Abstract Lymphomas are a heterogeneous group of malignancies, which usually have a good prognosis and high cure rates. Lymphomas are sensitive to chemotherapy and radiotherapy, and many patients can be cured even after a relapse, resulting in a need for effective follow-up. However, the cost-benefit ratio of radiological imaging in predicting the forthcoming relapses is poor. Consequently, there is a need for biological prognostic and predictive markers to distinguish patients at the highest risk of relapse at the time of diagnosis or during follow-up. Despite rapid progress in lymphoma treatments, some patients still die from lymphoma. Thus, more data on the basic biological features of lymphomas are also needed. Gelatinases (MMP-2 and MMP-9) and their tissue inhibitors (TIMP-1 and TIMP-2) have been found to play a role in the progression of solid tumours. TP53 is a tumour suppressor gene, the mutations and protein over-expression of which have been demonstrated to be associated with survival in most cancer types. There is also some evidence that these proteins could have prognostic significance in lymphomas as well. In the present study, the tissue expression, plasma concentrations and clinical value of gelatinases and their tissue inhibitors were evaluated in lymphomas. 249 primary tissue samples from patients with Hodgkin, follicular, or diffuse large B-cell lymphoma were analysed for expression of gelatinases and/or their inhibitors using immunohistochemistry. In follicular lymphoma, p53 protein expression was also investigated. The plasma samples of 126 lymphoma patients and a control group of 44 healthy volunteers were collected and studied by ELISA. TIMP-1 expression correlated with bulky tumour and nodular sclerosis subtype of Hodgkin lymphoma. In follicular lymphoma, p53 over-expression was an independent adverse prognostic factor for survival and a predictor of histological transformation. Plasma MMP-2-TIMP-2 complex appeared to be a potential follow-up marker predicting the risk of relapse in lymphoma patients. Plasma levels of the MMP-2-TIMP-2 complex, proMMP-2, TIMP-2 and proMMP-2/TIMP-2 ratio were at abnormal levels both in patients with newly diagnosed lymphoma and those in remission compared to healthy controls. The clinical significance of these markers needs further studies.

Introdução: O Carcinoma Urotelial de Bexiga (CUB) é o segundo tumor urológico mais prevalente no Brasil. Devido ao elevado custo médico no processo que envolve seu diagnóstico, tratamento e seguimento, o CUB é um dos tipos de tumores mais caros para os sistemas de saúde. Embora existam fatores prognósticos definidos, como o estadiamento patológico, a diferenciação histológica e a presença de invasão angiolinfática (IAL), os mesmos demonstram-se insuficientes para uma acurada definição de comportamento da doença. Com a evolução da pesquisa molecular um grande número de potenciais novos marcadores de agressividade tem surgido. As Metaloproteinases da matriz (MMP) sao proteínas teciduais, pertencentes à família das endoproteinases, que degradam vários componentes da matriz extracelular. A expressão de diversas MMPs, especialmente MMP-2 e MMP-9 (gelatinases), bem como seus ativadores e inibidores, tem sido estudada como potencial marcador de comportamento tumoral em várias neoplasias. Objetivos: Avaliarmos os níveis de expressão dos genes das gelatinases MMP-2 e MMP-9 no CUB, assim como proteínas envolvidas em suas vias de ativação e inibição (MMP-14, IL-8, TIMP-1, TIMP-2, RECK e TGF-! ). Material e Método: Estudamos pela técnica de qRT-PCR a expressão dos 8 genes em amostras de CUB de 40 pacientes submetidos a RTUb, tendo como grupo controle amostras de urotélio sem câncer de 6 pacientes submetidos a prostatectomia aberta por HPB, bem como sua relação com marcadores prognósticos clássicos (estádio, grau e IAL). Resultados: Houve uma superexpressão de MMP-9 na maioria das amostras de CUB, bem como subexpressão de MMP-2, TIMP-1, TIMP-2, MMP-14, IL-8, TGF-! e RECK. Comparando os níveis de expressão dos genes com o estádio patológico, houve uma superexpressão de MMP-9 nos tumores pT1-2, quando comparados com pTa (p=0,026), bem como maior expressão de IL-8 nos tumores pT1 e pT2 (p=0,015 e p=0,048, respectivamente). Embora estatisticamente nao significativa, houve uma superexpressão de MMP-14 nos tumores pT2, quando comparados aos demais (p=0,087). Com relação ao grau histológico, também identificamos superexpressão de MMP-9 nos tumores de alto grau, quando comparados aos de baixo grau (p=0,012), assim como maior expressão de IL-8 nos tumores de alto grau (p=0,003). Conclusão: Houve uma superexpressão de MMP-9 e uma subexpressão de MMP-2, TIMP-1, TIMP-2, MMP-14, RECK, IL-8 e TGF-! no CUB, quando comparado com o grupo controle. Também identificamos uma superexpressão de MMP-9 e IL-8 em tumores pT1-2 quando comparados com pTa e de alto grau quando comparados com baixo grau. A subexpressão dos principais inibidores da MMP-9 (TIMP-1 e RECK) pode explicar sua superexpressão no CUB, assim como a superexpressão de IL-8 nos tumores invasivos e de alto grau pode agir como fator ativador da MMP-9 nestes mesmos tumores / Introduction: Bladder cancer (BC) is the second most common urological tumor in Brazil. Because its high cost on diagnosis, treatment and follow-up, BC is one of the most expensive malignancies for health care providers. Although we have well-known prognostic factors, like pathological stage, histologic grade and lymphovascular invasion, they are insufficient to figure more accurate tumor aggressiveness. Recent molecular biology helped us to discover a huge amount of potential markers. Matrix metalloproteinases (MMP) are tissue endopeptidases that degrade components of extracellular matrix. Expression of several MMP, specially MMP-2 and MMP-9 (gelatinases), and their activators and inhibitors, are investigated as potential behavior markers in many neoplasms. Objectives: The aim of this study was to evaluate expression levels of gelatinases MMP-2 and MMP-9 genes by quantitative real-time polymerase chain reaction (qRT-PCR) in BC, as well as other proteins evolved in the activation and inhibition pathways (MMP-14, IL-8, TIMP-1, TIMP-2, RECK e TGF-b). Material and Method: Present study analyzed tissue expression of 8 genes in BC samples of transutethral resection of 40 patients by qRT-PCR, as well as their relation with current prognostic factors (stage, grade and LVI). The control group was composed of utothelial tissue from 6 patients with benign prostatic hyperplasia (BPH) treated surgically with retropubic prostatectomy. Results: In the tumor samples, MMP-9 presented an overexpression and MMP-2, TIMP-1, TIMP-2, MMP-14, RECK, IL-8, and TGF-b were underexpressed in BC tissue compared to control. Comparing gene level expression to pathologic stage, there was MMP-9 overexpression in pT1-2 tumors compared to pTa (p=0.026), as wall as IL-8 overexpression in pT1 and pT2 tumors (p=0.015 e p=0.048, respectively). Although not statiscally significant, there was MMP-14 overexpression in pT2 tumors in comparison to pTa-1 (p=0.087). About grade, there was MMP-9 overexpression in high-grade tumors compared to low-grade (p=0.012), as well as IL-8 overexpression in high-grade tumors (p=0.003). There was not relation of any gene expression to LVI. Conclusions: We found overexpression of MMP-9 and underexpression of MMP-2, TIMP-1, TIMP-2, MMP-14, RECK, TGF-b and IL-8 in BC compared with the control group. According to the prognostic factors we found increased levels of MMP-9 and IL-8 gene expression in pT1-2 compared to pTa tumors and high-grade compared to low-grade tumors. Underexpression of major MMP-9 inhibitors (TIMP-1 and RECK) could explain MMP-9 overexpression in BC, as well as IL-8 overexpression in high-grade and stage tumors could act as activation factor of MMP-9 in these tumors

Mammals cannot re-form heavily damaged bones as in large fracture gaps, whereas zebrafish efficiently regenerate bones even after amputation of appendages. However, the source of osteoblasts that mediate appendage regeneration is controversial. Several studies in zebrafish have shown that osteoblasts are generated by dedifferentiation of existing osteoblasts at injured sites, but other observations suggest that de novo production of osteoblasts also occurs. In this study, we found from cell-lineage tracing and ablation experiments that a group of cells reserved in niches serves as osteoblast progenitor cells (OPCs) and has a significant role in fin ray regeneration. Besides regeneration, OPCs also supply osteoblasts for normal bone maintenance. We further showed that OPCs are derived from embryonic somites, as is the case with embryonic osteoblasts, and are replenished from mesenchymal precursors in adult zebrafish. Our findings reveal that reserved progenitors are a significant and complementary source of osteoblasts for zebrafish bone regeneration.

Patients with cystic fibrosis who are infected with the pathogen Pseudomonas aeruginosa have shown favorable responses to the drug azithromycin (AZM). This drug works in an anti-inflammatory capacity, improving clinical outcomes and improving quality of life in this population. The drug has also been shown to affect macrophage polarization by shifting these cells away from an inflammatory phenotype toward an alternatively activated anti-inflammatory phenotype. The full impact of this phenotypic change is not well understood in the context of the response to P. aeruginosa infection, or the overall immune response in cystic fibrosis. To understand how the AZM-polarized macrophage affects other types of cells, we utilized a co-culture in vitro system, with macrophages and fibroblasts incubating together. In this system, we determined that AZM causes upregulation of the pro-fibrotic mediator transforming growth factor-β as well as the extracellular matrix (ECM) protein fibronectin. The mediator of ECM turnover, matrix metalloproteinase (MMP)-9 was upregulated in this system as well. In an in vivo model of P. aeruginosa infection, MMP- 9 and fibronectin were increased in the bronchoalveolar lavage 7 days post-infection in mice that were treated with AZM. This was accompanied by a decrease in damage to the lung tissue, determine by histological examination. To determine if these changes would continue in human subjects with cystic fibrosis, a clinical study was done in this population. Subjects with AZM treatment had decreased TGF-β levels, but no differences in MMP-9 or fibronectin. Interestingly, correlations between certain fibrotic mediators and inflammatory cytokines, specifically interleukin -1β, were different in subjects with AZM treatment compared to subjects without AZM therapy. Together, these data indicate that AZM alters the fibrotic response from the macrophages, as well as the interaction of the inflammatory response and fibrosis development.

Periodontal diseases may increase risk of vascular calcification in cardiovascular diseases but the potential mechanisms are not defined. Fetuin, a naturally-occurring serum glycoprotein in humans, protects against ectopic arterial calcification. We considered that patients with periodontitis could be at increased risk of developing calcifying atheromas because periodontal-disease associated enzymes may enter the circulation and subsequently degrade fetuin, thereby disrupting its ability to inhibit calcification. By in silico investigation, MMP -3 and -7 were predicted to cleave fetuin but only MMP-7 actually degraded human fetuin in vitro. MMP-7 degradation of fetuin was time- and concentration- dependent and was inhibited by an MMP Inhibitor. By mass spectrometry the presence of novel, MMP-7-mediated cleavage sites in fetuin were found. Fetuin bound tightly to MMP-7 (kd =2.96 x 10-9 M). The degradation of fetuin by MMP-7 could explain, at least in part, the apparent association between periodontal diseases and calcifying atheromas.

Periodontal diseases may increase risk of vascular calcification in cardiovascular diseases but the potential mechanisms are not defined. Fetuin, a naturally-occurring serum glycoprotein in humans, protects against ectopic arterial calcification. We considered that patients with periodontitis could be at increased risk of developing calcifying atheromas because periodontal-disease associated enzymes may enter the circulation and subsequently degrade fetuin, thereby disrupting its ability to inhibit calcification. By in silico investigation, MMP -3 and -7 were predicted to cleave fetuin but only MMP-7 actually degraded human fetuin in vitro. MMP-7 degradation of fetuin was time- and concentration- dependent and was inhibited by an MMP Inhibitor. By mass spectrometry the presence of novel, MMP-7-mediated cleavage sites in fetuin were found. Fetuin bound tightly to MMP-7 (kd =2.96 x 10-9 M). The degradation of fetuin by MMP-7 could explain, at least in part, the apparent association between periodontal diseases and calcifying atheromas.

Systemic hypoxia frequently occurs in patients with cardiopulmonary diseases. Maintenance of vascular reactivity and endothelial viability is essential to preserving oxygen delivery in these patients. The role of matrix metalloproteinase-2 (MMP-2) and heme oxygenase-2 (HO-2) in the vascular response to hypoxia were investigated. In the first part of the thesis, the role of MMP-2 in regulating systemic arterial contraction after prolonged hypoxia was investigated. MMP-2 inhibition with cyclic peptide CTTHWGFTLC (CTT) reduced phenylephrine (PE)-induced contraction in aortae and mesenteric arteries harvested from rats exposed to hypoxia for 7 d. Responses to PE were reduced in MMP-2-/- mice exposed to hypoxia for 7 d compared to wild-type controls. CTT reduced contraction induced by big endothelin-1 (big ET-1) in aortae harvested from rats exposed to hypoxia. Increased contraction to big ET-1 after hypoxia was observed in wild-type controls, but not MMP-2-/- mice. Rat aortic MMP-2 and MT1-MMP protein levels and MMP activity were increased after 7 d of hypoxia. Rat aortic MMP-2 and MT1-MMP mRNA levels were increased in the deep medial vascular smooth muscle. These results suggest that hypoxic induction of MMP-2 activity potentiates contraction in systemic conduit and resistance arteries through proteolytic activation of big ET-1. The second part of the thesis investigated oxygen regulation of HO-2 protein and whether it plays a role in preserving endothelial cell viability during hypoxia. HO-2, but not HO-1, protein level was maintained during hypoxia in human endothelial cells through enhanced translation of HO-2 transcripts. Inhibition of HO-2 expression increased the production of reactive oxygen species, decreased mitochondrial membrane potential, and enhanced apoptotic cell death and activated caspases during hypoxia, but not during normoxia. These data indicate that HO-2 is translationally regulated and important in maintaining endothelial viability and function during hypoxia. In summary, the thesis demonstrates the importance of MMP-2 and HO-2 in preserving vascular function during prolonged systemic hypoxia. These enzymatic pathways may, therefore, represent novel therapeutic targets that may be exploited to ameliorate the effects of hypoxia in patients with cardiopulmonary disease.

Fragment based drug discovery (FBDD) has been applied to two protease drug targets, MMP-12 and HIV-1 protease. The primary screening and characterization of hit fragments were performed with surface plasmon resonance -technology. Further evaluation of the interaction was done by inhibition studies and in one case with X-ray crystallography. The focus of the two projects was different. Many MMP inhibitors contain a strong zinc chelating group, hydroxamate, interacting with the catalytic zinc atom. This strategy may be the cause for the low specificity of MMP inhibitors. Using FBDD we found a fragment with an unusual strong affinity for MMP-12. An inhibition assay confirmed that it was an inhibitor but indicated a stoichiometry of 2:1. Crystallography data revealed that an adduct of the fragment was bound in the active site, with interactions both with the catalytic zinc and the S1’ pocket. This may present a new scaffold for MMP-12 inhibitors. For HIV-1 protease the focus was on identifying inhibitors not sensitive to current resistance mutations. A fragment library for screening with SPR-technology was designed and used for screening against wild type enzyme and three variants with resistance mutations. Many of the hits were promiscuous but a number of fragments with possible allosteric inhibition mechanism were identified. The temperature dependency of the dissociation rate and reported resistance mutations was studied with thermodynamics. A good, but not perfect correlation was found between resistance and both the dissociation data and the free energy for dissociation compared to data from wild type enzyme. However, the type of mutation also influenced the results. The flap mutation G48V displayed thermodynamic profiles not completely correlating with resistance. It was found that dissociation rate and thermodynamics may complement each other when studying resistance, but only one of them may not be enough.

Glioblastoma multiforme (GBM) is an aggressive central nervous system (CNS) cancer characterized by enhanced tumor cell motility, pernicious invasion into the normal brain, extensive tumor-induced angiogenesis, and adaptive resistance to current therapeutic paradigms. One of the difficulties associated with GBM is the ability of the tumor cells to infiltrate normal CNS tissue. Neurosurgeons can remove the primary tumor mass, but peripheral cells that are inaccessible will ultimately result in a secondary lesion that can lead to death. The matrix metalloproteinases (MMP) are well known for their abilities to facilitate processes of cellular motility and invasion through their clearance of extracellular matrix (ECM). A specific member of this family, MMP-1, is not observed in normal brain, yet its expression is a common characteristic of GBM. The various causes of MMP-1 expression, and its consequences in GBM cells are unknown. As such, functional studies were conducted related to the induction of MMP-1 expression via another molecule intrinsic to GBM, nitric oxide (NO). The exposure of GBM cell lines to nanomolar concentrations of NO produced significant inductions of MMP-1 expression and GBM cell motility. The specific removal of MMP-1 with siRNA elicited an abrogation of NO-stimulated motility, suggesting a pathological contribution by this enzyme. Furthermore, recent accumulating evidence suggests that MMP-1 contributes to tumor cell survival and related angiogenesis in other cancer settings. To investigate these capabilities in GBM, cell lines were stably engineered to have either MMP-1 over-expression or knock-down. Both tumor formation and size were significantly reduced with MMP-1 knock-down and significantly increased with over-expression. In a model of GBM cell-induced angiogenesis, the presence of MMP-1 contributed to an angiogenic phenotype. Further angiogenesis studies revealed a significant recruitment of host endothelium to the tumor interstitium in vivo. Proteomic studies suggest that one mechanism by which MMP-1 could influence angiogenesis is through the easement of the anti-angiogenic tissue inhibitor of metalloproteinases-4 (TIMP-4), since the removal of MMP-1 elicits a significant increase in TIMP-4 detection. Altogether, these functional data present MMP-1 as a promising target for future therapeutic investigation, because it is unique to the GBM environment and contributes to key overlapping GBM pathologies.

Traumatic brain injury (TBI) is a serious health concern, causing cognitive, motor, and sensory deficits, including olfactory dysfunction. This dissertation explores the effects of TBI on synaptic plasticity within the olfactory system, seeking to define mechanisms guiding postinjury sensory reinnervation. Physical forces induced by TBI can axotomize olfactory receptor neurons (ORNs), which innervate olfactory bulb (OB). These axons regenerate OB projections after injury, a process involving growth through a complex extracellular matrix (ECM). As such, we investigated a potential molecular mechanism capable of modifying local OB ECM to support postinjury synaptogenesis. Since matrix metalloproteinases (MMPs) and their ECM substrates are recognized for TBI therapeutic potential, we explored the role of MMP9 and its substrate osteopontin (OPN) in promoting ORN reinnervation of the OB after mild fluid percussion injury (FPI). First, we confirmed that FPI deafferented the mouse OB. In Chapter 2, we showed concurrent activation of neuroglia, elevated spectrin proteolysis and reduction in ORN-specific olfactory marker protein (OMP). As OMP normalized during regeneration, growth associated protein-43kD (GAP-43) peaked, marking OB entry of ORN growth cones. Ultrastructural analysis revealed ongoing ORN axon shrinkage and degeneration, glial phagocytosis of cellular debris, and a reorganization of synaptic structure. To explore ECM role in mediating postinjury OB reinnervation, we defined the time course of MMP9 activity and several downstream targets. Chapter 3 reports biphasic MMP9 activity increase during acute/subacute degeneration, accompanied by robust generation of 48kD OPN cell signaling peptide. OPN receptor CD44 also increased during the acute/subacute interval, suggesting potential interaction of the two proteins. Finally, we utilized MMP9 knockout (MMP9KO) mice to confirm MMP9 role in OB synaptogenesis. In Chapter 4, MMP9KO reversed FPI-induced lysis of 49kD OPN and altered postinjury expression of ORN axon degeneration marker OMP. Additional ultrastructural analysis verified delayed recovery of OB synaptic features within the injured MMP9KO. Overall, we demonstrated that mild FPI elicits ORN axotomy to induce OB reactive synaptogenesis, and that MMP9 supports reinnervation by processing OPN for activation of local glia, cells which reorganize the ECM for synapse regeneration.