Familial history is the strongest risk factor for developing ovarian cancer (OC), and a significant contributor to breast cancer risk. Most hereditary breast cancers and OCs are associated with mutation of the tumor suppressor Breast and Ovarian Cancer Susceptibility Gene 1 (BRCA1). Studying risk-associated BRCA1 truncation mutations, such as the founder mutation 185delAG, may reveal signaling pathways important in OC etiology. Recent studies have shown novel BRCA1 mutant functions that may contribute to breast and OC initiation and progression independent of the loss of wtBRCA1. Previously, we have found that normal human ovarian surface epithelial (HOSE) cells expressing the 185delAG mutant, BRAT ( BRCA1 185delAG Amino Terminal truncated protein), exhibit enhanced chemosensitivity and up-regulation of the OC-associated serpin, maspin. In the current study, I identify an additional target of the BRAT mutation, matrix metalloprotease 1 (MMP1), a key player in invasion and metastasis. BRAT-expressing HOSE cells exhibit increased MMP1 messenger RNA (mRNA) by real time PCR and protein by Western blotting. Pro-MMP1 levels are also higher in conditioned media of BRAT-expressing cells and HOSE cell lines derived from BRAT mutation carriers. c-Jun is critical for BRAT-mediated MMP1 up-regulation, as siRNA knockdown diminishes MMP1 levels. Luciferase reporter constructs reveal that activator Protein 1 (AP1) sites throughout the distal end of the promoter contribute to BRAT-mediated MMP1 expression, and basal activity is mediated in part by an AP1 site at (-72). Reporters containing a single nucleotide polymorphism (SNP) associated with OC risk and progression yield increased activity that is further enhanced in BRAT cells. Interestingly, BRAT-mediated changes in chemosensitivity and gene regulation are not recapitulated in a normal breast epithelial or breast cancer cell model. This suggests tissue-specific mutant BRCA1 functions may contribute to breast and ovarian tissue specificity of BRCA1 mutation-associated cancer risk and also to differential breast and ovarian cancer risk and penetrance associated with specific mutations. Early molecular and cellular changes such as MMP1 up-regulation in the ovarian surface epithelium of BRCA1 mutation carriers may promote OC initiation and progression and represent a step forward on the continuum of cellular malignancy. Further investigation is warranted, as elucidating these early changes will aid in identification of potential screening and treatment strategies.

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Quantum Dot (QD) nanobeacons distinguish themselves from molecular beacons with the promise of non-linear activation, tunability, and multi-functionality. These unique features make them highly attractive for cancer detection imaging with opportunities for increased signal-to-background ratio and tunable sensitivity. In this thesis, a nanobeacon was designed to target matrix metalloproteinase-7 (MMP-7), known to be over-expressed by a wide array of tumours. The nanobeacon is normally dark until specifically activated by MMP-7. The overall design strategy links single QDs to multiple energy acceptors by GPLGLARK peptides that can be cleaved specifically by MMP-7. However, design details such as the choice of energy acceptor and conjugation method was found to drastically alter the function of the nanobeacon. Studies of nanobeacons synthesized with Black Hole Quencher-1 or Rhodamine Red by either covalent conjugation or electrostatic self-assembly revealed that peptide conformation and bonding flexibility are both important considerations in nanobeacon design due to QD sterics.

As major effector cells of the innate immune response, macrophages must adeptly migrate from blood to infected tissues. Endothelial transmigration is accomplished by matrix metalloproteinase (MMP)-induced degradation of basement membrane and extracellular matrix components. The classical activation of macrophages with LPS and IFN-γ causes enhanced microtubule stabilization and secretion of MMPs. Macrophages upregulate MMP-9 expression and secretion upon immunological challenge, and require its activity for migration during inflammatory response. However, the dynamics of MMP-9 production and intracellular distribution, as well as the mechanisms responsible for its trafficking, are unknown. Using immunofluorescent imaging, we localized intracellular MMP-9 to small Golgi-derived cytoplasmic vesicles that contain calreticulin and PDI, in activated macrophages. Vesicular organelles of MMP-9 aligned along stable subsets of microtubules and colocalized with the anterograde molecular motor protein, kinesin. We demonstrated a functional contribution of stable MTs in the enhanced trafficking of MMP-9 extracellularly, and showed that heterogeneity exists in macrophage cell populations with respect to MMP-9 production.

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.

Quantum Dot (QD) nanobeacons distinguish themselves from molecular beacons with the promise of non-linear activation, tunability, and multi-functionality. These unique features make them highly attractive for cancer detection imaging with opportunities for increased signal-to-background ratio and tunable sensitivity. In this thesis, a nanobeacon was designed to target matrix metalloproteinase-7 (MMP-7), known to be over-expressed by a wide array of tumours. The nanobeacon is normally dark until specifically activated by MMP-7. The overall design strategy links single QDs to multiple energy acceptors by GPLGLARK peptides that can be cleaved specifically by MMP-7. However, design details such as the choice of energy acceptor and conjugation method was found to drastically alter the function of the nanobeacon. Studies of nanobeacons synthesized with Black Hole Quencher-1 or Rhodamine Red by either covalent conjugation or electrostatic self-assembly revealed that peptide conformation and bonding flexibility are both important considerations in nanobeacon design due to QD sterics.

As major effector cells of the innate immune response, macrophages must adeptly migrate from blood to infected tissues. Endothelial transmigration is accomplished by matrix metalloproteinase (MMP)-induced degradation of basement membrane and extracellular matrix components. The classical activation of macrophages with LPS and IFN-γ causes enhanced microtubule stabilization and secretion of MMPs. Macrophages upregulate MMP-9 expression and secretion upon immunological challenge, and require its activity for migration during inflammatory response. However, the dynamics of MMP-9 production and intracellular distribution, as well as the mechanisms responsible for its trafficking, are unknown. Using immunofluorescent imaging, we localized intracellular MMP-9 to small Golgi-derived cytoplasmic vesicles that contain calreticulin and PDI, in activated macrophages. Vesicular organelles of MMP-9 aligned along stable subsets of microtubules and colocalized with the anterograde molecular motor protein, kinesin. We demonstrated a functional contribution of stable MTs in the enhanced trafficking of MMP-9 extracellularly, and showed that heterogeneity exists in macrophage cell populations with respect to MMP-9 production.

No description available.