Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 95-113).

Diss. (sammanfattning) Stockholm : Karol. inst., 2006. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Washington, 2004. / Vita. Includes bibliographical references (leaves 106-122).

Doctor of Philosophy / Biochemistry and Molecular Biophysics Interdepartmental Program / Anna Zolkiewska / Cancer cells exploit key signaling pathways in order to survive, proliferate, and metastasize. Understanding the intricacies of the aberrant signaling in cancer may provide new insight into how to therapeutically target tumor cells. The goal of my research was to explore the role of two modulators of transmembrane signaling, the secretory pathway and cell surface proteolysis, in the aggressiveness of breast cancer cells. To study the role of the secretory pathway, I focused on the family of endoplasmic reticulum (ER) chaperones. I found that several ER chaperones were upregulated in breast cancer cells grown under anchorage-independent conditions as mammospheres versus those grown under adherent conditions. Furthermore, certain members of the protein disulfide isomerase (PDI) family were consistently upregulated in two different cell lines at both the mRNA and protein levels. Knocking down these PDIs decreased the ability of the cells to form mammospheres. I demonstrated that the requirement for PDI chaperones in mammosphere growth is likely due to an increased flux of extracellular matrix (ECM) components through the ER. Next, I examined the role of cell surface proteolysis in modulating the aggressiveness of breast cancer cells. Cell-surface metalloproteases release soluble growth factors from cells and activate the corresponding growth factor receptors. I determined that specific metalloproteases (ADAM9 or ADAM12), modulate the activation of Epidermal Growth Factor Receptor (EGFR). I demonstrated that EGFR activation enhances the CD44⁺/CD24⁻ cell surface marker profile, which is a measure of cancer cell aggressiveness. I found that the MEK/ERK pathway, which is a downstream effector of EGFR activation, modulates the CD44⁺/CD24⁻ phenotype. When DUSP4, a negative regulator of the MEK/ERK pathway, is lost, activation of EGFR by metalloproteases no longer plays a significant role in cancer cell aggressiveness. This indicates that the ligand dependent activation of the EGFR/MEK/ERK pathway is a critical step in DUSP4-positive aggressive breast cancer. Finally, I examined the importance of metalloproteases in the regulation of Programmed-death ligand 1 (PD-L1), a transmembrane protein expressed by some cancer cells that plays a major role in suppressing the immune system. I demonstrated that cell-surface metalloproteases have the ability to cleave PD-L1 and release its receptor-binding domain to the extracellular environment. Collectively, these data indicate that (a) ER chaperones support anchorage-independent cell growth, (b) metalloproteases are important in regulation of an aggressive phenotype through the EGFR/MEK/ERK pathway, and (c) metalloproteases cleave PD-L1, a key component of immunosuppression in cancer.

Stress, either physical or psychological, can have a dramatic impact on our immune system. There has been little progress, however, in understanding chronic stress-induced immunosuppression. Naive CD4+ T cells could modulate immune responses via differentiation to T helper (Th) cells. In this study, we showed that stress promotes the release of the Th1 cytokines interferon (IFN)-γ and tumor necrosis factor (TNF)-α, the Th2 cytokines interleukin (IL)-4 and IL-10 and the Th17 cytokine IL-17 of splenic naive CD4+ T cells. This suggests that stress promotes the differentiation of naive CD4+ T cells to Th1, Th2 and Th17 cells. Knockout strategies verified that TLR2 might modulate the differentiation of Th1/Th2 cells by inhibiting p38 mitogen-activated protein kinase (MAPK). Taken together, our data suggest that chronic stress induces immune suppression by targeting TLR2 and p38 MAPK in naive CD4+ T cells.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24525号 / 医博第4967号 / 新制||医||1065(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 渡邊 直樹, 教授 中川 一路, 教授 秋山 芳展 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

INTRODUÇÃO: O melanoma primário da cavidade oral é uma neoplasia agressiva, rara e originada a partir da proliferação de melanócitos malignos da mucosa. Ele representa aproximadamente de 0,2 a 8% de todos os melanomas. Estudos recentes apontam algumas vias moleculares tem sido encontradas por estarem envolvidas na patogenia dos melanomas. Dentre essas vias destaca-se a via proliferativa da MAPK (mitogen activated protein kinase), esta cascata de sinalização está envolvida no controle do crescimento celular, proliferação e migração, e tem sido relacionada com um papel importante no desenvolvimento e progressão do melanoma cutâneo. OBJETIVOS: Analisar a expressão proteica e mutação pontual dos componentes da via MAPK e correlacionar com os dados clínicos-histológicos. MATERIAL E MÉTODOS: Através da imunoistoquímica avaliar a expressão proteica dos anticorpos RAS; BRAF; MEK1; MEK2; ERK1 e ERK2 em 35 casos de melanomas orais organizados em matriz (TMA: Tissue Microarray) e através de pirosequenciamento avaliar a mutação pontual dos genes BRAF; NRAS; KRAS em 14 casos de melanomas orais. RESULTADOS: Idade dos pacientes entre 9 e 91 anos, sem predileção por sexo, 75% caucasianos, 71,42% acometeram o palato, 80% com aspecto histológico grau III. A análise da expressão proteica foi: RAS (28,57%); BRAF (82,85%); MEK1 (0%); MEK2 (51,43%); ERK1 (20%)e ERK2 (74,28%). Na análise molecular observamos mutações para BRAF (9/14 casos) e NRAS (2/14 casos). CONCLUSÃO: Todos os aspectos da via MAPK necessita de outras elucidações em melanomas de áreas foto-protegidas e melanomas de mucosa e comparando diferentes populações. Entretanto, os resultados deste presente estudo apontam importante alterações na cascata RAS-RAF-MEK-ERK e estes são indicadores de prognóstico ruim em melanomas primários da mucosa oral, independente da exposição solar / BACKGROUND: Primary melanoma of the oral cavity is an aggressive and rare neoplasm and originated from the proliferation of malignant melanocytes of the mucosa. It represents approximately 0.2 to 8% of all melanomas. Recent studies indicate some molecular pathways have been found to be involved in the pathogenesis of melanomas. Among these means there is a proliferative MAPK pathway (\"mitogen activated protein kinase\"), this signaling pathway is involved in controlling cell growth, proliferation and migration, and it has been associated with a role in the development and progression of melanoma skin. OBJECTIVES: To analyze protein expression and mutation of components of the MAPK pathway and to correlate with the clinical, histological data. MATERIALS AND METHODS: Using immunohistochemistry to evaluate the protein expression of RAS, BRAF, MEK1, MEK2, ERK1 and ERK2 antibodies in 35 cases of oral melanomas organized array (TMA: Tissue Microarray) and using pyrosequencing to assess the mutation of the BRAF, NRAS, KRAS in 14 cases of oral melanomas. RESULTS: Age of patients between 9 and 91 years, regardless of gender, 75% Caucasian, 71.42% in palate, 80% with histologic grade III. Analysis of protein expression was: RAS (28.57%); BRAF (82.85%); MEK1 (0%), MEK2 (51.43%); ERK1 (20%) and ERK2 (74.28%). Molecular analysis we found BRAF mutations (9/14 cases) and NRAS (2/14 cases). CONCLUSION: All aspects of the MAPK pathway requires further elucidation in melanomas of photo-protected areas and mucosal melanomas and comparing different populations. However, the results of this study indicate important changes in the cascade RAS-RAF-MEK-ERK and these are indicators of poor prognosis in primary melanomas of the oral mucosa, regardless of sun exposure

The mammalian Na+/H+ exchanger isoform 1 (NHE1) is a ubiquitously expressed membrane protein that regulates intracellular pH. In the myocardium, NHE1 has been implicated in ischemia/reperfusion (I/R) and cardiac hypertrophy (CH). Hormonal, autocrine and paracrine stimuli, acidosis, cardiotoxic metabolites released during I/R and CH increases NHE1 protein expression and activity. The involvement of NHE1 in CH and I/R has been further supported with the use of NHE1 inhibitors, which have been beneficial in the prevention/regression of several models of CH and I/R injury. Despite the fact that elevation of NHE1 expression and activity have been demonstrated in several models of heart disease, it was unclear whether elevation of NHE1 protein expression was sufficient to induce a specific cardiac pathology, or whether activation of the protein was required. To understand the direct role of NHE1 in CH and I/R, an in vivo and in vitro gain-of-function model, expressing varying levels and activities of NHE1 were examined. In vivo, our N-line mice expressed wild type NHE1 and our K-line mice expressed constitutively active NHE1. In vitro, neonatal rat ventricular cardiomyocytes were infected with the IRM adenovirus containing wild type NHE1 or the K-IRM adenovirus containing active NHE1. We demonstrated that expression of constitutively active NHE1 promotes CH to a much greater degree than expression of wild type NHE1 alone, both in vivo and in vitro. This NHE1-dependent hypertrophic response occurred independent of signaling pathways involved in CH including, mitogen activated protein kinases, p90 ribosomal S6 kinase, calcineurin and glycogen synthase kinase. The NHE1-dependent hypertrophic effect also occurred independent of gender. In addition, the expression of active NHE1 increased the susceptibility of intact mice to neurohormonal stimulation and progressed the hypertrophic response. When these hearts expressing active NHE1 were subjected to I/R using the ex vivo working heart perfusion model, fatty acid (FA) oxidation and glycolysis rates increased, thus generating greater ATP production rates. This was associated with cardioprotective effects in the myocardium, as well as a more energetically efficient myocardium. Expression of the endoplasmic reticulum (ER) stress response proteins, calreticulin and PDI were also shown to be increased relative to controls, and may contribute to the cardioprotection observed. We demonstrate that active NHE1 induces cardioprotection and alters cardiac metabolism in working hearts subjected to I/R. Overall, our results suggest that expression of active NHE1 has a double edged sword effect, on one side it induces CH while on the other side, it protects the heart against I/R injury.

No description available.