No description available.

Thesis (Ph. D.)--Mechanical Engineering, Georgia Institute of Technology, 2007. / Giddens, Don P., Committee Co-Chair ; Zhu, Cheng, Committee Chair ; Garcia, Andres J., Committee Member ; Smith, Marc K., Committee Member ; McEver, Rodger P., Committee Member.

Obesity and insulin resistance are characterized by elevated pro-inflammatory proteins in the blood and immune cell accumulation in the visceral adipose tissue. Resident leukocytes release tumor necrosis factor α (TNFα) and other inflammatory cytokines which stimulate adipocyte lipolysis, recruit leukocytes to adipose tissue, promote pro-inflammatory immune cell polarization, facilitate oxidative stress, and activate intracellular kinases which dull insulin signaling cascades in metabolic tissues. Immune cell mediated dysregulation of stromal and parenchymal cells has raised suspicion that insulin resistance is an immune disorder initiated by activated white blood cells with over-nutrition. Efforts to improve pathological metabolism by reducing inflammation have yielded mixed results in humans and animal models. The role of inflammation and immune cell accumulation in the visceral fat (VF) in the progression of insulin resistance remains presently debated. There is, however, a consensus that identifying the triggers for obesity and impaired insulin signaling is of the utmost importance. The goal of this report is to identify dietary fat absorption as a key initiator of inflammatory action and insulin desensitization which may be dampened by reducing immune cell accumulation in adipose tissue. To explore how lean, healthy organisms become obese and insulin resistant, we examined the inflammatory consequences of isocaloric but variable macronutrient loads in the VF of lean mice. Mice were administered single liquid meals composed of low-fat (10% fat) or high-fat (60% fat) diet and observed by intravital microscopy to quantify leukocyte-endothelium interactions in mesenteric postcapillary venules (MPCV) 1, 2, 3, and 4 hours after oral gavage. Leukocyte rolling and leukocyte adhesion were transiently elevated within 1 hour after feeding and returned to baseline levels 4 hours later. Endothelial cell surface expression of P-selectin (Psel), a rapidly activated cell adhesion molecule (CAM), confirmed that high-fat feeding induced Psel dependent leukocyte rolling through the VF microcirculation. Furthermore, leukocyte accumulation in the VF was modestly increased by a single high-fat meal (HFM). Repetitive high-fat diet (HFD) consumption for 24 hours prolonged elevated leukocyte-endothelium interactions and promoted neutrophil accumulation in the VF. The neutrophilic enzyme myeloperoxidase (MPO), a producer of the chlorinating agent hypochlorous acid, increased in abundance and activity in the VF of HFM fed mice. Elevated leukocyte-endothelium interactions, leukocyte infiltration, and MPO activity in VF were not observed in Psel deficient (Psel-/-) mice following lipid overload. To ascertain if MPO is required for sustained endothelial activation, leukocyte-endothelium interactions and leukocyte infiltration were monitored in high-fat fed MPO deficient (MPO-/-) mice. Similar to the Psel-/- mice, MPO-/- mice were protected from the inflammatory effects of high-fat feeding. Our data supports postprandial hyperlipemia as an inducer of transient and Psel dependent inflammatory reactions that are sustained by prolonged HFD consumption. To study whether early phase inflammatory interventions granted late phase metabolic improvements, wild-type (WT), Psel deficient (Psel-/-), and MPO deficient (MPO-/-) C57BL/6 mice were given ad libitum access to LFD (10% fat) or HFD (60% fat) for 12-16 weeks. All mouse groups given HFD became obese. Prolonged HFD consumption sustained elevated leukocyte-endothelium interactions in MPCVs and was accompanied by increased local and systemic TNFα in WT mice. High-fat fed WT mice were hyperglycemic, hyperinsulinemic, glucose intolerant, and insulin resistant compared to LFD fed controls. Psel-/- mice were protected from leukocyte-endothelium interactions as well as local and systemic TNFα accumulation despite extended HFD consumption. Surprisingly, high-fat fed Psel-/- mice were equally hyperglycemic, hyperinsulinemic, glucose intolerant, and insulin resistant as the inflamed, high-fat fed WT mice. MPO-/- mice were also protected from elevated systemic TNFα and gained slightly less weight than the other high-fat fed groups. While MPO-/- mice were hyperglycemic and glucose intolerant, they did have improved insulin stimulated glucose clearance. The data presented in this report demonstrates the pro-inflammatory nature of postprandial hyperlipemia and the insulin desensitizing nature of prolonged HFD consumption. Ablation of VF immune cell accumulation by Psel deletion is not sufficient for improving insulin signaling or glycemic control, which is consistent with prior reports. Deletion of MPO, however, did result in slightly less obesity and marginally improved insulin signaling. We conclude that while immune cell accumulation in the VF contributes to the progression of insulin resistance, it is not a prerequisite for metabolic pathology development. / Physiology

No description available.

This thesis describes structural studies on the interactions between the fucosylated chondroitin sulfate (fCS) oligosaccharides and human proteins known as selectins. fCS is a carbohydrate obtained from sea cucumbers, that can be classified as a branched glycosaminoglycan (GAG). It has attracted much attention due to its anti-coagulant, anti-inflammatory, antimetastatic and anti-HIV properties and its structure was previously determined by NMR. Selectins constitute a family of proteins involved in cell adhesion processes, such as inflammation, attachment of viral particles and migration of tumour cells. fCS oligosaccharides have been shown to bind to selectins, which is likely a reason behind their biological activity. However, the mechanism of this interaction is currently unknown. The initial part of the thesis describes the experimental work on expression and purification of the recombinant L- and P-selectin constructs in Pichia pastoris, Escherichia coli and HEK 293 cells. The aim of these experiments was to produce two constructs for each selectin, a single domain construct, consisting of the C-type lectin domain only, and a double domain construct, consisting of both the C-type lectin and the EGF-like domains. The intention was that the recombinant proteins would be labelled with 13C and 15N to allow for the in-depth structural NMR studies on the fCS-selectin interaction. Various experimental approaches have been explored, including the use of different cell lines, modifications to construct design, as well as alterations to expression and purification conditions. Although it was not possible to produce soluble selectin constructs in either bacterial or yeast cells, protein expression tests in HEK293 cells, performed in collaboration with the Oxford Protein Production facility (OPPF), led to production of a soluble L-selectin construct, consisting of the L-selectin C-type lectin domain. The produced L-selectin construct, as well as two commercially available constructs of the Land P-selectin extracellular domains, were used in the Saturation Transfer Difference (STD) NMR experiments to provide new information about the nature of the fCS-selectin binding. The STD experiments allowed to identify the regions within the fCS oligosaccharides that are in direct contact with the protein and likely play an important role in this interaction. Experiments on different protein constructs allowed the comparison of fCS binding to P-selectin and to two different recombinant constructs of L-selectin. Results of these studies suggest that the binding occurs via a similar mechanism for both L- and P-selectins and that the fCS oligosaccharides bind to one-domain L-selectin construct with similar affinity as to a larger construct, consisting of the entire extracellular region of the protein. Alongside the experimental work, theoretical in silico studies on the fCS-selectin binding were undertaken as part of this project. The existing X-ray structures of selectin complexes were subjected to Molecular Dynamics (MD) simulations, which allowed to explore the dynamic behaviour of E-selectin upon binding to sialyl Lewis x (sLex). It was found that sLex forms a more favourable interaction with the extended conformation of E-selectin and that the protein in this conformation is characterised by a high degree of interdomain flexibility, with a new type of interdomain movement observed in the MD studies on this complex. In further in silico studies, the fCS oligosaccharides were docked to the existing P-selectin structures. The docking tests were performed on the computationally produced fCS trisaccharides with fucose branches either 2,4 or 3,4-sulfated. Results were evaluated with MD simulations and analysed in the light of current knowledge of selectin-ligand binding and the STD NMR experimental results. The in silico studies allowed to identify a subset of P-selectin residues that are likely involved in the interaction with fCS oligosaccharides in vivo. The conformational behaviour of P-selectin upon binding to fCS was also explored and it was found that the interdomain hinge is flexible during this interaction and allows transition from bent to extended conformational state. Finally, a new NMR method was developed to facilitate the studies of complex carbohydrates, incorporating the concepts of G-matrix Fourier Transform (GFT) NMR into 2D HSQC and 2D HSQC-TOCSY experiments. The method allows to separate peaks in the regions of high spectral overlap, providing information that can simplify the assignment process. The new experiments facilitated the structural evaluation of a sample containing a mixture of oligosaccharides resulting from the depolymerisation of fCS polysaccharide.

During inflammation and thrombosis, leukocytes tether to and roll on vascular surfaces and platelets through selectin molecules under shear flow. This selectin family of cell adhesion molecules includes P-, E-, and L-selectin. The association and dissociation of two or more selectin-mediated bonds under mechanical load produce the rolling motion of the leukocytes. Although much has been uncovered about the properties of selectins, the complete story of the selectin-mediated adhesion process is yet to be told. The goal of this research is to gain a more quantitative understanding of this receptor-ligand binding through the study of the dissociation kinetics of E-selectin-mediated adhesion using flow chamber techniques. From transient tethering experiments, the dissociation rate of E-selectin-mediated adhesion was found to have a triphasic shear dependence at low shear stresses, where the bond transitioned from a slip to a catch then again to a slip bond. This trend was further supported by observations of the average rolling velocity of cells adhering to E-selectin at various shear stresses. A triphasic force dependence of the rolling velocity was revealed that showed that regions of increasing rolling velocity corresponded to the slip bond regime where tether lifetime decreased with increasing shear stress. Decreasing rolling velocity coincided with the catch bond regime, a regime of prolonged tether lifetime with increasing shear stress. An invertible flow chamber was used in hopes of directly quantifying the dissociation rate of rollingly adherent cells on E-selectin to compare it to the dissociation rate data obtained through transient tethering experiments. However, tether formation, which relates to the association rate, and its role in the stability of rolling seemed to be a key factor in the dissociation rate of rollingly adherent cells over the low shear stress range. Overall, these results provide supporting evidence of a shear threshold for E-selectin as well as data to suggest that tether formation, in coordination with off-rate, determine the rolling velocity behavior of cells on E-selectin substrates.

The human body is an intricate network of many highly regulated biochemical processes and cell adhesion is one of them. Cell adhesion is mediated by specific interactions between molecules on apposing cell surfaces and is critical to many physiological and pathological processes like inflammation and cancer metastasis. During inflammation, blood-borne circulating leukocytes regularly stick to and roll on the vessel walls, which consist in part, adhesive contacts mediated by the selectin family of adhesion receptors (P-, E- and L-selectin). This is the beginning of a multi-step cascade that ultimately leads to leukocyte recruitment in areas of injury or infection. In vivo, selectin-mediated interactions take place in a hydrodynamic milieu and hence, it becomes imperative to study these interactions under very similar conditions in vitro. The goal of this project was to characterize the kinetic and mechanical properties of selectin interactions with different physiologically relevant ligands and selectin-specific monoclonal antibodies (mAbs) under a mechanically stressful milieu, using atomic force microscopy (AFM). Elasticity studies revealed that bulk of the complex compliance came from the selectins, with the ligands or mAbs acting as relatively stiffer components in the stretch experiments. Furthermore, molecular elasticity was inversely related to selectin length with the Consensus Repeats (CRs) behaving as Hookean springs in series. Besides, monomeric vs. dimeric interactions could be clearly distinguished from the elasticity measurements. L-selectin dissociation studies with P-selectin Glycoprotein Ligand 1 (PSGL-1) and Endoglycan revealed that catch bonds operated at low forces while slip bonds were observed at higher forces. These results were consistent with previous P-selectin studies and suggested that catch bonds could contribute to the shear threshold for L-selectin-mediated rolling By contrast, only slip bonds were observed for L-selectin-antibody interactions, suggesting that catch bonds could be a common characteristic of selectin-ligand interactions. Force History studies revealed that off-rates of L-selectin-sPSGL-1 (or 2-GSP-6) interactions were not just dependent on applied force, as has been widely accepted but in fact, depended on the entire history of force application, thus providing a new paradigm for how force could regulate bio-molecular interactions. Characterizing selectin-ligand interactions at the molecular level, devoid of cellular contributions, is essential in understanding the role played by molecular properties in leukocyte adhesion kinetics. In this aspect, data obtained from this project will not only add to the existing body of knowledge but also provide new insights into mechanisms by which selectins initiate leukocyte adhesion in shear.

Diss. (sammanfattning) Linköping : Univ., 2003. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 83-95).

La glycosylation est un processus enzymatique permettant l'ajout de sucres à des composés (sucres, lipides ou protides), modifiant ainsi leurs propriétés. La glycosylation est impliquée dans la détoxification des xénobiotiques et des variations d'activité des enzymes responsables ont été identifiées comme facteur de risque de cancer en particulier dans les organes exposés aux xénobiotiques. Dans la première partie de notre travail nous étudions l'impact des polymorphismes génétiques de certaines enzymes responsables de la détoxification (UGT1A7, GST et XRCC1) sur le risque de carcinome hépatocellulaire. Nous montrons que la combinaison de certains polymorphismes génétiques peut entraîner une augmentation du risque de CHC. Des modifications d'expression des glycoprotéines de surface ont été observées dans les cellules cancéreuses jouant un rôle dans leurs interactions avec le microenvironnement. Dans la seconde partie, nous étudions l'effet de l'inhibition des interactions des cellules de CHC/cellules endothéliales par le blocage du couple sialyl Lewis x/E-sélectine sur la croissance tumorale. Ce blocage est obtenu, d'une part par transfert du gène de la Fucosyl-transferase I, inhibant l'expression de sLex à la surface des cellules de CHC, et d'autre part, par utilisation de cimétidine ou d'amiloride permettant une inhibition de l'expression de la E-sélectine par les cellules endothéliales. Nous obtenons une inhibition de la croissance tumorale in vivo par blocage de la néoangiogénèse. Ces travaux permettent donc d'identifier des facteurs de risque génétiques de CHC et d'envisager une autre voie de traitement du CHC. / Glycosylation is an enzymatic process that consists of the addition of glycosyl groups to compounds (sugars, lipids or proteins), thus modifying their properties. Glycosylation is involved in the detoxification of xenobiotics and variations in activity of enzymes responsible have been identified as a potential risk factor for cancer in particular in organs in contact with the external environment. In the first part of our work we study the impact of polymorphisms of detoxification enzyme (UGT1A7, GST and XRCC1) on the risk of hepatocellular carcinoma. We show that the combination of genetic polymorphisms of such enzymes may increase the risk of HCC. Modifications in the expression of surface glycoproteins have been observed in cancer cells and play a role in their interactions with the tumoral microenvironment. In the second part, we study the effect of inhibition of interactions of HCC cells / endothelial cells on tumor growth by blocking the interaction between sialyl Lewis x and E-selectin. First, we achieved the inhibition of the expression of sLex on the surface of HCC cells by introducing fucosyl transferase- I gene in HCC cells. In a second part of our work we used cimetidine and amiloride to inhibit the expression of E-selectin by endothelial cells. This approach resulted in inhibition of HCC cells / endothelial cells interaction and thereby tumor growth inhibition in vivo. This effect is mediated by an inhibition of tumor neoangiogenesis. This work therefore identifies genetic risk factors for HCC and allows considering another way of treatment of HCC.