Podocytes are highly differentiated glomerular epithelial cells that play an essential role in the establishment of the glomerular filtration barrier, a structural apparatus that selectively restricts the filtration of different macromolecules in the blood stream on the basis of their sizes, shape and charge. Podocyte dysfunction, one of the major causes of proteinuria, is of pathogenetic and prognostic significance in human glomerular disease. My study is focused on the investigation of novel pathways leading to podocyte dysfunction and proteinuria. In the first part, immunoblotting and quantitative reverse transcriptase PCR (RT-PCR) were used to demonstrate that LIM and senescent cell antigen-like domains 1 (PINCH1) is induced and undergoes nuclear translocation in podocytes after transforming growth factor, beta 1 (TGF-beta1) treatment. Bioinformatics analysis revealed the putative nuclear export signal/nuclear localization signal (NES/NLS) at the PINCH1 C-terminus which is required for its nuclear translocation. Immunoprecipitation and GST pull-down assay identified the interaction between PINCH1 and Wilms tumor 1 (WT1) which led to suppression of the WT1-mediated podocalyxin gene expression. In vivo, PINCH1 also underwent nuclear translocation and interacted with WT1 after TGF-beta1 stimulation. Our data identifies nuclear transcription factor WT1 as a novel binding partner for PINCH1, and provides novel insight into the mechanism of podocyte dysfunction under pathological conditions. In the second part, RT-PCR results revealed that treatment with TGF-beta1 induced gene expression of several wingless-type MMTV integration site family members (Wnts), predominantly Wnt1, and activated beta-catenin in mouse podocytes. Wnt antagonist Dickkopf-1 (DKK1) blocked TGF-beta1-induced beta-catenin activation and preserved nephrin expression. In vivo, ectopic expression of constitutively active TGF-beta1 induced Wnt1 expression, activated glomerular beta-catenin, upregulated its downstream target genes, and led to podocyte injury and proteinuria. Consistently, concomitant expression of DKK1 gene abolished beta-catenin activation in mouse glomeruli, inhibited TGF-beta1-triggered Wnt/beta-catenin target genes, and ameliorated proteinuria. These results establish a role for Wnt/beta-catenin signaling in the pathogenesis of podocyte injury and also suggest that this signaling pathway could be exploited as a therapeutic target for the treatment of proteinuric kidney diseases.

Metabolic networks provide a vital framework for understanding the cellular metabolism in both physiological and pathophysiological states, which will ultimately facilitate network analysis-based drug discovery. In this thesis, we aim to employ a metabolic network analysis approach to study cancer metabolism (a pathophysiological state) and the metabolism of the bacterial pathogen, S. aureus (a physiological state), in order to understand, predict, and ultimately target cell metabolism for drug discovery. Cancer cells have distinct metabolism that highly depend on glycolysis instead of mitochondrial oxidative phosphorylation alone, even in the presence of oxygen, also called aerobic glycolysis or the Warburg effect, which may offer novel therapeutic opportunities. However, the origin of the Warburg effect is only partially understood. To understand the origin of cancer metabolism, our theoretical collaborator, Prof. Alexei Vazquez, developed a reduced flux balance model of human cell metabolism incorporating the macromolecular crowding (MC) constraint and the maximum glucose uptake constraint. The simulations successfully captured the main characteristics of cancer metabolism (aerobic glycolysis), indicating that MC constraint may be a potential origin of the Warburg effect. Notably, when we experimentally tested the model with mammalian cells from low to high growth rates as a proxy of MC alteration, we find that, consistent with the model, faster growing cells indeed have increased aerobic glycolysis. Moreover, the metabolic network analysis approach has also been shown to be capable of predicting the drug targets against pathogen metabolism when completely reconstructed metabolic networks are available. We deduced common antibiotic targets in Escherichia coli and Staphylococcus aureus by identifying shared tissue-specific or uniformly essential metabolic reactions in their metabolic networks. We then predicted through virtual screening dozens of potential inhibitors for several enzymes of these reactions and demonstrated experimentally that a subset of these inhibited both enzyme activities in vitro and bacterial cell viability. Our results indicate that the metabolic network analysis approach is able to facilitate the understanding of cellular metabolism by identifying potential constraints and predicting as well as ultimately targeting the metabolism of the organisms whose complete metabolic networks are available through the seamless integration of virtual screening with experimental validation.

Prostate cancer is the second leading cause of cancer death in men. While prostate specific antigen (PSA) is currently the best biomarker available, its use has many limitations. This study investigates the biosynthesis, secretion and activation of PSA. PSA is secreted as a pro enzyme containing a seven amino acid activation peptide (APLILSR). Because APLILSR is removed extracellularly in vivo, the hypothesis was tested that it may be detected in the blood or urine. Our data indicates that APLILSR is filtered from the bloodstream by the kidney, and is detectable in the urine of patients with prostate cancer, but not controls. Idiopathic pulmonary fibrosis (IPF) is a fatal interstitial lung disease of unknown etiology. Matrix metalloproteinases (MMPs) are a family of proteinases that regulate extracellular matrix turnover and are believed to contribute to IPF. For this reason, the hypothesis that levels of MMP activation peptides will increase in patients with IPF was tested. To test these hypotheses, urine from mice were collected and an ELISA was used to quantify MMP activation peptides. These experiments show that the activation peptides of MMP-2, MMP-7, MMP-8 and MMP-9 are increased in mice with pulmonary fibrosis compared to control mice. The data also showed that that the activation peptides of MMP-2, MMP-7, and MMP-9 are increased in the urine of human patients with IPF compared to healthy controls. These data suggest that urine detection of MMP activation peptides is feasible and correlates with disease. Because urinary detection of the activation peptides of proteinases are indicative of proteinase activation in vivo, the new hypothesis that the accurate measurement of proteinase activation peptides will be relevant clinically arises, and that such measurements may aid in the diagnosis of disease and serve as a marker for following disease progression.

Immunophilins (IP) are receptors for immunosuppressive drugs like cyclosporin A, FK506, rapamycin and their analogs, which are collectively referred to as immunophilin ligands (IPL). The receptors for FK506 belong to the family of FK506- binding proteins (FKBP). Previous studies showed that FK506 augments neuronal growth in vitro. In animal models, the drug promotes morphologic and functional recovery following neuronal lesioning. Here we show for the first time that FK506 has neurotrophic effects in human brain primary cultures. Our data support a possible use of FK506 and its analogues in the treatment of neurodegenerative disorders and as adjuvants in neural tissue transplants. The effect of the drug in vivo, in patients, will ultimately depend on the presence and distribution of IP receptors in the normal and degenerating human brain. FKBP12, the archetypal member of the FKBP family, plays a role in protein folding, protein complexes assembly and intracellular calcium release. Crush injury of facial or sciatic nerves in rat leads to markedly increased FKBP12 levels in the respective nerve nuclei in parallel with nerve regeneration. The presence of FKBP IP has never been demonstrated in the human brain. Using light and immunofluorescent microscopy, laser confocal microscopy and western blotting, we studied FKBP12 expression in a set of archival brain material from Parkinsons disease, Alzheimers disease, dementia with Lewy bodies, encephalitic and non- encephalitic HIV- positive patients and age matched controls. We show that FKBP12 is present in the human brain, predominantly in neurons. Its levels and distribution are altered in the mid- frontal cortex, deep gray matter and midbrain of patients with neurodegenerative diseases. Moreover, it colocalizes with markers of pathology (Lewy bodies, neurofibrillary tangles and neuritic plaques) in areas of neurodegeneration. Disease- specific and region- specific changes are evident. Alterations in basal ganglia FKBP12 levels are also observed in MPTP- treated primates in association with dopaminergic loss (evidenced using PET functional imaging). We propose that the altered expression and distribution of FKBP12 is linked to abnormal protein folding and axonal transport. It may also reflect a compensatory regenerative response that renders immunophilins promising diagnostic and therapeutic targets.

The antioxidant enzyme extracellular superoxide dismutase (EC-SOD) is abundant in the lung and limits inflammation and tissue injury in response to many pulmonary insults. Previous studies reported a loss of full-length EC-SOD from the lung parenchyma with accumulation of proteolyzed EC-SOD in the airspace after interstitial lung injury. However, following airspace only inflammation (pneumonia), EC-SOD accumulates in the airspace without a loss from the interstitium, suggesting this antioxidant may be released from an extrapulmonary source. Because leukocytes are known to express EC-SOD and are prevalent in the bronchoalveolar lavage fluid after injury, it was hypothesized that these cells may transport and release EC-SOD into airspaces. To investigate this, bone marrow chimeras were generated using wild-type and EC-SOD knockout (KO) mice. Following intratracheal treatment with asbestos, reconstituted mice without pulmonary EC-SOD expression, but with EC-SOD in infiltrating and resident leukocytes did not have detectable levels of EC-SOD in the airspaces. In addition, leukocytederived EC-SOD did not significantly lessen inflammation or early stage fibrosis. Although these results indicate that leukocyte-derived EC-SOD is not influential in asbestos-induced interstitial lung injury, EC-SOD in these cells may play a role in attenuating pneumonias and other inflammatory diseases. To test this hypothesis, wild-type and EC-SOD KO mice were given Escherichia coli pneumonia. Notably, even though EC-SOD KO mice had greater pulmonary inflammation than wild-type mice, there was less bacterial clearance from their lungs following infection. While EC-SOD expression has been previously reported in macrophages and neutrophils, its function and subcellular localization in these inflammatory cells is unclear. In this study, EC-SOD was found to be in membrane bound vesicles of phagocytes. This finding led to the hypothesis that inflammatory cell EC-SOD may play a role in antibacterial defense. To investigate this, phagocytes from wild-type and EC-SOD KO mice were evaluated. While macrophages lacking EC-SOD produced more oxidants than EC-SOD expressing cells after stimulation, they had significantly impaired phagocytosis and bacterial killing ability. Overall, these studies suggest that while EC-SOD inside leukocytes does not contribute to interstitial lung injuries, it plays a central role in mediating bacterial infections by facilitating bacterial clearance and limiting inflammation by promoting phagocytosis.

Metastasis is a major contributor to breast cancer mortality, as currently available therapies are unable to ensure progression or disease-free survival. Little is known about the molecular pathogenesis of metastasis, and the role of the surrounding microenvironment is only beginning to be understood. In vitro studies have repeatedly shown that epithelial to mesenchymal transition (EMT) and loss of E-cadherin expression are critical events in the initiation of metastasis and can be induced by the microenvironment. However, metastases are often well-differentiated and epithelial in phenotype, suggesting that EMT is reversible. The role of E-cadherin expression and mesenchymal to epithelial reverting transition (MErT) in metastatic colonization of the secondary site remains ill-defined. Evidence for E-cadherin re-expression and partial MErT was observed in metastases of breast and prostate cancer patients, and suggests that MErT is unstable and reversible. MDA-MB-231 breast cancer cells cultured with hepatocytes also resulted in E-cadherin re-expression and partial MErT, suggesting that such phenotypic plasticity can be induced by the microenvironment of the liver, a key site of breast cancer metastases. Re-expression of E-cadherin following hepatocyte coculture not only results in heterotypic ligation between cancer and liver parenchymal cells, but also activates Erk survival signaling and increases resistance to nutrient-deprivation and chemotherapy. Taken together, our results indicate that the distant organ microenvironment may induce E-cadherin re-expression and partial MErT to enhance the survival of metastatic cancer cells at the secondary organ.

BACKGROUND: Human gliomas account for the most common and malignant tumors in the central nervous system (CNS). Despite optimal treatments, survival of patients with high-grade glioblastoma multiforme (GBM) remains poor. Recent coordinated genomic analyses of a large cohort of clinical GBM specimens identified frequent co-alterations of genes in three core pathwaysthe P53, retinoblastoma (RB), and receptor tyrosine kinase (RTK) pathways that are crucial in gliomagenesis. Further multi-institutional efforts have sub-classified GBMs into four clinical relevant subtypes based on their signature genetic lesions. Among them, PDGFRA overexpression is concomitant with a loss of CDKN2A locus (encoding P16INK4A and P14ARF) in a large number of tumors within one subtype of GBMs. To better understand and design therapeutic strategies against gliomas driven by abnormal platelet-derived growth factor (PDGF) signaling, functional studies using human or mouse models are needed. MAJOR FINDINGS: In order to establish a model that allows us to assess contributions of different signaling pathways to PDGFRα-induced glioma formation, we generated Ink4a/Arf-deficient primary mouse astrocytes (referred to as mAst hereafter) and human glioma cells that overexpress PDGFRα and/or PDGF-A. We found that activation of PDGFRα confers tumorigenicity to Ink4a/Arf-deficient mAst and human glioma cells in the brain. Restoration of p16INK4a but not p19ARF by retroviral transduction suppresses PDGFRα-promoted glioma formation. Mechanistically, abrogation of signaling modules in PDGFRα that lost capacity to bind to SH-2-containing phosphotyrosine phosphatase SHP-2 or Phosphoinositol 3'-Kinase (PI3K) significantly diminished PDGFRα-promoted tumorigenesis. Furthermore, inhibition of SHP-2 by shRNAs or pharmacological inhibitors disrupted the interaction of PI3K with PDGFRα, suppressed downstream AKT/mTOR activation, and impaired tumorigenesis of Ink4a/Arf-null cells, whereas expression of an activated PI3K mutant rescued the effect of SHP-2 inhibition on tumorigenicity. In clinical glioblastoma specimens, PDGFRα and PDGF-A are co-expressed and such co-expression is linked with activation of SHP-2/AKT/mTOR-signaling. Our data thus suggest that in glioblastomas with Ink4a/Arf deficiency, overexpressed PDGFRα promotes tumorigenesis through the PI3K/AKT/mTOR-mediated pathway regulated by SHP-2 activity. SIGNIFICANCE: We expect these findings will improve our understanding of the formation of the gliomas with PDGFRA and INK4A/ARF aberrations. There were studies that predicted SHP-2/PTPN11 as one of the linker genes in clinical GBMs that interact with multiple commonly altered genes. Our results functionally validate this hypothesis and identify SHP-2 as a converge point of several signaling pathways such as PDGFR, EGFR, PI3K, and mTOR that are frequently deregulated in GBMs. It thus represents a promising target for treatments against this fatal disease.

Biologic scaffolds composed of extracellular matrix (ECM) have been used to promote site-specific, functional remodeling of tissue in both preclinical animal models and human clinical applications. Although the mechanisms of action of ECM scaffolds are not completely understood, proteolytic degradation of the ECM scaffold and subsequent progenitor cell recruitment are thought to be important mediators of the constructive remodeling process. Proteolytic degradation of the ECM scaffolds results in the generation and release of cryptic peptides with novel bioactive properties not associated with their parent molecules such as angiogenic, antimicrobial, mitogenic, and chemotactic properties. While previous studies have suggested that degradation products of ECM scaffolds are chemotactic for progenitor cells in vitro, the present thesis expands upon these findings in vivo. In a non-regenerating model of mid-second phalanx digit amputation, treatment with ECM degradation rpodcuts resulted in the accumulation of a heterogeneous population of cells with in vitro differentiation potential along osteogenic, adipogenic, and neuroectodermal lineages. Focusing specifically on the Sox2+ population of cells found at the site of injury, work in the present thesis showed that Sox2+ cells co-express bone marrow and periosteal stem cell markers CD90 and Sca1, but not dermal stem cell marker CD133 or circulating stem cell marker c-kit (CD117). Additionally, bone marrow chimeric studies utilizing wild type C57/BL6 and Sox2 eGFP/+ mice showed that the Sox2+ cells are not derived from the bone marrow, but more likely from a local tissue source such as the periosteum. Fractionation of the ECM degradation products resulted in the identification of a highly conserved cryptic peptide derived from the C-terminal telopeptide of the collagen type IIIα molecule with chemotactic activity for multiple progenitor cells in vitro, IAGVGGEKSGGF. Administration of the cryptic peptide in a model of digit amputation resulted in the accumulation of Sox2+, Sca1+, Lin- cells at the site of amputation. Peptide treatment also resulted in the formation of a bone nodule at the site that coincided with the spatial location of Sox2+ cells. In vitro, the peptide accelerated osteogenesis of mesenchymal stem cells and increased the expression of osteogenic and chondrogenic genes. The result of this body of work shows that degradation products of ECM scaffolds contain cryptic peptides with the ability to influence chemotaxis and differentiation of progenitor cells in vitro and in vivo. The ability to influence stem cell phenotype and fate may be useful in designing new therapies for regenerative medicine approaches to complex, composite tissue reconstruction. Additionally, the findings of the present thesis may serve as the basis for future studies investigating the importance of ECM degradation in the downstream constructive remodeling events at a site of ECM implantation in soft tissue models of injury.

Background: The five year overall survival rate for colorectal cancer (CRC) patients varies between 38.8% and 59.9%. Selecting patients who are likely to respond to therapy remains a clinical and pathological challenge, hence the need for predictive and prognostic biomarkers. The objectives of this study were: 1) to establish which genes were differentially expressed with respect to sensitivity to treatment, 2) to integrate the list of differentially expressed genes with copy number to systematically identify predictive biomarkers, and 3) to establish which genes are commonly gained in the panel of CRC cell lines. As proof of concept of the approach the copy number variations of the identified genes were assessed in a cohort of Dukes’ A and B cancers, in order to analyse the likelihood of these genes acting as useful biomarkers. Methods: Cell viability assays were carried out on a panel 15 CRC cell lines. IC50s were measured for 5-fluoruracil (5-FU), oxaliplatin (L-OHP), and BEZ-235, a PI3K/mTOR inhibitor. We carried out a systematic array-based survey of gene expression and copy number variation in CRC cell lines, and compared these to responses to different treatments. Cell lines were profiled using array comparative genomic hybridisation (aCGH; NimbleGen 135k), Illumina gene expression analysis, reverse phase protein arrays (RPPA), and targeted sequencing of KRAS hotspot mutations. The associations between the biological variables and drug sensitivity were assessed using correlation coefficients, chi-square analysis, and the Mann Whitney-U test. Tissue microarrays (TMA) were constructed for a cohort of CRC patients (n=118) and TRIB1 and MYC amplifications were measured using fluorescence in situ hybridisation (FISH). The protein expression for trib1 and 14 associated biomarkers were investigated using Automated Quantitative Analysis (AQUA) and analysed using the Pearson’s correlation coefficient. Results: Twenty-three regions were frequently gained, and fourteen regions were lost across the cell line panel. Gains were observed at 2p, 3q, 5p, 7p, 7q, 8q, 12p, 13q, 14q, and 17q, and losses at 2q, 3p, 5q, 8p, 9p, 9q, 14q, 18q, and 20p. Frequently gained regions contained EGFR, PIK3CA, MYC, SMO, TRIB1, FZD1, and BRCA2, while frequently lost regions contained FHIT and MACROD2. Gene enrichment analysis showed that differentially expressed genes with respect to treatment response were involved in Wnt signalling, EGF receptor signalling, apoptosis, cell cycle, and angiogenesis. Stepwise integration of copy number and gene expression data yielded 47 candidate genes that were significantly correlated (corrected p-value ≤0.05). Differentially expressed genes common to all three treatment responses included AEBP2, DDX56, MRPL32, MRPS17, MYC, NSMCE2, and TBRG4. TRIB1 (n=76) and MYC (n=81) were amplified (FISH score ≥1.8) in 14.5% and 7.4% of the CRC cohort, respectively. TRIB1 and MYC amplifications were significantly correlated (corrected p-value ≤ 0.0001). Trib1 protein expression in the patient cohort was significantly correlated (corrected p-value ≤ 0.01) with protein expression of pErk, Akt, and Caspase 3. Conclusions: The CRC in-vitro model was used effectively in this study for discovery of both predictive and prognostic biomarkers. A set of candidate predictive biomarkers for 5-FU, L-OHP, and BEZ235 have been described, worthy of further study. Amplification of the putative oncogene TRIB1 has been assessed for the first time in a cohort of CRC patients. Inhibition of TRIB1 may be a synthetic lethal approach when MYC amplifications are present, which requires further clinical and experimental validation.

No description available.