In Canada, colorectal cancer is the second leading cause of cancer death for both men and women. There are many different factors that contribute to the progression and spread of the disease. However, increasing evidence now suggests that the tumor microenvironment plays a paramount role in these processes. CD26 is a multifunctional, cell-surface glycoprotein that has intrinsic enzyme activity, binds adenosine deaminase and interacts with the extracellular matrix. Through its various functions it serves to constrain cancer progression. For example, it is known to cleave CXCL12, the ligand for CXCR4. The CXCL12:CXCR4 axis is normally involved in cancer metastasis by promoting cancer cell migration, invasion and proliferation. Down-regulation of CD26 is observed in certain cancers - this has been shown in vitro to occur in response to certain soluble mediators. The first part of this study looked at the effects of glucose and its metabolic product lactate on CD26 expression in colorectal carcinoma cells. Our study showed that CD26 expression is lower in cancer cells that are grown in low-glucose, high-lactate conditions, which replicates the situation within a tumor. The second part of this study examined the effect of adenosine, a purine nucleoside, on colorectal carcinoma cells and supportive stromal cells - cancer-associated HS675.T fibroblasts (CAFs) and Met-5a mesothelial cells. Adenosine increased the proliferation of CAFs and increased CXCL12 mRNA in both stromal cell lines. It also increased MMP-13 mRNA in stromal cells as well as colorectal cancer cells, suggesting that adenosine may promote progression and metastasis through various mechanisms. The last section focused on the ability of cellular products and 3-dimensional tissue topology to coordinate and affect the behaviour of the different cell populations. Here we show that secretory products from colorectal cancer cells promote CAF proliferation but inhibit mesothelial cell proliferation, and are also able to modulate MMP-13 expression. Finally, certain responses are enhanced in multicellular spheroids. In conclusion, the tumor microenvironment represents a major consideration in the treatment of solid tumors. Our data suggest that various soluble mediators, such as adenosine, may have therapeutic implications in cancer treatment and might represent novel targets for future research.

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Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

This thesis will be organized into three chapters discussing the mechanism underlying the onset and progression of osteoarthritis (OA) in the temporomandibular joint (TMJ). Understanding the mechanism of OA development in the TMJ helps in understanding how OA progresses and how to treat this disease. The goal of this investigation is to examine the process of cartilage degeneration and OA biomarker expression in the TMJ to understand their role in TMJ OA onset and development.Chapter one covers mechanisms that are altered in TMJ OA during disease progression. Using animal models with different stressors such as mechanical disturbances, direct injury, and changes in the extracellular matrix composition revealed the role of the different mechanisms that are up-regulated and down regulated during cartilage destruction. Chapter two will cover a paper I wrote that introduces a novel non-invasive technique applied to mice, which induces an early onset of OA in the TMJ. I developed this technique with the aim to provide a new mouse model where the onset and progression of OA more closely mimic the natural TMJ OA progression in humans. The histopathological analysis of the cartilage demonstrates that onset of OA starts at 2 weeks after treatment induction and is aggravated by week eight. This data demonstrated the effectiveness of our technique in inducing OA in the TMJ. Chapter three will cover a second paper I wrote on the association of RAGE with the progression of OA in the TMJ of mice by using mice with and without RAGE expression. RAGE has been show to contribute to the progression of OA by releasing several pro-inflammatory and catalytic cytokines. Additionally, RAGE has been shown to modulate the expression of specific OA biomarkers, including HtrA-1, Mmp-13, and Tgf-β1 in knee cartilage. The objective of this study was to study the effect of knocking out RAGE on the expression of Mmp-1 3, HtrA-1, and Tgf-β1 in the TMJ. After histophatological and quantitative analysis of biomarkers expression, the results demonstrated for the first time that absence of RAGE expression in the TMJ provides a protective effect against development of TMJ OA in mice.

Equine laminitis is a devastating condition that results in the failure of the tissue responsible for suspending the skeleton within the hoof capsule. The digital laminae is composed of two interdigitated layers, the dermal lamellae surrounding the distal pedal bone, and the epidermal lamellae, which interfaces with the hoof wall. During laminitis, these layers separate, allowing for rotation and sinking of the pedal bone. While there are multiple diseases and physiological conditions associated with the development of laminitis, including sepsis, metabolic syndrome, and unequal weight bearing, the exact cause remains elusive. Prior work by our research group identified the metalloprotease ADAMTS-4 as a potential early instigator of disease. The data presented herein catalogs the distribution of the substrates of this enzyme, aggrecan and versican, the ramifications of ADAMTS-4-mediated versican loss in the laminae, and further expands into the repression of the canonical wnt signaling pathway and potential additional metalloprotease (MMP) involvement in disease, utilizing a model of acute, carbohydrate-induced laminitis. Additionally, samples from other models of laminitis induction and clinical samples were screened for differential expression of relevant gene markers, including versican, members of the canonical wnt signaling pathway, and MMP-1 and -13. Together, these data provide a characterization of laminar pathology in the carbohydrate-induced model, as well as highlighting key similarities and differences amongst multiple methods of disease development, and lay important groundwork for developing clinical therapeutic interventions.

<p>Giant cell tumour of bone (GCT) is an aggressive primary bone tumour with an unclear etiology that presents with significant local osteolysis due in part to the accumulation of multinucleated osteoclast-like giant cells. However, it is the neoplastic spindle-like stromal cells within GCT that largely direct the pathogenesis of the tumour. I hypothesize that parathyroid hormone-related protein (PTHrP) is a key mediator within GCT that promotes the characteristic osteolytic phenotype by stimulating both bone resorption and giant cell formation. The work presented in this thesis collectively demonstrates that the stromal cells express PTHrP and its receptor, the parathyroid hormone type 1 receptor (PTH1R), and that PTHrP acts in an autocrine/paracrine manner within the tumour to stimulate expression of factors that promote bone resorption. Data are presented that demonstrate that PTHrP stimulates stromal cell expression of the receptor activator of nuclear factor-κB ligand (RANKL), a known essential regulator of osteoclastogenesis, which results in increased formation of multinucleated cells from murine monocytes. Moreover, the GCT stromal cells express matrix metalloproteinase (MMP)-1 and MMP-13. These results suggest that the stromal cells may participate directly in bone resorption through the degradation of type I collagen, the promotion of osteoclast activity, or through a combination of these elements. PTHrP also regulates the expression of MMP-13 by the stromal cells. Experiments with CD40 ligand show that local factors present within the tumour can influence PTHrP expression by the stromal cells and potentiate its catabolic effects by stimulation of RANKL and MMP-13 expression. Together, this thesis presents evidence that suggests PTHrP is an important factor in the pathophysiology of GCT by its actions on promoting catabolism within the tumour. The role of PTHrP in normal physiology and the mechanisms of action presented here suggest that research into the effects of PTHrP within GCT may provide invaluable information that enhances our understanding of the biology of this particularly aggressive bone tumour.</p> / Doctor of Philosophy (PhD)

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L’arthrose (OA) est la maladie articulaire la plus répandue dans le monde faisant l’objet de nombreux travaux de recherche en raison de son lourd impact socioéconomique. Plusieurs travaux dans ce domaine ont pour objectif de déterminer les mécanismes moléculaires impliqués dans sa physiopathologie. Plusieurs travaux ont appuyés l’implication de la prostaglandine (E2) PGE2 dans sa physiopathologie, contrairement à la prostaglandine (D2) (PGD2) dont le rôle reste à déterminer. C’est pourquoi, nous nous sommes penchés dans cette thèse à l’étude de cette dernière molécule. Dans la première partie de nos travaux, nous avons montré que la PGD2 diminue au niveau du cartilage articulaire et au niveau niveau des explants de cartilage humains, la production des métalloprotéases-1(MMP-1) et MMP-13 induites par (Interleukine-1β) l’IL-1β. Cette diminution de la production protéique est accompagnée d’une diminution de l’expression au niveau de l’ARNm, et d’une diminution de l’activité du promoteur de MMP-1 et MMP-13. Cet effet est exercé via le récepteur D prostanoïde (DP1), bien que le Chemoattractant receptor expressed on Th2 cells (CRTH2) soit également exprimé chez les chondrocytes humains, mais ne semble pas être impliqué dans l’effet observé. Cette action inhibitrice se fait via la voie DP1/AMPc/protéine kinase A (AMPc/PKA). Dans la suite de nos travaux, nous avons montré pour la première fois l’expression des prostaglandines D-synthases responsables de la biosynthèse de la PGD2 au niveau des chondrocytes humains par immunohistochimie, avec des niveaux d’expression de l’ARNm plus élevés de la L-PGDS au niveau du cartilage OA comparativement au cartilage normal. L’IL-1β pourrait être responsable de cette augmentation via l’activation de la voie JNK et p38 MAPK, ainsi que par la voie NF-κB. L’ensemble de ces données indiquent que la modulation des niveaux de la PGD2 au niveau de l’articulation pourrait être pourvue d’un important potentiel thérapeutique. La L-PGDS pour sa part semble avoir un rôle important dans la physiopathologie de l’OA. / Osteoarthritis (OA) is the most common joint disease world wide, because of its higher socioeconomic impact it is one of the most studied joint diseases. The aims of these studies was to determine the molecular mechanisms involved in the pathophysiology of osteaarthritis. Previous studies have mainly focused on the involvement of prostaglandin (E2) PGE2 in contrast to PGD2 in the pathogenesis osteoarthritis as such the role of PGD2 remains unclear. In this thesis we examined the involvment of PGD2 in the pathogenesis of OA. In the first part of our work, we showed that in a dose dependent manner PGD2 decreased the interleukin-1β (IL-1β)–induced mettalloproteases (MMP-1) and MMP-13 expression both at protein and mRNA levels by supression of their promoter activity. The inhibitory effect was exerted via the D prostanoid receptor (DP1) and mediated through the cAMP/protein kinase A (PKA) signalling pathway. Although human chondrocytes do express the Chemoattractant Receptor Expressed on Th2 cells (CRTH2) the latter were not implicated in the inhibiton of MMP-1 and MMP-13. In the second part of our work, we showed the expression of prostaglandin D synthases (PGDS) responsible for the biosynthesis of PGD2 in human chondrocytes, with higher levels of mRNA expression of lipocaline type prostaglandin D-synthase (L-PGDS) in OA cartilage compared to normal cartilage. IL-1β may be responsible for this increase via the activation of Jun N-terminal kinase (JNK) and p38 mitogen activated protein kinase (MAPK), as well as the nuclear factor-κB (NF-κB). Together, these data indicate that modulation of the levels of PGD2 at the joint may be provided with an important therapeutic potential. L-PGDS in turn seems to have an important role in the pathogenesis of OA.

Made available in DSpace on 2015-03-03T15:38:43Z (GMT). No. of bitstreams: 1 PedroPAS_TESE_1-70.pdf: 4719637 bytes, checksum: 8f16cb0e2326a80cfc947b1ea2b89641 (MD5) Previous issue date: 2012-02-28 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / Myofibroblasts are cells that exhibit a hybrid phenotype, sharing the morphological characteristics of fibroblasts and smooth muscle cells, which is acquired during a process called differentiation. These cells then start to express -SMA, a marker that can be used for their identification. Studies suggest that myofibroblasts are related to the aggressiveness of different tumors and that TGF-1 and IFN- play a role in myofibroblast differentiation, stimulating or inhibiting this differentiation, respectively. The objective of this study was to investigate the role of myofibroblasts in epithelial odontogenic tumors, correlating the presence of these cells with the aggressiveness of the tumor. Immunohistochemistry was used to evaluate the expression of TGF-1 and IFN- in myofibroblast differentiation, as well as the expression of MMP-13, which is activated by myofibroblasts, and of EMMPRIN (extracellular matrix metalloproteinase inducer) as a precursor of this MMP. The sample consisted of 20 solid ameloblastomas, 10 unicystic ameloblastomas, 20 odontogenic keratocysts, and 20 adenomatoid odontogenic tumors. For evaluation of myofibroblasts, anti- -SMA-immunoreactive cells were quantified in connective tissue close to the epithelium. Immunoexpression of TGF-1, IFN-, MMP-13 and EMMPRIN was evaluated in the epithelial and connective tissue components, attributing scores of 0 to 4. The results showed a higher concentration of myofibroblasts in solid ameloblastomas (mean of 30.55), followed by odontogenic keratocysts (22.50), unicystic ameloblastomas (20.80), and adenomatoid odontogenic tumors (19.15) (p=0.001). No significant correlation between TGF-1 and IFN- was observed during the process of myofibroblast differentiation. There was also no correlation between the quantity of myofibroblasts and MMP-13 expression. Significant correlations were found between MMP-13 and TGF-1 (r=0.087; p=0.011), between MMP- 13 and IFN- (r=0.348; p=0.003), as well as between EMMPRIN and MMP-13 (r=0.474; p<0.001) and between EMMPRIN and IFN- (r=0.393; p=0.001). The higher quantity of myofibroblasts observed in solid ameloblastomas, odontogenic keratocysts and unicystic ameloblastomas suggests that these cells are one of the factors responsible for the more aggressive biological behavior of these tumors, although the myofibroblast population was not correlated with TGF-1, IFN-, MMP-13 or EMMPRIN. The correlation between MMP- 13 and TGF-1 suggests that the latter induces the expression of this metalloproteinase. The present results also support the well-established role of EMMPRIN as an inducer of MMP-13. Furthermore, the relationship between EMMPRIN and IFN- and between MMP-13 and IFN- suggests synergism in the antifibrotic effect of these markers / Os miofibroblastos s?o c?lulas que apresentam um fen?tipo h?brido exibindo caracter?sticas morfol?gicas de fibroblastos e de c?lulas musculares lisas, sendo a aquisi??o de tal fen?tipo denominada diferencia??o, passando ent?o a expressar a -SMA, a qual ? importante na identifica??o dessas c?lulas. Estudos t?m sugerido que os miofibroblastos apresentam rela??o com a agressividade de diversas les?es e que o seu processo de diferencia??o estaria relacionado ? express?o do TGF- 1 e do IFN- atuando, respectivamente, no est?mulo e na inibi??o dessa diferencia??o. O objetivo deste trabalho foi investigar o papel dos miofibroblastos em les?es odontog?nicas epiteliais, relacionando-os ? agressividade das les?es e analisar por meio da imuno-histoqu?mica, a express?o do TGF- 1 e IFN- no processo de diferencia??o, al?m da an?lise da MMP-13 que ? ativada por miofibroblastos e do indutor de metaloproteinases de matriz (EMMPRIN) como precursor desta MMP. A amostra foi constitu?da por 20 ameloblastomas s?lidos, 10 ameloblastomas unic?sticos, 20 ceratocistos odontog?nicos e 20 tumores odontog?nicos adenomat?ides. Para a avalia??o dos miofibroblastos, foram quantificadas as c?lulas imunorreativas ao anticorpo - SMA presentes no tecido conjuntivo, pr?ximo ao tecido epitelial. As express?es de TGF- 1, IFN- , MMP-13 e EMMPRIN, foram avaliadas no componente epitelial e no conjuntivo, estabelecendo-se o percentual de imunorreatividade e atribuindo-se escores de 0 a 4. A an?lise dos miofibroblastos evidenciou maior concentra??o nos ameloblastomas s?lidos (m?dia de 30,55), seguido pelos ceratocistos odontog?nicos (22,50), ameloblastomas unic?sticos (20,80) e tumores odontog?nicos adenomat?ides (19,15) com valor de p= 0,001. N?o foi encontrada correla??o significativa entre TGF- 1 e IFN- no processo de diferencia??o dos miofibroblastos, bem como na rela??o entre a quantidade de miofibroblastos e a express?o da MMP-13. Constatou-se, correla??o estat?stica entre MMP-13 e TGF- 1 (r= 0,087; p= 0,011) al?m de significante correla??o entre MMP-13 e IFN- (r=0,348; p=0,003). Entre EMMPRIN e MMP-13 verificou-se signific?ncia (r= 0,474; p<0,001) assim como entre EMMPRIN e IFN- (r=0,393; p=0,001). A maior quantidade de miofibroblastos evidenciada nos ameloblastomas s?lidos, ceratocistos odontog?nicos e ameloblastomas unic?sticos sugere que estas c?lulas podem ser um dos fatores respons?veis para um comportamento biol?gico mais agressivo destas les?es, embora a popula??o de miofibroblastos n?o tenha apresentado correla??o com TGF- - 1, IFN- ,MMP-13 e EMMPRIN. Quanto a correla??o evidenciada entre MMP-13 e TGF- 1, isto pode sugerir um papel indutor do TGF- 1 para a express?o da MMP-13, assim como os resultados deste estudo refor?am a rela??o bem estabelecida do EMMPRIN como indutor da MMP-13. Constatou-se tamb?m rela??o entre EMMPRIN e IFN- assim como entre MMP-13 e IFN- sugerindo, dessa forma, um sinergismo na a??o anti-fibr?tica desses marcadores

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.