The aim of the work presented in this thesis was to develop vectors for gene transfer, that are based on herpes simplex virus type 1, for possible application in the field of surgery.

No description available.

no / Prohormone convertases (PCs) are endoproteases that process many substrates in addition to hormone precursors. Although overexpression of PCs is linked to carcinogenesis in some solid tumors, the role of subtilisin-kexin isoenzyme-1 (SKI-1) in this context is unknown. We show that SKI-1 is constitutively expressed in human pigment cells with higher SKI activity in seven out of eight melanoma cell lines compared with normal melanocytes. SKI-1 immunoreactivity is also detectable in tumor cells of melanoma metastases. Moreover, tissue samples of the latter display higher SKI-1 mRNA levels and activity than normal skin. From various stimuli tested, 12-O-tetradecanoylphorbol-13-acetate and tunicamycin affected SKI-1 expression. Importantly, SKI-1 inhibition by the cell-permeable enzyme inhibitor decanoyl-RRLL-chloromethylketone (dec-RRLL-CMK) not only suppressed proliferation and metabolic activity of melanoma cells in vitro but also reduced tumor growth of melanoma cells injected intracutaneously into immunodeficient mice. Mechanistic studies revealed that dec-RRLL-CMK induces classical apoptosis of melanoma cells in vitro and affects expression of several SKI-1 target genes including activating transcription factor 6 (ATF6). However, ATF6 gene silencing does not result in apoptosis of melanoma cells, suggesting that dec-RRLL-CMK induces cell death in an ATF6-independent manner. Our findings encourage further studies on SKI-1 as a potential target for melanoma therapy.

From previous studies it has been demonstrated that the fusion of tumor cells with antigen-presenting cells generates hybrids that are known to induce anti-tumor immunity. With the advancement of scientific research and medicine, the need to produce cell-cell hybrids for cancer immunotherapy and for various other applications is substantial. Among the many methods used to generate these hybrid cells, electrofusion is a technique that is more widely used and recognized as a method to efficiently produce hybrids. Electrofusion requires two steps. In the first step, cells are brought into close adjacent contact either by a mechanical method like centrifugation or by dieletrophoresis using alternating current (AC). The second step includes the reversible breakdown and fusion of cell membranes induced by high voltage direct current (DC) pulses. The goal of this investigation was to study the use of electrical charge to bring cells into close contact with one another in the cell contact stage prior to delivering high voltage fusion pulses. The possibility of achieving considerable cell-cell contact was tested in two separate electrical systems. In the first system B16 murine melanoma cancer cells were subjected to a range of direct current (DC) voltages between 4 V/cm and 40 V/cm. With the use of DC from a small power source the response of the cells was tested in multiple fusion chambers consisting of two or four electrodes. The configurations of the chambers were varied by changing the distance between the electrodes, the thickness, material and type of coating on the electrodes. In the second system the movement of cells in the presence of corona charge was studied. B16 cells in a culture dish were confined by a circular grounded electrode and subjected to corona discharge for known periods of time. Application of corona charge (positive or negative) facilitated the contact of cells in the annular region between the two circular electrodes. After series of tests, final designs for fusion chambers to be used with DC and with corona were developed. Cell contact achieved with the DC fusion chamber was not substantial enough to produce a significant amount of fusion yield. The fusion chamber designed to be used with corona on the other hand produced exceptional cell contact results consequentially generating fusion yields as high as 40%.

Hyaluronan (HA) and its principal receptor CD44 are known to be involved in regulating tumor cell dissemination and metastasis. It is hypothesized that the CD44-HA interaction regulates proliferation and invasion of tumor cells in dependence on the molecular weight and the presentation form of HA. To address this hypothesis, we reconstituted 3D collagen (Coll I) matrices and functionalized them with HA of molecular weight of 30-50 kDa (low molecular weight; LMW-HA) and 500-750 kDa (high molecular weight; HMW-HA). A post-modification strategy was applied to covalently immobilize HA to reconstituted fibrillar Coll I matrices, resulting in a non-altered Coll I network microstructure and stable immobilization over days. Functionalized Coll I matrices were characterized regarding topological and mechanical characteristics as well as HA amount using confocal laser scanning microscopy, colloidal probe force spectroscopy and quantitative Alcian blue assay, respectively. To elucidate tumor cell behavior, BRO melanoma cell lines with and without CD44 receptor expression were used for in vitro cell experiments. We demonstrated that only soluble LMW-HA promoted cell proliferation in a CD44 dependent manner, while HMW-HA and immobilized LMW-HA did not. Furthermore, an enhanced cell invasion was found only for immobilized LMW-HA. Both findings correlated with a very strong and specific adhesive interaction of LMW-HA and CD44+ cells quantified in single cell adhesion measurements using soft colloidal force spectroscopy. Overall, our results emphasize the importance of presentation mode and molecular weight specificity in biomaterial studies on the impact of HA on cell behavior.

No description available.

京都大学 / 0048 / 新制・課程博士 / 博士(人間健康科学) / 甲第18196号 / 人健博第13号 / 新制||人健||1(附属図書館) / 31054 / 京都大学大学院医学研究科人間健康科学系専攻 / (主査)教授 齋藤 邦明, 教授 足立 壯一, 教授 長田 重一 / 学位規則第4条第1項該当 / Doctor of Human Health Sciences / Kyoto University / DFAM

No description available.

Cell migration is an essential element in the immune response on the one hand and in cancer metastasis on the other hand. The architecture of the actin network in lamellipodia determines the elasticity of the leading edge and contributes to the regulation of migration. We have implemented a new method for the analysis of actin network morphology in the lamellipodia of B16F1 mouse melanoma cells. This method is based on fitting multilayer geometrical models to electron microscopy images of lamellipodial actin networks. The chosen model and F-actin concentrations are thereby deterministic parameters. Using this approach, we identified distinct structural features of actin networks in lamellipodia. The mesh size which defines the elasticity of the lamellipodium was determined as 34 and 78 nm for a two-layer network

Submitted by Erika Demachki (erikademachki@gmail.com) on 2014-10-22T16:44:26Z No. of bitstreams: 4 Tese - Débora Cristina Silva dos Passos - 2013 - Parte 01.pdf: 10357491 bytes, checksum: 1678b4aad93bca319ee5ddb93d3808bc (MD5) Tese - Débora Cristina Silva dos Passos - 2013 - Parte 02.pdf: 9533486 bytes, checksum: ad5e21870e09f4f46a3045a69996aed3 (MD5) Tese - Débora Cristina Silva dos Passos - 2013 - Parte 03.pdf: 8695341 bytes, checksum: 5f8d696c0a73f923cb129f3c0aa1e4b7 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Jaqueline Silva (jtas29@gmail.com) on 2014-10-22T19:00:24Z (GMT) No. of bitstreams: 4 Tese - Débora Cristina Silva dos Passos - 2013 - Parte 01.pdf: 10357491 bytes, checksum: 1678b4aad93bca319ee5ddb93d3808bc (MD5) Tese - Débora Cristina Silva dos Passos - 2013 - Parte 02.pdf: 9533486 bytes, checksum: ad5e21870e09f4f46a3045a69996aed3 (MD5) Tese - Débora Cristina Silva dos Passos - 2013 - Parte 03.pdf: 8695341 bytes, checksum: 5f8d696c0a73f923cb129f3c0aa1e4b7 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2014-10-22T19:00:24Z (GMT). No. of bitstreams: 4 Tese - Débora Cristina Silva dos Passos - 2013 - Parte 01.pdf: 10357491 bytes, checksum: 1678b4aad93bca319ee5ddb93d3808bc (MD5) Tese - Débora Cristina Silva dos Passos - 2013 - Parte 02.pdf: 9533486 bytes, checksum: ad5e21870e09f4f46a3045a69996aed3 (MD5) Tese - Débora Cristina Silva dos Passos - 2013 - Parte 03.pdf: 8695341 bytes, checksum: 5f8d696c0a73f923cb129f3c0aa1e4b7 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2013-05-16 / Melanoma is a type of cancer that arises from melanocytes and is notoriously resistant to radiation and chemotherapy. The thiosemicarbazones are synthetic compounds with marked biological properties such as antibacterial, antiviral, antiprotozoal and antitumor and previous studies have demonstrated cytotoxic activity against the human melanoma cells, so in this study, we evaluated the antiproliferative activity, the enzymatic activity of Caspases 2, 3, 6, 8, 9, the effect on the cell cycle gene expression levels of caspases 2, 3, 6, 8, 9, Apaf-1 and microscopic morphological changes in human melanoma cells (SK -MEL-37) twenty one monoterpene derived from natural thiosemicarbazone (-) - camphene: camphene, benzaldehyde, benzophenone, menthone, ethyl pyruvate, p-nitroacetophenone, pchloroacetophenone, p-methoxyacetophenone, p-methylacetophenone, p fluoracetofenona-phidroxiacetofena, furan, 3-methoxy-4-hydroxybenzaldehyde, p-fluorbenzaldehyde, 2- hydroxybenzaldehyde, cinnamic aldehyde, thiophene-2-carboxaldehyde, 1-H-imidazole-4- carboxaldehyde, tiossemicaroazida and six montoterpeno natural R-(+)-limonene: benzaldehyde, thiosemicarbazide, o-nitro, m-nitro, p-nitro, p-hydroxy and p-dimethylamino. The values found for the inhibitory concentration for 50% of cells (IC50) were between 12 μM and 55 μM. The percentage of cells in phase and in phase G0/G1 decreased SG2 / M increased after forty-eight hours of incubation with benzaldehyde thio-camphene, limonene thio-benzaldehyde, m-nitro, p-hydroxy and thiosemicarbazide increased indicating that the growth inhibitory effect might be also due to arrest of cells at S-G2/M phase. We observed increased activity of caspase 3 (m-nitro thio-limonene), 6 (camphene thio-benzaldehyde and p-hydroxy thio-limonene) and 8 (thio-benzaldehyde limonene). Late apoptotic features were detected in 62% of cells treated with benzaldehyde thio-camphene and morphological changes typical of apoptosis were visualized by fluorescence microscopy and scanning electron microscopy (SEM) after treatment with benzaldehyde thio-camphene chosen due to their low IC50 value (12 mM). It was observed gene expression of caspases 2, 3, 6, 8 and Apaf-1 in cells treated with benzaldehyde thio-camphene indicating the participation of these enzymes in the anti-proliferative effect observed. Our results indicate that the thiosemicarbazones derivatives can inhibit proliferation, regulate cell cycle, induce apoptosis of human melanoma cells (SK-MEL-37) and could be an candidate for future preclinical in vivo studies. / O melanoma é um tipo de câncer que surge nos melanócitos e é notoriamente resistente à radioterapia e quimioterapia. As tiossemicarbazonas são compostos sintéticos com marcantes propriedades biológicas tais como antibacteriana, antiviral, antiprotozoária e antitumoral e em estudos anteriores demonstraram ação citotóxica frente à celulas de melanoma humano, por isso, neste estudo, foi avaliada a atividade anti-proliferativa, a atividade enzimática das caspases 2, 3, 6, 8, 9, o efeito no ciclo celular, os níveis de expressão gênica das caspases 2, 3, 6, 8, 9, Apaf-1 e as alterações morfológicas por microscopia em células de melanoma humano (SK-MEL-37) de vinte e uma tiossemicarbazonas derivadas do monoterpeno natural (-)- canfeno: canfeno, benzaldeído, benzofenona, mentona, etil piruvato, acetofenona, pnitroacetofenona, p-cloroacetofenona, p-metoxiacetofenona, p-metilacetofenona, pfluoracetofenona, p-hidroxiacetofena, furano, 3-metóxi-4-hidroxibenzaldeído, pfluorbenzaldeído, 2-hidroxibenzaldeído, aldeído cinâmico, tiofeno-2-carboxialdeído, 1-Himidazol- 4-carboxialdeído, tiossemicaroazida, bem como seis do montoterpeno natural R-(+)- limoneno: benzaldeído, tiossemicarbazida, o-nitro, m-nitro, p-nitro, p-dimetilamino e phidróxi . Os valores encontrados para a concentração inibitória para 50% das células (IC50) situaram-se entre 12 μM e 55 μM. A porcentagem de células na fase G0/G1diminuiu e na fase SG2/M aumentou após quarenta e oito horas de incubação com o benzaldeído tio-canfeno, benzaldeído tio-limoneno, m-nitro, p-hidróxi e tiossemicarbazida, indicando que o efeito antiproliferativo observado pode ser devido a uma interrupção das células na fase SG2/M. Observou-se uma maior atividade de caspase 3 (m-nitro tio-limoneno), 6 (benzaldeído tiocanfeno e p-hidróxi tio-limoneno) e 8 (benzaldeído tio-limoneno). Características apoptóticas tardias foram detectados em 62% das células tratadas com benzaldeído tio-canfeno e as alterações morfológicas típicas de processo de apoptose foram visualizadas através da microscopia de fluorescência e de microscopia eletrônica de varredura (MEV) após tratamento com o benzaldeído tio-canfeno escolhido devido ao seu baixo valor de IC50 (12 μM). Observou-se a expressão gênica das caspases 2, 3, 6, 8 e o apaf-1 nas células tratadas com benzaldeído tio-canfeno indicando a participação dessas enzimas no efeito antiproliferativo observado. Os resultados indicam que as tiossemicarbazonas derivadas de canfeno e limoneno podem inibir a proliferação celular, regular o ciclo celular e induzir apoptose nas células de melanoma humano (SK-MEL-37), portanto, podem ser considerados candidatos para futuros ensaios pré-clínico in vivo.