No description available.

No description available.

No description available.

<p>Biotherapeutic compounds such as monoclonal antibodies help millions of people worldwide.  Currently, one of the most popular ways to deliver these compounds is via subcutaneous (SC)  injection. While it is understood that SC drug delivery does change with respect to injection  location, it is not understood why, as how the composition of SC changes as a function of location  is unknown. In this study, liquid chromatography mass spectrometry was used to understand and  describe how the SC tissue space changes on a molecular level. SC tissue from three different  locations, belly, breast, and behind the ear, of Yucatan minipigs was harvested and analyzed to  understand if and how SC tissue changes when anatomical location changes. It was determined  that there were distinct differences between the proteins identified in the three anatomical  locations. These differences included differences in relative cell populations, indicating that  different anatomical locations of SC tissue have different functions. Additionally, an ex vivo human SC tissue model was used to identify a core human proteome, as well as determine  compositional differences between female and male SC tissues. This model was also compared to  the Yucatan minipig model to determine compositional similarities between all groups. Finally,  proteomics were also used to ascertain whether the mass of SC tissue used affected the proteomic  results of the sample. These results indicated that human SC identifies the same number of proteins  down to samples of 10mg. This information can be used to design a proteomic experiment that  uses core needle biopsies to determine what gauge needle should be used in a wide scale clinical  study characterizing the human SC proteome. </p>

<p> Musculoskeletal injuries are one of the leading causes of human disability. Tissue engineers aim to restore damaged musculoskeletal tissues by creating scaffolds that promote cellular adhesion, proliferation, and eventual differentiation into functional tissue. It is known that the extracellular matrix (ECM) regulates cellular behavior and is often used as a basis for biological scaffolds; however, current scaffolds often mimic the ECM of adult, homeostatic tissue and frequently lead to poor tissue restoration. What is rarely taken into consideration is that the ECM undergoes extensive remodeling during development to facilitate growth.</p> <p>In the musculoskeletal system, myogenic progenitors (<em>Pax3</em>+) and connective tissue cells (<em>Prx1</em>+) proliferate and differentiate into muscle, tendon, cartilage, and conjoining interfaces (<em>e.g.</em> myotendinous junction), while depositing and remodeling the ECM. As tissues mature, cells continue to refine ECM networks to withstand the functional demands to facilitate movement. The ECM composition and architecture of adult musculoskeletal tissues have been studied individually and are thought to be distinct; however, there has yet to be a comprehensive comparative analysis of the ECM in adult muscle, tendon, and the myotendinous junction (MTJ) in a single study. Additionally, how the matrisome of adult musculoskeletal system compares to the ECM dynamics during forelimb development, remain largely unknown due to lack of techniques to analyze embryonic matrisome composition and synthesis. </p> <p>To address these research gaps, we (1) used quantitative proteomics to map the matrisome composition in the mature murine MTJ, relative to the tendon and muscle; (2) adapted tissue fractionation and biorthogonal non-canonical amino acid tagging techniques to embryonic tissues as a method to quantify the global and nascent embryonic matrisome; and (3) subsequently used these techniques to establish a baseline of ECM dynamics during forelimb morphogenesis (embryonic day, E11.5-E14.5) and growth (postnatal day, P3 and P35). We hypothesized that proteomic evaluation of ECM composition and synthesis in developing and adolescent limbs would resolve differences between embryonic and growing tissues. Indeed, we saw significant differences in global and nascent matrisome composition between embryonic and adolescent forelimbs. Notably, the relative abundance and ratios of collagens associated with type I fibrillogenesis (I, III, and V) were significantly different as a function of development embryogenesis and across the adult muscle, MTJ, and tendon.</p> <p>Type I collagen fibrils are critical for tissue architecture and function. Using genetic mouse models, the regulatory roles of COL5A1 in the initiation of type I collagen fibrillogenesis, and organization of subsequent fibrils, have been well characterized in tendons and ligaments; however, is it unknown which cell types contribute COL5A1 to the ECM in the forelimb. To identify the functional contribution of COL5A1 by myogenic or connective tissue cell populations, we generated conditional (cre-flox) knock-out mouse models to inactivate <em>Col5a1</em> using <em>Pax3</em>- or <em>Prx1</em>-drivers, respectively. Haploinsufficiency of <em>COL5A1</em> in humans is associated classical Ehlers-Danlos syndrome, characterized by skin fragility and join instability; similar, albeit more severe, phenotypes were present in <em>Prx1Cre/+;Col5a1fl/fl</em> mutants, but not in <em>Pax3Cre/+;Col5a1fl/fl</em> mutants or controls. Interestingly, THBS4+ and COL22A1+ networks at the MTJ were morphologically affected in <em>Prx1Cre/+;Col5a1fl/fl</em> limbs. Additional work needs to be conducted to characterize the systematic phenotypes observed in <em>Prx1Cre/+;Col5a1fl/fl</em> limbs.</p> <p>Together, our results indicate that there are distinct, complex ECM dynamics, originating from distinct cell-types, that drive musculoskeletal morphogenesis in the forelimb. Further, the tools developed here will serve as a foundation for quantitative proteomic analyses of the matrisome composition in embryonic tissues. Collectively, this work provides a baseline of ECM protein dynamics during musculoskeletal morphogenesis, a helpful guide for tissue engineers in designing scaffolds to promote restoration of damaged tissues, with enhanced integration into the host tissue.</p>

Breast cancer therapy resistance and disease recurrence are driven by an infrequent population of stem-like tumor cells, termed breast cancer stem cells or tumor-initiating cells (BTIC). Whereas drugs that target BTIC could be combined with conventional therapies to provide durable remissions, identifying such agents has been difficult. To achieve the latter, our lab screened more than 35,000 compounds for their capacity to reduce the activity of BTIC-enriched mouse mammary tumorspheres, wherein we identified numerous antagonists of multiple serotonin receptors (HTRs). The serotonergic antagonist that prevented sphere formation with the highest potency is a highly selective antagonist of HTR5A, SB-699551. We subsequently demonstrated that this agent affects BTIC activity in breast tumor cell lines representative of all clinical and molecular subtypes of breast cancer. Whereas the primary target of SB-699551 is known, the downstream signaling pathways responsible for its anti-BTIC effect remains enigmatic. The goal of this thesis work was to elucidate the signaling pathways downstream of HTR5A in human breast tumor cell lines. We used a phospho-proteomic approach to establish that treatment of human SB-699551 affects the phosphorylation of proteins involved in the Gi-coupled and the PI3K/AKT/mTOR signaling axes. Moreover, we demonstrated that selective antagonists of PI3K, AKT, and mTOR phenocopied the effect of SB-699551 in tumorsphere forming assays. Taken together, our data suggests that SB-699551 elicits its effect through the PI3K/AKT/mTOR signaling pathways downstream of HTR5A. / Thesis / Master of Health Sciences (MSc) / Accumulating data suggests that the progression of breast cancer is driven by a rare population of breast tumor-initiating cells (BTIC). BTIC lie dormant during conventional therapy and initiate recurrence after such therapies are withdrawn. Hence, there is an urgent need to develop drugs that target BTIC that can be combined with the current standard of care to improve the durability of remission. With the latter objective in mind, our lab previously determined that antagonists of serotonin signaling target BTIC. One of the agents that we identified in our screen inhibits the activity of serotonin receptor 5A (HTR5A). The exact signaling mechanism whereby inhibition of HTR5A leads to a loss in BTIC activity was enigmatic. Hence, this thesis aims to elucidate the signaling pathways downstream of HTR5A in breast cancer. Knowledge of the latter will help identify a plausible mechanism in addition to identifying biomarkers of therapy efficacy.

The canonical Wnt/β-catenin signaling pathway is essential for the proper regulation of cell-fate decisions throughout embryogenesis and in adult issues. Activation of the Wnt signaling pathway allows for nuclear localization of the cell adhesion protein β-catenin, which then interacts primarily with members of the T-Cell Factor/Lymphoid Enhancer Factor (TCF/LEF) transcription factor family to modulate gene activity. The TCF/LEF family includes TCF7, TCF7L1, TCF7L2, and LEF1. While all four family members share a common DNA binding consensus sequence, their expression throughout embryogenesis and adult stem cell populations is unique, with their misexpression commonly occurring in Wnt related cancers and correlating strongly with metastasis and poor patient outcomes. TCF/LEF exchange at target gene loci is a key feature of mediating context-specific cellular responses to Wnt signaling and can be observed to occur in a variety of populations throughout development and in adult stem cell populations. To model TCF/LEF exchange in vitro we have optimized a micropatterning fabrication and culture protocol capable of identifying and isolating discrete LEF1-only and TCF7L1-only populations during gastrulation-like processes. To characterize how complements of TCF/LEFs change during cellular divisions we have developed a novel mitotic chromatin proteomic technique. This method identifies LEF1 as the only TCF/LEF to remain associated with mitotic chromatin in Wnt-activated conditions in mouse embryonic stem cells that are transitioning out of pluripotency as a consequence of removing leukemia inhibitory factor from their culture medium. Additionally, gene targeting techniques were used to label endogenous LEF1 and TCF7L1 with different fluorescent proteins in a single mouse embryonic stem cell line, allowing us to use TCF/LEF protein expression as a reporter of Wnt/β-catenin pathway status, which we found to be capable of identifying a unique set of compounds that are undetected by traditional Wnt activity (TOP-Flash) reporter screens. By using gene editing technology, and novel applications of proteomic and cell culture techniques, we have been able to investigate the mechanisms driving TCF/LEF expression and exchange in mouse embryonic stem cells to identify potentially clinically relevant therapeutic targets for their potential use in addressing TCF/LEF dysregulation in cancer. We have identified a novel mechanism through which TCF/LEFs maintain cell fate over cellular division; presented a novel live-cell drug screening platform capable of identifying compounds missed by existing platforms; and presented an optimized cell culture technique for the isolation of TCF/LEF exchange events. Taken together, the work in this thesis provides new insights into the mechanisms through which TCF/LEFs regulate their gene targets during cell fate transitions and throughout mitosis. / Thesis / Doctor of Science (PhD) / Throughout development and adult life cells are in constant communication, using a variety of cell signaling pathways to maintain adult stem cell populations and to pattern tissues throughout the body. Communication between cells often requires one cell to release a protein molecule (called a ligand) that is recognized by a receptor molecule on the surface of another cell. These cell surface receptors, when bound by the signaling ligand become activated and often set of a cascade of internal cellular events that ultimately result in changes in gene transcription in the nucleus. These transcriptional changes are toggled by proteins known as sequence-specific transcription factors that are able to selectively regulate expression of target genes. The net effect of combinations of extracellular ligands binding cell surface receptors determines the selective recruitment of specific transcription factors that activate a cell’s transcriptional program, in turn defining its fate and function. A very important developmental signaling pathway is the Wnt signaling pathway, which employs a family of secreted Wnt molecules as ligands. The Wnt pathway is critical at all stages of organismal development and plays an essential role in tissue maintenance in mature animals. However, due to its critical role in stem cell maintenance, when mutations occur in Wnt signaling components it can have dire consequences. Wnt signaling has been found to be disrupted in more than 70-80% of all cancers. One major feature among these Wnt-related cancers is the inappropriate expression and mobilization of Wnt transcription factors. While the expression and activity of Wnt transcription factors – known as T-Cell Factor/Lymphoid Enhancer Factors (TCF/LEFs) – changes throughout development and stem cell maintenance, their inappropriate expression is frequently associated with metastasis and poor patient outcomes. We have used mouse embryonic stem cells (mESCs) as a model system with which to study the mechanisms employed by TCF/LEFs to regulate their target genes. Through a number of approaches, which include adding fluorescent tags to TCF/LEF factors to track their intercellular locations and expression levels or enzymatic tags to identify proteins that interact with individual TCF/LEFs during a snapshot of cell activity, we have gained new knowledge about how these critical transcription factors regulate Wnt-regulated transcriptional programs. We also describe a method for generating micropatterned growth surfaces for mESCs that forces clusters of cells to grow within small circular shapes with a diameter of 1 mm or less. We show that mESCs confined to circular micropatterns differentiate in a highly reproducible manner that allows us to study the cell populations undergoing differentiation with a focus on cell fate determination mechanisms.

No description available.

Oxidative stress, described as the state of disturbed intracellular redox balance, has been associated with several human conditions including ageing, apoptosis, cancer, autoimmune and neuro-degenerative diseases. Stress studies have shown that reactive oxygen species (ROS) and reactive nitrogen species (RNS) along with its intermediates can attack essential cell targets such as: DNA, proteins, lipids and carbohydrates, leaving behind dysfunctional biologic molecules. In effect, a cell's primary response is to involve several defense mechanisms that are under a complex and intricate regulatory control to repair any damages that may have occurred. Although several stress studies have been conducted in the past that have approached this biologically complex process step by step, application of a Systems Biology towards a comprehensive understanding is still emerging. The current objective of this project is to identify proteins that change in response to cumene hydroperxoide (CHP) treatment and in parallel make an attempt to uncover events and processes that are a part of CHP-induced oxidative stress response. From a systems biology viewpoint, the Yeast Oxidative Stress project will monitor response at three different levels: transcriptomics, proteomics and metabolomics, with dynamic changes being measured from 3 to 120 min after CHP addition. Data collected from the different levels will be integrated to accomplish a holistic viewpoint of stress response in the given system and to develop mathematical tools for modeling biochemical networks. Saccharomyces cerevisiae was chosen as a model, based on its availability of a completely mapped genome sequence with a collection of null mutants that was relevant to our fundamental research of stress response mechanism. Yeast, a simple unicellular eukaryote has been extensively used for applied studies and has proven to be indispensable for stress research. Information derived from this project can reveal response mechanisms used by higher eukaryotes, especially if via analogous signaling cascades that are comparable between organisms. Current research investigates an optimal workflow for generating 2D gel based protein expression data and identifying proteins that are induced by cumene hydroperoxide treatment. A non-targeted protein profiling followed by a 2-way ANOVA analysis provided a list of proteins that differ significantly between treatments. Protein identification provided relevant information on which proteins are affected by CHP induced stress response, including posttranslational modifications of peroxiredoxins. Redox active protein, Ahp1, was regulated post-translationally with sulfonic acid modification observed for its active Cys(62) residue. / Ph. D.

Legionella pneumophila is a pathogen that can proliferate in premise (i.e., building) plumbing and, when aerosolized during water use, infect the lungs of exposed individuals and cause a deadly form of pneumonia known as Legionnaires' disease. Given that it is one of the primary sources of tap-water associated disease throughout much of the world, this organism has been the subject of intense research, ranging from aiming to understand key aspects of its physiology that allow it to proliferate in premise plumbing, to the specific virulence factors that make it so infectious to humans. The work presented here starts with a comprehensive review of published studies related to the L. pneumophila proteome, i.e., the set of expressed proteins associated with a given strain under a given set of environmental conditions, showing how the field has progressed in parallel to improvements in mass spectrometry technologies and how proteomics can be used as a tool to understand this unique and important organism. Copper is a natural antimicrobial that can be present in drinking water due to passive release from copper pipes or intentionally dosed (e.g., copper-silver ionization systems) for microbial control. However, some L. pneumophila strains have recently been found to exhibit copper resistance, an adaptive process that is not fully understood at the physiological level. Chapter Two describes the copper survivability of three outbreak-associated strains of L. pneumophila and examines the copper-induced proteome of QC1, a strain found to display high resistance to copper. Pairwise comparisons of the proteomes of copper-resistant and copper sensitive strains indicated that L. pneumophila QC1 adapts to copper exposure via the induction of redox and metal homeostasis proteins, while concomitantly inducing motility and pathogenesis related proteins, suggestive that copper induces a search for a host protozoan strain for protection. In 2014 and 2015, Flint, Michigan experienced the largest per capita community-wide Legionnaires' Disease outbreak in US history. The outbreak was associated with a change in the source of the municipal drinking water supply from Detroit water, which was sourced from the Great Lakes and subject to appropriate corrosion control, to the Flint River, which was not appropriately controlled for corrosivity. The underlying drivers of this outbreak have been debated and include: elevated iron in the water serving as a nutrient for L. pneumophila, diminished chlorine in the water due to reactions with iron, reduced copper in the water due to shifts in pH influencing release from copper pipes, and shifts in potentially key components of the microbial community. In Chapter Three of this dissertation, we employ controlled microcosm studies to establish a fundamental understanding of interactive effects of pipe material and water of varying iron bioavailability (ferric chloride, ferrous chloride and ferric pyrophosphate) on the microbial community and its relationship with L. pneumophila numbers. The combination of copper pipes and Flint River water decreased the diversity of the microbial community to a larger degree than copper pipes with Detroit water, implying greater copper bioavailability in the former condition. Several Order were found to be significantly associated with high or low numbers of culturable L. pneumophila recovered from the microcosms. Most notably, the Order Pseudomonadales was significantly associated to the reactors with low culturable L. pneumophila. This order contains Pseudomonas species known to inhibit the growth of L. pneumophila. The findings reported in this dissertation can be used to develop more informed management practices for drinking water systems to reduce the risk of Legionnaires' Disease outbreaks associated with premise plumbing. Specifically, 1) copper might be inducing a more pathogenic form of copper resistant L. pneumophila, 2) the use of corrosive control in municipal water systems goes beyond the influence on lead and copper pipes, but also on the microbial community, which in part influences L. pneumophila, and 3) there are organisms, such as Pseudomonadales species, associated with environments with low culturable L. pneumophila which might be introduced to the drinking water systems as probiotics. / Doctor of Philosophy / Legionella pneumophila is a microbe found in drinking water plumbing systems. This organism causes Legionnaires' Disease, a severe form of pneumonia that particularly affects immunocompromised individuals. Due to its health and economic impact, there are worldwide efforts to understand the biology of this organism, from the conditions that allows it to grow in the drinking water plumbing, to the specific components that allows it to infect humans. In this dissertation, we first review the published studies related to the L. pneumophila proteome, a powerful tool used to functionally describe biological organisms. This first chapter showed how proteomics can be used to understand this unique and important organism. In the next chapter we studied how copper metals may influence the proteome of L. pneumophila. Copper pipes have been extensively used to control the growth of microorganisms in drinking water systems, however some studies have reported that copper may promote the growth of L. pneumophila. In this chapter, we showed that a copper resistant strain of L. pneumophila adapts to copper exposure by inducing motility and pathogenesis related proteins, suggesting that it might be more infectious. In the last chapter of this dissertation, we investigated the combined effect of pipe material and water chemistry, on the microbial community and its relationship with L. pneumophila. The combination of copper pipes and a more corrosive water decreased the diversity to a larger degree, in comparison to the other evaluated conditions. Several organisms were also identified to be significantly associated with the high or low culturable L. pneumophila. This is of particular interest because they might be used as potential probiotics to control the growth of L. pneumophila. The findings reported in this dissertation can help to better understand the significance of water chemistry and pipe material, particularly copper pipes, for the purpose of reducing risk of Legionnaires' Disease outbreaks associated with drinking water systems.