Lipoprotein lipase (LPL) is an enzyme to hydrolyze triglycerides in lipoproteins and thereby make the fatty acids available for cellular metabolic reactions. Short-term fasting down-regulates LPL activity in adipose tissue. This regulation is through post-translational mechanism. The objective of this work was to investigate (1) The molecular mechansim for regulation of LPL activity in adipose tissue; (2) The basis for the tissue-specific regulation of LPL in adipose tissue, heart and skeletal muscle. LPL in adipose tissue can be found both inside (intracellular) and outside adipocytes (extracellular). Within adipocytes, neither LPL mass nor the distribution of LPL between active and inactive forms changed on fasting. Extracellular LPL mass also did not change significantly, but shifted from predominantly active to predominantly inactive. Activie, extracellular LPL was distributed in a similar way in the two nutritional states. The down-regulation during fasting is due to a decline of extracellular LPL activity. The up-regulation of LPL activity induced by re-feeding did not need new mRNA. The down-regulation of LPL activity induced by fasting did not occur when mRNA synthesis was inhibited. LPL activity in adipose tissue from fasted rats was fully restored by actinomycin. So fasting switches on a gene, whose product suppresses LPL activity. Similar results were also obtained in experiments on mice. When food was removed from young rats in the early morning, adipose tissue TNF-α activity increased and LPL activity decreased within six hours. There was a negative correlation between TNF-α and LPL activities. Pentoxifylline, that inhibits biosynthesis of TNF-α, almost abolished the rise of TNF-α and the decrease of LPL activity. Actinomycin D virtually abolished the response of LPL activity to fasting or exogenous TNF-α. This study suggests that fasting signals via TNF-α to a gene whose product causes a rapid shift of newly-synthesized LPL molecules towards an inactive form.

During the past decade clinical islet transplantation has become a viable strategy for curing type 1 diabetes. The limited supply of organs, together with the requirement for islets from multiple donors to achieve insulin independence, has greatly limited the application of this approach. The islets are infused into the liver via the portal vein, and once exposed to the blood, the grafted tissue has been shown to be damaged by the instant blood-mediated inflammatory reaction (IBMIR), which is characterized by coagulation and complement activation as well as leukocyte infiltration into the islets. Islet revascularization is a subsequent critical step for the long-term function of the transplanted graft, which may partially be impeded by the IBMIR. In this thesis, we have explored novel strategies for circumventing the effects of the IBMIR and facilitating islet revascularization. Systemic inhibitors of the IBMIR are typically associated with an increased risk of bleeding. We therefore evaluated alternative strategies for modulating the islets prior to transplantation. We demonstrated, using an adenoviral vector, that a high level of expression and secretion of the anticoagulant hirudin could be induced in human islets. An alternative approach to limiting the IBMIR was developed in which anticoagulant macromolecular heparin complexes were conjugated to the islet surface. This technique proved effective in limiting the IBMIR in both an in vitro blood loop model and an allogeneic porcine model of islet transplantation. An increased adhesion of endothelial cells to the heparin-coated islet surface was demonstrated, as was the capacity of the heparin conjugate to bind the angiogenic factors VEGF and FGF; these results have important implications for the revascularization process. The outcome of the work in this thesis suggests that modulation of the islet surface is an attractive alternative to systemic therapy as a strategy for preventing the IBMIR. Moreover, the same techniques can be employed to induce revascularization and improve the engraftment of the transplanted islets. Ultimately, improved islet viability and engraftment will make islet transplantation a more effective procedure and increase the number of patients whose diabetes can be cured.

Long-term survival of implanted cells requires oxygen and nutrients, the need for which is met by vasculari- zation of the implant. The use of scaffolds with surface-attached heparin as anchoring points for angiogenic growth factors has been reported to improve this process. We examined the potential role of surface modification of gelatin scaffolds in promoting endothelial cell infiltration by using a unique macromolecular conjugate of heparin as a coating. Compared to other heparin coatings, this surface modification provides flexible heparin chains, representing a new concept in heparin conjugation. In vitro cell infiltration of scaffolds was assessed using a three-dimensional model in which the novel heparin surface, without growth factors, showed a 2.5-fold increase in the number of infiltrating endothelial cells when compared to control scaffolds. No additional improvement was achieved by adding growth factors (vascular endothelial growth factor and/or fibroblast growth factor-2) to the scaffold. In vivo experiments confirmed these results and also showed that the addition of angiogenic growth factors did not significantly increase the endothelial cell infiltration but increased the number of inflammatory cells in the implanted scaffolds. The endothelial cell-stimulating ability of the heparin surface alone, combined with its growth factor-binding capacity, renders it an interesting candidate surface treatment to create a prevascularized site prepared for implantation of cells and tissues, in particular those sensitive to inflammation but in need of supportive revascularization, such as pancreatic islets of Langerhans. / <p>De två sista författarna delar sistaförfattarskapet.</p>

Relative changes are detectable in the blood of end-stage renal disease (ESRD) patients during hemodialysis (HD) treatment using optical spectroscopy. However, the potential impacts of several confounding factors that could affect the detection of these changes have not been evaluated. The objectives of this thesis were to: 1) investigate how the variations and/or changes in acid-base and oxygen parameters during HD treatment can affect the optical signature of whole blood of ESRD patients, 2) to investigate the effect of heparin on the optical properties of whole blood and its impact on our method. Blood samples were drawn from 23 ESRD patients at 5 time points during a 4 hour HD treatment and sent for blood gas and blood spectroscopy analyses. No significant correlations were found between the changes in the blood transmittance spectra and acid-base and oxygen parameters. This indicates that the perturbations in these parameters due to HD procedures do not confound the detection of changes in the blood transmittance spectra of ESRD patients during HD treatment. Additionally, the effect of heparin in modifying the optical properties of whole blood does not confound the detection of changes in the blood of ESRD patients due to HD treatment using whole blood-based optical spectroscopy. ANOVA revealed significant (P<0.05) measurable changes in the blood transmittance spectra of ESRD patients during HD treatment. Significant spectral differences (P<0.05) were found between ESRD patients. The lack of uniform spectral characteristics across patients is

Relative changes are detectable in the blood of end-stage renal disease (ESRD) patients during hemodialysis (HD) treatment using optical spectroscopy. However, the potential impacts of several confounding factors that could affect the detection of these changes have not been evaluated. The objectives of this thesis were to: 1) investigate how the variations and/or changes in acid-base and oxygen parameters during HD treatment can affect the optical signature of whole blood of ESRD patients, 2) to investigate the effect of heparin on the optical properties of whole blood and its impact on our method. Blood samples were drawn from 23 ESRD patients at 5 time points during a 4 hour HD treatment and sent for blood gas and blood spectroscopy analyses. No significant correlations were found between the changes in the blood transmittance spectra and acid-base and oxygen parameters. This indicates that the perturbations in these parameters due to HD procedures do not confound the detection of changes in the blood transmittance spectra of ESRD patients during HD treatment. Additionally, the effect of heparin in modifying the optical properties of whole blood does not confound the detection of changes in the blood of ESRD patients due to HD treatment using whole blood-based optical spectroscopy. ANOVA revealed significant (P<0.05) measurable changes in the blood transmittance spectra of ESRD patients during HD treatment. Significant spectral differences (P<0.05) were found between ESRD patients. The lack of uniform spectral characteristics across patients is

<p>Mast cells, best known for their pathologic role in allergy, have recently been shown to have key roles in the initiation of adaptive immune responses. These cells are located throughout the body just beneath barriers separating host from environment, possess multiple pathogen recognition systems, and store large quantities of fully active inflammatory mediators. These key features make them uniquely situated to act as sentinels of immunity, releasing the very earliest alarm signals when a pathogen is present. As a testament to the importance of these cells, mast cell-deficient mice have suboptimal immune responses, and mast cell activators can act as potent adjuvants for experimental immunizations. Specifically, mast cells have been shown to enhance the number of naive lymphocytes in infection site-draining lymph nodes, and to encourage the migration of dendritic cells to responding lymph nodes.</p><p>Although infections usually occur at peripheral sites, adaptive immune responses are initiated in distant lymph nodes. Despite the distance, signals from the site of infection result in dramatic, rapid reorganization of the node, including massive recruitment of naive lymphocytes from the circulation and extensive vascular restructuring to accommodate the increase in size. How such signals reach the lymph node is not well understood.</p><p>When mast cells degranulate, in addition to releasing soluble mediators such as histamine, they expel large, stable, insoluble particles composed primarily of heparin and cationic proteins. The work presented herein demonstrates that these particles act as extracellular chaperones for inflammatory mediators, protecting them from dilution into the interstitial space, degradation, and interaction with non-target host cells and molecules. The data show clearly that mast cells release such particles, that they are highly stable, that they contain tumor necrosis factor (a critically important immunomodulator), and that they can traffic from peripheral sites to draining lymph nodes via lymphatic vessels. Furthermore, extensive biochemical characterization of purified mast cell-derived particles was performed. Finally, evidence is presented that such particles can elicit lymph node enlargement, an infection-associated phenomenon that favors the development of adaptive immunity, by delivering peripheral TNF to draining lymph nodes. </p><p>This signaling concept, that particles may chaperone signals between distant sites, also has important implications for adjuvant design. The evidence presented here shows that encapsulation of TNF into synthetic particles similar to mast cell-derived particles greatly enhances its potency for eliciting lymph node enlargement, an indication that adaptive immunity may be improved. This delivery system should ensure that more adjuvant arrives in the draining lymph node intact, where it would lead to changes favorable to the development of the immune response. Such a system would also facilitate the delivery of multi-component adjuvants that would act synergistically at the level of the lymph node when gradually released from microparticle carriers. An additional advantage of microparticle encapsulation is that vaccine formulations of this type may require much lower doses of expensive antigen and adjuvants.</p><p>The delivery of inflammatory mediators to lymph nodes during immune responses may be an important general feature of host defense. Although the action of mediators of peripheral origin on draining lymph nodes has been described before, this is the first demonstration of a specific adaptation to deliver such mediators. Not only is the characterization of mast cell-derived particles important to basic immunology, but mimicking this adaptation may also lead to improved therapeutics.</p> / Dissertation

Thrombus formation and blood coagulation is a major problem associated with blood contacting products such as catheters, vascular grafts and artificial hearts. An intense research is being conducted towards the synthesis of new hemocompatible materials and mdifications of surfaces with biological molecules. In this study, polyurethane (PU) films were synthesized in medical purity from diisocyanate and polyol without using any other ingredients and the chemical, thermal and mechanical properties were characterized by solid state NMR, FTIR, GPC, mechanical tests, DMA and TGA. The surfaces of PU films were modified by covalent immobilization of different molecular weight heparins / low molecular weight heparin (LMWH) and unfractionated heparin (UFH) and these surfaces were examined by ESCA, ATR-FTIR, AFM and contact angle goniometer. Cell adhesion studies were conducted with whole human blood and examined by SEM. The effects of different types of heparins on blood protein adsorption and on platelet adhesion were analyzed by electrophresis and SEM, respectively. The surfaces of the UFH immobilized polyurethane films (PU-UFH) resulted in lesser red blood cell adhesion in comparison to LMWH immobilized polyurethane film surfaces (PU-LMWH). When the PU films were treated with blood plasma, the surfaces modified with two different heparin types showed a clearly different protein adsorption behavior especially in the early stage of blood plasma interaction. PU-LMWH samples showed about three times less protein adsorption compared to PU-UFH samples. The morphologies of platelets adhered on material surfaces demonstrated differences / such as PU-UFH had clusters with some pseudopodia extensions, while PU-LMWH had round shaped platelets with little clustering. PU surfaces modified by immobilization of LMWH and UFH, demonstrated promising results for the improvement of non-thrombogenic devices and surfaces.

The desirable properties of polyurethanes (PUs) such as mechanical flexibility associated with chemical versatility make these polymers attractive in the development of biomedical devices. In this study, various segmented polyurethanes were synthesized through polymerization reactions between polycaprolactone (PCL) diol or triol and excess hexamethylene diisocyanate (HDI) with varying NCO/OH ratios and the effect of composition on the properties of the resultant polyurethane films were examined. Initially, isocyanate terminated prepolymers were synthesized through one-shot polymerization, and then these prepolymers were cured by introducing crosslinkages into the structure and thus PUs were obtained. In order to enhance biocompatibility and hydrophilicity of the resulting polymers, heparin was added into the prepolymer before the curing process. The influence of excess HDI as a crosslinker on the degree of H-bond formation between hard-hard segments or hard-soft segments was examined by using Fourier transform infrared-Attenuated total reflectance spectroscopy (FTIR-ATR). Also the effects of HDI content on the chemical, physical and mechanical properties of the polyurethanes were examined with differential scanning calorimetry (DSC), X-Ray diffraction spectroscopy (XRD), dynamic mechanical analyzer (DMA), mechanical tester and goniometer. FTIR- ATR, DSC and DMA analyses showed that use of triol resulted in better network formation and homogenous distribution of hard segments within soft segment matrix. Incorporation of heparin into the polymer matrix produced more hydrophilic films (water contact angle reduced from 80 to 60). Polyurethanes from PCL and HDI in the absence of any solvent, initiator, catalyst or chain extender were successfully synthesized and this kind of synthesis enhanced biocompatibility and increased the potential of polymers for use in biomedical applications.

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It is the first time in Lithuania that practical application of semen filtration by Sephadex G-15 for the needs of artificial insemination industry was assessed. The efficacy of semen filtration was assessed applying a battery of sperm quality assays to study bovine spermatozoa quality before and after filtration, as well as after cryopreservation. The estimation of effect of different concentrations of progesterone, oestradiol, heparin separately or in combination, on different functional parameters of bull spermatozoa after thawing was performed. These are the first published studies applying integrated approach to study the effects of sperm challenge with several biologically active substances.