experiencing behavioural problems in their schools. Qualitative interviews with 16 principals across 4 school districts were conducted to explore how these inclusive minded principals conceptualize and understand the needs of this particular group of students, and what they consider to be their roles and responsibilities as principals in meeting these needs. The data suggest that despite the number of barriers that serve to hamper principals’ efforts to develop the ideal inclusive school, there are a great many strategies principals intentionally use to facilitate change toward more inclusive school cultures and pedagogy. These strategies emanate from, and are reflective of, an inclusive philosophy that is common among participants. Principals’ individual philosophies and ideologies serve as a compass in guiding decision-making and actions that affect staff, students, and the wider school community. In an inclusive school, these ideologies are reflective of the principles of inclusion, such as the need to create a culture of care wherein all students feel valued, supported, and experience a sense of belonging and individual self worth. The implications of this research toward improving the schooling experiences of students with behavioural problems as well as other marginalized groups of learners are discussed in the context of the call for a re-culturing of schools toward more inclusive environments.

Die Arbeit beschäftigt sich mit dem Problem der Fettlebererkrankung. In der Einleitung wird auf die aktuelle Relevanz der Gesundheitsstörung und Therapiemöglichkeiten eingegangen, insbesondere durch das, in der Therapie des Diabetes mellitus Typ 2 gebräuchliche Biguanid Metformin. Der Bezug zu molekularbiologischen Signalwegen wird hergestellt und verschiedene in vitro Modellsysteme werden vorgestellt. Anschließend wird auf die Herkunft und genetische Besonderheiten der verwendeten primären Maushepatozyten und Hepatomzellen eingegangen, bevor die angewandten Methoden vorgestellt werden. Zum Einsatz kam in dieser Arbeit vor allem die Lipidmessung mittels Fettrot, um das Ausmaß an Steatosis quantifizierbar zu machen. Im Ergebnisteil folgen zuerst Versuche zur Zytotoxizität der einzelnen Chemikalien und deren Einfluss auf intrazelluläre Energieniveaus, bevor der Einfluss auf die hepatozellulären Fetteinlagerungen im Detail untersucht wird. Unterstützt werden die Ergebnisse durch mikroskopische Bilder der Hepatozyten, welche die beschriebenen Effekte verdeutlichen. Insgesamt konnten folgende Thesen aufgestellt werden: • Zwischen primären Hepatozyten von Wildtyp- und Knockout-Mäusen, bestehen nach 24 stündiger Kultivierung Unterschiede bezüglich des intrazellulären Lipidgehaltes, welche sich nach 72 stündiger Kultivierungszeit nivellieren. • Metformin- und Fructoseinkubation senken den intrazellulären ATP-Gehalt, gleichzeitige Anwesenheit von Metformin und Glucose vermindern den Effekt. • Durch 72-stündige Inkubation der primären Hepatozyten und Behandlung mit Metformin konnte der intrazelluläre Lipidgehalt um circa 40% gesenkt werden. • Durch 72-stündige Inkubation der primären Hepatozyten mit Glucose konnte der intrazelluläre Lipidgehalt um circa 100% gesteigert werden. • Bei humanen Hepatomzellen (HuH7) konnte kein Metformin- und kein Glucoseeffekt beobachtet werden. • Der LXR-Agonist TO901317 wirkt auf den intrazellulären Lipidgehalt Metformin entgegen.

Background: Mesenchymal stromal cells (MSCs) are multipotent cells that can differentiate into various cell types and possess immunomodulatory and anti-inflammatory effects, making them interesting candidates for therapeutic applications. MSCs are present in small quantities in tissues like bone marrow and therefore need to be expanded while preserving their essential characteristics. They should adhere to plastic, differentiate into osteocytes, adipocytes and chondrocytes and express specific cell surface markers. Currently, the “golden standard” culture media supplement is fetal bovine serum (FBS). However, there is a potential contamination risk of MSCs by xenogeneic and zoonotic infectious agents, which can trigger an immune response. As an alternative, xeno-free serum supplements derived from human sources, e.g., human serum (HS) can be used.  Aim: This study aimed to identify and characterize human bone marrow derived MSCs and examine the effects different supplements have on the cells.  Methods: MSCs were cultured in 10% FBS, 2% FBS and 10% HS for 20 -21 days. Differentiation was induced and the potential was detected with immunocytochemistry. Cell surface markers CD73, CD90, CD105 and CD45 were identified with flow cytometry.  Results and Conclusion: There was no significant difference in morphology, differential potential or immunophenotype between the different serum conditions. However, HS-supplemented culture media resulted in a significantly higher number of cells with 1 x 107 cells after 20 days without affecting their differentiation potential and immunophenotype in comparison to 10% FBS with 2.2 x 106 cells (p=0.0004). MSCs cultured in 2% FBS resulted in the least number of cells (9.9 x 105) after 21 days of expansion.

Actinomycosis is an infrequent bacterial infection involving Actinomyces spp and related organisms which may occur at many body sites. It can also be found in the microbiota. Actinomyces spp are described as gram-positive bacilli whereas some species grow strictly anaerobically and some facultative. Culturing is a standardized method when suspecting actinomycosis and can be a diagnostic challenge because of inhibition of microbiota and slow growth. Enriched agar plates are used when culturing fastidious bacteria and may be more selective when including antibiotics. The aim of this project was to evaluate if using antibiotic disc facilitates identification of Actinomyces spp when mixed with microbiota. A mix of microbiota was made by pooling together several species. The susceptibility of different isolates and microbiota was analysed using antibiotic discs to determine which disc to use in a trial. A trial was done by inoculating the isolates with the microbiota on agar plates, dispensing ciprofloxacin, and trimethoprim discs. A control group without antibiotic discs were also tested. Results showed variance for most isolates susceptibility. No disc performed superior in the trial, but ciprofloxacin on FAA plates incubated anaerobically gave slightly higher recovery. Both discs facilitated identification of some isolates by supressing much microbiota. Considering that the isolates had varying susceptibility it may be problematic to find one common disc. This study has given new insights on what may facilitate identification. Further studies are needed to determine if antibiotic discs could facilitate identification of Actinomyces and needs testing on clinical samples using larger sample size.