As metaloproteases da matriz (MMPs) são enzimas proteolíticas que participam da degradação da matriz extracelular no organismo de vertebrados. Estudos mostram a grande importância dessas enzimas no processo de remodelação do tecido cardíaco, além de sugerirem a participação da MMP-2 em doenças cardiovasculares. Em estudo recente foi demonstrado que as MMPs clivam o receptor ?2-adrenérgico, contribuindo para o aumento do tônus arteriolar de ratos espontaneamente hipertensos (SHR). Acredita-se que processo semelhante possa ser verificado em relação ao receptor ?-1 adrenérgico e proteínas das junções, que são fundamentais para o funcionamento do coração. As análises in sílico realizadas mostraram regiões prováveis de clivagem pela metaloprotease da matriz 2 humana recombinante (rhMMP-2) na porção extracelular, especificamente na região Nterminal deste receptor, no entanto, as análises de comparação de similaridade de substratos não apresentaram resultados significativos, embora os resultados preliminares obtidos no teste in vitro mostraram que houve hidrolise logo no início do peptídeo sintético ASPPASLLPPAS, entre os resíduos alanina e serina, entre as duas prolinas e por fim entre o resíduo de prolina e alanina, regiões com grandes chances de ocorrer a hidrólise, pois o substrato nativo desta enzima é o colágeno que é composto por uma cadeia polipeptídica com uma sequência de repetições onde geralmente temos glicina-X-Y, onde X normalmente é uma prolina e Y frequentemente uma hidroxiprolina, e raramente lisina e hidroxilisina, no entanto a replicação deste experimento não apresentou o mesmo resultado. Já os resultados obtidos no western blotting mostraram que a expressão do receptor é diminuída quando os cardiomiócitos são previamente tratados com 40mM e 120mM de rhMMP- 2 e esse efeito tem uma reversão significativa quando as células são previamente tratadas com inibidores doxiciclina ou ONO-4817, corroborando com os trabalhos apresentados na literatura em que a rhMMP-2 atua no receptor ?1adrenérgico. / Matrix metalloproteinases (MMPs) are proteolytic enzymes that participate in the degradation of the extracellular matrix in the vertebrate organism. Studies show the great importance of these enzymes in the remodeling process of cardiac tissue, besides suggesting the participation of MMP-2 in cardiovascular diseases. In a recent study, MMPs were shown to cleave the ?2-adrenergic receptor, contributing to the increase in arteriolar tone of spontaneously hypertensive rats (SHR). It is believed that a similar process can be verified also in the ?-1 adrenergic receptor and junction proteins, which are fundamental to the heart function. The in situ analyzes performed revealed sections prone to be cleaved by matrix metalloproteinase 2 recombinant human (rhMMP-2) in the extracellular portion, specifically in the Nterminal region of this receptor, however, the comparative analyzes of the similarity of substrates did not present significant results, but those obtained in the in vitro test showed that there was hydrolysis right at the beginning of the synthetic peptide ASPPASLLPPAS, between alanine and serine residues, between the two proline and finally between the proline residue and alanine. Hydrolysis among proline residues was expected, even though it was not predicted for in silica cleavage, since the native substrate of this enzyme is collagen, which is composed of a polypeptide chain with a sequence of repetitions in which it usually has glycine-XY, where X usually is a proline and Y often a hydroxyproline, and rarely lysine and hydroxylysine, however the replication of this experiment di not present the same result. The obtained results in western blotting have presented that receptor expression is decreased when cardiomyocytes are pretreated with 40mM and 120mM rhMMP-2 and this effect has a significant reversion when cells are pretreated with doxycycline or ONO-4817 inhibitors, supporting previous studies which demonstrate that rhMMP- 2 acts on the ? 1 adrenergic receptor.

As metaloproteases da matriz (MMPs) são enzimas proteolíticas que participam da degradação da matriz extracelular no organismo de vertebrados. Estudos mostram a grande importância dessas enzimas no processo de remodelação do tecido cardíaco, além de sugerirem a participação da MMP-2 em doenças cardiovasculares. Em estudo recente foi demonstrado que as MMPs clivam o receptor ?2-adrenérgico, contribuindo para o aumento do tônus arteriolar de ratos espontaneamente hipertensos (SHR). Acredita-se que processo semelhante possa ser verificado em relação ao receptor ?-1 adrenérgico e proteínas das junções, que são fundamentais para o funcionamento do coração. As análises in sílico realizadas mostraram regiões prováveis de clivagem pela metaloprotease da matriz 2 humana recombinante (rhMMP-2) na porção extracelular, especificamente na região Nterminal deste receptor, no entanto, as análises de comparação de similaridade de substratos não apresentaram resultados significativos, embora os resultados preliminares obtidos no teste in vitro mostraram que houve hidrolise logo no início do peptídeo sintético ASPPASLLPPAS, entre os resíduos alanina e serina, entre as duas prolinas e por fim entre o resíduo de prolina e alanina, regiões com grandes chances de ocorrer a hidrólise, pois o substrato nativo desta enzima é o colágeno que é composto por uma cadeia polipeptídica com uma sequência de repetições onde geralmente temos glicina-X-Y, onde X normalmente é uma prolina e Y frequentemente uma hidroxiprolina, e raramente lisina e hidroxilisina, no entanto a replicação deste experimento não apresentou o mesmo resultado. Já os resultados obtidos no western blotting mostraram que a expressão do receptor é diminuída quando os cardiomiócitos são previamente tratados com 40mM e 120mM de rhMMP- 2 e esse efeito tem uma reversão significativa quando as células são previamente tratadas com inibidores doxiciclina ou ONO-4817, corroborando com os trabalhos apresentados na literatura em que a rhMMP-2 atua no receptor ?1adrenérgico. / Matrix metalloproteinases (MMPs) are proteolytic enzymes that participate in the degradation of the extracellular matrix in the vertebrate organism. Studies show the great importance of these enzymes in the remodeling process of cardiac tissue, besides suggesting the participation of MMP-2 in cardiovascular diseases. In a recent study, MMPs were shown to cleave the ?2-adrenergic receptor, contributing to the increase in arteriolar tone of spontaneously hypertensive rats (SHR). It is believed that a similar process can be verified also in the ?-1 adrenergic receptor and junction proteins, which are fundamental to the heart function. The in situ analyzes performed revealed sections prone to be cleaved by matrix metalloproteinase 2 recombinant human (rhMMP-2) in the extracellular portion, specifically in the Nterminal region of this receptor, however, the comparative analyzes of the similarity of substrates did not present significant results, but those obtained in the in vitro test showed that there was hydrolysis right at the beginning of the synthetic peptide ASPPASLLPPAS, between alanine and serine residues, between the two proline and finally between the proline residue and alanine. Hydrolysis among proline residues was expected, even though it was not predicted for in silica cleavage, since the native substrate of this enzyme is collagen, which is composed of a polypeptide chain with a sequence of repetitions in which it usually has glycine-XY, where X usually is a proline and Y often a hydroxyproline, and rarely lysine and hydroxylysine, however the replication of this experiment di not present the same result. The obtained results in western blotting have presented that receptor expression is decreased when cardiomyocytes are pretreated with 40mM and 120mM rhMMP-2 and this effect has a significant reversion when cells are pretreated with doxycycline or ONO-4817 inhibitors, supporting previous studies which demonstrate that rhMMP- 2 acts on the ? 1 adrenergic receptor.

Methotrexate (MTX) is an antimetabolite anticancer agent that is used in treatment of multiple cancers, such as acute lymphoblastic leukaemia and osteosarcoma. A lack of selective tumour toxicity is one of the major problems associated with MTX chemotherapy, especially when given at high doses, as in high dose MTX (HDMTX) therapy. MTX causes various toxicity problems including life-threatening nephrotoxicity, haematological toxicity and neurotoxicity. Overcoming this toxicity is of great importance and has been attempted in various ways, not least via the design of prodrugs. The concept of tumour protease, and specifically matrix metalloproteinase (MMP), activated prodrugs was the focus of the work described in this thesis. This concept relies upon attachment of an MMP-sensitive peptide sequence to a specific site in a drug structure, so as to inactive it. The activity of the parent drug is restored once it is activated by the MMPs in the tumour microenvironment. In this work, different MMP-sensitive peptide sequences linked to MTX were synthesised, resulting in 63 MTX prodrugs. The MMP-mediated activation of these conjugates in tumour tissues (specifically HT1080 homogenates) ex vivo was assessed and the results were compared to the activation of these conjugates in various normal tissues specifically liver, kidney and lung. Specific criteria were established for the selection of promising conjugates for more detailed study. From 7 promising compounds, compound 75 was identified as the lead prodrug, demonstrating selective MMP activation, as indicated by inhibition of its activation by broad spectrum MMP inhibitor ilomastat. The pharmacokinetics of compound 75 was studied in tumour (HT1080) xenograft-bearing mice and the results were compared to those obtained from administration of equimolar doses of conventional MTX. Compound 75 led to enhanced tumour concentrations of MTX, with reduced exposure to normal tissues in vivo compared to conventional MTX therapy. Furthermore, the efficacy of equimolar doses of compound 75 and directly dosed MTX in reduction of HT1080 volume were compared. Superior antitumour activity was observed with compound 75 compared to MTX treatment. Compound 75 is the first example of an MMP-activated prodrug to be reported with enhanced therapeutic index, as evidenced by a full in vivo pharmacokinetic analysis and normal tissue metabolism data. The data presented in thesis support the concept of MMP-activated prodrug development, and form a strong foundation upon which to develop a clinicallyuseful MTX prodrug, with the potential to enhance efficacy and reduce toxicity to the patient.

Angiogenesis, the formation of new vessels from a preexisting vasculature, is critical for supplying a healing myocardium with oxygen and nutrients to sustain metabolism post myocardial infarction (MI). Interleukin-1β (IL-1β), a proinflammatory cytokine increased in the heart post-MI, is considered essential for angiogenesis in tumor growth and metastasis, arthritis, endometriosis, and wound healing. Matrix metalloproteinases (MMPs) are implicated in angiogenesis because of their ability to selectively degrade components of the extracellular matrix. Vascular endothelial growth factors (VEGFs) play a vital role in angiogenesis because of their involvement in the recruitment and proliferation of endothelial cells. The current study explores IL-1β-stimulated regulation of angiogenic genes in cardiac microvascular endothelial cells (CMECs), the signaling mechanisms involved, and the implications in the processes of angiogenesis. DNA microarray analysis indicated IL-1β modulates the expression of numerous angiogenesis-related genes, notably upregulating MMP-2 and downregulating VEGF-D expression. RT-PCR and Western blot analyses confirmed the differential expression in response to IL-1β. In-gel zymographic analysis demonstrated IL-1β-stimulated increase in MMP-2 activity. IL-1β activated ERK1/2 and JNKs, not p38 kinase, and activated PKCα/β1 independent of MAPKs. IL-1β inactivated GSK3β via ERK1/2. Pharmacological inhibition of these signaling cascades indicated IL-1β-stimulated regulation of MMP-2 and VEGF-D occurs via ERK1/2, JNKs, and PKCα/β1-dependent mechanisms. In addition, inactivation of GSK3β inhibited basal VEGF-D expression. H2O2 significantly increased MMP-2 protein levels while IL-1β-induced VEGF-D downregulation was further potentiated by ROS scavenging compounds and inhibition of NF-κB. Phalloidin-FITC stain indicated a sharp reduction in fibrillar actin in the cytoskeleton of IL-1β-stimulated cells. Wounding assays revealed that IL-1β induced CMEC migration but prevented cell-to-cell contact and restoration of the monolayer. Flow cytometric analysis revealed a G0/G1 phase cell cycle arrest in IL-1β-stimulated cells, indicative of decreased proliferation. IL-1β inhibited three-dimensional in vitro tube formation by CMECs. Lastly, IL-1β inhibited microvessel sprouting from aortic rings, an assay examining the collective response of multiple cell types. Collectively, the data presented in this study provide evidence that IL-1β differentially regulates important angiogenesis-related genes in CMECs. This differential regulation may lead to interruptions in the processes of angiogenesis, ultimately creating a dysfunctional phenotype for myocardial vessel formation.

Current hydrophilic resin adhesives undergo hydrolytic degradation and show a decrease in bond strength over time. Nanoleakage and ultrastructure studies suggest that inadequately infiltrated collagen leads to enzymatic degradation and resin-dentin bond failure. Adequate degree of conversion (DC) of resin adhesives is also critical to resin-dentin bond strength and durability. The long-term goal of this dissertation is the realization of durable resin-dentin bond. It is hypothesized that ethanol-wet bonding technique (EW) may effectively facilitate the infiltration of hydrophobic monomers into hydrophilic acid-etched dentin by maintaining interfibrillar spacing, stiffening collagen matrix, and improving adhesive resin-demineralized dentin matrix miscibility. Chlorhexidine (CHX), Matrix Metalloproteinase-inhibitor (MMP-inhibitor), should further preserve collagen integrity and resin-dentin bond strength. Moreover, efficient photoinitiator systems that broaden light absorptivity and provide more reactive radicals may enhance polymerization. In this dissertation, a clinically-relevant EW protocol, 3×15s absolute ethanol rinsing, provided significantly higher microtensile bond strength (πTBS) of a hydrophobic resin (70%BisGMA/30%TEGDMA) to dentin as compared to water-wet bonding (WW). All groups showed no significant drop of πTBS after 1-year storage except EW without CHX application, showing marginally significant reduction in πTBS (p=0.0558) suggesting MMP-inhibition by CHX in EW. These results were consistent with subsequent experiments. EW maintained interfibrillar width and hybrid layer thickness for resin infiltration and retention. Monomer molar concentration across the hybrid layer was significantly higher in EW than WW. An application of 2% CHX diacetate further preserved collagen banding in EW. WW showed more generalized spotted nanoleakage, while EW presented localized reticular nanoleakage. The use of Irgacure 819 (BAPO) alone and in combination with benzoyl peroxide (BPO) or camphorquinone (CQ) increased DC of hydrophobic and hydrophilic resins over resins containing the CQ/amine (4E) control. Only BAPO and BAPO/BPO demonstrated significantly higher immediate shear bond strength than CQ/4E. Within the limitations of these studies, EW improved resin-dentin bond durability by maintaining collagen interfibrillar spaces for efficient infiltration of a hydrophobic BisGMA/TEGDMA resin resulting in significantly higher πTBS and monomer molar concentrations with less nanoleakage distribution within the hybrid layer than WW. CHX further maintained collagen integrity and πTBS in EW. BAPO is a potential alternative photoinitiator of dental resins.

No description available.

<p>Extracellular matrix degrading proteases from the plasminogen (plg) activator (PA) and the matrix metalloproteinase (MMP) systems have been implicated as important mediators of ovulation and corpus luteum (CL) formation and regression. The aim of this thesis was to investigate the expression and regulation of PAs and MMPs in the ovary and to examine their functional roles for CL formation and function. </p><p> The expression of membrane-type MMP-1 (MT1-MMP) and its substrate gelatinase A (MMP-2) mRNAs was studied during pregnant mare serum gonadotropin (PMSG)/human chorionic gonadotropin (hCG)-induced ovulation in immature rats. These proteases were coordinately regulated so that both were highly expressed in the theca cells of large preovulatory follicles. This suggests that MT1-MMP activates gelatinase A in preovulatory follicles to degrade the follicular wall during ovulation. </p><p> In pseudopregnant (psp) rats, MT1-MMP mRNA was expressed in the CL throughout the luteal phase. Tissue inhibitor of metalloproteases type-1 (TIMP-1) mRNA was expressed during CL formation and regression. MMP-2 and collagenase-3 mRNAs were expressed during CL formation and regression, respectively. When the luteal phase was artificially prolonged or shortened, TIMP-1 and collagenase-3 mRNAs were induced only after the serum progesterone levels had decreased, indicating a close association with luteolysis in the rat. </p><p> In psp mice, the expression of mRNAs coding for both PAs, seven MMPs, and five protease inhibitors was studied. Most of the studied molecules were coordinately expressed during formation or regression of the CL. However, uPA, MT1-MMP, and TIMP-3 mRNAs were expressed throughout the luteal phase. The role of uPA was examined in psp uPA deficient mice. These mice displayed no abnormalities in luteal function or vascularity. The role of uPA is thus either not essential or can be compensated by other proteases in the absence of uPA. </p><p> In order to control the timing of the CL formation, a mouse model for PMSG/hCG-induced CL formation was developed. Five different protocols were evaluated. One of them provided CL that were stable for six days. In that protocol the mice were treated with prolactin (PRL), twice daily from day 2 of CL life onward. The expression of the steroid acute regulatory protein (StAR) mRNA in the psp CL was also characterized to assess its use as a molecular marker for CL development and regression. It was highly expressed in the forming and functional CL and downregulated at a late stage of CL regression.</p><p> The functional role of plg and MMPs for CL formation and function was investigated in plg deficient mice treated with the MMP inhibitor galardin (GM6001). Both psp mice and PMSG/hCG +PRL-induced CL formation were used. Several molecular markers for CL development and regression were used to evaluate the health status of the CL. Our data showed that healthy and vascularized CL formed even in plg deficient mice treated with the inhibitor. However, serum progesterone levels were significantly reduced in these mice, an effect that was mainly attributable to the plg deficiency. In conclusion, neither plg nor MMPs, alone or in combination, seem to be essential for the development of a functional CL.</p>

Extracellular matrix degrading proteases from the plasminogen (plg) activator (PA) and the matrix metalloproteinase (MMP) systems have been implicated as important mediators of ovulation and corpus luteum (CL) formation and regression. The aim of this thesis was to investigate the expression and regulation of PAs and MMPs in the ovary and to examine their functional roles for CL formation and function. The expression of membrane-type MMP-1 (MT1-MMP) and its substrate gelatinase A (MMP-2) mRNAs was studied during pregnant mare serum gonadotropin (PMSG)/human chorionic gonadotropin (hCG)-induced ovulation in immature rats. These proteases were coordinately regulated so that both were highly expressed in the theca cells of large preovulatory follicles. This suggests that MT1-MMP activates gelatinase A in preovulatory follicles to degrade the follicular wall during ovulation. In pseudopregnant (psp) rats, MT1-MMP mRNA was expressed in the CL throughout the luteal phase. Tissue inhibitor of metalloproteases type-1 (TIMP-1) mRNA was expressed during CL formation and regression. MMP-2 and collagenase-3 mRNAs were expressed during CL formation and regression, respectively. When the luteal phase was artificially prolonged or shortened, TIMP-1 and collagenase-3 mRNAs were induced only after the serum progesterone levels had decreased, indicating a close association with luteolysis in the rat. In psp mice, the expression of mRNAs coding for both PAs, seven MMPs, and five protease inhibitors was studied. Most of the studied molecules were coordinately expressed during formation or regression of the CL. However, uPA, MT1-MMP, and TIMP-3 mRNAs were expressed throughout the luteal phase. The role of uPA was examined in psp uPA deficient mice. These mice displayed no abnormalities in luteal function or vascularity. The role of uPA is thus either not essential or can be compensated by other proteases in the absence of uPA. In order to control the timing of the CL formation, a mouse model for PMSG/hCG-induced CL formation was developed. Five different protocols were evaluated. One of them provided CL that were stable for six days. In that protocol the mice were treated with prolactin (PRL), twice daily from day 2 of CL life onward. The expression of the steroid acute regulatory protein (StAR) mRNA in the psp CL was also characterized to assess its use as a molecular marker for CL development and regression. It was highly expressed in the forming and functional CL and downregulated at a late stage of CL regression. The functional role of plg and MMPs for CL formation and function was investigated in plg deficient mice treated with the MMP inhibitor galardin (GM6001). Both psp mice and PMSG/hCG +PRL-induced CL formation were used. Several molecular markers for CL development and regression were used to evaluate the health status of the CL. Our data showed that healthy and vascularized CL formed even in plg deficient mice treated with the inhibitor. However, serum progesterone levels were significantly reduced in these mice, an effect that was mainly attributable to the plg deficiency. In conclusion, neither plg nor MMPs, alone or in combination, seem to be essential for the development of a functional CL.

This thesis focuses on applying microfluidic techniques to analyze two carbon management methods; underground carbon sequestration and enhanced oil recovery. The small scale nature of microfluidic methods enables direct visualization of relevant pore-scale phenomena, enabling elucidation of parameters such as diffusion coefficients and critical compositions. In this work, a microfluidic platform was developed to control a two-phase carbon dioxide (CO2)-water interface for diffusive quantification with fluorescent techniques. It was found that the diffusion coefficient of CO2 in pure water was constant (1.86 [± 0.26] x10-9 m2/s) over a range of pressures. The effects of salinity on diffusivity were also measured in solutions, it was found that the diffusion coefficient varied up to 3 times. A microfluidic technique able to determine the critical composition of a model ternary mixture was also successfully implemented. Results indicate potential application of this approach to minimum miscibility pressure measurements used in enhanced oil recovery.

Angiogenesis, the growth of new blood vessels, is a complex process involving several cell types and molecular signals. Excessive vascular growth is a problem in tumors, and insufficient vascularization hampers the function of transplanted insulin-producing pancreatic islets. Understanding the mechanisms behind blood vessel growth generates increased means to control angiogenesis. In this thesis a model of pancreatic islet transplantation to muscle has been used to study the involvement of leukocytes in the development of new vasculature. Transplantation of isolated islets of Langerhans into mouse muscle promoted revascularization of the grafts to a level comparable to native islets in the pancreas. The complete and functional vascular restoration resulted in improved blood glucose control compared to the clinical standard implantation site, the liver. This proved muscle as a transplantation site to be a clinically relevant option for the treatment of type 1 diabetes. The rapid islet revascularization process was found to be dependent on a distinct subset of neutrophils characterized by high expression of the chemokine receptor CXCR4 and the enzyme matrix metalloproteinase 9 (MMP-9). These cells were recruited to recently transplanted and hypoxic grafts by islet-secreted vascular endothelial growth factor A (VEGF-A). Leukocyte migration and interactions in the engraftment area were monitored using a high-speed confocal microscope followed by software tracking. New software was developed to visualize migration statistics. This tool revealed areas around the islet graft where neutrophil gathering coincided with sites of angiogenesis. Macrophages in the engraftment area positioned themselves close to the newly formed vasculature and were shown to have a stabilizing effect on the vessels. When macrophages were removed, no pericytes were recruited to the forming vasculature. The perivascular macrophages also began to express a pericyte marker when in the graft, suggesting a close relationship between these cell types or macrophage plasticity. In conclusion, this thesis presents muscle as a proangiogenic transplantation site for pancreatic islets for the treatment of type 1 diabetes, where the revascularization of the grafts was dependent on the recruitment and actions of specialized immune cells.