No description available.

Epidermal Growth Factor (EGF) is a member of the EGF-like family. EGF binding to the Epidermal Growth Factor Receptor (EGFR) affects cell survival as well as proliferation, migration, and tissue differentiation. Over-expression along with ligand-induced activation of EGFR has been correlated with increased in vivo invasiveness of tumor cells and enhanced in vitro migration of cell lines. This in turn makes EGFR a very important target for cancer therapy. In our investigation we have discovered that the cytoplasmic domain of proEGF (proEGFcyt) has the ability to decrease proliferation and migration in thyroid carcinoma cell lines. We illustrated that proEGFcyt causes specific alterations in the microtubular (MT) phenotype and the composition of MT-associated proteins (MAP) in FTC-133 overexpressing proEGFcyt, a finding not observed in FTC-133 over-expressing a novel splice form of proEGFcyt with a deletion of the complete exon 23 (proEGFdel23) (Pyka et al., 2004). Here, we demonstrate that proEGFcyt suppresses motility and elastinolytic activity in human thyroid Ca cells as a result from reduced secretion of cath-L. This impaired the ability of thyroid Ca cells to penetrate elastin matrices. The reduction in cath-L secretion was as a result of an up-regulation of SNAP25, a member of the t-SNARE plasma membrane complex, which is involved in Ca2+-dependent exocytosis. Furthermore, we demonstrated that proEGFcyt-mediated silencing of UCH-L1 causes the decrease in EGFR due to enhanced EGFR ubiquitination. This correlated with altered proteasomal degradation and provides a unique new mechanism on how proEGFcyt can affect cell proliferation in human thyroid cancer cells. These studies identified novel functions of human proEGF as a bidirectional signaling molecule consisting of the known extracellular EGF domain which functions as the classical ligand and activator of EGFR-mediated cell growth and the proEGF cytoplasmic domain which has the ability to suppress migration and growth of human thyroid carcinoma cells.

In search of thyroid carcinoma biomarkers, proteins secreted by thyroid cancer cell lines, papillary-derived TPC-1 and anaplastic-derived CAL62, were analyzed using liquid chromatography-tandem mass spectrometry. Of forty six high-confidence identifications, six proteins were considered for verification in thyroid cancer patients’ tissues and blood. The localization of two proteins, nucleolin and prothymosin-alpha (PTMA), was confirmed in TPC-1 and CAL62 by confocal microscopy and immunohistochemically in xenografts of TPC-1 cells and human thyroid carcinomas. Increased nuclear and cytoplasmic expression of PTMA was observed in anaplastic carcinomas compared to normal thyroid tissues, papillary and poorly differentiated carcinomas. Importantly, six proteins were detected in thyroid cancer patients’ sera, warranting future analysis to confirm their potential as blood-based thyroid cancer markers. Herein we demonstrate the ability of secretome analysis of thyroid cancer cell lines to identify proteins that may be studied for application in management of thyroid carcinomas upon future validation.

In search of thyroid carcinoma biomarkers, proteins secreted by thyroid cancer cell lines, papillary-derived TPC-1 and anaplastic-derived CAL62, were analyzed using liquid chromatography-tandem mass spectrometry. Of forty six high-confidence identifications, six proteins were considered for verification in thyroid cancer patients’ tissues and blood. The localization of two proteins, nucleolin and prothymosin-alpha (PTMA), was confirmed in TPC-1 and CAL62 by confocal microscopy and immunohistochemically in xenografts of TPC-1 cells and human thyroid carcinomas. Increased nuclear and cytoplasmic expression of PTMA was observed in anaplastic carcinomas compared to normal thyroid tissues, papillary and poorly differentiated carcinomas. Importantly, six proteins were detected in thyroid cancer patients’ sera, warranting future analysis to confirm their potential as blood-based thyroid cancer markers. Herein we demonstrate the ability of secretome analysis of thyroid cancer cell lines to identify proteins that may be studied for application in management of thyroid carcinomas upon future validation.

No description available.

Epidermal Growth Factor (EGF) is a member of the EGF-like family. EGF binding to the Epidermal Growth Factor Receptor (EGFR) affects cell survival as well as proliferation, migration, and tissue differentiation. Over-expression along with ligand-induced activation of EGFR has been correlated with increased in vivo invasiveness of tumor cells and enhanced in vitro migration of cell lines. This in turn makes EGFR a very important target for cancer therapy. In our investigation we have discovered that the cytoplasmic domain of proEGF (proEGFcyt) has the ability to decrease proliferation and migration in thyroid carcinoma cell lines. We illustrated that proEGFcyt causes specific alterations in the microtubular (MT) phenotype and the composition of MT-associated proteins (MAP) in FTC-133 overexpressing proEGFcyt, a finding not observed in FTC-133 over-expressing a novel splice form of proEGFcyt with a deletion of the complete exon 23 (proEGFdel23) (Pyka et al., 2004). Here, we demonstrate that proEGFcyt suppresses motility and elastinolytic activity in human thyroid Ca cells as a result from reduced secretion of cath-L. This impaired the ability of thyroid Ca cells to penetrate elastin matrices. The reduction in cath-L secretion was as a result of an up-regulation of SNAP25, a member of the t-SNARE plasma membrane complex, which is involved in Ca2+-dependent exocytosis. Furthermore, we demonstrated that proEGFcyt-mediated silencing of UCH-L1 causes the decrease in EGFR due to enhanced EGFR ubiquitination. This correlated with altered proteasomal degradation and provides a unique new mechanism on how proEGFcyt can affect cell proliferation in human thyroid cancer cells. These studies identified novel functions of human proEGF as a bidirectional signaling molecule consisting of the known extracellular EGF domain which functions as the classical ligand and activator of EGFR-mediated cell growth and the proEGF cytoplasmic domain which has the ability to suppress migration and growth of human thyroid carcinoma cells.

No description available.

The normal function of the thyroid hormone (TH) system is essential for growth, development and metabolism in humans as well as in other species. The action of TH is dependent on its binding to thyroid hormone receptors (THR) found in the cell nucleus. In some situations, chemicals with structural similarities to TH can bind to these receptors and disrupt their normal function. It has been previously demonstrated that environmental contaminants including, carbamazapine, nonlyphenol (NP), bisphenol A (BPA), and several others are able to bind to the THR as either agonists or antagonists and modulate downstream biochemical responses. Municipal wastewater effluent (MWWE) is a major source of these contaminants entering aquatic environments. Recently extracts of MWWE have been shown to contain chemicals that are capable of binding to THRs. However, MWWE is a complex mixture of chemicals and the specific chemicals have not been identified. In this thesis, a proof of concept was developed for using an Effects Directed Assessment (EDA) approach to isolate thyroid receptor active compounds in MWWE. An EDA is a technique created to extract and identify chemicals from complex mixtures, using various fractionation methods. Once these chemicals have been identified, they are further reviewed for biological relevance. A competitive binding assay for THR was developed and applied to determine the relative binding affinity of known environmental contaminants to THR. Nuclear thyroid hormone receptors were isolated from rainbow trout liver by differential centrifugation. This method involved liver tissue homogenization and subsequent centrifugations to separate the nuclear fraction containing the receptors. The binding characteristics of the isolated THR were evaluated using the thyroid hormones triiodothyronine (T3) and thyroxine (T4) in a competitive binding assay. Minimal binding affinity was present in this assay and future studies should validate the assay further and assure that it is comparable to literature values. Environmental contaminants, including BPA, NP were also tested to determine their relative binding affinity to the THRs compared to the endogenous hormones. High concentrations of both BPA and NP bound to the thyroid hormone receptor, displacing radiolabeled T3 from its binding site. The rainbow trout competitive binding assay was also used to test the binding affinities of extracts from two municipal wastewater effluents collected in the Grand River watershed in southern Ontario. Effluents were extracted using a solid phase adsorbent (HLB Oasis cartridge), eluted with methanol, taken to dryness then reconstituted in ethanol for use in the assay. Both effluent extracts displaced the binding of radiolabeled T3 to the thyroid receptors. The studies demonstrate that a competitive THR assay can be used to detect chemicals in complex mixtures with the potential to interact with THRs. The next step should be to apply the assay using an EDA approach to isolate and identify specific chemicals in effluents that are not yet known to bind to the THR. Interference with the normal function of the TH system has the potential to disrupt normal growth, development and metabolism in aquatic organisms in the receiving environments.

Perfluorinated acids (PFAs) are widespread global and human blood organohalogen contaminants. These monomer decomposition products used in surface treatment products and in fluoropolymer manufacturing and fire fighting may disrupt maternal thyroid hormone homeostasis given that animal studies demonstrate an apparent hypothyroxinemic condition upon PFA exposure. Firstly, we developed a method for properly quantifying perfluorohexane sulfonate (PFHxS), a PFA suspected of overreporting in past literature. We then investigated whether perfluorooctanoate (PFOA), PFHxS and perfluorooctane sulfonate (PFOS) were determinants of maternal hypothyroxinemia in a pregnant women population from Edmonton using a case-control design. Free thyroxine (fT4) and thyroid stimulating hormone (TSH) were screened in 974 women collected during 15-20 weeks of pregnancy. Cases (n=96, hypothyroxinemic: normal TSH and fT4: lowest 10th percentile) and controls (n=175, fT4: 50th and 90th percentile) were matched based on age and physician. Conditional logistic regression indicated that these PFAs are not associated with maternal hypothyroxinemia. / Environmental Health Sciences

Thesis (M.Sc.)--York University, 2006. Graduate Programme in Biology. / Typescript. Includes bibliographical references. Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&res_dat=xri:pqdiss&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&rft_dat=xri:pqdiss:MR19698