<p> Regulation of barrier function is critical for patients who suffer from inflammatory diseases such as acute respiratory distress syndrome (ARDS) and sepsis. A major regulator of endothelial barrier function is vascular endothelial cadherin (VE-cad). Cellular levels of VE-cad are known to be regulated by p120 catenin. Loss of p120 leads to decreased barrier function as a result of the endocytosis of VE-cad. However, recent work from our lab shows that expression of an endocytic defective VE-cad mutant was not able to rescue barrier function, as measured using transendothelial electrical resistance (TEER). In contrast, expression of a non-phosphorylatable VE-cad mutant was able to restore barrier function independent of p120 binding. These results suggest that endocytosis is not the only mechanism regulating VE-cad localization to the cell-cell junctions, but rather the phosphorylation state of the protein may play a more critical role to stabilizing VE-cad at the junction. In order to investigate junctional stability of VE-cad, we created a recombinant form of VE-cad by cloning mEos2 into a plasmid containing the VE-cad gene. This fluorophore is photoconvertible, thus allowing for tracking protein movement at the cell-cell junction. The VE-cad proteins, labeled with mEos2 at the C-terminus, were introduced via adenoviral infection into human umbilical vein endothelial cells (HUVEC). Initially, mEos2 fluoresces green, in order to induce photoconversion, a 405nm laser is directed in a specific region of interest (ROI) at the junction. A conformational change in the mEos2 protein will cause irreversible red fluorescence. Tracking the change in fluorescence intensity in the ROI will provide insight into the localization of VE-cad at endothelial cell junctions. We now have a model that can be used to test junctional localization and stability of endocytic defective and non-phosphorylatable mutants of VE-cad.</p><p>

<p> Each time a cell divides, the microtubule cytoskeleton self-organizes into the metaphase spindle: an ellipsoidal steady-state structure that holds its stereotyped shape despite microtubule turnover and internal stresses. This ellipsoidal architecture, in which microtubule minus-ends are focused into two poles, is essential to the spindle&rsquo;s function of accurately segregating chromosomes. In this work, I ask how the spindle forms and holds its steady-state shape. I report that the molecular motor dynein and the microtubule binding-protein NuMA are essential for mammalian spindles to reach and hold a steady-state geometry. In their absence, the kinesin-5 Eg5 powers a turbulent microtubule network that can drive flow of cytoplasmic organelles and whole-cell movement. Dynein and NuMA were previously known to be essential for spindle pole formation, but we did not know their contribution to shape stabilization at the whole-spindle scale&mdash;nor did we know how and where they pull on microtubules to build poles. Using quantitative live imaging and laser ablation, I show that dynein pulls specifically on microtubule minus-ends, rapidly transporting them towards poles. Dynein localization to microtubule minus- ends depends on NuMA, which recruits the dynein adaptor dynactin to minus-ends. Contrary to previous models, NuMA localization to minus-ends is independent of dynein and involves a C-terminal region outside its canonical microtubule-binding domain. Thus, NuMA serves as a mitosis-specific minus-end cargo adaptor, targeting dynein activity to minus-ends to cluster spindle microtubules into poles. This microtubule end-clustering compacts the spindle microtubule network to a defined geometry and suppresses network turbulence, maintaining a steady-state spindle shape over long timescales.</p><p>

<p> The purpose of these studies was to determine the role of Interleukin-17A (IL-17A) in the immune response to respiratory mycoplasma infection. Serum levels of IL-17A increase in disease-susceptible BALB/c mice, but not disease-resistant C57BL/6 mice, infected with <i>Mycoplasma pulmonis</i>. Increased serum IL-17A was associated with mycoplasma pathology during infection in BALB/c mice, including: the presence of pulmonary neutrophils, progressive weight loss, and the development of inflammatory lung lesions. </p><p> Neutralizing the function of IL-17A using monoclonal anti-IL-17A antibodies during mycoplasma infection reduced disease severity in disease-susceptible BALB/c mice, but not disease-resistant C57BL/6 mice. Providing daily intra-peritoneal injections of anti-IL-17A antibodies to BALB/c mice infected with <i> M. pulmonis</i> was effective at reducing weight loss, the prevalence of clinical signs, and the incidence of gross lesions. Histological lesions, characterized by the presence of pulmonary neutrophils, were also lower in infected BALB/c mice receiving anti-IL-17A antibodies daily. Bacterial burden remained unaffected in mice regardless of treatment. Neutralizing IL-17A throughout infection was effective at reducing late mycoplasma pathology, a period influenced by the actions of adaptive immunity and this is supported by a reduction in disease severity when infected BALB/c mice were provided intra-peritoneal injections of anti-IL-17A antibodies only after T-cells infiltrate the lungs. </p><p> Pulmonary T-cells, specifically CD4⁺ T-helper (Th17) cells, were the primary source of IL-17A throughout infection with <i>M. pulmonis </i> in disease-susceptible BALB/c mice. Although Th17 cells increased in the lung after infection, the Th17 response did not reach its peak until the later stages of infection and coincided with when the neutralization of IL-17A started to reduce the severity of disease. IL-17A⁺ T-cells did not express Retinoic Acid Related (RAR) Orphan Receptor-&gamma;t (ROR&gamma;t), a signature Th17 transcription factor, after infecting BALB/c mice with <i> M. pulmonis</i> and suggests that ROR&gamma;t is not a suitable marker to identify the IL-17A⁺ T-cells worsening mycoplasma disease. </p><p> The effect of neutralizing IL-17A was mimicked in disease-susceptible BALB/c mice depleted of neutrophils during <i>M. pulmonis</i> infection. Depleting neutrophils in BALB/c mice infected with <i>M. pulmonis</i> abrogated weight loss while reducing the appearance of both clinical signs and gross lesions. IL-17A promotes pathology during disease utilizing various mechanisms, one of which is to mobilize and activate neutrophils; however, the IL-17A failed to worsen mycoplasma disease in the absence of neutrophils during <i>M. pulmonis</i> infection in BALB/c mice. These results suggest that IL-17A relies only upon neutrophil recruitment and activation to exacerbate mycoplasma disease. Supporting this, combining the neutralization of IL-17A with the depletion of neutrophils failed to lessen disease severity beyond what either treatment could achieve alone. These findings underscore IL-17A or neutrophils as targets for inhibition to reduce the severity of disease during mycoplasma infection. </p><p> Both IL-4 and IL-17A increase in the lungs of BALB/c mice infected with <i> M. pulmonis</i> and there are Th17 cells that secrete IL-4. In STAT6 KO mice that respond poorly to IL-4 and generate defective Th2-mediate immunity, neutralizing IL-17A also reduced inflammatory damage during <i> M. pulmonis</i> infection. Treating STAT6 KO mice with anti-IL-17A antibodies during <i>M. pulmonis</i> infection reduced weight loss, the prevalence of clinical signs, and incidence of inflammatory lesions. Like wild-type mice, the pathologic effect of IL-17A manifested during the later stages of <i> M. pulmonis</i> infection in STAT6 KO mice and coincided with the activation of adaptive immunity. Neutralizing IL-17A also failed to change mycoplasma numbers during infection in STAT6 KO mice. IL-17A is highlighted as an independent contributor to mycoplasma pathology with no impact on mycoplasma clearance; inhibiting the activation of Th2- and Th17-mediated immune responses could increase resistance by permitting the development of protective responses during infection. </p><p> This work emphasizes the importance of IL-17A and Th17 cells as an autonomous immune response worsening neutrophil-mediated pathology during late mycoplasma infection in susceptible mice. Monoclonal antibodies that neutralize the function of IL-17A could reduce the severity of disease during mycoplasma infection in man and animals. Directly targeting neutrophils may also lessen the negative impact IL-17A has on mycoplasma pathology. Vaccines that do not activate IL-17A-mediated immunity could reduce the susceptibility to mycoplasma infection and allow for the development of immune responses that lead to mycoplasma clearance. IL-17A functions to worsen disease severity without impacting mycoplasma clearance, and so IL-17A is identified as a contributor to pathology during infection. </p><p>

Two possible mechanisms involved in the facilitated transfer of glucose through red cell and other membranes have been investigated. In the first section, the hypothesis that glucose transfer involves phosphorylation was tested by estimating phosphatase activities in human red cells, adult and foetal guinea-pig red cells and kidney homogenates, and the effects upon them of known inhibitors of glucose transfer. No correlation between phosphatase activity and glucose transfer in the red cell could be found. Histochemical studies are presented to show the distribution of acid and alkaline phosphatases in the adult and foetal guinea-pig kidney, and the placenta. In the second section, the possibility is explored that glucose forms a reversible complex with a phospholipid component of the cell membrane. Methods were developed to extract phospholipids from sheep placental tissue and human and sheep red cells, to separate them chromatographically and to isolate the glucose-lipid complex. Uptake of the irreversible inhibitor 2,4-dinitrofluorobenzene (DNFB) labelled with was measured, and calculated to be 400 million molecules per red cell to give full inhibition of glucose transfer. The lipids extracted from the DNFB inhibited red cells contained radioactivity equivalent to 10-20 million molecules DNFB per red cell, which considerably exceeds the number of carrier sites thought to be involved in glucose transfer. A large proportion of the radioactivity was found in the cephalin fractions, and the possibility is discussed that phosphatidyl ethanolamine is the membrane carrier component. The technique of monolayers was used to study the surface behaviour of certain phospholipids in the presence of glucose, and the preliminary results obtained were discussed.

The fine structure of the hinge cells resembled the fine structure of the medullary cells of the hinge region, and of plant or animal tissue capable of active ion transport. There were abundant mitochondria, vesicles and ER. The hinge cell walls possessed numerous plasmodesmata, whose possible functions were compared with those suggested for the Pacinian cerpuscles, where degree of stimulus is proportional to the area of membrane distorted. Closure of traps is a result of bending the sensory hinge, which caused the release of an action potential. Bending the hair caused swelling of organelles, and the appearance of myelin forms in hinge cells, as shown in EM photographs of the hinge region. These effects were considered on the basis of the LUCY model (1964, 1968) for altered membrane phospholipid orientation. A muscle contracting substance (MCS) was looked for, because acetyloholine has the property of contracting frog rectus muscle, occurs in vesicles in animal synapses as a chemical transmitter of excitation by action potentials, and as well is present in hairs of the plant Urtica. The MCS was indentified as LONG'S (1967) 'B' lysophosphatidic acid, resulting from water-soluble phospholipase D acting on lysolecithin. An action potential is supposed to alter membrane potential and release calcium ions; both would change the activity of phospholipase D. The results following indicated an activated phospholipase from an action potential caused bending the hair, caused increased membrane permeability, and trap closure. Application of chlor-choline-chloride to traps with hairs, suggested activation of lecithin synthesis via activated choline kinase, and reversal of this activation by gibberellic acid. Current hypotheses, such as an ATP use 'mechanoenzyme' membrane complex altering permeability, the water-filled, phospholipid lined hypothesis of Glauert (1968), and the Mitchell hypothesis (1961), were considered in relation to the results. A model is presented of Dionaea trap cell with membranes in dynamic equilibrium, a phospholipase destroying, and a choline kinase complex reconstituting the plasma membrane.

<p> Blood supply is crucial for tumor growth and metastasis. However, current anti-angiogenic therapy is not as effective as predicted, thus a better understanding of the tumor angiogenic process and new anti-angiogenic agent are urgently required. Anthrax lethal toxin (LeTx) has an anti-angiogenic effect on tumors. Tumors treated with LeTx are smaller, paler, and have lower mean vessel density compared to control treated tumors. Most interestingly, compared to current anti-angiogenic treatment, LeTx does not cause normalization of tumor vessels. Instead, tumors treated with LeTx have massive hemorrhages, pointing to a potential alternative mechanism to inhibit tumor angiogenesis. I hypothesize that instead of causing &ldquo;normalization&rdquo; of tumor vasculature, LeTx&rsquo;s anti-angiogenic effects works in a manner similar to a hemorrhagic toxins. To test this hypothesis, I compared the effect of LeTx to snake venom metalloproteinase, a known hemorrhagic toxin, in tumor vasculature. Quantified by Nuance multispectral imaging system, both LeTx and SVMP caused an increase in tumor hemorrhage. Futher analysis of vasculature integrity using continued vessel length showed disruption of vessels by LeTx and SVMP. With these results, I conclude that the anti-angiogenic effects of LeTx are due to its hemorrhagic nature, and not due to normalization of tumor vasculature. Further understanding of LeTx mechanism can help design novel anti-angiogenic agent that compliments current therapy.</p><p>

<p> The maintenance of cell surface proteins is critical to the ability of a cell to sense and respond to information in its environment. As such, modulation of cell surface composition and receptor trafficking is a potentially important target of control in virus infection. Sorting endosomes (SEs) are control stations regulating the recycling or degradation of internalized plasma membrane proteins. Here we report that human cytomegalovirus (HCMV), a ubiquitous beta herpesvirus, alters the fate of internalized clathrin-independent endocytosis (CIE) cargo proteins, retaining them in virally reprogrammed SEs. We show that the small G protein ARF6, a regulator of CIE trafficking, is highly associated with SE membranes, relative to uninfected cells. This finding suggests that ARF6 and CIE cargo egress from the SE is diminished by infection. Over expression of the ubiquitin specific protease (USP) 6, also known as TRE17, was sufficient to restore ARF6 and some ARF6 cargo trafficking to the cell surface in infected cells. The USP-activity of TRE17 is required to rescue both ARF6 and associated cargo from SE retention in infection. Intriguingly, TRE17 expression does not affect all CIE cargos retained at SEs in infection. Although TRE17 mediates the trafficking of internalized major histocompatibility complex type I (MHCI) to the cell surface in uninfected cells, MHCI is insensitive to TRE17-mediated trafficking in the context of HCMV infection. These findings demonstrate a reprogramming of endocytic trafficking by HCMV infection and suggests that HCMV hijacks the normal sorting machinery and selectively sorts specific cargos into endocytic micro-domains that are subject to alternate sorting fates.</p><p>

<p> Pancreatic ductal adenocarcinoma (PDAC) is a notoriously lethal disease characterized by prominent stromal involvement, which plays complex roles in regulating tumor growth and therapeutic response. The extracellular matrix (ECM)-rich stroma has been implicated as a barrier to drug penetration, although stromal depletion strategies have had mixed clinical success. It remains less clear how biophysical interactions with the ECM regulate invasive progression and susceptibilities to specific therapies. Here, an integrative approach combining 3D cell culture and quantitative imaging techniques is used to evaluate invasive behavior and motility as determinants of response to classical chemotherapy and photodynamic therapy (PDT), in which light activated agents induce site-directed cell death by generating reactive oxygen species. The 3D culture protocol developed for these studies with transplanted multicellular PDAC spheroids in rheologically characterized ECM shows that in invasion-promoting ECM environments, PDT response is markedly enhanced in the most motile populations while the same cells exhibit chemoresistance. Conversely, drug-resistant sublines with characterized increase in invasive potential were generated to compare differential treatment response in identical ECM conditions, monitored by particle-tracking microrheology measurements of matrix remodeling. In both scenarios, ECM infiltrating cells exhibit increased PDT sensitivity, whether invasion is consequent to selection of chemoresistance, or whether chemoresistance is correlated with acquisition of invasive behavior. However, while ECM-infiltrating, chemoresistant cells exhibit mesenchymal phenotype, EMT induction in monolayers lacking ECM is not sufficient to enhance PDT sensitivity, yet does impart chemoresistance as expected. In further experiments seeking to elucidate intertwined roles of mechanical and biochemical interactions with ECM components, invasive progression and response to therapeutics were evaluated using ECM protein admixtures and collagen hydrogels with varying extent of crosslinking. In these studies, increased collagen stiffness or presence of laminin-rich ECM both inhibit invasion of PDAC cells, although cells that do infiltrate into ECM nevertheless exhibit chemoresistance and enhanced PDT sensitivity, independent of their ECM environment. In addition to containing platform development with broader applicability to inform microenvironment-dependent therapeutics, results of this work collectively reveal the efficacy of PDT for targeting the most aggressive, chemoresistant, and invasive PDAC cell populations associated with dismal outcomes for this disease.</p><p>

<p> Arteriogenesis, the expansion of collateral arteries, is vital process for compensatory blood flow to tissues in response to vascular occlusions. In patients with peripheral arterial disease (PAD), arteriogenesis is crucial for overcoming limb ischemia, but for many, further treatment is required. In the United States alone, approximately 80,000 individuals lose limbs to this disease each year. Thus, it is critical to understand the mechanisms regulating arteriogenesis. Indirect evidence suggests that mitogen-activated protein kinase kinases 1 and 2 (MEK1 and 2) are involved in arteriogenesis, but this has not been directly tested. To do this, we used a mouse model of hindlimb ischemia, femoral artery ligation (FAL), treated with a specific allosteric MEK1 and 2 inhibitor (PD0325901). Whereas control mice showed increased myogenesis, angiogenesis, and arteriogenesis, mice dosed daily with PD0325901 failed to recover. In order to examine for any temporal regulation, mice were treated days 8 to 28 post surgery. Interestingly, mean arterial luminal area increased. In a complimentary experiment, mice were treated out to 7 days post surgery, treatment was stopped, and tissues were collected at day 28 post surgery. Although muscle tissue had recovered by this time, mean arterial luminal area remained low relative to controls, suggesting a critical window of MEK1 and 2 signaling being necessary for recovery. Surprisingly, results from MEK2^{&ndash;/&ndash;} mice also fail to undergo arteriogenesis after surgery, indicating this effect may be specific to MEK2 alone. BrDU-injected mice co-stained with either CD31 or &alpha;SMA show that loss of MEK2 predominantly affects endothelial cells within the arteries. Lastly, equivalent results are shown in mice lacking tumor endothelial marker 8 (TEM8). These results indicate MEK2 activity is required for arteriogenesis, and show the first known physiological role for TEM8. In addition, the results have implications in the current use of MEK1 and 2 inhibitors for anti-cancer therapy, as these drugs may affect remodeling arteries. Our results also have potential implications for future therapies for PAD, as MEK2 activation after blockage could stimulate arterial growth, preventing the need for amputation.</p><p>

<p> Integrins are trans-membrane receptors that form focal contacts and mediate the attachment of the smooth muscle cell cytoskeleton to the extracellular matrix. Focal adhesion kinase (FAK) regulates the recruitment and assembly of focal adhesion proteins. It has been established that integrin proteins and FAK play important roles in cell adhesion, tension generation, and mechanotransduction. Integrins and FAK are abundant in smooth muscles; however, the function of FAK in gastric smooth muscle cell contractile regulation remains unclear. To better understand the role of FAK in gastric fundus smooth muscle contractile regulation, we performed two major projects: (1) addressing gastric smooth muscles contractile regulation in cholinergic neurotransmission by FAK phosphorylation (Chapter 2) and (2) characterizing the change in Ca²⁺ sensitization proteins distribution at focal adhesion sites under electric field stimulation induced cholinergic neurotransmission and FAK phosphorylation regulation (Chapter 4). In carrying out the first project, the contractile responses of murine gastric smooth muscles were determined using standard myobath &ndash; isometric force transducer techniques and phosphorylated or total proteins determined by automated capillary electrophoresis and immunoblotting by Wes Simple Western. For the second project, we developed smooth muscle tissue <i>in situ </i> proximity ligation assay (PLA) for the quantitative PLA analysis of protein-protein interaction and protein phosphorylation (Chapter 3). From these studies, we revealed a novel role of FAK in gastric fundus smooth muscle contractile regulation by cholinergic stimulation. We also demonstrate quantifiable tissue level PLA that can be extended and applied to studies of protein-protein interaction, and protein phosphorylation in various tissues and signaling pathways.</p><p>