Early detection of prostate cancer is problematic due to the lack of a marker that has high diagnostic sensitivity and specificity. The prostate specific antigen test, in combination with digital rectal examination, is the gold standard for prostate cancer diagnosis. However, this modality suffers from low specificity. Therefore, specific markers for clinically relevant prostate cancer are needed. Our objective was to proteomically characterize the conditioned media from human prostate cancer cell lines to identify secreted proteins that could serve as novel prostate cancer biomarkers. An initial proof of principle study of the PC3 prostate cancer cell line was conducted. From this study over 200 proteins were identified in the conditioned media. Through gene ontology analysis and literature searches Mac-2 binding protein was selected as a candidate biomarker for validation in the serum of prostate cancer patients. A preliminarily validation showed that Mac-2 binding protein has discriminatory ability in prostate cancer diagnosis. However, an extended validation did not confirm this. Based on our proof of principle study we optimized our workflow and extended our analysis by culturing three different prostate cell lines [PC3 (bone metastasis), LNCaP (lymph node metastasis), and 22Rv1 (localized to prostate)]. We conducted a bottom-up analysis of each cell line by 2-dimensional liquid chromatography and tandem mass spectrometry. Of the 2124 proteins identified, 12% (329) were classified as extracellular and 18% (504) as membrane-bound. Among the identified proteins were known prostate cancer biomarkers such as PSA and KLK2. To select the most promising candidates for further investigation, tissue specificity, biological function, disease association based on literature searches, and comparison of protein overlap with the proteome of seminal plasma and serum were examined. Based on these results, several candidates were selected for validation in serum of patients with and without prostate cancer. Of these four novel candidates: follistatin, chemokine (C-X-C motif) ligand 16, pentraxin 3 and spondin 2 showed discriminatory ability. Of the four candidates, follistatin was further studied in an extended validation in serum of patients with biopsy confirmed prostate cancer and tissues of prostate cancer patients of low and high grade tumours by immunohistochemistry. In addition, follistatin was also investigated in the tissue of colon and lung cancer where intense staining was observed in one specimen of lung squamous carcinoma.

Reproductive diseases, mainly those resulting in the infertility affect the chances of human being to reproduce. On the contrary, the heart disease, cancer and degenerative diseases currently account for majority of deaths in the world. Usually, these lifestyle diseases need longer lifespan to become the cause of death. The proteins secreted by cells carry important information about the cell's well-being, as well as about the condition of the tissues formed by these cells. Once secreted, these proteins may also be transferred throughout the body by means of body fluids, many of which are easily accessible for further 'in-depth' studies. Cellular and secreted proteins are often a focus of studies using proteomic means and the revelation of protein alterations can lead us to new ideas about the molecular mechanisms of diseases as well as possible identification of proteins that may be used as new targets for pharmaceutical intervention or molecules that could be used for diagnostic or prognostic purposes. Taking into consideration the above aspects, this research was undertaken to find proteins that could: (a) characterise the human follicular fluid as microenvironment of the maturing oocyte, to increase understanding of reproductive processes to improve the techniques of assisted repro- duction;...

Cancer metastasis is the critical event leading to 90% of cancer related death. Although significant improvement in our understanding on cancer metastasis has been made through years of research, the fundamental mechanism behind this process is still not fully elucidated. For cancer researchers, the “gold standard” for metastasis studies has traditionally been the use of tissue culture and mouse models. Tissue culture offers the simplest system and ease of control but is not able to recapitulate many of the features found in an in vivo tumor microenvironment. On the other hand, mouse model systems offer the most sophisticated and physiologically relevant platforms for studying cancer. However, the lack of control over the in vivo environment in these mouse models and inherent discrepancies from human physiology make results from these models difficult to be translated to clinical trials. The advancement in microfabrication techniques and cancer models developed based on these techniques has shown potential in addressing the gap between in vitro tissue culture and mouse models. Microscopic tumor microenvironments could be built in these in vitro systems to study behavior of human cancer cells. However, the expertise involved in and extra instrumentation needed for implementing these systems have prevented their widespread use by general cancer researchers. In this dissertation, we developed two simple microfabricated systems and demonstrated their application in two aspects of cancer research. The first system is a microfabricated cell patterning stencil, where paracrine signaling can be established and its impact can be measured based on cell migration. Using this tool, we investigated the interaction between melanoma and microenvironmental cells from their common metastasis target organ. Through these simple patterning techniques, we observed significant effects that a given microenvironmental cell line had on the two different melanoma lines, as well as how melanoma affected different microenvironmental cell lines. The second system, a microfluidic device, is able to present individual soluble factors to cancer cells in order to test the response of cancer cells to these physiologically relevant factors. Through this stand-alone system, we found that breast cancer metastasis is influenced by the protein molecules secreted by themselves as well as the local glucose level. Through these findings we believe that our microfabricated systems can benefit the general cancer research community in which a complicated problem can be broken down into manageable pieces and studied on a simple platform in a controlled way. Observation made through these systems can inspire general cancer researchers to form new hypotheses and eventually lead to new findings. / 2017-02-17T00:00:00Z

The nematode Caenorhabditis elegans is a model organism for understanding sensory molecules of multicellular organisms. Ovulating hermaphrodites produce putative pheromone(s) that cause male attraction. Because pheromones are produced in such small quantities, adult conditioned-media from large-scale synchronous culture is necessary to analyze these pheromones. Current protocols for culture synchronization have volume constraints that limit large-scale synchronous cultures and current methodology for adult conditioned-media production is impractical. Modification of Tangential Flow Filtration (TFF) systems was investigated for use as a method to increase the volume limits of bleach egg harvest for C. elegans culture synchronization. Also, an adult retention device built within the culture vessel was investigated to optimize the environment for aseptic conditioned-media production from dense large-scale C. elegans cultures. During this investigation, we have shown that synchronous C. elegans cultures for adult conditioned-media production can be grown at scales larger than reported before, with potential for further scale up. Our growth methodologies have also yielded denser cultures than previously achieved at large scales. Since rapid bleach harvesting appears to be the bottleneck for large-scale production of synchronous C. elegans cultures, our approach of using modified TFF systems with mesh to retain C. elegans eggs increased the amount of eggs that could be bleach harvested at one time. Using this method we have been able to achieve up to 5x103 synchronous C. elegans per mL at a 50L scale. Since scale-up of TFF is straightforward, our results suggest that the technique reported here can easily be applied to larger scale systems for production of adult conditioned-media for C. elegans. Further, the adult retention device within the culture vessel can ensure that the whole process remains aseptic.

Novel serological biomarkers to aid in the detection and clinical management of pancreatic cancer patients are urgently needed. In the present study, we performed in-depth proteomic analysis of conditioned media from six pancreatic cancer cell lines (MIA-PaCa2, PANC1, BxPc3, CAPAN1, CFPAC1 and SU.86.86), the normal pancreatic ductal epithelial cell line HPDE, and pancreatic juice samples from cancer patients for identification of novel biomarker candidates. Using 2D-LC-MS/MS, a total of 3479 non-redundant proteins were identified with ≥2 peptides. Subsequent label-free protein quantification and integrative analysis of the biological fluids resulted in the generation of candidate biomarkers, of which five proteins were shown to be significantly elevated in plasma from pancreatic cancer patients in a preliminary assessment. Further verification of two of the proteins in ~200 serum samples demonstrated the ability of these proteins to significantly improve the area under the receiver operating characteristic curve of CA19.9 from 0.84 to 0.91.

Early detection of prostate cancer is problematic due to the lack of a marker that has high diagnostic sensitivity and specificity. The prostate specific antigen test, in combination with digital rectal examination, is the gold standard for prostate cancer diagnosis. However, this modality suffers from low specificity. Therefore, specific markers for clinically relevant prostate cancer are needed. Our objective was to proteomically characterize the conditioned media from human prostate cancer cell lines to identify secreted proteins that could serve as novel prostate cancer biomarkers. An initial proof of principle study of the PC3 prostate cancer cell line was conducted. From this study over 200 proteins were identified in the conditioned media. Through gene ontology analysis and literature searches Mac-2 binding protein was selected as a candidate biomarker for validation in the serum of prostate cancer patients. A preliminarily validation showed that Mac-2 binding protein has discriminatory ability in prostate cancer diagnosis. However, an extended validation did not confirm this. Based on our proof of principle study we optimized our workflow and extended our analysis by culturing three different prostate cell lines [PC3 (bone metastasis), LNCaP (lymph node metastasis), and 22Rv1 (localized to prostate)]. We conducted a bottom-up analysis of each cell line by 2-dimensional liquid chromatography and tandem mass spectrometry. Of the 2124 proteins identified, 12% (329) were classified as extracellular and 18% (504) as membrane-bound. Among the identified proteins were known prostate cancer biomarkers such as PSA and KLK2. To select the most promising candidates for further investigation, tissue specificity, biological function, disease association based on literature searches, and comparison of protein overlap with the proteome of seminal plasma and serum were examined. Based on these results, several candidates were selected for validation in serum of patients with and without prostate cancer. Of these four novel candidates: follistatin, chemokine (C-X-C motif) ligand 16, pentraxin 3 and spondin 2 showed discriminatory ability. Of the four candidates, follistatin was further studied in an extended validation in serum of patients with biopsy confirmed prostate cancer and tissues of prostate cancer patients of low and high grade tumours by immunohistochemistry. In addition, follistatin was also investigated in the tissue of colon and lung cancer where intense staining was observed in one specimen of lung squamous carcinoma.

Novel serological biomarkers to aid in the detection and clinical management of pancreatic cancer patients are urgently needed. In the present study, we performed in-depth proteomic analysis of conditioned media from six pancreatic cancer cell lines (MIA-PaCa2, PANC1, BxPc3, CAPAN1, CFPAC1 and SU.86.86), the normal pancreatic ductal epithelial cell line HPDE, and pancreatic juice samples from cancer patients for identification of novel biomarker candidates. Using 2D-LC-MS/MS, a total of 3479 non-redundant proteins were identified with ≥2 peptides. Subsequent label-free protein quantification and integrative analysis of the biological fluids resulted in the generation of candidate biomarkers, of which five proteins were shown to be significantly elevated in plasma from pancreatic cancer patients in a preliminary assessment. Further verification of two of the proteins in ~200 serum samples demonstrated the ability of these proteins to significantly improve the area under the receiver operating characteristic curve of CA19.9 from 0.84 to 0.91.

Macrophage-derived inflammation contributes to chronic inflammation in adipose tissue in obesity and is also linked to the development of skeletal muscle (SM) insulin resistance. The long-chain n-3 PUFA have been shown to modulate cytokine secretion from macrophages, though subsequent effects on SM inflammation and function are unknown. A model of macrophage conditioned media (MCM) was used to examine effects of n-3 PUFA on macrophage inflammation and consequent effects on SM cells. Treatment of RAW 264.7 macrophages with long-chain n-3 PUFA decreased LPS-induced MCP-1 and IL-6 gene expression and MCP-1 secreted protein. In turn, MCM from n-3 PUFA-treated macrophages decreased TNF-α and IL-6 gene expression in LPS-stimulated L6 SM cells, but did not affect insulin-stimulated pAkt content. Long-chain n-3 PUFA did not affect gene expression of inflammatory signaling intermediates NF-κB and TLR4. Overall this thesis suggests that long-chain n-3 PUFA are important nutritional strategies for reducing macrophage-derived inflammation, with ensuing benefits in SM inflammation. / NSERC-CGS, Ontario Graduate Scholarship

Made available in DSpace on 2014-08-20T13:32:54Z (GMT). No. of bitstreams: 1 dissertacao_regis_sturbelle.pdf: 477794 bytes, checksum: d7692995394f46dd2524f4a667bb1ce6 (MD5) Previous issue date: 2009-10-29 / One of the most important sanitary problems that cause economic losses in swine industry is the diarrhea caused by Enterotoxigenic Escherichia coli (ETEC). It is the main infectious cause of mortality for animals in suckling and post-weaning periods. Their primary factors of pathogenicity are the fimbriae, which bond the bacteria to specific receptors of the enterocytes. This causes a succession of events, brought about by the enterotoxins, the stable toxins (ST) and heat-labile toxins (LT), leading to diarrhea. Another important structure is the flagellum, which has an important role in cell stimulation to produce pro-inflammatory cytokines through the interaction with Toll-like receptors (TLR5), signaling cell recruitment and activation, thus increasing local inflammation as well as antigen presentation to lymphocytes. Quorum Sensing is a signaling system among bacteria that uses substances denominated autoinducers (AI). When the auto-inducers reach a certain concentration, due to an increase of cell density, there is an activation of transcriptional factors, which regulate the gene expression. The catecholamines (adrenaline and noradrenalin), produced by nervous cells, use the auto-inducer type 3 (AI-3) pathway. These have a significant role in gene expression as they stimulate the growth and the expression of virulence factors of the Escherichia coli. The goal of the present study was to produce experimental vaccines containing total cultures of ETEC cultivated in different induction conditions, mediated by the conditioned media (mc) so as to study the expression of vaccine antigens. Different concentrations of mc were used, with the 50% of conditioned media added with 500 μM of adrenaline having the major initial growth and antigen expression. In the motility assay, a sixteen-fold increase was observed in comparison to the control. By the ELISA, hemoagglutination, hemoagglutination inhibition and Dot Blot techniques, a higher expression of fimbriae (F4) was observed, whereas by Dot Blot, ELISA and RT-PCR techniques an increase of LT expression was observed. Based on this data a bacterin was prepared using 50% of mc and 500 μM of adrenaline, with Al(OH)3 as an adjuvant. Five weeks old Swiss female mice were vaccinated twice with 250 μL subcutaneously on days 0 and 8 14. The vaccine immune response was evaluated by ELISA and hemoagglutination inhibition. The mc bacterin vaccinated group respond with a seroconversion significantly higher that the control group. These results suggest that the presence of mc with adrenaline is capable of activating the expression of important vaccine antigens, therefore making bacterin more efficient. / Um dos maiores problemas sanitários que causam prejuízo à suinocultura é a diarréia causada por Escherichia coli Enterotoxigênica (ETEC). Esta é a causa infecciosa mais comum de mortalidade em animais em aleitamento e no período pós-desmame. Os fatores primários de patogenicidade desta bactéria são as fímbrias, que as ancoram a receptores específicos dos enterócitos permitindo que as enterotoxinas, toxinas termo estáveis (ST) e toxinas termo lábeis (LT) desencadeiem uma sucessão de eventos levando à diarréia. Outra estrutura é o flagelo que possui um papel na estimulação de células na produção de citocinas pró-inflamatórias através da interação com receptores Toll-like 5 (TLR5), sinalizando o recrutamento e ativação de células, desta forma ampliando a inflamação local bem como a apresentação de antígeno aos linfócitos. Quorum Sensing é um sistema de sinalização entre as bactérias, os quais produzem substâncias denominadas de auto-indutores (AI). Quando os auto-indutores alcançam uma determinada concentração, decorrente do aumento da densidade celular ocorre uma ativação de fatores transcricionais que acabam regulando a expressão gênica. As catecolaminas (adrenalina e noradrenalina), produzidas pelas células nervosas, utilizam a via de sinalização do auto-indutor tipo 3 (AI-3) que têm um papel significante na expressão gênica, estimulando o crescimento e a expressão de fatores de virulência de Escherichia coli. O objetivo deste estudo foi utilizar o sistema de sinalização Quorum Sensing para induzir a expressão in vitro de antígenos vacinais de Escherichia coli Enterotoxigênica cultivadas em diferentes condições de indução mediada pelos meios condicionados (mc). Diferentes concentrações de mc foram utilizadas, sendo que o maior crescimento inicial e a maior expressão de antígenos de interesse foram obtidos com 50% de meio condicionado adicionado de 500 μM de adrenalina. No ensaio de motilidade foi observado um aumento de 16 vezes em relação ao controle. Pelas técnicas de ELISA, hemaglutinação, inibição da hemaglutinaçõa e Dot blot foi observado uma maior expressão de fimbria (F4) e pelas técnicas de Dot blot, ELISA e RT-PCR foi observado aumento da expressão de LT. Baseado nesses dados 6 testou-se uma bacterina utilizando 50% de mc e 500 μM de adrenalina com Al(OH)3 como adjuvante. Foram utilizados camundongos Swiss fêmeas com 5 semanas de idade que foram vacinados por via subcutânea com 250 μL nos dias 0 e 14. A avaliação da resposta imune foi realizada através de soroconversão frente aos antígenos estudados por meio de ELISA e inibição da hemaglutinação. O grupo vacinado com a bacterina com mc obteve uma soroconversão significativamente superior ao grupo controle. Estes resultados sugerem que a presença de mc com adrenalina são capazes de ativar a expressão de importantes antígenos vacinais, tornando desta forma a bacterina mais eficiente.