Cell culture models and novel gene therapeutic strategies for colorectal cancer

The development of relevant cellular model systems for colorectal cancer is of utmost importance for an improved in vitro assessment of therapeutic strategies against colorectal cancer. Recently published low passage colon cancer cell lines that closely reflect the characteristics of the respective parental in vivo tumor cells represent very promising cell culture models and were therefore used for the investigations in the present thesis. To provide an in vitro model system that also recapitulates the three-dimensional structure of in vivo tumors, these low passage cell lines were cultivated as multicellular spheroids. Compared to monolayer cultures the multicellular spheroids exhibited a wide variety of changes in their expression patterns. The differential expression includes proteins that are involved in growth signaling (15-hydroxyprostaglandin dehydrogenase), protein biosynthesis (acidic ribosomal protein P0), and regulation of the cyto- or nucleoskeleton (acidic calponin and LMNA protein). These proteins were identified by 2D electrophoresis and subsequent MALDI-TOF mass spectrometry. Both methods were established in the lab in the context of this work. Chemotherapy with 5-fluorouracil (5-FU) represents the traditional treatment of colorectal cancer. However, in many patients the efficiency of this therapeutic strategy is often limited by the development of chemoresistance against 5-FU. Therefore, it was an aim of this thesis to detect novel proteins involved in 5-FU chemoresistance that were previously not ascribed to resistance against this chemotherapeutic drug. A chemoresistant subline of a colon cancer cell line was generated by long-term treatment with 5-FU and served as a model for the investigation of 5-FU chemoresistance. This subline exhibited resistance against both 5-FU-induced inhibition of proliferation and apoptosis. Differences in the expression of cytokeratin 18, heat shock protein 27 and aldehyde dehydrogenase 1B1 between the chemoresistant subline and parental cells were detected by 2D electrophoresis. These findings imply that the cytoskeleton plays a role in the development of chemoresistance against 5-FU. Furthermore, processes located to the mitochondria seem to be involved in this resistance, since heat shock protein 27 and aldehyde dehydrogenase 1B1 are associated with this subcellular organelle. The biological relevance of the findings made in the present PhD thesis has to be determined in further studies. Gene therapy represents a promising alternative strategy for the treatment of colorectal cancer. A novel nonviral gene transfer system was developed by combination of DNA with the polycation PEI25br and the cationic lipids DOCSPER or DOSPER to form lipopolyplexes. These lipopolyplexes enabled enhanced gene transfer in vitro and are promising for in vivo applications, since the established lipopolyplexes preserved their small size at physiological conditions; a property essential for a successful in vivo application. Furthermore, the lipopolyplexes exhibited the capability to efficiently transfect three-dimensional multicellular spheroids. The potential of lipopolyplexes for therapeutic applications was further increased by the utilization of the artificial promoter CTP4 which enables highly specific gene expression in cancer cells with mutations in the Wnt signaling pathway by transcriptional targeting. In addition to its high specificity, this promoter enabled high gene expression levels that were comparable to expression levels obtained by the strong, but unspecific CMV promoter. The efficiency of the CTP4 promoter was demonstrated in seven low passage colon cancer cell lines and also in multicellular spheroids. The transcriptional targeted lipopolyplexes not only enabled high tumor specific expression of reporter genes like luciferase or EGFP but also the expression of a therapeutic gene, interleukin-2 (IL-2). Furthermore, tumor specific expression of cytotoxic protease 2A in combination with IL-2 was possible by using a novel bicistronic construct. The expression of the rhinoviral protease 2A led to efficient reduction of overall cap-dependent gene expression levels and therefore also the proliferation of the transfected cells, while continued IL-2 expression was guaranteed by an IRES element enabling cap-independent gene expression in the presence of protease 2A. In summary, the present results provide a promising basis for the development of novel potent strategies in the treatment of colorectal cancer

Thermal Lattice Boltzmann Methods for the Simulation of Turbulent Flows with Conjugate Heat Transfer – Application to Refrigerated Vehicles

In dieser Arbeit wird eine thermische Lattice-Boltzmann-Methode (TLBM) für die instationäre Simulation turbulenter Strömungen mit natürlicher Konvektion und konjugierter Wärmeübertragung vorgestellt. Turbulente Strömungen mit ihren chaotischen Druck- und Geschwindigkeitsschwankungen stellen eine besondere Herausforderung für numerische Simulationen dar, wobei turbulente Strömungen, angetrieben durch thermische Auftriebskräfte, eine besonders schwierige Aufgabe darstellen. Wie in dieser Arbeit gezeigt wird, ermöglicht TLBM Large Eddy Simulationen (LES) solcher Probleme im industriellen und technischen Maßstab unter Verwendung eines Smagorinsky-Feinstruktur-Modells und unter Ausnutzung seiner intrinsischen Parallelisierbarkeit sowie der Möglichkeit, mehrere tausend Prozessorkerne zu verwenden. Die Eignung der vorliegenden Methode wird in dieser Arbeit anhand von Anwendungen zur Simulation der Innenluftströmung und der Isolationseffizienz eines Kühlwagens, des Wärmetransports im Luftspalt zwischen Rotor und Stator bei Elektromotoren, der Weiterentwicklung hocheffizienter Isolation auf der Basis von Vakuumisolationspaneelen (VIP) und Latentwärmespeichern sowie deren Anwendung in Kühlwagen gezeigt. Eine umfassende Validierung der Methode und ihrer Implementierung im Open-Source-Framework OpenLB wird durchgeführt. Gitterkonvergenz zweiter Ordnung wird gegen das analytische Porous Plate Problem demonstriert, während stabile Simulationen auch bei grober Diskretisierung mit hohen Reynolds- und Rayleigh-Zahlen erreicht werden. Eine sehr gute Übereinstimmung wird für natürliche Konvektion in einem quadratischen Hohlraum, ein bekannter Benchmark-Fall, vom laminaren zum turbulenten Regime mit 10^3 <= Ra <= 10^10 und bei Auflösungen von y+ ~ 2 gezeigt. Im ersten Teil der Ergebnisse werden Simulationen eines leeren Kühlaufbaus für einen Kühllastwagen vorgestellt. Das Strömungsfeld und der Wärmeübergang innerhalb eines gegebenen Kühllastwagens zeigt eine sehr gute Übereinstimmung mit den Messergebnissen, insbesondere den experimentellen Daten für ein Kühlfahrzeug bei Re ~ 53000 an vier charakteristischen Geschwindigkeits- und 13 Temperaturpositionen im Lastwagen. Die Wärmeübertragung durch die Wände wird in den Simulationen durch konjugierte Wärmeübertragung aufgelöst. Dies ermöglicht nun die präzise Vorhersage von Wärmeströmen nahe von Nusselt-Korrelationen für den gegebenen Aufbau, aber - im Gegensatz zu gewöhnlichen Nusselt-Korrelationen - wird der Wärmestrom in der Simulation räumlich aufgelöst. Im zweiten Teil der Ergebnisse wird die Strömung und der Wärmeübergang in einem Ringspalt mit innen rotierendem Zylinder untersucht. Die besondere Herausforderung bei der Simulation dieser Taylor-Couette-Strömung ist die Bildung von Taylor-Wirbeln, die durch ihre Rotation senkrecht zur Hauptströmungsrichtung den entsprechenden Wärmeübergang deutlich erhöhen. Detaillierte instationäre Simulationen werden über einen weiten Drehzahlbereich von fast schleichender Strömungen bis hin zum Auftreten von Taylor-Wirbeln durchgeführt. Es wird eine gute Übereinstimmung mit bisherigen Ergebnissen für die Strömungsstrukturen und die Verbesserung des Wärmeübergangs durch Taylor-Wirbel festgestellt. Insbesondere wird die vorliegende Methode mit Messungen, einer Korrelation und Simulationen unter Verwendung des Scherspannungstransport-Turbulenzmodells (SST) verglichen. Besonderes Augenmerk wird auf die Vorhersage der kritischen Taylor-Zahl gelegt. Während direkte numerische Simulationen (DNS) mit LBM die kritische Taylor-Zahl aus den Experimenten nahezu identisch vorhersagen, wird sie von LBM-LES leicht und vom SST-Modell weiter überschätzt, was auf die übermäßig dissipative Natur der Turbulenzmodelle für die Transition zurückzuführen ist. Im dritten Teil der Ergebnisse werden innovative Konzepte für verbesserte, nachhaltigere Kühlfahrzeuge numerisch untersucht. Um den Kraftstoffverbrauch und die damit verbundenen Emissionen zu reduzieren, werden zwei Ansätze als vielversprechend angesehen: (a) der Einbau von Vakuum-Isolationspaneelen (VIP) in die Wände des Kühlkoffers und (b) die Einführung eines Latentwärmespeichers (LHS) zum Austausch der kraftstoffbetriebenen Klimaanlage (AC). Die Verwendung des vorliegenden TLBM erlaubt in den Simulationen die Auflösung der durch die AC und die natürliche Konvektion induzierten turbulenten Luftströmung, des Wärmeflusses innerhalb der Isolierwände und der tiefgefrorenen Ladung. Dies liefert neue Erkenntnisse über den Einfluss der Konzepte auf die Wärmeübertragung in verschiedenen Kühlaufbauten. Die Simulationen zeigen einen stark reduzierten und homogenisierten einströmenden Wärmestrom für das kombinierte PUR- und VIP-Isoliermaterial im Vergleich zu einer reinen PUR-Isolierung. Die Dämmung des Kühlaufbaus mit VIPs halbiert daher die erforderliche Kühlenergie. Dies ermöglicht den Ersatz der AC durch einen LHS in Dachnähe und ein zusätzliches Lüftungssystem mit deutlich geringerer Gesamtleistung. Unter Berücksichtigung der Temperaturhomogenität von Tiefkühlprodukten wird eine leichte Umströmung des Kühlgutes als notwendig erachtet. Die maximal zulässige Ausfallzeit der AC wird in den Simulationen mit jeweils ca. 3,3 min (PUR), 8 min (PUR+VIP) und 11 min (PUR+VIP+LHS) ermittelt. Im vierten Teil der Ergebnisse wird eine LBM zur Simulation des Schmelzens und des konjugierten Wärmeübergangs auf der Basis des Transports der Gesamtenthalpie vorgestellt, welche bei Validierung gegen die analytische Lösung des zeitabhängigen Stefan-Problems präzise Ergebnisse liefert. Die in dieser Arbeit entwickelte Methode zeigt geringe Grenzflächendiffusion für einen weiten Bereich von Relaxationszeiten und Stefan-Zahlen. Weiterhin wird eine enge Übereinstimmung für das Schmelzen von Gallium einschließlich der natürlichen Konvektion in 2D und 3D mit Messungen und Simulationen mit unterschiedlichen Ansätzen gezeigt. Das Modell wird ferner auf das Schmelzen von Paraffin in zwei komplexen Metallschaumgeometrien angewendet. Es wird eine Voxel-basierte parallele Vernetzung vorgestellt, die eine schnelle und automatisierte Verarbeitung der komplexen Geometrie in wenigen Minuten ermöglicht. Die Simulationen erfassen erfolgreich den materialübergreifenden Wärmetransfer in 3D, wobei die Wärmeleitfähigkeit des Schaums mehr als 1000-mal größer als die des Paraffins ist. Die Form der Schmelzfront und der Einfluss der spezifischen Oberfläche der verschiedenen Metallschäume stehen in enger Übereinstimmung mit früheren Simulationen

TRX Suspension Training: A New Functional Training Approach For Older Adults - Development, Training Control And Feasibility

International Journal of Exercise Science 8(3): 224-233, 2015. Because of its proximity to daily activities functional training becomes more important for older adults. Sling training, a form of functional training, was primarily developed for therapy and rehabilitation. Due to its effects (core muscle activation, strength and balance improvements), sling training may be relevant for older adults. However, to our knowledge no recent sling training program for healthy older adults included a detailed training control which is indeed an essential component in designing and implementing this type of training to reach positive effects. The purpose of this study was to develop a TRX Suspension Training for healthy older adults (TRX-OldAge) and to evaluate its feasibility. Eleven participants finished the 12 week intervention study. All participants trained in the TRX-OldAge whole-body workout which consists of seven exercises including 3-4 progressively advancing stages of difficulty for every exercise. At each stage, intensity could be increased through changes in position. Feasibility data was evaluated in terms of training compliance and a self-developed questionnaire for rating TRX-OldAge. The training compliance was 85 %. After study period, 91 % of the participants were motivated to continue with the program. The training intensity, duration and frequency were rated as optimal. All participants noted positive effects whereas strength gains were the most. On the basis of the detailed information about training control, TRX-OldAge can be individually adapted for each older adult appropriate to its precondition, demands and preference

Patterns of sickness absence from a secondary hospital in Melbourne: A 10-year longitudinal study

Background/aims: There has been significant concern in recent years regarding increases in absenteeism in the healthcare sector, leading to lost productivity and projected workforce shortages. This study aimed to identify patterns of sickness absence over a 10-year period in a single-site secondary hospital in Melbourne, Australia. Methods: Data regarding sickness absences were extracted from anonymised payroll records from 2007 to 2016. The patterns of sickness absence analysed included seasonality, amount of leave and category of leave. These were explored both for individuals and in the aggregate. Results: Compared to the Australian average of 9.7 days, this cohort of employees took less sick leave, averaging at 8.81 days each. As a group, a consistent proportion of staff took no sick leave, 1–3 days, 4–6 days, or 7–9 days each year in the 10-year timespan. Only a small proportion took more than 9 days of sickness leave per year. Conclusions: The pattern of leave-taking was consistent for the group as a whole, however, individual leave patterns vary

As Relações intersetoriais nos conselhos municipais de saúde: um estudo de caso nos Conselhos de Joinville, Jaraguá do Sul e Balneário Barra do Sul - SC

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Filosofia e Ciências Humanas, Programa de Pós-Graduação em Sociologia Política, Florianópolis, 2011Os conselhos municipais de saúde (CMS) são resultado importante do processo de lutas pelo Sistema Único de Saúde no Brasil, sistema este que prevê os conselhos, juntamente com as conferências de saúde, como espaços de participação e controle social. Apesar dos avanços conquistados ao longo dos anos, os CMS ainda encontram dificuldades para a consolidação de suas atribuições. A hipótese principal desta pesquisa é a de que a articulação intersetorial é uma das ações capazes de fortalecer os CMS. A pesquisa parte de duas frentes: uma teórica, que traça uma aproximação entre o conceito de intersetorialidade e a noção de interdependência proposta por Norbert Elias, e outra empírica, através da pesquisa de campo em três CMS do estado de Santa Catarina. Os resultados reafirmam a hipótese inicial, tendo claro que a articulação intersetorial fortalece um CMS mas não é isoladamente garantia de sucesso, visto a importância de outros fatores, como o reconhecimento das ações do conselho por parte dos gestores municipais e a garantia de uma estrutura mínima de trabalho, tanto material quanto humana.The municipal health councils (CMS) are important outcome of the process of struggle by the Unified Health System in Brazil, a system that provides advice, along with health conferences as forums for participation and social control. Despite the advances made over the years, CMS is still difficult to consolidate their authority. The main hypothesis of this research is that inter-sector collaboration is one of the actions that can strengthen the CMS. The research on two fronts: a theory, which draws a connection between the concept of intersectionality and the notion of interdependence proposed by Norbert Elias, and other empirical, through field research in three CMS in the state of Santa Catarina. The results confirm the initial hypothesis, having clear that a stronger intersectoral linkages but CMS is not alone guarantee success, given the importance of other factors such as the recognition of the council's actions on the part of municipal managers and ensuring a minimum framework of work, both material and human

Effect of Hydrophobic Mutations in the H2-H3 Subdomain of Prion Protein on Stability and Conversion In Vitro and In Vivo

Prion diseases are fatal neurodegenerative diseases, which can be acquired, sporadic or genetic, the latter being linked to mutations in the gene encoding prion protein. We have recently described the importance of subdomain separation in the conversion of prion protein (PrP). The goal of the present study was to investigate the effect of increasing the hydrophobic interactions within the H2-H3 subdomain on PrP conversion. Three hydrophobic mutations were introduced into PrP. The mutation V209I associated with human prion disease did not alter protein stability or in vitro fibrillization propensity of PrP. The designed mutations V175I and T187I on the other hand increased protein thermal stability. V175I mutant fibrillized faster than wild-type PrP. Conversion delay of T187I was slightly longer, but fluorescence intensity of amyloid specific dye thioflavin T was significantly higher. Surprisingly, cells expressing V209I variant exhibited inefficient proteinase K resistant PrP formation upon infection with 22L strain, which is in contrast to cell lines expressing wild-type, V175I and T187I mPrPs. In agreement with increased ThT fluorescence at the plateau T187I expressing cell lines accumulated an increased amount of the proteinase K-resistant prion protein. We showed that T187I induces formation of thin fibrils, which are absent from other samples. We propose that larger solvent accessibility of I187 in comparison to wild-type and other mutants may interfere with lateral annealing of filaments and may be the underlying reason for increased conversion efficiency

Estágio supervisionado no formato de curso de Extensão na licenciatura em EPT do IFSC

This report provides information about a supervised training experience carried out in the format of extension courses, by students of the undergraduate course in Professional and Technological Education at  Instituto Federal de Santa Catarina. This practice occurred during the social isolation caused by the pandemic of COVID-19, when  the face-to-face activities of educational institutions.came to a halt. The courses were part of an extension project coordinated by the tutors of the supervised training, and were planned and executed by undergraduate students, targeted at high school students. Featuring professional choice and the world of work as a general theme, the courses started from the training and careers of the extension students themselves, and resulted in quite significant experiences, not only  for the students involved, but also for the community and the institution itself. The experience provides room for reflection on the possibilities of relating internship and extension, stimulating an even greater presence of extension opportunities in the teacher training offered by Institutos Federais.Este relato traz informações sobre uma experiência de estágio supervisionado realizado em formato de cursos de extensão por estudantes da Licenciatura em Educação Profissional e Tecnológica do Instituto Federal de Santa Catarina. Tal prática se deu durante o isolamento social causado pela pandemia de COVID-19, quando as instituições de ensino paralisaram suas atividades presenciais. Os cursos fizeram parte de um projeto de extensão coordenado pelos professores de estágio e foram planejados e executados pelos discentes da referida licenciatura, tendo como público-alvo estudantes de nível médio. Apresentando como tema geral a escolha profissional e o mundo do trabalho, os cursos partiram das formações e carreiras dos próprios discentes extensionistas e resultaram em vivências bastante significativas, tanto para os estudantes envolvidos quanto para a comunidade e para a própria instituição. Tal experiência abre espaço para a reflexão sobre as possibilidades de relacionar estágio e extensão, estimulando uma presença ainda maior de oportunidades da extensão na formação docente ofertada pelos institutos federais