No description available.

Fibrillin-1 is the main component of the 10-12 nm microfibrils, which are found in several elastic and non-elastic tissues. Human fibrillin-1 contains multiple calcium-binding epidermal growth factor-like (cbEGF) domains interspersed with transforming growth factor β-binding protein-like (TB) domains. TB4 domain contains a flexible RGD loop which mediates cell adhesion via αVβ3, α5β1 and αVβ6 integrins. Mutations which introduce amino acid substitutions into TB4 are associated with a wide spectrum of diseases such as Marfan syndrome (MFS), ectopia lentis, Stiff skin syndrome (SSS). Amino acid substitutions such as W1570C, C1564S and C1577G in the TB4 domain have been found to cause SSS. The upstream TB5 domain has been predicted to modulate integrin binding and a deletion in the domain has been found in Weill-Marchesani syndrome (WMS), phenotype of which is similar to SSS (skin fibrosis and short stature), thereby suggesting that the underlying pathogenic mechanism might be similar. This study has used cellular, biochemical and biophysical methods to investigate the effects of SSS substitutions C1564S and W1570C on domain structure and function and compared it to a MFS substitution C1564Y in the TB4 domain and WMS deletion in the TB5 domain. Effects of the SSS mutations on structure of the domains were studied using limited proteolysis, nuclear magnetic resonance spectroscopy and calcium chelation experiments. Subsequently, the ability of human fibroblasts to secrete wild-type and mutant fibrillin-1 was examined to identify the effect of the mutations on the trafficking of the protein. Finally, cell binding assays and SPR was employed to investigate the effect of disease-causing mutations on fibrillin-1/integrin interactions. The results demonstrate that the SSS mutations affect TB4-cbEGF23 interface and calcium-binding to cbEGF23 but do not alter secretion of recombinant fibrillin-1 mutant fragments from the cell. On the other hand, intracellular retention was observed for MFS substitution C1564Y which was shown to be more susceptible to proteolysis than SSS substitution C1564S. WMS deletion also gives rise to partial retention of the recombinant fragment, suggesting a different pathogenic mechanism for these disorders. Cell binding assays and surface plasmon resonance (SPR) experiments show that SSS mutations affect binding to αvβ3 integrin, but not αvβ6 integrin suggesting that selectively impaired integrin interactions may contribute to pathogenesis of SSS.

No description available.

Cells experience force throughout their lifetimes. Cells sense force via adhesion receptors, such as the cadherins, which anchor cells to neighboring cells, and integrins, which tether cells to the underlying matrix. Both adhesion receptors respond to force by activating signaling pathways inside the cell. These pathways trigger growth of adhesion complexes and reinforcement of the cytoskeleton in order to resist the force. These activities are energetically costly. Thus, mechanisms are needed to couple force transmission and energy production. In this thesis, I demonstrated force on cadherins activates a master regulator of energy homeostasis known as AMP-activated kinase (AMPK). In response to force, AMPK was recruited to the cadherins. AMPK promoted growth of the adhesion complex and cytoskeletal reinforcement by stimulating energy production in the cell. Additionally, AMPK formed a complex with vinculin—a protein that is recruited to both cadherins and integrins. I observed AMPK activation of vinculin dictates whether vinculin joins the cadherin complex. Conversely, AMPK activation has no bearing on vinculin recruitment to integrins. This work provides three novel contributions: (1) the first link between energy production and force transmission, (2) a molecular mechanism for how AMPK increases adhesion complex growth, and (3) an explanation for how vinculin discriminates between cadherins and integrins.

Copy of author's previously published article in pocket on back end-paper. Includes bibliography (leaves 135-185). Aims to characterise the expression of PETA-3 (Platelet Endothelial Tetraspan Antigen-3), CD9, CD63 and ?gb?s1 integrins in normal human tissue ; to determine the subcellular localisation in endothilial cells and platelets ; to investigate protein-protein interactions involving PETA-3 ; and to examine the effects of anti-PETA-3 monoclonial antibodies on platelet and endothilial cell function.

Thesis (Ph. D.)--University of Rhode Island, 2006. / Typescript. Includes bibliographical references (leaves 104-121).

The body is under constant attack from pathogens trying to slip by our immune defence. If the barrier is breached, invading pathogens enter the tissues and cause inflammation. During this process neutrophils, constituting the first line of defence, leave the bloodstream and seek out and kill the invading pathogens. The mechanisms leading to activation of receptors on neutrophils must be closely orchestrated. Pro- and anti-inflammatory substances can influence the outcome of the inflammation process by affecting the involved players. If not well balanced, inflammatory diseases, such as atherosclerosis and rheumatoid arthritis, can be the outcome. The aim of this thesis was to elucidate the effect of pro- (fMLP, Leukotriene B4, and Interleukin-8) and anti- (lipoxins, aspirin and statins) inflammatory substances on the β2 integrins, mediating adhesion of neutrophils both under “normal” conditions and during coronary artery disease. More specifically, the effect of these substances on the β2 integrins were studied in regard to: i) the activity (i.e. affinity and avidity) of β2 integrins, ii) the signalling capacity of β2 integrins (i.e. detected as release of arachidonic acid, and the production of reactive oxygen species, and iii) the signal transduction mediated by the β2 integrins (i.e. phosphorylation of Pyk2). The pro-inflammatory substances belong to the family of chemoattractants that induces transmigration and chemotaxis. A hierarchy exists between the different family members; the end-target chemoattractants (e.g. fMLP) being more potent than intermediary chemoattractants (e.g. IL-8 and LTB4). It was found that intermediary chemoattractants regulate β2 integrins by mainly affecting the avidity of β2 integrins. End-target chemoattractants on the other hand, affected the β2 integrins by increasing the avidity and the affinity, as well as their signalling capacity. The anti-inflammatory substances used in this study were the exogenous aspirin and statins, and the endogenous lipoxins. In the presence of aspirin, stable analogues of lipoxin (i.e. epi-lipoxins) are formed in a trans-cellular process. Lipoxin inhibited the signalling capacity of β2 integrins mediated by intermediary chemoattractants, as well as the signal transduction induced by end-target chemoattractants. Moreover, the signalling capacity of β2 integrins in neutrophils from patients suffering from coronary artery disease (CAD) was impaired. Arachidonic acid, the precursor for both pro- and anti-inflammatory eicosanoid, induced an increase in the β2 integrin activity (both affinity and avidity), but had no effect on the signal transduction. In conclusion, different “roles” were observed for end-target and intermediary chemoattractants in the regulation of β2 integrins. The inhibitory effects of the anti-inflammatory lipoxins support earlier studies suggesting that these agents function as “stop signals” in inflammation. This is also confirmed by our findings in CAD patients, who have elevated levels of epi-lipoxins due to aspirin treatment. Moreover, Pyk2 was identified as a possible target for the inhibitory effect of anti-inflammatory drugs.

Fundamental questions related to the locations of Cerebrospinal Spinal Fluid (CSF) absorption deficit and causes of the pressure gradients that expand the ventricles with hydrocephalus remain largely unanswered. Work in the Johnston lab over a 15 year period has demonstrated that CSF moves through the cribriform plate foramina in association with the olfactory nerves and is absorbed by a network of lymphatic vessels located within the olfactory turbinates. A kaolin-based rat model of communicating hydrocephalus was developed as a collaborative effort with Drs. McAllister, Wagshul and Li. After developing a method to quantify lymphatic CSF uptake in rats, we examined and observed that the movement of a radioactive tracer into the nasal turbinates was significantly reduced in the kaolin-injected animals compared to saline injected controls. However, it was possible that while lymphatic CSF uptake was compromised, other CSF absorption pathways may have compensated. To answer this, we measured the CSF outflow resistance (Rout) and observed it to be significantly greater in the kaolin group compared with animals receiving saline and there was a significant positive correlation between CSF Rout and ventricular volume. Nonetheless, it is not clear how impaired CSF clearance could lead to a dilation of the ventricles since the ventricular and subarachnoid compartments are in communication with one another and pressure would likely increase equally in both. At this point, we came across a theoretical paper that postulated that a drop in periventricular interstitial fluid pressure might provide an intraparenchymal pressure gradient favouring ventricular expansion. In addition, studies in non-CNS tissues indicated that a disruption of beta-1 (β1) integrin-matrix interactions could lower tissue pressure. Based on these suppositions and data, we examined if these concepts had relevance to the brain. For this, we measured pressure in the brain and observed a decline in periventricular pressures to values significantly below those monitored in the ventricular system following the injection of the anti integrin antibodies. Many of the animals developed hydrocephalus over 2 weeks post antibody injection. These data provide a novel mechanism for the generation of intraparenchymal pressure gradients that is likely contributing to ventricular expansion.

Leukocyte adhesion to cerebral endothelial cells plays a critical role in the inflammatory response following transient global cerebral ischemia but its contribution to delayed neuronal cell death is not completely understood. We compared ischemic mice treated with a monoclonal antibody to β2-integrin adhesion receptors (anti-CD18) or a non-binding control antibody following ischemia. Inflammation was characterized by increased CD18 expression on leukocytes and inflammatory mediators in the peripheral blood and brain tissue. Notably, interleukin-1β, which has been shown to mediate cell death in neurons, was elevated in the blood and brain. Anti-CD18 blocked leukocyte adhesion as well as the inflammatory responses, including interleukin-1β expression in neurons. Blocking leukocyte adhesion protected the structural integrity of the hippocampus, cerebral cortex and thalamus, and preserved spatial. Leukocytes adhesion to endothelial cells plays an important role in the evolution of neurological deficit in global cerebral ischemia despite the lack of transmigration of leukocytes across blood-brain-barrier.

Leukocyte adhesion to cerebral endothelial cells plays a critical role in the inflammatory response following transient global cerebral ischemia but its contribution to delayed neuronal cell death is not completely understood. We compared ischemic mice treated with a monoclonal antibody to β2-integrin adhesion receptors (anti-CD18) or a non-binding control antibody following ischemia. Inflammation was characterized by increased CD18 expression on leukocytes and inflammatory mediators in the peripheral blood and brain tissue. Notably, interleukin-1β, which has been shown to mediate cell death in neurons, was elevated in the blood and brain. Anti-CD18 blocked leukocyte adhesion as well as the inflammatory responses, including interleukin-1β expression in neurons. Blocking leukocyte adhesion protected the structural integrity of the hippocampus, cerebral cortex and thalamus, and preserved spatial. Leukocytes adhesion to endothelial cells plays an important role in the evolution of neurological deficit in global cerebral ischemia despite the lack of transmigration of leukocytes across blood-brain-barrier.