Staphylococcus aureus causes nosocomial- and community- acquired infections. This versatile pathogen expresses virulence factors (VF) that enhance establishment of infection and immune evasion. Our research focused on defining the roles of S. aureus VF on host immune responses during intracellular or extracellular infections. Accessory gene regulator (agr) controls VF expression and intracellular survival. Our goal was to determine mammary epithelial cells (MEC) responses to intracellular infection and subsequent polymorphonuclear leukocyte (PMN) responses. Intracellular S. aureus increased thrombomodulin expression by MEC and activated protein C (APC) production. APC inhibited PMN chemotaxis. Findings depicted an indirect role for VF on PMN responses, so next we determined signaling pathways and cytokine responses of PMN to S. aureus toxins. Live S. aureus infections increased activation of stress signaling pathways and highlighted a role for agr-regulated genes in MAPK p38 phosphorylation and α-hemolysin in ERK phosphorylation and IL-8 expression in PMN. Continuing our studies of VF, chemotaxis inhibitory protein of S. aureus (CHIPS) inhibits monocyte chemotaxis. We hypothesized that CHIPS inhibited C5a receptor (C5aR) signaling. Monocytes pretreated with CHIPS did not inhibit C5aR signaling. Nevertheless, signaling pathways can reduce PMN function in models such as glucocorticoid treatment. Immunosuppressive effects of glucocorticoids on PMN are restored with OmniGen-AF® supplementation. Glucocorticoid receptor and Toll-like receptor signaling potentially crosstalk to restore PMN function. OmniGen-AF® supplementation restored dexamethasone-induced immunosuppression in a MyD88-dependent manner. Overall, this research focused on characterizing immune responses to S. aureus infections and PMN signaling pathways and how it is key to understanding pathogenesis. / Ph. D.

The unfolded protein response (UPR) is a cellular mechanism whose primary functions are to sense perturbations in the protein-folding capacity of the endoplasmic reticulum and to take corrective steps to restore homeostasis. Although the UPR is conserved across all eukaryotic cells, it is considerably more complex in mammalian cells, due to the presence of three interconnected pathways triggered by separate sensor proteins, a translation attenuation mechanism, and a negative feedback loop. The mechanisms of these interacting biochemical pathways in the mammalian UPR allow for a better fine-tuning of the response than in the case of lower eukaryotes, such as yeasts. The present thesis develops a quantitative mathematical model for the dynamics of the UPR in mammalian cells, which incorporates all the proteins and interactions between them that are known to play a role in this response. This model can be used to provide quantitative information about the levels of its components throughout the response, and to analyze the ramifications of perturbations of the UPR. The model uses a system of ordinary nonlinear differential equations based on biochemical rate equations to describe the dynamics of the UPR as a network of interacting proteins and mRNAs. An early model is presented as a first attempt to investigate the UPR network and construct an inclusive wiring diagram, as well as suggesting a framework to model the differential equations. Then, a refined, quantitative model is designed based on experimental data collected on Mouse Embryonic Fibroblasts treated with Thapsigargin to induce stress and trigger the UPR. The model defines the differential equations and determines the unknown kinetic parameters by optimizing the fit of the system's solution to the experimental data. It includes the UPR's intrinsic feedback loops and allows for the integration of various forms of external stress signals. To the best of our knowledge, it is the first, data-validated, quantitative model in the literature for the UPR in mammalian cells. The last chapters of the thesis address, from a modeling point of view, two important questions for the UPR: (1) cell survival versus apoptosis; and (2) incompleteness of the biological wiring diagram. Recent experimental results show that the UPR is capable of producing qualitatively different results leading to cell survival or death depending on the nature, strength, and persistence of the inducing stress. This thesis proposes several approaches by which the equations can be modified to model the transition from adaptation to apoptosis as a dynamic switch, while taking into account the various hypotheses of cell death mechanisms. Finally, we use recently-developed computational algebra techniques to infer an optimal structure of the UPR network, based solely on the experimental data; the resulting wiring diagram provides insights on elements of the structure of the model that may have been overlooked during the classical (mechanistic) approach to our original data-based model.

Although numerous transcription factors (TFs) are expressed by oligodendrocytes (OGs), the role(s) of most of these TFs in oligodendrogenesis remains to be elucidated. One such TF is Hoxa2, which was recently shown to be expressed by O4-positive (+) pro-OGs. Hence, the main objectives of this thesis were to determine the expression profile and function(s) of Hoxa2 during OG development. Immunocytochemical analysis of primary mixed glial cultures demonstrated that Hoxa2 is expressed throughout oligodendrogenesis, diminishing only with the acquisition of a myelinating phenotype. Subsequently, immunohistochemical analysis suggested that Hoxa2 is expressed by migratory oligodendroglial cells in the embryonic spinal cord. However, double immunofluorescent analysis of Hoxa2 transgenic knockout mice showed that OG specification and early maturation proceed normally in the absence of Hoxa2 in the spinal cord. As Hoxa2 is one of 39 murine Hox genes, which exhibit extensive overlapping expression profiles in the spinal cord, we decided to examine the expression of an additional Hox TF, Hoxb4, during OG development. Immunocytochemical analysis of primary mixed glial cultures demonstrated that Hoxb4 is also expressed throughout OG development. Furthermore, comparison of the expression profiles of Hoxb4 and Olig2 suggested that Hoxb4 is expressed by oligodendroglial cells in the spinal cord. Hence, Hoxb4, as well as other Hox TFs could compensate for Hoxa2 in the spinal cord in its absence. As the anterior boundary of most Hox genes has been found to be in the hindbrain or spinal cord, we decided to look at the telencephalon which would be less likely to have compensatory mechanisms. Our results showed that similar to the spinal cord, Hoxa2 is expressed by oligodendroglial cells in the telencephalon. Subsequently, it was found that over-expressing Hoxa2 in CG4 cells, an oligodendroglial cell line derived from the perinatal rat cerebral cortex, impairs their differentiation. In an attempt to determine the mechanism by which it accomplishes this, we examined the expression of polysialylated neural cell adhesion molecule (PSA-NCAM), which has been implicated in this process. Contrary to our expectations, however, it was found that over-expressing Hoxa2 in CG4 cells results in significantly fewer PSA-NCAM+ cells. Hence, the results suggest that Hoxa2s effect on OG differentiation is independent of its effect on PSA-NCAM expression. The expression of Hox genes is enhanced by retinoic acid (RA), which, in turn, both inhibits, as well as promotes OG differentiation. Although the reason for these opposing roles is uncertain, examination of the experimental protocols utilized by different research groups reveals disparities in age, CNS region, as well as RA concentration. As a result, RAs effect on oligodendrogenesis could be stage- and/or concentration-dependent. In order to determine which of these factors could impact RAs effect on OG differentiation we treated CG4 cells with two different concentrations of RA at two distinct time points. The results showed that both factors (concentration and time/stage) can impact RAs effect on CG4 cell differentiation. In an attempt to determine the mechanism by which it accomplishes this, we examined the expression of PSA-NCAM. Contrary to our expectations, the results suggest that RAs effect on CG4 differentiation is independent of its effect on PSA-NCAM expression. The results of this thesis suggest that Hoxa2 and RA could play multiple roles in OG development. Although these roles appear to be similar, further research will be needed to determine whether Hoxa2 acts a downstream effector in the RA signaling pathway in oligodendroglial cells.

Cancer is an ongoing problem all over the world. To find a cure to this disease, both clinicians and scientists are looking for a reasonable treatment method. According to Hanahan and Weinberg, one of the hallmarks of cancer is evasion of programmed cell death, referred to as apoptosis. Apoptosis is an important cellular process, and is regulated by many different pathways. Proteins in these pathways contribute to either cell death or cell survival depending on the cell stresses. Much research in systems biology has been devoted to understanding these pathways at the molecular level. In this study a mathematical model is built to describe apoptosis, and the pathways involving the related proteins p53 and Akt. The primary purpose of the construction of the kinetic model is to verify that this network can exhibit bistability between cell survival and cell death. Sensitivity and bifurcation analysis are conducted to determine which parameters have the greatest effect on the system behavior.

Although numerous transcription factors (TFs) are expressed by oligodendrocytes (OGs), the role(s) of most of these TFs in oligodendrogenesis remains to be elucidated. One such TF is Hoxa2, which was recently shown to be expressed by O4-positive (+) pro-OGs. Hence, the main objectives of this thesis were to determine the expression profile and function(s) of Hoxa2 during OG development. Immunocytochemical analysis of primary mixed glial cultures demonstrated that Hoxa2 is expressed throughout oligodendrogenesis, diminishing only with the acquisition of a myelinating phenotype. Subsequently, immunohistochemical analysis suggested that Hoxa2 is expressed by migratory oligodendroglial cells in the embryonic spinal cord. However, double immunofluorescent analysis of Hoxa2 transgenic knockout mice showed that OG specification and early maturation proceed normally in the absence of Hoxa2 in the spinal cord. As Hoxa2 is one of 39 murine Hox genes, which exhibit extensive overlapping expression profiles in the spinal cord, we decided to examine the expression of an additional Hox TF, Hoxb4, during OG development. Immunocytochemical analysis of primary mixed glial cultures demonstrated that Hoxb4 is also expressed throughout OG development. Furthermore, comparison of the expression profiles of Hoxb4 and Olig2 suggested that Hoxb4 is expressed by oligodendroglial cells in the spinal cord. Hence, Hoxb4, as well as other Hox TFs could compensate for Hoxa2 in the spinal cord in its absence. As the anterior boundary of most Hox genes has been found to be in the hindbrain or spinal cord, we decided to look at the telencephalon which would be less likely to have compensatory mechanisms. Our results showed that similar to the spinal cord, Hoxa2 is expressed by oligodendroglial cells in the telencephalon. Subsequently, it was found that over-expressing Hoxa2 in CG4 cells, an oligodendroglial cell line derived from the perinatal rat cerebral cortex, impairs their differentiation. In an attempt to determine the mechanism by which it accomplishes this, we examined the expression of polysialylated neural cell adhesion molecule (PSA-NCAM), which has been implicated in this process. Contrary to our expectations, however, it was found that over-expressing Hoxa2 in CG4 cells results in significantly fewer PSA-NCAM+ cells. Hence, the results suggest that Hoxa2s effect on OG differentiation is independent of its effect on PSA-NCAM expression. The expression of Hox genes is enhanced by retinoic acid (RA), which, in turn, both inhibits, as well as promotes OG differentiation. Although the reason for these opposing roles is uncertain, examination of the experimental protocols utilized by different research groups reveals disparities in age, CNS region, as well as RA concentration. As a result, RAs effect on oligodendrogenesis could be stage- and/or concentration-dependent. In order to determine which of these factors could impact RAs effect on OG differentiation we treated CG4 cells with two different concentrations of RA at two distinct time points. The results showed that both factors (concentration and time/stage) can impact RAs effect on CG4 cell differentiation. In an attempt to determine the mechanism by which it accomplishes this, we examined the expression of PSA-NCAM. Contrary to our expectations, the results suggest that RAs effect on CG4 differentiation is independent of its effect on PSA-NCAM expression. The results of this thesis suggest that Hoxa2 and RA could play multiple roles in OG development. Although these roles appear to be similar, further research will be needed to determine whether Hoxa2 acts a downstream effector in the RA signaling pathway in oligodendroglial cells.

<p>Learning and memory is one of the critical components of the human experience. In one model of memory, hippocampal LTP, it is believed that the trafficking of AMPA receptors to the synapse is a fundamental process, yet the spatiotemporal kinetics of the process remain under dispute. In this work, we imaged the trafficking of AMPA receptors by combining two-photon glutamate uncaging on single spines with a fluorescent reporter for surface AMPA receptors. We found that AMPA receptors are trafficked to the spine at the same time as the spine size is increasing. Using a bleaching protocol, we found that the receptors that reach the spine come from a combination of the surface and endosomal pools. Imaging exocytosis in real time, we found that the exocytosis rate increases briefly (~1 min.), both in the spine and neighbouring dendrite. Finally, we performed pharmacological and genetic manipulations of signaling pathways, and found that the Ras-ERK signaling pathway is necessary for AMPAR exocytosis.</p> <p>In a set of related experiments, we also investigated the capacity of single spines to undergo potentiation multiple times. By stimulating spines twice using glutamate uncaging, we found that there is a refractory period for synaptic plasticity in spines during which they cannot further be potentiated. We furthermore found that inducing plasticity in a given spine inhibits plasticity at nearby spines.</p> / Dissertation

Plants sense light intensity, quality and direction through a group of photoreceptors to modulate their growth and development. One family of photoreceptor is called phytochromes (phys) that perceives red and far red light. Phys transduce light signals via a sub-family of the basic Helix-Loop-Helix (bHLH) transcription factors called Phytochrome Interacting Factors (PIFs). PIFs function as negative regulators in the phy-mediated light signaling pathways. In darkness, PIFs regulate downstream gene expressions to inhibit photomorphogenesis. Upon light exposure, PIFs are phosphorylated and poly-ubiquitylated prior to their rapid degradation through the 26S proteasome pathway. One of the PIFs, PIF1, has the highest affinity for both phyA and phyB and also displayed the fastest degradation kinetics under both red and far red light. Here we showed that PIF1 directly and indirectly regulates key genes involved in chlorophyll biosynthesis to optimize the greening process in Arabidopsis. PIF1 binds to a G-box (CACGTG) DNA sequence element present in its direct target genes (e.g., protochlorophyllide oxidoreductase C, PORC) in darkness and regulates their expression. Structure-function studies revealed two separate regions called APB and APA necessary for binding to phyB and phyA, respectively, located at the amino-terminus and a novel phosphorylation site at the carboxy-terminus of PIF1. Both amino- and carboxy-terminal regions are necessary for the light-induced degradation of PIF1. However, the DNA binding is not necessary for the light-induced degradation of PIF1. Using a targeted systems biology approach, we identified new factors, HECATE proteins that promote photomorphogenesis by negatively regulating the function of PIF1. Moreover, we employed an unbiased genetic screening using luciferase imaging system to identify new mutants defective in the light-induced degradation of PIF1. The cloning and characterization of these mutants will help identify the factors, such as the kinase and E3 ligase, responsible for the light-induced degradation of PIF1. Taken together, these data revealed detail mechanisms of how PIF1 negatively regulates photomorphogenesis and how light induces rapid degradation of PIF1 to promote photomorphogenesis. / text

Cancer is an ongoing problem all over the world. To find a cure to this disease, both clinicians and scientists are looking for a reasonable treatment method. According to Hanahan and Weinberg, one of the hallmarks of cancer is evasion of programmed cell death, referred to as apoptosis. Apoptosis is an important cellular process, and is regulated by many different pathways. Proteins in these pathways contribute to either cell death or cell survival depending on the cell stresses. Much research in systems biology has been devoted to understanding these pathways at the molecular level. In this study a mathematical model is built to describe apoptosis, and the pathways involving the related proteins p53 and Akt. The primary purpose of the construction of the kinetic model is to verify that this network can exhibit bistability between cell survival and cell death. Sensitivity and bifurcation analysis are conducted to determine which parameters have the greatest effect on the system behavior.

CD97 gehört zur Familie der Adhäsions-G-Protein gekoppelten Rezeptoren (aGPCR), die aus einem langen extrazellulären N-terminalen Fragment (NTF) und einem nicht-kovalent gekoppelten C-terminalen Fragment (CTF) mit der sieben-transmembranären (TM7) Region und dem intrazellulären Teil bestehen. CD97 wird in malignen Tumoren exprimiert. In der humanen Fibrosarkomzelllinie HT1080 steigert die stabile Überexpression von CD97 die ungerichtete zweidimensionale (2D) Migration einzelner Zellen. Eine Verkürzung von CD97 im CTF auf zwei transmembranäre (TM2) Domänen führt zu einer Suppression der 2D-Migration im Vergleich zu stabil mock-transfektierten HT1080 Kontrollzellen. Wahrscheinlich supprimiert CD97/TM2 die endogene CD97-Wirkung. Unbekannt ist, welche Signalwege durch CD97-Überexpression in HT1080 reguliert werden und welche Signalwege für die Migrationssteigerung von HT1080 verantwortlich sind. Die Klärung dieser Signalwege ist Gegenstand der vorliegenden Arbeit. Die Phosphorylierung von Proteinkinasen ist eine posttranslationale Modifikation zur Regulation der Kinaseaktivität mit nachfolgender Aktivierung oder Inaktivierung eines Signalweges. Daher sind Expression und Phosphorylierung der Proteinkinasen zur Identifikation regulierter Signalwege interessant. Dazu wurden in Lysaten von CD97/TM7, CD97/TM2 und mock-transfektierten HT1080 mittels Kinetworks Phosphosite Screen KPSS 1.3 Profiling (Multi-Immunoblot™) 37 verschiedene Proteinphosphorylierungen untersucht und regulierte Signalwege identifiziert. An 25 Phosphorylierungsstellen erfolgt eine Regulation durch CD97. Anschließend wurden die Ergebnisse der interessantesten Proteine hinsichtlich ihrer Expression und Phosphorylierung im Western Blot verifiziert und um Proteine erweitert, die klassisch an der Regulation der Zellmigration beteiligt sind. Es zeigt sich eine Aktivierung des PI3-Kinase/Akt-Signalweges und eine Inhibierung von Src durch CD97. 2D-Migrationsversuche von HT1080 CD97/TM7, CD97/TM2 und mock mit spezifischen Inhibitoren gegen den PI3-Kinase/Akt-Signalweg und gegen Src bestätigen, dass diese Kinasen an der CD97-induzierten Steigerung der 2D-Migration beteiligt sind. Weiterhin finden sich Hinweise, dass in HT1080 CD97 die Apoptose hemmt und die Proliferation reguliert. Insgesamt wird in dieser Arbeit ein Überblick über die durch CD97 regulierten Signalwege gegeben. Die CD97-gesteigerte 2D-Migration von HT1080 wird durch eine Aktivierung des PI3-Kinase/ Akt-Signalweges und Inhibierung von Src vermittelt.

abstract: Proper cell growth and differentiation requires the integration of multiple signaling pathways that are maintained by various post-translational modifications. Many proteins in signal transduction pathways are conserved between humans and model organisms. My dissertation characterizes four previously unknown manners of regulation in the Drosophila Decapentaplegic (Dpp) pathway, a pathway within TGF-beta family. First, I present data that the Dpp signal transducer, Mothers Against Dpp (Mad), is phosphorylated by Zeste-white 3 (Zw3), a kinase involved in the Wingless pathway. This phosphorylation event occurs independently of canonical phosphorylation of Mad by the Dpp receptor. Using ectopic expression of different alleles of Mad, I show that Zw3 phosphorylation of Mad occurs during the cell cycle in pro-neuronal cells and the loss of phosphorylation of Mad by Zw3 results in ectopic neuronal cells. Thus, Mad phosphorylation by Zw3 is necessary for cell cycle control in pro-neuronal cells. Second, I have shown that the regulator dSno, which has previously been shown to be a TGF-beta antagonist and agonist, is also a Wingless pathway antagonist. Loss of function flip-out clones and ectopic expression of dSno both resulted in changes of Wingless signaling. Further analysis revealed that dSno acts at or below the level of Armadillo (Arm) to inhibit target gene expression. Third, I have demonstrated that the protein Bonus, which is known to be involved in chromatin modification, is required in dorsal-ventral patterning. Further experiments discovered that the chromatin modifier is not only a necessary Dpp agonist, but it is also necessary for nuclear localization of Dorsal during Toll signaling. Last, I showed that longitudinal lacking-like (lola-like) is also required in dorsal-ventral patterning. The loss of maternally expressed lola-like prevents dpp transcription. This shows that lola-like is integral in the Dpp pathway. The study of these four proteins integrates different signaling pathways, demonstrating that the process of development is a web of connections rather than a linear pathway. / Dissertation/Thesis / Doctoral Dissertation Molecular and Cellular Biology 2014