Orientadores: Luiz Paulo Kowalski, Oslei Paes de Almeida / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba. / Made available in DSpace on 2018-08-12T21:46:34Z (GMT). No. of bitstreams: 1 Mariano_FernandaViviane_M.pdf: 2260766 bytes, checksum: 9d916cd6b44b24dfe9804dc33b654b5a (MD5) Previous issue date: 2009 / Resumo: As neoplasias de glândulas salivares são raras com incidência anual de 0,4 a 3,5 casos por 100.000 pessoas. Correspondem a cerca de 2 a 6,5% de todos os tumores da egião de cabeça e pescoço. A freqüência de neoplasias malignas varia de 0,4 a 2,6 casos por 100.000 pessoas e o tipo mais comum é o carcinoma mucoepidermóide. O local mais freqüentemente acometido é a glândula parótida, seguida das glândulas salivares menores, submandibular e sublingual. A probabilidade de pacientes com tumores de glândulas salivares desenvolverem metástases à distância está associada com o tipo histológico dos tumores, sendo mais comumente relacionada ao carcinoma adenóide cístico, carcinoma mucoepidermóide de alto grau, carcinoma de ducto salivar e tumores localizados na glândula submandibular, base de língua e tumores faringeanos. O objetivo deste estudo foi avaliar as características clínicas e histopatológicas de carcinomas de glândulas salivares maiores tratados no Hospital A.C. Camargo, São Paulo, de 1953 a 2004, a fim de identificar fatores de risco associados ao desenvolvimento de metástases à distância e sua importância prognóstica. Os dados clínicos foram obtidos através dos prontuários médicos dos pacientes e transferidos para fichas clínicas padronizadas para o estudo. O número total de casos estudados foi de 255, dos quais 57 pacientes desenvolveram metástases à distância, sendo a glândula submandibular o local primário mais relacionado e o pulmão o sítio metastático mais comum. Não houve diferença estatística entre os gêneros. O tipo histológico mais encontrado nos casos com metástase foi o carcinoma adenóide cístico. Os fatores associados a pior prognóstico foram: ocorrência de paralisia facial, invasão de estruturas adjacentes, tumores localizados na glândula submandibular, comprometimento linfonodal, estádio clínico avançado, presença de tumor residual e desenvolvimento de metástase à distância. Os resultados deste trabalho permitiram obter informações sobre as principais características clínicas e patológicas dos carcinomas de glândulas salivares maiores e identificar que indivíduos portadores de carcinoma adenóide cístico apresentam o maior risco de desenvolvimento de metástase à distância. / Abstract: Salivary gland tumors are rare with annual incidence around of 0.4 to 13.5 cases per 100,000 people. They correspond about 2 to 6.5% of all tumors from head and neck region. The frequency of malignant tumors varies from 0.4 to 2.6 cases per 100,000 people and the more common type is the mucoepidermoid carcinoma, occurring preferentially in the parotid gland, followed of minor salivary glands, submandibular and sublingual. The probability of patients with salivary gland tumors to develop distant metastasis is associate with histology type of tumor, being more commonly related with adenoid cystic carcinoma, high-grade mucoepidermoid carcinoma, salivary duct carcinoma and tumors localized in the submandibular gland, base of the tongue, and pharynx. The objective of this study was to evaluate the clinical characteristics of patients with carcinomas of major salivary glands treated in the A.C. Camargo Hospital, São Paulo, from 1953 to 2004, to identify risk factors associated to the development of distant metastasis and its prognostic importance. The clinical data was obtained reviewing the medical charts of the patients and transferred to the standardized data collection forms. The total number of studied cases was 255, of these 57 patients had developed distant metastasis, being the submandibular gland the primary tumor site most frequently related. The lung was the most common metastatic site. There was no statistical difference between genders. The histological type more frequently associated with distant metastasis was adenoid cystic carcinoma. The factors associated with poor outcomes were: facial paralysis, invasion of adjacent structures, tumors from submandibular, positive lymph nodes, advanced clinical stage, presence of residual tumor, and distant metastasis. The results of this work had allowed to get information concerning the main clinical and pathological characteristics of major salivary glands carcinomas and to identify that individuals with adenoid cystic carcinoma had the highest risk of development of distant metastasis. / Mestrado / Estomatologia / Mestre em Estomatopatologia

Natural killer cells are part of the innate immune response, and get their name ‘natural killer’ due to their unique ability to recognize and kill aberrant cells without prior sensitization. The Ly49 receptors on NK cells are involved in both their education process and mediating their ability to recognize aberrant cells. Virally-infected and tumor cells have been shown to exhibit decreased levels of MHC-I on their cell surface. As MHC-I is expressed on all nucleated cells, the Ly49 receptors on NK cells recognize this loss of MHC-I on the surface as a kill signal, leading to direct cytotoxicity through the release of perforin and granzymes. This recognition process by NK cells is termed “missing-self.” The interactions between MHC-I and Ly49 receptors are integral to NK cell function. The response of the NK cell to cells which it encounters is dependent on a balance of activating and inhibitory receptors. This signal in turn is dependent on the engagement and interactions of various ligands, such as MHC-I, by the activating and inhibitory Ly49s on the NK cells. In this compilation of my three main research projects, the interactions and binding capacity between the different MHC-I molecules and different members of the Ly49 receptor family are explored. In addition, the importance of the Ly49 receptors in NK cell-mediated cancer immunosurveillance is studied.

Metastasis persists as a significant unsolved hurdle in cancer treatment, with greater than 90% of cancer-related deaths attributed to metastasis. In order for cells to successfully metastasize, they must dynamically regulate cell adhesion. However, cell adhesion molecules are rarely mutated or deleted genetically in cancer, indicating that tumor cells are able to co-opt intrinsic regulatory mechanisms of adhesion to drive metastasis. We previously identified Activated Leukocyte Cell Adhesion Molecule (ALCAM) as a clinically relevant driver of metastasis and hypothesized that tunable regulation of its function contributes to tumor cell adhesion and metastasis. We tested this hypothesis through two channels. We identified ALCAM as a novel binding partner of tetraspanin CD151, a known regulator of cell adhesion and motility. We previously demonstrated that clustering of integrin-free CD151 (CD151free) increased cell adhesion, decreased cell motility, and inhibited metastasis. Here, we identified ALCAM as a novel CD151 partner required for CD151free to control adhesion. Biochemical analyses revealed that CD151free is coupled to ALCAM by the scaffolding protein syntenin-1. Additionally, we show that the intracellular domain of ALCAM (ALCAM-ICD) is susceptible to ɣ-secretase cleavage, which releases a PDZ-binding peptide capable of disrupting the CD151/syntenin-1/ALCAM complex. Disruption of this complex impedes CD151free-mediated regulation of tumor cell adhesion and metastasis, demonstrating that CD151free controls tumor cell migration through a trimeric complex of CD151/syntenin-1/ALCAM. Further evaluate of ALCAM revealed a potential alternative splicing which we predicted to control proteolytic shedding of its extracellular domain. We demonstrate that the loss of the membrane-proximal exon13 generates an ALCAM splice variant (ALCAM-Iso2) that enhances metastasis four-fold. Mechanistic studies identified a novel MMP14-dependent, membrane distal cleavage site in ALCAM-Iso2, which increases shedding ten-fold, thereby decreasing cellular cohesion and promoting motility. ALCAM-Iso2-expression was greatly increased in bladder cancer, further emphasizing that ALCAM alternative splicing can contribute to clinical disease progression. The requirement for both the loss of exon 13 and the gain of metalloprotease activity suggests that ALCAM shedding and concomitant regulation of dissemination is a locally tunable process. In summary, this dissertation presents two mechanisms by which tumor cells are able to dynamically regulate cell adhesion to modulate migration and metastasis.

Cell motility is a process tightly linked to tumor metastasis. The p21-activated kinases (PAKs) are modulators of cell motility commonly overexpressed and hyperactive in various malignancies. We investigated whether increased PAK2 expression and phosphorylation levels enhance cancer cell motility, thus worsening disease prognosis in non-small cell lung cancer (NSCLC). In silico analyses of publically available databases was accessed to determine the clinical relevance of PAK2 gene expression in NSCLC patient outcomes. Total and phospho-specific PAK1 and PAK2 antibodies were used to screen a panel of NSCLC cell lines. shRNA and small molecule inhibitors were utilized to assess the role of PAK1/2 kinases in augmenting tumor metastatic capacity in vitro by two and three-dimensional cell migration assays. Increased tumor PAK2 expression was associated with significantly worse survival in two unique cohorts of patients with resected lung adenocarcinoma. Phosphorylated PAK1/2 isoforms were frequently but variably expressed across 31 NSCLC cell lines, and at higher levels than in immortalized normal bronchial epithelial cells. We observed a dependency on PAK2 for cell motility specifically in cell lines with phosphorylated PAK1/2. Additionally, use of two PAK small molecule inhibitors recapitulated the effects observed in our PAK2 shRNA mediated knockdown studies. Our findings demonstrate the importance of PAK2 signaling in promoting lung cancer cell motility and are the first to link high PAK2 expression to poor clinical prognosis in lung adenocarcinoma. These data suggest that targeting PAK2 may cause an anti-migratory effect and potentially improve survival for NSCLC patients with elevated tumor PAK2 expression levels.

Ewing sarcoma is a highly aggressive pediatric bone tumor that is characterized EWS/FLI1 transcription factor. Ewing sarcoma tumors require the continued activity of EWS/FLI1 to sustain a gene signature that promotes proliferation and blocks differentiation leading to tumorigenesis and disease progression. Inhibition of EWS/FLI1 activity is not compatible with cell proliferation, which creates an attractive drug target. While no EWS/FLI1 targeted therapies have been translated into the clinic, trabectedin has demonstrated activity in early phase clinical trials. In this report, we optimize trabectedin therapy for the treatment of Ewing sarcoma. We identify the mechanism by which trabectedin inhibits EWS/FLI1 activity and demonstrate that inhibition can be achieved at clinically relevant concentrations. We use this novel mechanism of action to further optimize the schedule of administration and show that maximum EWS/FLI1 inhibition is obtained following a short term, high concentration treatment with trabectedin. Additionally, we characterize a second-generation analog of trabectedin, lurbinectedin, which has an improved pharmacokinetic profile that allows much higher serum concentrations to be achieved. In addition to being a bonafide EWS/FLI1 inhibitor, lurbinectedin can be combined with SN38 (in vitro) or irinotecan (in vivo) to augment the suppression of EWS/FLI1 target genes. Ultimately, we show that this combination strategy decreases tumor growth, extends lifespan, and leads to the differentiation of xenograft mouse models of Ewing sarcoma.

Evasion of cell death is essential to every step of tumorigenesis. Anti-apoptotic Bcl-2 family proteins are master inhibitors of cell death, and thus are often overexpressed in cancers. In particular, Bcl-2, Bcl-xL, and Mcl-1 are highly expressed in Estrogen Receptor-alpha positive (ERα+) breast cancers. However, one anti-apoptotic Bcl-2 family protein, Mcl-1, is understudied in ERα+ breast cancers. Herein we show that Mcl-1 is essential to ERα+ breast tumor cell survival, and is a more potent tumor cell survival factor than other family members, like Bcl-2 and Bcl-xL. Interestingly, Mcl-1 expression and activity increased upon Bcl-2/Bcl-xL dual inhibition with ABT-263, mediated by increased Mcl-1 cap-dependent translation. Blockade of cap-dependent translation, through inhibition of mTORC1 signaling, resulted in tumor cell killing in cell culture and in vivo, phenocopying ablation of Mcl-1 by RNA-interference. Mcl-1 depletion in combination with ABT-263 restored sensitivity to Bcl-2/Bcl-xL blockade, suggesting that Mcl-1 is a primary resistance factor to Bcl-2/Bcl-xL inhibition in ERα+ breast cancers. Importantly, preliminary studies suggest that anti-apoptotic Bcl-2 family proteins can promote resistance to standard of care breast cancer therapies. Mcl-1 inhibition, but not Bcl-2/Bcl-xL blockade, promoted cell death in a model of anti-estrogen resistance, long term estrogen deprivation (LTED). Mcl-1 inhibition using polymeric nanoparticles containing Mcl-1 siRNA (si-NPs), increased tumor cell death in combination with LTED and after treatment with the selective estrogen receptor downregulator fulvestrant. Therefore, Mcl-1 is a dominant tumor cell survival factor in ERα+ breast cancers that is rapidly and potently upregulated in response to targeted therapies. However, dependence on Mcl-1 for tumor cell survival may be clinically thwarted using mTOR inhibitors or si-NPs.

The skeleton is a common site for breast cancer metastasis. Although significant progress has been made to manage osteolytic bone lesions caused by breast tumors, patients still die of the disease, and the current treatments for these lesions are palliative. Thus, there is a need to identify the early determinants of the breast cancer bone metastatic process in order to treat these lesions more effectively. Progression and recurrence of breast cancer, as well as reduced survival of patients with breast cancer, are associated with chronic stress, a condition known to stimulate sympathetic nerve outflow. Interestingly, the bone is innervated by nerves of the sympathetic nervous system (SNS), and previous data from our lab show that chronic SNS stimulation promotes breast cancer colonization of bone. What has not been explored is how chronic SNS activity alters the bone vasculature, a key component in successful osteotropic breast cancer metastases. The research presented in this dissertation shows that stimulation of the beta 2-adrenergic receptors (b2AR) specifically on osteoblasts alters bone vascular density and the adhesion protein profile of bone marrow endothelium via increased osetoblastic Vegf-a and Il-6. These results support the notion that chronic stress affects not only colonization of bone by breast cancer cells, but also the interaction between endothelium and tumor cells that occurs during extravasation. Potential clinical benefits of beta-blockers, anti-angiogenics, and inhibitors that target adhesion need to be investigated further regarding breast cancer bone metastases.

Magister Chirurgiae Dentium - MChD / The aim of the study is to compare the palliative efficacy of two saliva substitutes (Sinspeek and Xerostom) in patients during radiotherapy for cancer of the head and neck. This crossover randomised controlled clinical trial was carried out on twenty-five patients with malignant tumours of the head and neck, following four weeks of radiotherapy at tygerberg hospital. The benefit of saliva substitutes to ameliorate the effects of xerostomia is well established and proper advice and access to relevant preparations is essential. / South Africa

No description available.

Major Histocompatibility Complex (MHC) Class I molecules present peptides to CD8⁺ T cells and are essential for most adaptive immune responses. The first described-spliced tapasin (“isoform 1”) plays a critical role in MHC-I antigen presentation by facilitating peptide loading onto MHC-I molecules in the endoplasmic reticulum (ER). This thesis examines the expression, localization and function of two novel, alternatively-spliced isoforms of human tapasin that lack exon 7 (“isoform 2”) or both exons 6 and 7 (“isoform 3”). Isoform 1 contains a di-lysine ER-retention motif; the two novel isoforms encode different carboxy (C) termini that lack this motif. It was hypothesized that isoforms 2 and 3 would function in MHC-I cross-presentation of exogenous antigens in non-ER compartments. Isoform 2, like isoform 1, was found to be mainly ER-localized; however, both these isoforms were also found to co-localize in smaller amounts with the trans Golgi network and endo/lysosomes by confocal microscopy. Isoform 3 lacks a transmembrane domain and was found to be secreted from cells as well as being found within the ER. All isoforms were widely expressed at the RNA level in many tissues and cell types; however, mature dendritic cells (DCs) expressed the highest levels of all three isoforms, consistent with the high cross-presenting abilities of DCs. Both isoform 1 and 2 stabilized the transporters associated with antigen processing (TAP) in murine tapasin-/- cells, but isoform 3 did not due to its missing transmembrane domain. Isoform 1 and 2 mediated very similar effects on endogenous MHC-I presentation of self and viral peptides, on surface MHC-I thermostability, and on MHC-I maturation rates. Isoform 3 was found to decrease loading of exogenous peptides onto MHC-I. None of the isoforms influenced cross-presentation of the soluble antigen ovalbumin in a mouse dendritic cell line. This thesis also examines the effect of antigen presentation machinery (APM) re-expression in MHC-I-deficient tumor cell lines, B16F10 and CMT.64, which are deficient in TAP and tapasin. Virally-driven TAP1 and Tapasin expression increased MHC-I expression in the tumor cell lines, augmented tumor cell immunogenicity, and decreased tumor growth in vivo due to increased tumor cell elimination by the immune system. / Science, Faculty of / Zoology, Department of / Graduate