Mammalian reovirus (MRV) is a prototype virus of Reoviridae family. MRV virions are composed of two concentric protein capsids that surround a genome of 10 segments of dsRNA. It has been shown that MRV can manipulate host gene expression and further induce apoptosis and cell cycle arrest in various cell lines. However, the detailed molecular mechanisms by which MRV regulates the expression of host cells are largely unknown. P53 is an important transcriptional factor which modulates the expression of more than 130 genes controlled in cell stress-response. We aimed to examine the molecular mechanisms underlying the effect of MRV infection on the expression of host genes and the possible role of p53 in the interaction of MRV and host cells. Prototype serotype 3 reovirus strain Dearing (T3D) and serotype 1 strain Lang (T1L) were used to infect different cell lines, respectively, H1299 (p53-null and p53 positive), HT1080 (p53 mutant and p53 positive) and HCT116 (p21 deficient and 14-3-3σ deficient). By comparing the virus replication curve of T1L and T3D in these cell lines, we found that MRV can replicate with a similar pattern in both p53-defective and p53-positive cell lines which indicated that p53 does not have significant impact on MRV replication in these cell lines. We further found that the level of Bcl-xL protein, which has been shown to be able to inhibit apoptosis, was increased in H1299 cell lines (both p53-null and p53 positive) infected by T3D, but decreased in the same cell lines infected by T1L. A similar change of Bcl-xL protein was not observed In HCT116 and HT1080 cell lines with MRV infection. Fifty four T1L×T3D reassortants were used to map which gene or gene combination was responsible for the changes of Bcl-xL protein. We found that the expression of Bcl-xL protein in H1299 cell line infected by MRV was majorly controlled by the S1 gene segment which encodes the σ1 cell attachment protein and the σ1s non structural protein, while minorly controlled by L3 gene segment of MRV.

Mammalian reovirus (MRV) is a prototype virus of Reoviridae family. MRV virions are composed of two concentric protein capsids that surround a genome of 10 segments of dsRNA. It has been shown that MRV can manipulate host gene expression and further induce apoptosis and cell cycle arrest in various cell lines. However, the detailed molecular mechanisms by which MRV regulates the expression of host cells are largely unknown. P53 is an important transcriptional factor which modulates the expression of more than 130 genes controlled in cell stress-response. We aimed to examine the molecular mechanisms underlying the effect of MRV infection on the expression of host genes and the possible role of p53 in the interaction of MRV and host cells. Prototype serotype 3 reovirus strain Dearing (T3D) and serotype 1 strain Lang (T1L) were used to infect different cell lines, respectively, H1299 (p53-null and p53 positive), HT1080 (p53 mutant and p53 positive) and HCT116 (p21 deficient and 14-3-3σ deficient). By comparing the virus replication curve of T1L and T3D in these cell lines, we found that MRV can replicate with a similar pattern in both p53-defective and p53-positive cell lines which indicated that p53 does not have significant impact on MRV replication in these cell lines. We further found that the level of Bcl-xL protein, which has been shown to be able to inhibit apoptosis, was increased in H1299 cell lines (both p53-null and p53 positive) infected by T3D, but decreased in the same cell lines infected by T1L. A similar change of Bcl-xL protein was not observed In HCT116 and HT1080 cell lines with MRV infection. Fifty four T1L×T3D reassortants were used to map which gene or gene combination was responsible for the changes of Bcl-xL protein. We found that the expression of Bcl-xL protein in H1299 cell line infected by MRV was majorly controlled by the S1 gene segment which encodes the σ1 cell attachment protein and the σ1s non structural protein, while minorly controlled by L3 gene segment of MRV.

Cell migration is an important component of many physiological and pathological processes such as tissue and organ morphogenesis during development, wound healing, inflammatory immune response, and tumor metastasis. The actin cytoskeleton is the basic engine driving cell migration. In the present study, we elucidate the role of an important actin interacting proteins, Adducins, in motility of normal lung epithelium and lung cancer cells. Adducins are the family of cytoskeleton protein capping the fast growing end and facilitating the bundling of actin filaments. Adducins are encoded by the three closely related genes namely alpha (ADD1), beta (ADD2) and gamma (ADD3) Adducin. ADD1 and ADD3 are ubiquitously expressed, whereas ADD2 is most abundant in brain and erythrocytes. Adducins are also involved in recruiting spectrin to the actin filaments forming spectrin-actin membrane skeletal network. Its role in cell motility remains controversial. In this study, we observed that CRISPR/Cas9 mediated stable knockout of ADD1 and ADD3 in 16HBE normal lung epithelium cells significantly increases transfilter migration of cells. On the other hand, stable overexpression of ADD1 in H1299 Non-Small Cell lung cancer cells significantly decreases wound healing, transfilter migration and Matrigel invasion of the cells. Importantly, the effects of Adducin depletion and overexpression on cell motility were not due to altered cell proliferation. ADD1 overexpressed H1299 cells were characterized by the increased adhesion and spreading on the collagen matrix. Fluorescence microscopy revealed alterations in their cortical actin cytoskeleton that was manifested in the assembly of peripheral F-actin bundles and formation of filopodia-like protrusions. These findings suggest that Adducins are negative regulators of motility of normal lung epithelial and lung cancer cells that act by altering the architecture of submembranous actin cytoskeleton and modulating cell adhesion to the extracellular matrix.

Bacteria typically adhere to various cell surfaces present in the human body to colonise or invade human tissues. Staphylococcus aureus (S. aureus) can express the fibronectin-binding proteins A and B (FnBP-A, FnBP-B) that can facilitate the binding of multiple copies of fibronectin (Fn). In addition, Fn bound to the bacterium trigger activation of α5β1 integrins found on the cells and facilitate invasion of human cells. Although the invasion mechanisms regarding signalling pathways and overall host cell interactions have been defined, the quantitative relationship between the mediators of invasion and the temporal kinetics has not yet been elucidated. In this thesis, newly developed microscopy-based methods have been used to quantify the interactions between H1299 cells and S. aureus at various Fn concentrations. After an approximate Fn concentration of 15 μg/ml, the S. aureus bacteria strains become saturated both for the wildtype and the negative control strains. Additionally, using the step-by-step protocol developed during this study, adhesion of the wildtype strain of S. aureus with 15 μg/ml Fn is occurring on the H1299 cells. Although adjustments to the protocol are needed, this adhesion mechanism will lead to an internalisation of the S. aureus strains to the H1299 cells.

No description available.

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

Background: Aspirin, the pro-drug of salicylate, is associated with reduced incidence of death from cancers and is commonly prescribed in combination with metformin in individuals with type 2 diabetes. Salicylate activates the AMP-activated protein kinase (AMPK) via Ser108 of the AMPK β1 subunit, a mechanism that is distinct from metformin, which increases AMP:ATP. Many cancers have high rates of fatty acid synthesis and AMPK inhibits this pathway through phosphorylation of acetyl-CoA carboxylase (ACC). It is unknown if targeting the AMPK-ACC-lipogenic pathway using salicylate and metformin may be effective for inhibiting cancer cell survival. Results: Salicylate suppresses clonogenic survival of prostate and lung cancer cells at therapeutic concentrations of aspirin. These clinically achievable concentrations of salicylate activated AMPK per the increasing phosphorylation of ACC and suppressing the activity of mTOR effectors kinase p70-S6 kinase and S6; effects that were enhanced with the addition of metformin and blunted in mouse embryonic fibroblasts (MEFS) deficient in AMPK β1. MEF cells deficient in AMPK β1 were more resistant to salicylates inhibitory effect on proliferation. Supplementation of media with fatty acids and mevalonate reverses the suppressive effects on cell survival indicating the inhibition of de novo lipogenesis is likely important. Conclusions: Salicylate increases ACC phosphorylation, reduces phosphorylation of mTOR targets and inhibits de novo lipogenesis in prostate and lung cancer cells, with concentrations of salicylate achievable through the ingestion of Aspirin (0.25-1.0mM) these effects are blunted in AMPK β1 deficient cells. Effects on AMPK activity via ACC phosphorylation as well as reductions in mTOR signalling targets and de novo lipogenesis are enhanced when used in combination with metformin. Suppressive effects on prostate and lung cancer cell survival are ameliorated when media is supplemented with mevalonate and fatty acids. Pre-clinical studies evaluating the use of salicylates alone and with metformin to inhibit de novo lipogenesis and the growth of prostate and lung cancers are warranted. / Thesis / Master of Science (MSc)