O câncer de mama é uma neoplasia altamente maligna e continua a ser a segunda principal causa de mortalidade entre as mulheres. Os efeitos antitumorais de metaloproteinases e desintegrinas de veneno de serpentes têm sido investigados em vários tipos de células tumorais. Neste estudo, foram avaliados os efeitos antitumorais e anti-angiogênicos induzidos pela Bothropoidina, uma metaloproteinase desintegrina-like isolada da peçonha de Bothrops pauloensis em células de câncer de mama humano MDA-MB-231 e células endoteliais. Após 24 horas de tratamento com 100pg/mL de Bothropoidina foi constatado um efeito citotóxico moderado de 30% em MDA-MB-231 contra 10% de citotoxicidade em MCF10A (uma linha de células da mama não tumorigênica), uma diferença significativa que sugere uma possível preferência desta proteína por alvos em células tumorais. Observou-se apoptose e apoptose tardia após tratamento com Bothropoidina (10pg/mL e 40pg/mL) em células MDA-MB-231. Além disso, esta toxina não só inibiu a adesão de células MDA-MB-231 de uma forma dose dependente, como a migração celular em aproximadamente 45%. Bothropoidina reduziu a viabilidade e adesão de células endoteliais em Matrigel e inibiu a angiogênese in vitro estimulada por bFGF em Matrigel, mostrando um número de vasos formados significativamente menor em relação ao controle. Os resultados demonstraram que Bothropoidina tem um potente efeito antitumoral e antiangiogênico in vitro, representando uma ferramenta biotecnológica para elucidar o efeito antitumoral de metaloproteinases desintegrinas-like em células cancerígenas. / Breast cancer is a highly malignant carcinoma and remains the second leading cause of mortality among women. The antitumor effects of metalloproteinases and disintegrins from snake venom on various types of cancer cells have been investigated. In this study, we evaluated the antitumor and antiangiogenic effects on MDA-MB-231 human breast cancer cells and endothelial cells induced by Bothropoidin, a disintegrin-like metalloproteinase isolated from Bothrops pauloensis snake venom. At 24h after treatment at 100pg/mL, Bothropoidin exerted a moderate cytotoxic effect of 30% on MDA-MB-231 versus 10% cytotoxicity against MCF10A (a non-tumorigenic breast cell line), a significant difference that suggests a possible preference by this protein for targets in cancer cells. Early and late apoptosis of MDA-MB-231 was observed after Bothropoidin treatment (10gg/mL and 40gg/mL). Furthermore, this toxin inhibited not only the adhesion of MDA-MB-231 cells in a dose-dependent manner but also cell migration by approximately 45%. In addition, Bothropoidin decreased endothelial cells viability and adhesion in Matrigel and inhibited in vitro angiogenesis in Matrigel stimulated by bFGF, showing significantly fewer formed vessels. The results demonstrated that Bothropoidin has potent in vitro antitumor and antiangiogenic effect and represents a biotechnological tool for elucidating the antitumor effect of disintegrins-like metalloproteinases in cancer cells. / Dissertação (Mestrado)

In this thesis I explore the emergence of the Christian triad with reference to two contemporary movements: Middle Platonism and Gnosticism. The earliest Christian writer to enumerate the three constituents of what would become the Christian Trinity is Justin. In addition to his three extant works, Justin’s triadology can be diagnosed from those he directly influenced – Tatian and Athenagoras – who I have (somewhat artificially) grouped under the heading the “school of Justin”. The ontological triad adopted by these Christian thinkers is compared with the triads of Middle Platonism and Gnosticism, both in terms of their structure and in terms of the function and ontological status of the individual constituents of these triads. In this thesis I propose that a liturgical triad of primitive Christianity, the trine baptismal formula, was conflated by the “school of Justin” with the ontological triad of Middle Platonism, resulting in three referents of the baptismal formula being embued with new functions and ontological status. Whilst emerging as a hierarchical triad, the logic of Platonic ontology when combined with Christian tradition required the sharp distinction between God, as Being, and all other things resulting in a Christian triad that was also a unity. This new triad became fixed as a central tenet of Christianity. I find no plausible connection between any known Gnostic triad and the triad of the “school of Justin”. There is some interaction between Gnostic and Platonic thought during this period. It is possible that the Triple-Powered One pre-empted the Being-Mind-Life triad of Neoplatonism.

Although originally designed as an extension for the Unifi ed Modeling Language (UML), today, the Object Constraint Language (OCL) has been broadly adopted in the context of both UML and other modeling and domain-specifi c languages. However, appropriate tooling, supporting modelers and software developers on using OCL is still scarce and lacks important features such as debugging support. As OCL constraints are likely to become rather complex for real world examples, it is hard to comprehend the in uence of single OCL expressions and subexpressions on the result of a completely evaluated OCL constraint in the context of speci fic constrained objects. Therefore, debugging is of topmost importance for both constraint comprehension and maintenance. Thus, the major task of this work is to develop a graphical debugger integrated into Dresden OCL and the Eclipse Integrated Development Environment (IDE) to fill this gap.

Der Große Beleg dokumentiert die Entwicklung eines Java Code-Generators für Dresden OCL2 for Eclipse. Schwerpunkt der Arbeit liegt dabei auf der Abbildung der Object Constraint Language auf die Programmiersprache Java mit Hilfe von AspectJ.

Previous studies have shown that the upregulation of CD151 and β1 is associated with poor prognosis in many cancers such as breast cancer. Studies have provided evidence that these proteins are associated with the adhesion and migration of tumor cells. In this study, a microfluidic flow chamber was utilized to determine how CD151 and β1 affected the firm and initial adhesion of metastatic breast cancer cells to a planar endothelial monolayer under shear stress. This system mimicked the adhesion of metastatic breast cancer cells to the endothelial cells of the circulatory system. CD151 and β1 increased the firm adhesion of metastatic breast cancer cells onto an endothelial monolayer when subjected to high shear stresses. CD151 and β1 increased the initial adhesion of metastatic breast cancer cells onto an endothelial monolayer. A transwell assay was utilized to determine how CD151 and β1 affected random migration through different matrixes and random transendothelial migration. CD151 and β1 decreased the random migration of metastatic breast cancer cells through matrices. Additionally, background information is provided related to the metastatic cascade, how it can be modeled with microfluidics, and how CD151 and β1 have been known to effect cancer and metastasis.

Made available in DSpace on 2014-06-12T15:54:21Z (GMT). No. of bitstreams: 2 arquivo5215_1.pdf: 1381913 bytes, checksum: 3a78f7ed849513c01bc3e4ced1760801 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2006 / O ácido úsnico é um metabólito de líquen que apresenta uma grande variedade de atividades biológicas, dentre as quais, citotoxidade frente a células oriundas de tumores malignos humanos. Apesar da existência de revisões recentes sobre a atividade citotóxica do ácido úsnico, o mecanismo de ação desta droga ainda não foi completamente elucidado. Não existe na literatura referência ao envolvimento do gene supressor de tumor p53 com os efeitos do ácido úsnico. Na sua forma normal, a proteína p53 atua em resposta a diferentes estresses celulares levando à transcrição de genes que induzem a retenção do ciclo celular ou apoptose. Entre as formas de atuação do p53 está a repressão de genes que codificam proteínas associadas à polimerização e estabilização de microtúbulos. Estas funções são perdidas quando ocorrem mutações em sua via metabólica, o que acontece em mais de 50% dos tumores cancerosos humanos. O objetivo deste trabalho foi investigar se o mecanismo da ação anticancerígena do ácido úsnico envolve a ativação da via metabólica p53. Para estudos da sensibilidade de linhagens cancerígenas ao ácido úsnico, foram realizados ensaios pelo método colorimétrico do MTT [3-(4,5- dimethylthiazol-2-yl)-2,5diphenyl-tetrazolium bromide], utilizando-se várias concentrações do fármaco (1 a 60 μM) por 72h, frente às seguintes linhagens de células malignas humanas: MCF7(câncer de mama, positiva para receptores de estrogênio, p53 normal), MDA-MB-231(câncer de mama, negativa para receptores de estrogênio, p53 inativo), H1299 (câncer de pulmão, nula para p53). Para determinar o envolvimento do p53 na ação citotóxica do ácido úsnico, os níveis das proteínas p53 e p21 (um inibidor de quinases dependentes de ciclinas cuja expressão é controlada pelo p53) em células MCF7 tratadas com 29 μM de ácido úsnico por 24h foram determinados utilizando-se ensaios western blot com o anticorpo monoclonal DO-12 (específico para p53) e WAF1 (específico para p21). Para verificar se a ação anticancerígena do ácido úsnico resulta em dano ao DNA celular, a fosforilação da SER15 do p53 (um marcador para danos em DNA) foi investigada, após tratamento de células MCF7 com 29 μM de ácido úsnico por 24h. Nestes estudos, ensaios western blot foram realizados com o anticorpo policlonal FOSFO-SER15, específico para serina fosforilada. Para verificar se o aumento nos níveis da proteína p53 detectados após o tratamento com ácido úsnico eram acompanhados por um aumento em sua atividade transcricional, foram executados ensaios com ß-Gal. Nesta metodologia utilizaram-se fibroblastos T22 de camundongos, portadores do plasmídeo RGDFos-LacZ (contendo o resíduo de 36 pb do sítio de ligação para o p53), tratados com diferentes concentrações de ácido úsnico. Para a investigação dos efeitos do ácido úsnico na formação e estabilização de microtúbulos, células MCF7 foram tratadas com 29 μM de ácido úsnico por 24h, fixadas em metanol e tratadas com anticorpo monoclonal anti-ß-tubulina. O ácido úsnico mostrou atividade citotóxica frente às várias linhagens celulares oriundas de tumores malignos humanos, promovendo elevação nos níveis das proteínas p53 e p21. Entretanto, este aumento não foi acompanhado de incremento na atividade transcricional nem da fosforilação da SER15 do p53. Também não foram detectadas modificações na formação dos microtúbulos. As propriedades anticancerígenas do ácido úsnico como agente não genotóxico que atua de uma forma independente do p53 fazem dele um candidato em potencial para novas terapias de câncer

Philosophiae Doctor - PhD / Globally, breast cancer is the most common cancer affecting women and it is predicted that in 2030 about 12 million deaths will occur with approximately 21.7 million new cases [2]. Genetic risk factors as well as race and ethnicity, account for about 5-10% of all breast cancer occurrences. Triple negative breast cancer (TNBC), tumors that tested negative for oestrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2), contribute to 10-20% of all breast carcinomas [3,4] and is known to be a more aggressive type of cancer with varying degree of response to chemotherapeutic and radiation therapy [5,6] / 2022-02-24

Utilization of potential synergistic interactions among different bioactive agents is a promising approach to inhibit complex diseases such as cancer. Nobiletin (NBT) and tangeretin (TAN) are major polymethoxyflavones (PMFs) found in citrus fruits. Herein, we studied NBT and TAN in combination with atorvastatin (ATST, Lipitor, a cholesterol-lowering drug) in MDAMB231 and MCF-7 human breast cancer cells. Both NBT/ATST and TAN/ATST combinations at low doses produced much stronger inhibitory effect on cancer cell viability in comparison to those produced by NBT, TAN, or ATST alone at much higher doses. Isobologram analysis confirmed that both NBT/ATST and TAN/ATST combinations produced strong synergy in inhibiting the growth of two breast cancer cell lines. Flow cytometry analysis showed that both NBT/ATST and TAN/ATST combinations caused significant cell cycle arrest at G0/G1 phase in MDAMB231 cells (ER+). Consistent with these results, PMFs and ATST combinations decreased expression levels of phospho Rb, cyclin D1, and CDK4. Further experiments showed that the combination treatment induced autophagy and late apoptosis in MDA-MB-231 cells. Meanwhile, co-treatment of PMFs and ATST induce G2/M phase in MCF-7 (ER+) cells.. The combination of PMFs and ATST also caused autophagy in MCF-7 cells, which was evidenced by activation of LC3B and P62. In conclusion, our result demonstrated strong synergy between two major citrus PMFs (NBT and TAN) and ATST in inhibiting human breast cancer cell growth.

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Cancer progression and physiological changes within the cells are accompanied by alterations in the biophysical properties. Therefore, the cell biophysical properties can serve as promising markers for cancer detection and physiological activities. To aid in the investigation of the biophysical markers of cells, a microfluidic chip has been developed which consists of a constriction channel and embedded microelectrodes. Single-cell impedance magnitudes at four frequencies and entry and travel times are measured simultaneously during their transit through the constriction channel. This microchip provides a high-throughput, label-free, automated assay to define biophysical signatures of malignant cells and monitor the therapeutic efficacy of drugs. Here, we monitored the dynamic cellular biophysical markers in response to sphingosine kinase inhibitors (SphKIs), and compared the effectiveness of drug delivery using Poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) loaded with SphKIs versus conventional delivery. Cells treated with SphKIs showed significantly higher impedance magnitudes at all four frequencies. The bioelectrical parameters extracted using a model also revealed that the highly aggressive breast cells treated with SphKIs shifted electrically towards that of a less malignant phenotype; SphKI-treated cells exhibited an increase in cell-channel interface resistance and a significant decrease in specific membrane capacitance. Furthermore, SphKI-treated cells became slightly more deformable as measured by a decrease in their channel entry and travel times. We observed no significant difference in the bioelectrical changes produced by SphKI delivered conventionally or with NPs. However, NPs-packaged delivery of SphKI decreased the cell deformability. In summary, the results showed that while the bioelectrical properties of the cells were dominantly affected by SphKIs, the biomechanical properties were mainly changed by the NPs. / Master of Science