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Athman, Jaffre JosephMembrane vesicle trafficking of immune modulatory stimuli during Mycobacterium tuberculosis infection
Doctor of Philosophy, Case Western Reserve University, 2017, Pathology
Mycobacterium tuberculosis (Mtb) is an intracellular pathogen that infects lung macrophages. On macrophages and T cells, Mtb lipoproteins and lipoglycans regulate many co-stimulatory and inhibitory effects, but the mechanisms for their release and trafficking from infected cells during infection are poorly understood. Here, we demonstrated that Mtb infected macrophages release two distinct populations of extracellular vesicles. One population contains host cell content, while the other contains Mtb molecules which we determine are derived by bacterial vesicle budding. Release of bacterial vesicles is an active process dependent on bacteria viability, not a by-product of Mtb degradation within phagosomes. Inhibition or loss of the host proteins CD14 or CD36 or the Mtb lipoprotein LprG does not affect release of Mtb content from infected macrophages. We propose that bacterial vesicles are secreted by Mtb within infected macrophages and subsequently released into the extracellular environment. Suppression of CD4+ T cell responses by Mtb contributes to immune evasion. Mtb cell envelope lipoglycans inhibit T cell receptor signaling. We show that bacterial vesicles released from Mtb infected macrophages inhibited activation of CD4+ T cells, demonstrating a mechanism for trafficking of inhibitory lipoglycans to T cells. Utilizing axenic derived bacterial vesicles, we show that lipoglycans from Mtb bacterial vesicles are transferred to T cells membranes. Primary stimulation of naive CD4+ T cells in the presence of BVs induced a state of T cell anergy. We show that Th1 cells, vital for limiting Mtb infection, are inhibited in their activation by bacterial vesicles, but do not have lasting impairment to restimulation. These studies suggest that bacterial vesicles are the primary mechanism for the export of lipoglycans and lipoproteins from Mtb. These vesicles serve to impair effector functions within infected macrophages, and circulate bacterial components beyond the site of infection to regulate immune responses by uninfected cells. Further, we demonstrate a novel mechanism for the direct regulation of CD4+ T cells by Mtb lipoglycans conveyed by bacterial vesicles that are produced by Mtb and released from infected macrophages. These lipoglycans are transferred to T cells to inhibit T cell responses, providing a mechanism that may promote immune evasion.

Committee:

Clifford Harding, MD/PhD (Advisor); George Dubyak, PhD (Committee Chair); W Henry Boom, MD (Committee Member); Roxana Rojas, MD/PhD (Committee Member); Pamela Wearsch, PhD (Committee Member); Liem Nguyen, PhD (Committee Member); Clive Hamlin, PhD (Committee Member)

Subjects:

Immunology; Microbiology; Molecular Biology

Keywords:

tuberculosis; Mycobacterium tuberculosis; T-cells; bacterial vesicle; exosome; immune evasion; LAM; lipoarabinomannan; membrane vesicle; anergy

Givens, Adam ThomasThe Air Close to the Trees: Evolution and Innovation in U.S. Army Assault Helicopter Units during the Vietnam War
Master of Arts (MA), Wright State University, 2011, History
Throughout the Vietnam War the United States Army‘s use of assault helicopters was unprecedented in modern warfare. Although planners originally anticipated their utilization on a European battlefield rather than against an insurgency, Army Aviation adapted, allowing them to overcome an uncertain future. Due to the unconventional nature of the conflict, continual revisions in tactics, techniques, and procedures ensured that assault helicopter doctrine was never concrete, but always shifting. Multiple factors influenced these developments, and manifold channels of dissemination allowed combat knowledge ultimately to influence training and doctrine. This thesis finds that previous works focus too heavily upon the initial large-scale airmobile battle, ignoring the more profound aspects of later experiences. Using memoirs, official Army documents, service journals, and personal papers, it argues that innovation amongst assault helicopter units occurred throughout the entirety of the American involvement in Vietnam, signifying a youthful Army Aviation that was amenable to varied and innovative thinking from within its ranks.

Committee:

Jonathan Winkler, PhD (Advisor); Paul Lockhart, PhD (Committee Member); Edward Haas, PhD (Committee Member)

Subjects:

American History; Armed Forces; History; Military History; Military Studies

Keywords:

United States Army; Vietnam War; assault helicopter; Huey; airmobility; air mobility; air assault; 1st Cavalry Division; 1st Aviation Brigade; Ia Drang; Lam Son 719; counterinsurgency; Seneff; Howze; Tolson; Oden; McNamara

Sande, Obondo JamesImmune Evasion by Mycobacterium tuberculosis: Mannose-Capped Lipoarabinomannan Induces GRAIL and CD4+ T cell Anergy
Doctor of Philosophy, Case Western Reserve University, 2016, Pathology
Mycobacterium tuberculosis (Mtb) persists and survives in the host in the face of many T cells subsets recognizing a range of Mtb antigens due to its ability to evade innate and adaptive immune responses. CD4+ T cells and infected antigen presenting cells (APC) are central for control of Mtb but also targets of its immune evasion strategies. The major Mtb cell wall glycolipid, mannose-capped lipoarabinomannan (LAM) is one of the molecules involved in immune evasion. Previous studies determined that LAM can inhibit phagosome maturation and antigen processing in macrophages and thus indirectly affect memory and effector CD4+ T cell function. LAM is trafficked from Mtb-infected macrophages via bacterial vesicles into the microenvironment of the infected site, where it can bind to uninfected APC and T cells. We proposed that this provides a mechanism for direct delivery of LAM to surrounding T cells, thereby further regulating their function. Earlier studies determined that LAM directly inhibits polyclonal murine CD4+ T cell activation by blocking ZAP-70 phosphorylation. In the first part of this thesis we extended our observation of direct inhibition of T cell activation by LAM in two directions. First we determined if LAM inhibition of murine primary CD4+ T cells could be extended to antigen-specific CD4+ T cell activation by antigen presenting cells and whether human CD4+ T cells were similarly inhibited. Second, we determined the mechanism of LAM-mediated inhibition of TCR signaling in terms of its effect on Lck and LAT phosphorylation and lipid raft integrity. We found that LAM inhibited antigen-specific murine CD4+ T cells and primary human T cells as well. In addition to ZAP-70, LAM inhibited phosphorylation of Lck and LAT. Inhibition of proximal TCR signaling was temperature sensitive, suggesting that LAM insertion into T cell membranes was required. We established that direct interaction of LAM with T cells inhibits antigen-specific CD4+ T cell activation by interfering with very early events in TCR signaling through LAM’s insertion in T cell membranes. Previous studies show that inhibition of proximal TCR signaling is associated with induction of T cell anergy. In the second focus of this thesis, we tested if LAM-induced inhibition of CD4+ T cell activation results in T cell anergy. We found that LAM induces anergy in P25 TCR transgenic CD4+ T cells (specific for P25 peptide of the 38 kDa antigen 85B of Mtb). Anergy induction required the presence of LAM in the T cell membrane during primary T cell activation. Once anergy was induced, LAM was no longer required in the T cell membrane, as removal of LAM did not affect the T cell anergic state. The induction of anergy was due to up-regulation of GRAIL (Gene Related to Anergy In Lymphocytes) protein in both murine and human CD4+ T cells. We further determined that exogenous IL-2 reversed LAM-induced anergy by downregulating GRAIL expression. We propose that LAM inhibits CD4+ T cell activation and up-regulates GRAIL expression to induce anergy in Mtb-reactive CD4+ T cells. Together, the studies described in this thesis determined that induction of CD4+ T cell anergy by LAM may represent one mechanism by which Mtb evades T cell recognition.

Committee:

Henry Boom, M.D. (Advisor); Alan Levine, Ph.D (Committee Chair); Roxana Rojas, MD, PhD (Committee Member); Clifford Harding, MD, PhD (Committee Member); Clive Hamlin, Ph.D (Committee Member)

Subjects:

Immunology; Microbiology; Molecular Biology

Keywords:

Mycobacterium tuberculosis; LAM; CD4 T cells; GRAIL; Anergy; Immune Evasion

Makiewicz, Kurt TimothyDevelopment of Simultaneous Transformation Kinetics Microstructure Model with Application to Laser Metal Deposited Ti-6Al-4V and Alloy 718
Master of Science, The Ohio State University, 2013, Materials Science and Engineering
Laser based additive manufacturing has become an enabling joining process for making one-of-a-kind parts, as well as, repairing of aerospace components. Although, the process has been established for more than a decade, optimization of the process is still performed by trial and error experimentation. At the same time, deployment of integrated process-microstructure models has remained as a challenge due to some of the reasons listed below: (1) lack of good process models to consider the laser-material interactions; (2) inability to capture all the heat transfer boundary conditions; (3) thermo-physical-mechanical properties; and (4) robust material model. This work pertains to the development of robust material model for predicting microstructure evolution as a function of arbitrary thermal cycles (multiple heating and cooling cycles) that can be integrated into a process model. This study focuses on the development of a material model for Ti-6Al-4V and Alloy 718. These two alloys are heavily used in turbine engines and undergo complex phase transformations, making them suited to developing a material model for laser metal deposition (LMD). The model uses simultaneous transformation kinetics (STK) theory to predict the transformation of one parent phase into several products. The model uses calculated thermodynamic properties of the alloys for portions of the respective transformation characteristics. Being a phenomenological model there are several user defined calibration parameters to fit the predicted output to experimental data. These parameters modify the nucleation and growth kinetics of the individual transformations. Analyses of experimental LMD builds are used to calibrate the material model. A Ti-6Al-4V build made on a room temperature substrate showed primarily colony alpha morphology in the bottom half of the build with a transition to basketweave alpha in the top half. An increase in hardness corresponding to the microstructural transition was observed. This sample had an average of 340 HV hardness. Analysis of the calculated thermal profiles at the location of the morphology transition showed a transition from cooling below the beta transus to cooling above the beta transus. The Ti-6Al-4V STK model was calibrated using the experimental data from this sample. The substrate of a second build was heated above the Ti-6Al-4V beta transus. This build showed predominantly basketweave alpha without a microstructural transition. Large prior beta grains (>1mm) were observed growing epitaxially from the substrate. These large grains promoted the basketweave formation. Hardness testing showed an average of 344 HV. Samples built in this way were also fatigue tested in the as built condition. Results show that they match previous builds that had been stress relieved. A third build was performed at room temperature on a substrate with large prior beta grains. This build showed basketweave morphology like the second build even though the substrate was not thermally controlled. The hardness for this build averaged 396 HV which is ~50 HV higher than the previous two. This build shows that it may be possible to produce better mechanical properties by controlling the beta grain size rather than heating the substrate. Eighteen Alloy 718 builds were made using proprietary processing conditions. All of these builds were analyzed for nano-scale γ’ and γ’’ precipitates. Two of the builds were similar but had different laser powers. The low laser power build did not show nano-scale precipitates. The higher power build did show small amounts (<3%) of nano-scale precipitates and a corresponding increase in hardness at their locations. The higher power build was used to develop the STK model for Alloy 718. Sixteen of these builds were part of a design of experiments and are referred to as DOE samples. Eight of them have a single layer while the other eight have multiple layers. They were examined for nano-scale precipitates. The amounts of precipitates were correlated to hardness values and thermal profiles.

Committee:

Sudarsanam Babu (Advisor); Wolfgang Windl (Committee Member)

Subjects:

Aerospace Materials; Materials Science; Metallurgy

Keywords:

Simultaneous Transformation Kinetics; STK; Microstructure Modeling; Laser Additive Manufacturing; Laser Metal Deposition; aerospace repair; Ti-6Al-4V; Inconel 718; Alloy 718; Additive Manufacturing; LAM; LMD;