<p>Phosphorus load reduction and dreissenid invasion are the two most important factors that influence europhication dynamics in the Great Lakes. The 1978 amendments to the Great Lakes Water Quality Agreement (GLWQA) between the United States and Canada established target phosphorus loads for the lakes, leading to reductions in external phosphorus loading to the Great Lakes. With diminished phosphorus levels, further nutrient management was a minor concern until the proliferation of invasive dreissenid mussels. Dreissenid mussels were first documented in the Laurentian Great Lakes in the late 1980s. Zebra mussels (<italic>Dreissena polymorpha</italic>) spread quickly into shallow, hard-substrate areas; quagga mussels (<italic>Dreissena rostriformis bugensis</italic>) spread more slowly and are currently colonizing deep, offshore areas. These mussels have the potential to modify biogeochemical processes and food web structure, altering nutrient cycling and availability. Following the mussel invasion, cyanobacterial blooms and nuisance benthic algal growth have reappeared in many nearshore areas of the Great Lakes.</p><p>This dissertation characterizes long-term patterns of phosphorus loading and mussel populations for Saginaw Bay, and estimates the effects of load reductions and dreissenid invasion on several aspects of pelagic water quality, focusing on phosphorus flux and cycling in Saginaw Bay. Bayesian approaches were used to quantify the impacts of load reduction and mussel invasion, while at the same time addressing model parameter uncertainty and prediction uncertainty associated with long-term observational data. Annual total phosphorus load estimates suggest a decreasing trend until the late 1970s to early 1980s, reflecting the effectiveness of point source controls implemented pursuant to GLWQA. Despite the decrease, however, the annual loads have not likely met the 440 tonne yr-1 target established for Saginaw Bay. In 1990 zebra mussels were discovered in the bay and by 1992 they were widespread and peaked with densities of >30,000 m<super>-2</super>. Following the peak, mean densities dropped and modeling results predict that the density will reach equilibria at ~600 m<super>-2</super>. When mussels appeared, the proportion of tributary phosphorus retained in Saginaw Bay increased from ~0.5 to ~0.7, reducing phosphorus export to the main body of Lake Huron. The combined effects of increased phosphorus retention and decreased phosphorus loading have caused an ~60% decrease in phosphorus export from Saginaw Bay to Lake Huron. The analysis of long-term patterns of pelagic water quality highlights the sustained effects of mussel invasion on altering water quality parameters in Saginaw Bay; there was a consistent decrease in chlorophyll concentrations by ~46%, and total phosphorus concentrations by ~25%, and an increase in secchi depths by ~15% over ~20 year invasion of mussels. A comparison of chlorophyll-phospohrus relationship between pre- and post-invasion periods suggest the reduced chlorophyll yield for a given phosphorus concentration after the mussel invasion. Further, decreases in both total phosphorus and chlorophyll concentrations were found in the majority of 24 mussel-invaded US lakes in addition to Saginaw Bay, and modeling results predict less chlorophyll yields per unit phosphorus level that ranges from oligo- to mesotrophic conditions. All lines of evidence presented in the dissertation point to the important roles of load reductions and invasive mussels affecting eutrophication dynamics in lake ecosystems.</p> / Dissertation

Ovarian cancer is a fatal disease because of the lack of symptoms and markers for early detection. Most ovarian neoplasms resemble and are classified according to the complex characteristics of Mullerian duct epithelia. We tested the hypothesis that Mullerian epithelial characteristics influence early ovarian neoplastic progression. The most common type of ovarian cancer is the serous carcinoma which resembles Mullerian-derived oviductal epithelium. We discovered that oviduct-specific glycoprotein (OVGP1), a tubal differentiation marker, was present in inclusion cysts, which are the preferential sites for malignant transformation, and in most low grade serous tumors, but absent in ovarian surface epithelium and most high grade carcinomas. OVGP1 was almost entirely limited to ovarian neoplasms with the notable exception of endometrial hyperplasia and carcinoma. A new antibody against OVGP1 detected elevated serum levels from most women with low grade ovarian cancers compared to normal controls. OVGP1 also identified a subset of patients with high grade serous carcinomas who had a more favorable outcome. To examine whether the differentiated phenotype of early ovarian neoplasms alters invasiveness, we established the first permanent cell line for serous borderline ovarian tumors (SBOT), which are differentiated but noninvasive. The results revealed a striking phenotypic similarity between two lines regardless of their cytogenetic diversity. They retained Mullerian epithelial characteristics in vitro, as demonstrated by their morphologic appearance and the differentiation markers keratin, E-cadherin, CA125 and OVGP1. Neither disruption of the growth pattern nor manipulations of the cadherin profile induced invasivenesss. Induction of invasiveness by SV40 early genes was associated with a loss in morphologic differentiation and of differentiation markers but increased motility. MMP secretion was independent of the invasion status. Our findings indicate that OVGP1 is an indicator of early ovarian epithelial neoplasia. It can be detected in the sera from women with early ovarian cancer, and thus, may be a new promising diagnostic marker for the early detection of ovarian cancer. In addition, the results show that Mullerian differentiation does not directly prevent invasiveness, but it diminishes in parallel with invasion caused by other factors. The lack of invasiveness by SBOT cells may depend on factors that regulate motility.

Cronobacter species cause serious infections such as meningitis and enteritis in newborns and neonates, with the major vehicle being contaminated powdered infant formula. The main objectives of this study were i) to identify potential virulence factors, such as enterotoxin production; ii) characterize the gene(s) involved in adhesion and invasion of the human brain microvascular endothelial cells (HBMEC); and iii) determine whether strains from clinical, food, and environmental sources differ in their ability to produce surface-attached bacterial aggregates, known as biofilms. Random transposon mutagenesis was used on strains demonstrating the best adherence and invasion to blood- brain barrier cell lines (BBB). Isogenic mutants were then screened for increased or decreased adherence and invasion. Screening of the transposon library identified one isogenic mutant of a clinical strain which lost the ability to adhere to BBB cells. The transposon rescue revealed the insertion site to be within a diguanylate cyclase (DGC) gene. The major function of DGC in many Gram-negative bacteria is to synthesize cyclic diguanylate (c-di-GMP), a secondary bacterial metabolite known for regulating biofilm formation, motility, and virulence or aspects of microbial pathogenicity. Based on the findings of this study, DGC appears to play an important role in Cronobacter species’ ability to produce biofilms and may also have a role of the pathogenicity in the microorganism.

In response to growing levels of resistance to currently used antimalarials, there is an urgent need to develop drugs that exhibit novel mechanisms to kill Plasmodium parasites. The objective of this study was to examine the antiparasitic activity of newly synthesized compounds based on imidazolium and triazolium rings. According to our structure/activity relationship studies the key components appear to be their positively charged rings and hydrophobic side groups, and bivalent compounds, which incorporate two positively charged rings, show even greater potency than monovalent compounds. Depending on the concentration used, our compounds appear to primarily inhibit intracellular parasite development or invasion into red blood cells. Selected compounds have been tested in vivo using a P. berghei ANKA murine model. Together, our findings demonstrate that small imidazolium- and triazolium-based compounds display both in vitro and in vivo activity through a novel mechanism of action that may involve inhibition of erythrocyte invasion.

In response to growing levels of resistance to currently used antimalarials, there is an urgent need to develop drugs that exhibit novel mechanisms to kill Plasmodium parasites. The objective of this study was to examine the antiparasitic activity of newly synthesized compounds based on imidazolium and triazolium rings. According to our structure/activity relationship studies the key components appear to be their positively charged rings and hydrophobic side groups, and bivalent compounds, which incorporate two positively charged rings, show even greater potency than monovalent compounds. Depending on the concentration used, our compounds appear to primarily inhibit intracellular parasite development or invasion into red blood cells. Selected compounds have been tested in vivo using a P. berghei ANKA murine model. Together, our findings demonstrate that small imidazolium- and triazolium-based compounds display both in vitro and in vivo activity through a novel mechanism of action that may involve inhibition of erythrocyte invasion.

The genus Bartonella comprises emerging pathogens that are causative agents of a wide range of clinical manifestations such as cat scratch disease, bacillary angiomatosis, and Carrion’s disease. All species are transmitted by blood-sucking arthropods and infect erythrocytes and endothelial cells of hosts. Carrion’s disease is a bi-phasic infection caused by Bartonella bacilliformis which is characterized by hemolysis of infected erythrocytes followed by invasion of the vascular endothelium. This provokes pronounced cellular proliferation, angiogenesis and skin eruptions called verruga peruana. Endothelial cells are thought to be the primary niche wherein bacteria reside between inoculation and erythrocyte infection. This study aims to elucidate some of the endothelial factors involved during the verruga peruana phase of Carrion’s disease. In order to adhere to and invade human microvascular endothelial cells (HMEC-1), B. bacilliformis engages a family of cell receptors called integrins. We used anti-integrin antibodies to show that the primary integrin involved is the fibronectin receptor á5â1, although the vitronectin receptor áVâ3 also plays a minor role. We show B. bacilliformis invasion is also dependent on integrin ligands, fibronectin and vitronectin as antibodies against these proteins decreased invasion and attachment, whereas pre-treatment of the bacteria with these molecules enhanced infection of endothelial cells. Bacterial uptake requires various host cytoplasmic signaling pathways to work in tandem, and our study identified three mitogen activated protein kinases involved. Apart from MAPKs, phosphotidylinositol 3 kinase plays a role during invasion and cell survival. PI3K inhibitors blocked bacterial internalization and B. bacilliformis infected cells showed accelerated apoptosis. Lastly, microarray analysis was performed to study the gene expression profile of B. bacilliformis infected HMEC-1 cells. Numerous molecules of the integrin signaling pathways are involved, suggesting integrins as the major receptor recruited for the successful infection by B. bacilliformis. In summary this is the first study to demonstrate the role of integrins as B. bacilliformis receptors and integrin ligands as facilitators of infection. Gene expression analysis suggests the possibility that integrin mediated signaling pathways are the key modulators of cellular alterations during B. bacilliformis infection. This hypothesis is supported by the identification of some members of the integrin signaling pathway necessary for B. bacilliformis entry into endothelial cells.

Le médulloblastome, un type hautement agressif de tumeur cérébrale pédiatrique, est l'un des cancers dont le pronostic est le plus sombre dû à la difficulté de s'y attaquer directement et efficacement. L'avenir de la recherche en oncologie repose sur notre capacité à mieux comprendre les mécanismes moléculaires et cellulaires impliqués dans la progression d'un néoplasme afin d'identifier les joueurs clés impliqués dans le phénotype invasif tumoral ainsi que de nouvelles cibles nous permettant de tirer profit de ces failles. L'une de celles-ci est la métalloprotéase MMP-9, qui est surexprimée de manière constitutive dans la plupart des cas de cancers et qui contribue fortement au phénomène d'angiogénèse ainsi qu'à la migration invasive des cellules tumorales. Une des voies de signalisation régulant l'expression de la MMP-9 passe par le facteur nucléaire NF-kB, qui est aussi intimement liée à la régulation de COX-2, un médiateur important de l'inflammation dans le microenvironnement tumoral. Sachant que le ciblage pharmacologique de chacune de ces fonctions contribue à la régression tumorale, il apparait pertinent d'évaluer le caractère thérapeutique in vitro de deux agents pharmacologiques, le propranolol ainsi que le lupeol, dont les potentiels anti-tumoraux et anti-inflammatoires respectifs ont été rapportés. Les mécanismes moléculaires à la base de leur activité demeurent cependant peu documentés. Nous avons donc émis l'hypothèse que, premièrement, le propranolol puisse inhiber la sécrétion de MMP-9 indispensable à la cancérogénèse en bloquant la voie de signalisation NF-kB et que, dans un deuxième temps, le lupeol modulerait négativement l'inflammation au site tumoral en bloquant la synthèse de COX-2 possiblement via la voie NF-kB. Pour ce faire, nous stimulerons in vitro la voie de signalisation NF-kB à l'aide d'un agent pro-carcinogène, le PMA, dans un modèle cellulaire tumoral de type DAOY. Les phénotypes invasifs et inflammatoires résultants seront par la suite évalués en réponse aux traitements pharmacologiques. Nos résultats démontrent que le propranolol inhibe l'activation de la voie NF-kB induite par un agent carcinogène de manière dépendante de la dose, diminuant conséquemment la sécrétion de MMP-9. Le mécanisme de notre agent affecte aussi deux autres voies de signalisation cruciales à la cancérogénèse, soit la voie des MAPK et de PI3K/AKT, sans toutefois induire l'apoptose des cellules traitées. Notre seconde hypothèse s'est cependant avérée inexacte puisque, bien que le lupeol module effectivement la voie NF-kB et l'expression de COX-2, elle le fait à la hausse en synergie avec notre agent carcinogène, le PMA. Ces recherches nous ont par contre permis de comprendre une nouvelle modulation du lupeol dans notre modèle néoplasique et d'analyser l'axe de signalisation MT1-MMP, dont la contribution était suspectée, à l'inflammation. De plus, nous avons associé une autre partie du mécanisme moléculaire du lupeol via l'expression de la protéine chaperonne HuR indispensable à la stabilisation du transcrit primaire de MMP-9. En conclusion, nous avons démontré un nouvel axe signalétique par lequel ces agents pharmacologiques affectent le développement tumoral. Ces données dénotent l'importance de cibler la voie NF-kB afin d'atténuer à plusieurs niveaux le phénotype tumoral et proposent l'implication de cibles thérapeutiques originales telles que les récepteurs β-adrénergiques dans la cancérogénèse. ______________________________________________________________________________ MOTS-CLÉS DE L’AUTEUR : Médulloblastome, MMP-9, NF-kB, propranolol, récepteurs β-adrénergiques, COX-2, inflammation, lupeol

ZNF217, un nouveau membre de la famille krüppel-like, est un facteur de transcription qui interagit avec des co-répresseurs et des protéines modifiant les histones suggérant que ZNF217 ferait partie d'un complexe répresseur de la transcription. ZNF217 serait un oncogène mais ses fonctions sont encore mal connues à l'heure actuelle. Les objectifs de ce travail ont été d'étudier le rôle de ZNF217 dans l'échappement tumoral et sa valeur pronostique dans le cancer du sein. Ainsi, nous avons montré que de forts niveaux d'expression de ZNF217 sont associés à : (i) une augmentation de la prolifération cellulaire in vitro et de la croissance tumorale in vivo, (ii) la stimulation de l'invasion et de la migration cellulaire, (iii) l'induction de l'EMT via la voie du TGF-β. De plus, les cellules surexprimant ZNF217 sont résistantes au paclitaxel et cette résistance est associée à la dérégulation de l'expression des membres de la famille Bcl-2 et d'Aurora-A. Enfin, nous avons montré pour la première fois que des forts niveaux d'expression d'ARNm de ZNF217 représentent un nouveau marqueur de mauvais pronostic dans le cancer du sein et sont associés au développement de métastases. ZNF217 semble donc jouer un rôle important dans la cancérogénèse mammaire et des stratégies thérapeutiques ciblant directement ZNF217 ou ciblant ses médiateurs (Aurora-A ou TGF-β) pourraient être utilisées en clinique dans le traitement des tumeurs mammaires surexprimant ZNF217

<p>Breast cancer remains among the most common cancers of the developed world. Despite advances in treatment modalities, deaths due to breast cancer are the second leading cause of cancer death among women. The transforming growth factor-beta (TGF-&beta;) pathway is an important modulator of breast cancer progression, acting in a tumor suppressing fashion in early carcinogenesis but switching in a poorly understood fashion to a promoter of cancer progression in later stages. Mutations and loss of function of TGF-&beta; components are common across a variety of cancers. In particular, the expression of the type III TGF-&beta; receptor (T&beta;RIII) is decreased with cancer grade and clinical progression in prostate, lung, ovarian, and pancreatic cancers. In an effort to enhance our understanding of the biology of TGF-&beta; on carcinogenesis, this dissertation looks at the role of T&beta;RIII in breast cancer progression.</p><p>Through an examination of clinical specimens, loss of T&beta;RIII was seen at both the message and protein levels with increasing tumor grade. Analysis of correlated patient outcomes showed that low T&beta;RIII expression was predictive of a shorter time to recurrence, demonstrating clinical relevance for T&beta;RIII expression. The contribution of T&beta;RIII to tumor progression was further examined by examining known TGF-&beta; functions, including proliferation, apoptosis, migration, and invasion. T&beta;RIII had no effect on proliferation or apoptosis, but had a suppressive effect on metastasis <italic>in vivo</italic>, as mammary cancer cells stably expressing T&beta;RIII that were orthotopically injected exhibited lower metatstatic burden and local invasion. <italic>In vitro</italic>, breast cancer cells exhibited suppression of migration and invasion in transwell assays. Finally, soluble T&beta;RIII (sT&beta;RIII) was shown to recapitulate the suppressive effects on invasion.</p><p>To further explore other potential mechanisms by which T&beta;RIII may be mediating its tumor suppressive effects, I examined the contribution of the cytoplasmic domain of T&beta;RIII, which is known to be critical in the regulation of T&beta;RIII cell surface expression and downstream signaling. <italic>In vitro</italic>, I demonstrated that abrogation of the cytoplasmic domain attenuates the T&beta;RIII-mediated suppression of migration and invasion. T&beta;RIII's suppressive effects are also concomitant with loss of TGF-&beta; signaling, as abrogation of the cytoplasmic domain failed to attenuate TGF-&beta; signaling while the full length receptor was able to do so. <italic>In vivo</italic>, I also showed that in the absence of the cytoplasmic domain, T&beta;RIII is unable to suppress metastasis and local invasion. Finally, a closer dissection of the cytoplasmic domain revealed that abolishing the interaction of T&beta;RIII with the scaffolding protein GIPC also attenuated T&beta;RIII's ability to dampen TGF-&beta; signaling and invasion.</p><p>In sum, T&beta;RIII was established as a prognostic marker for recurrence-free survival of breast cancer patients and as a suppressor of metastasis, migration, and invasion. Furthermore, several mechanisms contribute to T&beta;RIII's tumor suppressive effects, namely the generation of sT&beta;RIII and the interaction of T&beta;RIII with GIPC. Taken together, these studies further demonstrate the importance of TGF-&beta; signaling in cancer biology, elucidate mechanisms by which T&beta;RIII suppresses breast carcinogenesis, and expand upon our understanding of the emerging roles of T&beta;RIII in regulating tumor biology in general.</p> / Dissertation

The common type of myxoid soft tissue sarcomas is myxofibrosarcoma. Clinically, increased tumor grading and staging are frequently observed in myxofibrosarcomas after relentless local recurrences, which may eventually lead to metastatic diseases. However, metastatic myxofibrosarcomas are often refractory to current treatment strategies and constitute the primary cause of sarcoma-related death. Immunohistochemistry staining was applied to analyze myxoid tumors of soft tissue in our previous studies, and p21 protein (Cdc42/Rac)-activated kinase 1 (PAK1) was identified to be significantly upregulated in myxoid soft tissue sarcomas. The PAK1 is a pivotal serine/threonine kinase, which integrates stimuli from various signaling pathways to regulate cell survival, mitosis and cytoskeletal remodeling, etc. We first examined the endogenous PAK1 mRNA and total PAK1 protein levels in various myxoid sarcoma cell lines, including OH931, NMFH1 and NMFH2. This initial screening detected that upregulated PAK1 expression in OH931 and NMFH1, whereas downregulated PAK1 in NMFH2 cells. By wound healing and matrigel transwell assay, we further found that transfection of the expression plasmid carrying wild-type PAK1 gene or PAK1 T423E mutant promoted cell migration and invasion abilities in NMFH2 cells. On the other hand, knockdown of the PAK1 gene by short hairpin RNA interference inhibited the migration rate and invasion ability in NMFH1 cells. By 5-bromo-2-deoxyuridine assay and colony formation assay, we found that either exogenous expression of PAK1 protein or knockdown of PAK1 gene affected cell proliferation and transformation. Interestingly, immunofluorescence demonstrated that treatment with hepatocyte growth factor induced phosphorylation of PAK1 (Thr212) and promoted its nuclear import in NMFH2 cells. In summary, PAK1 plays oncogenic roles in myxoid sarcoma carcinogenesis.