Indiana University-Purdue University Indianapolis (IUPUI) / In mammalian cells DNA double strand breaks (DSBs) are highly variable with respect to sequence and structure all of which are recognized by the DNA- dependent protein kinase (DNA-PK), a critical component for the resolution of these breaks. Previously studies have shown that DNA-PK does not respond the same way to all DSBs but how DNA-PK senses differences in DNA substrate sequence and structure is unknown. Here we explore the enzymatic mechanism by which DNA-PK is activated by various DNA substrates. We provide evidence that recognition of DNA structural variations occur through distinct protein-protein interactions between the carboxy terminal (C-terminal) region of Ku80 and DNA-dependent protein kinase catalytic subunit (DNA-PKcs). Discrimination of terminal DNA sequences, on the other hand, occurs independently of Ku 80 C-terminal interactions and results exclusively from DNA-PKcs interactions with the DNA. We also show that sequence differences in DNA termini can drastically influence DNA repair through altered DNA-PK activation. Our results indicate that even subtle differences in DNA substrates influence DNA-PK activation and ultimately Non-homologous End Joining (NHEJ) efficiency.

Indiana University-Purdue University Indianapolis (IUPUI) / IL-17-producing T cells are critical to the development of pathogen and tumor immunity, but also contribute to the pathology of autoimmune diseases and allergic inflammation. CD8+ (Tc17) and CD4+ (Th17) IL-17-secreting T cells develop in response to a cytokine environment that activates Signal Transducer and Activator of Transcription (STAT) proteins, though the mechanisms underlying Tc17/Th17 development and stability are still unclear. In vivo, Tc17 cells clear vaccinia virus infection and acquire cytotoxic potential, that is independent of IL-17 production and the acquisition of IFN-γ-secreting potential, but partially dependent on Fas ligand, suggesting that Tc17-mediated vaccinia virus clearance is through cell killing independent of an acquired Tc1 phenotype. In contrast, memory Th cells and NKT cells display STAT4-dependent IL-23-induced IL-17 production that correlates with Il23r expression. IL-23 does not activate STAT4 nor do other STAT4-activating cytokines induce Il23r expression in these populations, suggesting a T cell-extrinsic role for STAT4 in mediating IL-23 responsiveness. Although IL-23 is important for the maintenance of IL-17-secreting T cells, it also promotes their instability, often resulting in a pathogenic Th1-like phenotype in vitro and in vivo. In vitro-derived Th17 cells are also flexible when cultured under polarizing conditions that promote Th2 or Th9 differentiation, adopting the respective effector programs, and decreasing IL-17 production. However, in models of allergic airway disease, Th17 cells do not secrete alternative cytokines nor adopt other effector programs, and remain stable IL-17-secretors. In contrast to Th1-biased pro-inflammatory environments that induce Th17 instability in vivo, during allergic inflammatory disease, Th17 cells are comparatively stable, and retain the potential to produce IL-17. Together these data document that the inflammatory environment has distinct effects on the stability of IL-17-secreting T cells in vivo.

Indiana University-Purdue University Indianapolis (IUPUI) / The islet beta cell is unique in its ability to synthesize and secrete insulin for use in the body. A number of factors including proinflammatory cytokines, free fatty acids, and islet amyloid are known to cause beta cell stress. These factors lead to lipotoxic, inflammatory, and ER stress in the beta cell, contributing to beta cell dysfunction and death, and diabetes. While transcriptional responses to beta cell stress are well appreciated, relatively little is known regarding translational responses in the stressed beta cell. To study translation, I established conditions in vitro with MIN6 cells and mouse islets that mimicked UPR conditions seen in diabetes. Cell extracts were then subjected to polyribosome profiling to monitor changes to mRNA occupancy by ribosomes. Chronic exposure of beta cells to proinflammatory cytokines (IL-1 beta, TNF-alpha, IFN-gamma), or to the saturated free fatty acid palmitate, led to changes in global beta cell translation consistent with attenuation of translation initiation, which is a hallmark of ER stress. In addition to changes in global translation, I observed transcript specific regulation of ribosomal occupancy in beta cells. Similar to other privileged mRNAs (Atf4, Chop), Pdx1 mRNA remained partitioned in actively translating polyribosomes during the UPR, whereas the mRNA encoding a proinsulin processing enzyme (Cpe) partitioned into inactively translating monoribosomes. Bicistronic luciferase reporter analyses revealed that the distal portion of the 5’ untranslated region of mouse Pdx1 (between bp –105 to –280) contained elements that promoted translation under both normal and UPR conditions. In contrast to regulation of translation initiation, deoxyhypusine synthase (DHS) and eukaryotic translation initiation factor 5A (eIF5A) are required for efficient translation elongation of specific stress relevant messages in the beta cell including Nos2. Further, p38 signaling appears to promote translational elongation via DHS in the islet beta cell. Together, these data represent new insights into stress induced translational regulation in the beta cell. Mechanisms of differential mRNA translation in response to beta cell stress may play a key role in maintenance of islet beta cell function in the setting of diabetes.

Indiana University-Purdue University Indianapolis (IUPUI) / It is increasingly recognized that mechanical properties of substrates play a pivotal role in the regulation of cellular fate and function. However, the underlying mechanisms of cellular mechanosensing still remain a topic of open debate. Traditionally, advancements in this field have been made using polymeric substrates of adjustable stiffness with immobilized linkers. While such substrates are well suited to examine cell adhesion and migration in an extracellular matrix environment, they are limited in their ability to replicate the rich dynamics found at cell-cell interfaces. To address this challenge, we recently introduced a linker-functionalized polymer-tethered multi-bilayer stack, in which substrate stiffness can be altered by the degree of bilayer stacking, thus allowing the analysis of cellular mechanosensitivity. Here, we apply this novel biomembrane-mimicking cell substrate design to explore the mechanosensitivity of C2C12 myoblasts in the presence of cell-cell-mimicking N-cadherin linkers. Experiments are presented, which demonstrate a relationship between the degree of bilayer stacking and mechanoresponse of plated cells, such as morphology, cytoskeletal organization, cellular traction forces, and migration speed. Furthermore, we illustrate the dynamic assembly of bilayer-bound N-cadherin linkers underneath cellular adherens junctions. In addition, properties of individual and clustered N-cadherins are examined in the polymer-tethered bilayer system in the absence of plated cells. Alternatively, substrate stiffness can be adjusted by the concentration of lipopolymers in a single polymer-tethered lipid bilayer. On the basis of this alternative cell substrate concept, we also discuss recent results on a linker-functionalized single polymer-tethered bilayer substrate with a lateral gradient in lipopolymer concentration (substrate viscoelasticity). Specifically, we show that the lipopolymer gradient has a notable impact on spreading, cytoskeletal organization, and motility of 3T3 fibroblasts. Two cases are discussed: 1. polymer-tethered bilayers with a sharp boundary between low and high lipopolymer concentration regions and 2. polymer-tethered bilayers with a gradual gradient in lipopolymer concentration.

Indiana University-Purdue University Indianapolis (IUPUI). / Tumor necrosis factor-induced protein 6 (TSG-6) has been shown to mitigate inflammation. Its presence in the secretome of adipose stem / stromal cells (ASC) and its role in activities of ASC have been overlooked. This thesis described for the first time the release of TSG-6 from ASC, and its modulation by endothelial cells. It also revealed that protection of endothelial barrier function was a novel mechanism underlying the anti-inflammatory activity of both ASC and TSG-6. Moreover, TSG-6 was found to inhibit mitogen-activated lymphocyte proliferation, extending the understanding of its pleiotropic effects on major cell populations involved in inflammation. Next, enzyme-linked immunosorbent assays (ELISA) were established to quantify secretion of TSG-6 from human and murine ASC. To study the importance of TSG-6 to specific activities of ASC, TSG-6 was knocked down in human ASC by siRNA. Murine ASC from TSG-6-/- mice were isolated and the down-regulation of TSG-6 was verified by ELISA. The subsequent attempt to determine the efficacy of ASC in ameliorating ischemic limb necrosis and the role of TSG-6, however, was hampered by the highly variable ischemic tissue necrosis in the BALB/c mouse strain. Afterwards in a mouse model of cigarette smoking (CS), in which inflammation also plays an important role, it was observed, for the first time, that 3-day CS exposure caused an acute functional exhaustion and cell cycle arrest of hematopoietic progenitor cells; and that 7-week CS exposure led to marked depletion of phenotypic bone marrow stem and progenitor cells (HSPC). Moreover, a dynamic crosstalk between human ASC and murine host inflammatory signals was described, and specifically TSG-6 was identified as a necessary and sufficient mediator accounting for the activity of the ASC secretome to ameliorate CS-induced myelotoxicity. These results implicate TSG-6 as a key mediator for activities of ASC in mitigation of inflammation and protection of HSPC from the myelotoxicity of cigarette smoke. They also prompt the notion that ASC and TSG-6 might potentially play therapeutic roles in other scenarios involving myelotoxicity.

Indiana University-Purdue University Indianapolis (IUPUI) / Mammary ductal epithelial cell growth is controlled by microenvironmental signals in serum under both normal physiological settings and during breast cancer progression. Importantly, the effects of several of these microenvironmental signals are mediated by the activities of the tumor suppressor protein kinases of the Hippo pathway. Canonically, Hippo protein kinases inhibit cellular growth through the phosphorylation and inactivation of the oncogenic transcriptional co-activator Yes-Associated Protein (YAP). This study defines an alternative mechanism whereby Hippo protein kinases induce growth arrest via the phosphorylation of the long isoform of Angiomotin (Amot130). Specifically, serum starvation is found to activate the Hippo protein kinase, Large Tumor Suppressor (LATS), which phosphorylates the adapter protein Amot130 at serine-175. Importantly, wild-type Amot130 potently inhibits mammary epithelial cell growth, unlike the Amot130 serine-175 to alanine mutant, which cannot be phosphorylated at this residue. The growth-arrested phenotype of Amot130 is likely a result of its mechanistic response to LATS signaling. Specifically, LATS activity promotes the association of Amot130 with the ubiquitin ligase Atrophin-1 Interacting Protein 4 (AIP4). As a consequence, the Amot130-AIP4 complex amplifies LATS tumor suppressive signaling by stabilizing LATS protein steady state levels via preventing AIP4-targeted degradation of LATS. Additionally, AIP4 binding to Amot130 leads to the ubiquitination and stabilization of Amot130. In turn, the Amot130-AIP4 complex signals the ubiquitination and degradation of YAP. This inhibition of YAP activity by Amot130 requires both AIP4 and the ability of Amot130 to be phosphorylated by LATS. Together, these findings significantly modify the current view that the phosphorylation of YAP by Hippo protein kinases is sufficient for YAP inhibition and cellular growth arrest. Based upon these results, the inhibition of cellular growth in the absence of serum more accurately involves the stabilization of Amot130 and LATS, which together inhibit YAP activity and mammary epithelial cell growth.

Cette thèse vise à expliquer la problématique de l’échec répété de la mise en oeuvre des politiques publiques de l’eau au Mali, notamment celles relatives à l’implantation du modèle de gestion intégrée des ressources en eau (GIRE). En effet, ces politiques publiques de l’eau peinent à s’implanter au Mali étant donné qu’elles sont conçues le plus souvent dans le cadre d’initiatives extérieures financées par la coopération internationale au développement. Ainsi, pour sa mise en oeuvre, le modèle global dominant de gestion intégrée de l’eau conçu dans ce cadre se trouve confronté à la réalité des contextes locaux de gestion de l’eau prévalant au Mali dans le delta intérieur du Niger (DIN). C’est donc à partir du point de vue de ces réalités contextuelles locales de gestion de l’eau dans le delta intérieur du Niger que cette recherche vise à étudier la manière dont les acteurs façonnent leurs politiques publiques de l’eau et les mettent en oeuvre. Pour ce faire, la thèse se focalise sur l’analyse du discours des acteurs, de leurs stratégies et logique d’action, ainsi que de leurs pratiques de gestion de l’eau en lien avec la mise en oeuvre du modèle de gestion intégrée de l’eau. L’analyse des données recueillies à diverses échelles d’interaction, tant au niveau institutionnel que territorial et infrastructurel, met en évidence plusieurs limites qui contraignent l’implantation de la GIRE au Mali. Ces limites portent notamment sur les pratiques de l’État à travers le déploiement de stratégies de contrôle par bricolage micro-institutionnel et par territorialisation, ainsi que par la normalisation de règles d’accès à l’eau et à la terre, combinés à une mise en application de dispositifs juridico-administratifs de conduite de soi et de contrôle de l’autre. / This thesis aims to explain the problem of the repeated failure of the implementation of public water policies in Mali, particularly those relating to the implementation of the integrated water resources management (IWRM) model. Indeed, these public policies related to water are struggling to take root in Mali given that they are most often designed within the framework of external initiatives financed by international development cooperation. Thus, for its implementation, the dominant global model of integrated water resource management designed in this context is confronted with the reality of the local water management contexts that prevail in Mali in the Inner Niger Delta (DIN). It is therefore from the perspective of these local contextual realities of water management in the Inner Niger Delta that this research aims to study the way in which actors shape their public policies related to water and implement them. To do so, the thesis focuses on the analysis of the actors' discourse, their strategies and their logic of action, as well as on their water resource management practices in relation to the implementation of the integrated water management model. The analysis of the data collected at different scales of interaction, at the institutional, territorial and infrastructural levels, highlights several limitations that hinder the implementation of IWRM in Mali. These limits concern in particular the practices of the State through the deployment of control strategies through micro-institutional tinkering and territorialization, as well as through the standardization of the rules of access to water and land, combined with the application of legal and administrative mechanisms for self-management and control of the other.

This study emanates from the worldwide issue of climate change, as well as the need for all nations to make an effort to reduce their carbon emissions and move towards greener economies. It delves into South Africa's current command-and-control environmental enforcement regime and highlights the pitfalls that allow major air polluters to avoid sanction of any form in many instances. This poor environmental enforcement and compliance effectively means that South Africa is unlikely to be capable of meeting targets set under international agreements. The study confirms that environmental enforcement is inadequately addressed in South Africa. This is attributable to the inherent shortcomings of command-and-control approaches, including that they are inflexible and offer few incentives for firms to modify behaviour to reduce emissions. Poor enforcement of environmental legislation results in negative externalities caused by air pollution being borne by people who did not create such pollution. The study therefore advocates the use of market-based mechanisms as an alternative to traditional command-and-control approaches to environmental enforcement. In light of the South African government's recent publication of the Draft Carbon Tax Bill, carbon taxes as a subset of market-based environmental enforcement mechanisms have the potential to better enforce the polluter pays principle. Mexico, arguably the most forward-thinking developing nation in terms of climate change mitigation, has taken numerous steps towards meeting international commitments, including the implementation of a carbon tax. While South Africa's proposed carbon tax does differ from Mexico's in some respects, the basic premise remains the same and some comparisons can be made in this regard, with accompanying lessons to be learned. Such lessons include that it is imperative that the carbon tax must be set at a high enough rate to meet international commitments whilst at the same time avoiding adverse economic effects, maintaining social welfare and stabilising economic output levels. The proposed carbon tax, while unable to achieve this on its own, is a good place to start and should be utilised in conjunction with the Draft Climate Change Bill to effectively and efficaciously bring about the required change. The proposed carbon tax undoubtedly has the potential to better hold major air polluters responsible for their CO2 and other GHG emissions. / Private Law / LL. M.