Thesis (Ph. D.)--Case Western Reserve University, 2005. / [School of Medicine] Department of Physiology and Biophysics. Includes bibliographical references. Available online via OhioLINK's ETD Center.

Thesis (Ph. D.)--University of Virginia, 2009. / Title from title page. Includes bibliographical references. Also available online through Digital Dissertations.

Thesis (Ph. D.)--University of Virginia, 1999. / Includes bibliographical references (p. 171-256). Also available online through Digital Dissertations.

Thesis (Ph. D.)--Cornell University, May, 2007. / Vita. Includes bibliographical references.

Thesis (Ph. D)--University of Washington, 2005. / Vita. Includes bibliographical references (leaves 115-142).

Thesis (Ph.D.)--McMaster University, 2004. / Advisor: William J. Muler. Includes bibliographical references (leaves 171-199). Also available online.

Made available in DSpace on 2015-05-14T12:56:08Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 842543 bytes, checksum: dcc4a89c58b1a2bed72a86aa5e76dab2 (MD5) Previous issue date: 2008-12-05 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The aim of this study was to assess salivary EGF concentration in head and neck cancer patients and its correlation with oral mucositis. It was an observational, transversal, control-case study. Whole stimulated saliva was collected in this groups: G1 control (n=9), G2 diagnostic moment (n=13), G3 before therapy (n=11), G4 during therapy (n=13) and G5 after therapy (n=11). The samples were centrifuged and measured. The supernatant was obtained and stored at -80° C until assayed. Salivary EGF content was measured with a commercial ELISA kit (Quantikine, R & D Systems, Minneapolis, MN, E.U.A.). The salivary flow means to G1, G2, G3, G4, G5 were 1,27; 1,58; 1,70; 0,77 and 0,99 ml/min. respectively. Differences on salivary flow did not reach statistical significance in the groups (p=0,064) but there was a strong reduction in G4 and G5. In relation to EGF levels, it was seen 1624,08; 1816,63; 1905,47 and 1457,12 pg/ml, respectively. There was no significant difference in the salivary EGF levels of this patients (p=0,686) but the decreased levels in G4 was evident. There was a positive correlation between salivary flow and EGF levels in G4 and G5, but it was not statistically significant, G4 (p=0,446) and G5 (p=0,258). There was not association between EGF concentration and oral mucositis development (p=0,037), but it was significant between decreased salivary flow and mucositis (p< 0,001). It was shown that EGF levels were not influenced for the use of alcohol (p=0,792), tobacco (p=0,706) and the patient performance status (p=0,213). It was possible to conclude that radiation therapy induces reduction in salivary flow and EGF levels over time. That EGF levels decreased is influenced by low salivary flow, but there was no association between oral mucositis development and salivary EGF levels. / O presente estudo teve como objetivo verificar a concentração do EGF na saliva de pacientes com neoplasias malignas em região de cabeça e pescoço e sua possível relação com a mucosite oral e o fluxo salivar. Constituiu-se em um estudo transversal, observacional, do tipo caso-controle. As amostras de saliva estimulada foram obtidas nos seguintes grupos: G1-controle (n=9), G2- momento do diagnóstico (n=13), G3-antes da terapia (n=11), G4-durante a terapia (n=13) e G5-após a terapia (n=11), sendo as mesmas centrifugadas e mensuradas. O sobrenadante foi então armazenado a temperatura de -80 º C e, posteriormente, descongelado a temperatura ambiente e submetido a imunoensaio do tipo sanduíche, onde a concentração salivar de EGF foi determinada por um kit comercial de ELISA (Quantikine, R & D Systems, Minneapolis, MN, E.U.A.). Verificou-se médias de fluxo salivar para G-1, G-2, G-3, G-4 e G-5 de 1,27; 1,58; 1,70; 0,77 e 0,99 ml/min, respectivamente. Segundo o teste F (ANOVA) não houve diferença estatisticamente significativa entre os fluxos nesses grupos (p=0,064), embora tenha havido redução significativa no G4 e G5. Em relação ao EGF, observou-se, consecutivamente, as seguintes médias: 1624,08; 1816,63; 1905,47; 1414,87 e 1457,12 pg/ml, também não sendo observada significância estatística nas diferenças entre os grupos (p=0,686), percebendo-se diminuição em seus níveis no G4. Por meio da correlação de Pearson, observou-se correlação positiva entre as medidas de fluxo salivar e as concentrações de EGF no G4 e G5, embora sem significância estatística, G4 (p=0,446) e G5 (p=0,258). Não houve associação entre as concentrações de EGF e o desenvolvimento da mucosite oral (p=0,337), mas houve entre o fluxo salivar diminuído e a mucosite (p< 0,001). Além disso, os níveis de EGF na saliva não foram influenciados pelos hábitos de etilismo (p=0,792), tabagismo (p=0,706), ou ainda, pelo estado de saúde geral do paciente (p=0,213), segundo o t-Student. Com base nos resultados obtidos, podemos concluir que a radioterapia promoveu diminuição no fluxo salivar e nos níveis de EGF, que os níveis de EGF foram influenciados pela diminuição do fluxo salivar e que não houve associação entre o desenvolvimento da mucosite oral e a concentração de EGF na saliva.

Colorectal cancer (CRC), which includes cancer of the large bowel and rectum is the third most common cancer in men and the second in women and there is a poorer survival rate in less developed regions of the world such as West Africa mainly due to the ‘out of reach’ costs of chemotherapy. Evidence suggests that aspirin, a non-steroidal anti-inflammatory drug (NSAID) has the potential to decrease incidence of, or mortality from, a number of cancers including CRC through several mechanisms of action. However, this evidence is dampened by aspirin’s gastrointestinal (GI) toxicity, which have been found to be mostly age-dependent. The search for potential aspirin-related compounds with the same or better cytotoxic effects against cancer cells accompanied by a safer toxicity profile has been ongoing over the years and led to us to synthesise a number of novel aspirin analogues. One of the mechanisms of action suggested for the anticancer property of aspirin is the COX-dependent pathway. In this thesis SW480 cell line, a CRC cell line that is COX-2 negative and mismatch repair (MMR) proficient was used to study the possible COX-independent mechanism of action for aspirin, its analogues and diflunisal at 0.5 mM. Diflunisal was included in this study because it is also a salicylate with reports of having cytotoxic effects. OE33 and FLO1 oesophageal cancer cells were also employed in the epidermal growth factor receptor (EGFR) and synergy experiments to show effects were not just specific to SW480 cells alone. These aspirin analogues were synthesised, identified using nuclear magnetic resonance (NMR) and infra-red (IR) spectroscopy, and tested for purity using thin layer chromatography (TLC) and melting point. The findings of this study suggest that these compounds breakdown into salicylates and perturb epidermal growth factor (EGF) internalization with PN517 (fumaryldiaspirin) and PN590 (ortho-thioaspirin) also driving EGF co-localization with early-endosome antigen-1 (EEA1). The perturbation of the internalization of EGF by aspirin and PN517 was also observed by a time-lapse assay using live confocal imaging. These compounds also had specific effects on different tyrosine phosphorylation sites of the EGFR, with none but PN590 inhibiting 4 phosphorylation at Y1068, and all but PN502 (ortho-aspirin), PN548 (meta-aspirin) and PN549 (para-aspirin) inhibiting phosphorylation at Y1045 and Y1173. Given that the EGF internalization assay involved the cells being treated with compounds for 2 h, cells were also treated for this same time period and probed with pEGFR 1045, which resulted in the compounds having no significant effect on phosphorylation at that site which is responsible for the ubiquitination of the EGFR. Most of these compounds were apoptotic with some showing a combination of apoptosis and necrosis. Aspirin and its isomers drove apoptotic cell death in SW480 cells via the BCL2-BAX pathway while the thioaspirins appear to follow the p21 pathway by decreasing the expression of the protein. In addition, it was shown that PN502 (aspirin), PN517 and PN590 had synergistic effects when used in combination with oxaliplatin at ED50, ED75 and ED90 in SW480 CRC cells. The cytotoxicity of these compounds individually or in combination was determined using MTT assay followed by the use of the CompuSyn and CalcuSyn software to calculate combination index (CI), which indicated whether a drug combination was synergistic, antagonistic or additive. PN517 and PN524 were synergistic when used in combination with cisplatin in OE33 oesophageal cancer cells. Effect of these compounds on the EGFR indicates a delay or disruption of the signalling pathway involved in the proliferation of cancer cells, thus, translating into protection against tumour formation or progression while the synergistic effects of these compounds when used in combination with platinum compounds can provide patients with less toxic chemotherapeutic regimen especially in patients with CRC tumours that harbour mutant TP53 gene and normally resistant to oxaliplatin. It is therefore proposed that the perturbation of EGF internalization is a novel mechanism of action for aspirin and its analogues in cancer therapy. These positive findings shed light on the understanding of the possible mechanism of action for aspirins and gives hope for a more affordable, less toxic therapy for the prevention, treatment and management of cancer.

Introduction: The incidence of oesophageal cancer (OC) is increasing. Targeted therapies are being investigated, as chemotherapy in advanced OC achieves only 50% response rates and confers significant toxicity. TRANS-COG is a sub-study of COG, the only randomised trial of the Epidermal Growth Factor Receptor (EGFR) tyrosine kinase inhibitor (TKI) gefitinib versus placebo in patients with advanced OC. Aim and Methods: The aim of TRANS-COG is to identify biomarkers for predicting response to gefitinib by investigating the frequency of mutations of EGFR pathway members and EGFR gene copy number gain (CNG) by Fluorescence In Situ Hybridisation (FISH). Results: No EGFR mutations were identified, frequency of other pathway member mutations are as follows: KRAS 3.6%, PI3KCA 3.2%, BRAF 0.6%. The frequency of EGFR CNG was 13.5%. There were two cases of combined EGFR CNG and KRAS mutation 0.3%.Patients with EGFR CNG tumours had improved OS with gefitinib compared to placebo, log rank p=0.033, HR 0.523 (95%CI 0.285-0.962). Those with EGFR high CNG (amplification) also had improved OS with gefitinib, median OS 4.40 months vs median OS 1.71 months for placebo log rank p=0.004, HR 0.184 (95% CI 0.052-0.653). Mutation of KRAS conferred poor prognosis, independent of treatment received; median OS 3.614 months in KRAS wild type vs 1.741 months in KRAS mutated tumours, log rank p=0.023, HR 1.8153 (95% CI 1.078 3.187). A novel KRAS codon 61 mutation, Q61L, was also identified. Conclusion: 21.18% of patients have dysregulation of the EGFR pathway, these abnormalities represent realistic therapeutic targets. This analysis demonstrates clinical utility of EGFR CNG as a predictive biomarker of gefitinib response.

Endocytosis is important for the regulation of signal transduction and for the movement of essential cellular components from outside the cell to their appropriate intracellular compartment(s). Two established mechanisms of endocytosis are clathrinmediated (CME) and clathrin-independent endocytosis, and they are responsible for internalization of different ligands. In this study, the newly established technique of total internal reflection fluorescent microscopy (TIRF-M) was used, along with standard biochemical and molecular biological tools, to systematically study the sorting and early trafficking of two established ligands of endocytosis, transferrin (Tf) and epidermal growth factor (EGF). TIRF-M studies revealed that Tf binds its receptor that is located in large clathrin arrays positioned just below the surface of the cell and that these large clathrin platforms serves as the major site of CME at the plasma membrane. EGF endocytosis is very different and occurs as follows 1) the liganded EGFR recruits Rab5 to the plasma membrane, 2) Rab5 concentrates around vesicles containing liganded EGFR and 3) these vesicles co-localize with EEA1 enriched endosomes. EEA1 was shown to play a pivotal role in EGF endocytosis, establishing a new role for EEA1 in vesicle trafficking in addition to its role in tethering and fusion. Finally, WDFY2, a new FYVE domain protein was shown to decorate a specific subset of vesicles, upstream of the EEA1 vesicle pool that appear to participate in Tf endocytosis. These studies establish new functions and components of endocytosis that enhances our understanding of this complex process.