This thesis examined the function of the urinary bladder in vivo in the freshwater rainbow trout. In the first part of the study two new techniques were developed to examine the possible urine storage and ionoregulatory roles of the bladder in vivo. An indirect approach, using non-catheterized fish, involved "spot sampling" from the bladder to determine urine composition, and measurement of the appearance of ^3H polyethylene glycol-4000 (a glomerular filtration marker) in surrounding water to quantify urination events. The direct approach employed a new external catheterization technique to collect naturally discharged urine. Both methods demonstrated that resting trout urinate in intermittent bursts at 20-30 min intervals, and that natural urine flow rate (U.F.R.) is at least 20 % lower and urinary Na^+ and Cl^- excretion rates at least 40% lower than determined by the traditional internal bladder catheter technique. The urine is stored for approximately 25 min prior to discharge, and significant reabsorption of water and ions (Na^+, Cl^-, K^+, urea, and possibly other substances) occurs via the bladder epithelium during this period; a small residual volume is likely always maintained. The second part of the study employed the new external catheter and the traditional internal catheter to quantify the responses of the bladder, relative to those of the kidney, to two experimental disturbances. Chronic (32 h) infusion with 140 mM NaCl produced isosmotic volume loading without a change in plasma [Na^+], [Cl^-], or acid-base status. The kidney responded with a large increase in glomerular filtration rate (G.F.R.), a smaller increase in U.F.R., and increased reabsorption of water and ions. The bladder responded with a small increase in urination burst volume, a larger increase in burst frequency, and therefore a decreased urine storage time. Despite this increased throughput, Na^+ and Cl^- reabsorption rates across the bladder epithelium actually increased. Reabsorption of urea and K^+ remained constant, despite expected decreases due to decreased urine storage time. A similar infusion with 140 mM NaHCO_3 produced isosmotic volume loading together with metabolic alkalosis reflected m increased blood pH, increased plasma [HCO_3^-], decreased plasma [Cl^-], with no change in plasma [Na^+]. The response of the kidney was similar, though HCO_3^- filtration, reabsorption, and excretion rates all increased, while rates for Cl^- were proportionately lowered; renal Na^+ handling was unaffected. Bladder urination patterns and Na^+ reabsorption were also similar, but there was no evidence of bladder involvement in HCO_3^- secretion or reabsorption (ie. in acid-base regulation). It is concluded that previous studies using internal catheterization have greatly underestimated the ionoregulatory effectiveness of the entire renal system by negating bladder function. The external catheterization technique developed in this thesis provides researchers with a method to collect naturally vented urine, and thereby evaluate the role of the entire renal system, including the bladder, in response to experimental manipulations. / Thesis / Master of Science (MS)

The use of polymeric biomaterials in regenerative medicine and drug delivery is a continually growing practice. Alginic acid (alginate) is widely used in these fields because of its beneficial properties from an engineering and mechanical perspective. Still, alginate has not yet been fully investigated from a biological perspective. For disciplines that anticipate in vivo use of their devices, it is crucial to understand the biological interactions between the device and the host. In this project, the in vitro and in vivo immunological effects of alginate are examined in two model systems: one with a protein antigen and one with a xenogeneic cell antigen. The former system is used as a proof of principle study for alginate's immunological effect on simple protein-based systems, similar to those found in protein/drug deli very applications and certain types of vaccines. This model uses bovine serum albumin (BSA) as the protein antigen. The latter system is used to demonstrate alginate's effect on more complex antigens, such as whole cells. Thus, Chinese hamster ovary (CHO) cells are used as the as the cell antigen. This model represents a system that may be found in tissue engineering applications, where whole cells are delivered with a biomaterial scaffold. Antibody production from blood serum indicated that alginate solution has adjuvant abilities while alginate microspheres do not. Thus, alginate solution possesses great potential in the field of vaccines. In addition, in vivo alginate challenges were found to have effects on second-set responses of splenocytes to in vitro alginate and antigen challenges. Splenocytes from alginate-injected mice were overall equally or less responsive to in vitro challenges than splenocytes without previous alginate immunization. Therefore, alginate solution may also have immunosuppressive effects, although the results from this project merely speculate on this possibility. Still, this ability would be helpful in overcoming current transplantation problems as well as certain tissue engineering hurdles. / Thesis / Master of Science (MS)

Aluminum is a neurotoxin and has been recognized as a causative agent for dialysis encephalopathy and renal osteodystrophy, as well as possibly being related to Alzheimer's disease. General public exposures to aluminum have increased in the modern, industrial age stimulating scientists to inquire into the degree of risk associated with such widespread use of aluminum. Aluminum is thought to be stored in bone, therefore, development of an in vivo method for the determination of aluminum a in human bone, suitable for routine monitoring of patients and population is the goal of this study. Using neutron activation analysis, low-energy neutrons are produced on the KN accelerator inducing the 27Al(n,y)28AI reaction in an irradiated site. Two different shapes (cylindrical and flat) of aluminum doped tissue equivalent phantoms, simulating both bone and soft tissue, have been built. Calibration lines, detection limits and doses delivered with the different shapes of phantom have been discussed, and compared to the previously published results. Two detection systems. an assembly of two large Nal{Tl) detectors and a hyperpure germani Jm detector, have been compared as well. The results achieved suggest that this technique may provide an alternative choice to painful bone biopsy for the in vivo monitoring of aluminum intoxication from long-term exposure. / Thesis / Master of Science (MS)

The purpose of this research was to further test a stable isotope based approach as a more reliable in vivo method to determine amino acid bioavailability from a variety of ingredients. The method was used to assess feather meal (FM), blood meal (BM), soybean meal (SBM), and a rumen protected amino acid (RPAA). An abomasal infusion of raw EAAs (isoleucine, leucine and methionine) and an abomasal infusion of sodium caseinate were used as control treatments to test the accuracy of the technique. The isotope-based results were then compared to in situ, in vitro and in vivo test results. The isotope-based technique provided AA bioavailability values for five non-essential AA and seven essential AA. The raw EAA infusion had the greatest AA recovery in plasma with an estimated absorbed RUP value of 93.4± 7.35% followed by the casein infusion (86.7 ± 4.81%), SBM (54.8 ± 5.19%), FM (52.7 ± 4.81%) and BM (47.5 ± 4.81%). The RPAA treatment had the lowest bioavailability at 9.9 ± 12.73%. Numerically, SBM supplied the most absorbable EAA of the 4 feed ingredients, but was not significantly different from that of BM and FM. Simply based on the control treatments in this research (raw EAA and casein), this isotope method was a more accurate method in determining AA bioavailability values with relatively low standard errors. Ingredients are exposed to all aspects of natural digestive processes and the method is able to determine AA appearance in the blood with no use of in situ or in vitro measurements. / Master of Science

Marine mammals are known for their low susceptibility to mercury toxicity, and it was hypothesized that selenium may play a role in protection against mercury toxicity. To gain insight into the mechanisms of the low susceptibility of cetaceans, we investigated the in vitro effects (1) of mercuric chloride (HgCl₂) on the ultrastructure and cell death of Atlantic spotted dolphin renal cells (Sp1K cells), (2) of HgCl₂ on the cell proliferation and cell cycle status of Sp1K and Rhesus monkey renal cells (MK2), and (3) of sodium selenite (Na₂SeO₃) on cell proliferation and cell death of control and HgCl₂-treated Sp1K cells. HgCl₂ affected multiple organelles and nuclei in Sp1K cells, and induced apoptosis in a time-and dose-dependent manner. Both ultrastructural changes and induction of apoptosis were milder than seen in other cell types in previous publications. In addition, Sp1K cells were able to proliferate at 25 µM HgCl₂ while MK2 cells were killed at 15 µM HgCl₂. An increase in percentage of cells in the G0/G1 phase in the cell cycle and a decrease in S, and G2/M phase cells were seen in Sp1K cells exposed to more than 10 uM HgCl₂ more than 72 hours. MK2 cells showed cell cycle changes only at 24 hours exposure, and may be due to a sensitive subgroup. These data suggested that Sp1K cells were less susceptible than other cell types in a cell-specific way, which was independent of selenium protection. Concurrent exposure to Na₂SeO₃ provided protection against the HgCl₂-induced decrease in cell proliferation of Sp1K. The protective effects were greater if Na₂SeO₃ and HgCl₂ were premixed, but disappeared if exposures did not overlap. Although pretreatments with Na₂SeO₃ alone did not provide protection, they increased the protection of selenium administered later. Furthermore, Na₂SeO₃ decreased HgCl₂-induced apoptosis. These data demonstrated the Na₂SeO₃ protection against HgCl₂ toxicity in Sp1K cells in terms of cell proliferation and apoptosis. This study is the first report that reveals the existence of mercury-selenium antagonism in cultured cetacean cells. The data supported the hypothesis that selenium protection against mercury toxicity is, at least partially, through competition of binding sites and formation of mercury-selenium complex. / Master of Science

Many medical therapies, such as thermal tumor detection and hypothermia cancer treatments, utilize heat transfer mechanisms of the body. The focus of this work is the development and experimental validation of a method to simultaneously estimate thermal conductivity, volumetric specific heat, and perfusion of in-vivo tissue. The heat transfer through the tissue was modeled using a modified Pennes' equation. Using a least-squares parameter estimation method with regularization, the thermal properties could be estimated from the temperature response to the known applied heat flux. The method was tested experimentally using a new agar-water tissue phantom designed for this purpose. A total of 40 tests were performed. The results of the experiments show that conductivity can be successfully estimated for perfused tissue phantoms. The values returned for volumetric specific heat are lower than expected, while the estimated values of perfusion are far greater than expected. It is believed that the mathematical model is incorrectly accounting between these two terms. Both terms were treated as heat sinks, so it is conceivable that it is not discriminating between them correctly. Although the method can estimate all three parameters simultaneously, but it seems that the mathematical model is not accurately describing the system. In the future, improvements to the model could be made to allow the method to function accurately. / Master of Science

No / Purpose: The hollow fiber assay is used successfully as a routine in vivo screening model to quantitatively define anticancer activity by the National Cancer Institute. This study investigates whether the hollow fiber assay can be used as a short-term in vivo model to demonstrate specific pharmacodynamic end points, namely microtubule and cell cycle disruption. Experimental Design: The growth of A549 cells was characterized within hollow fibers over 5 days in vivo at both subcutaneous (s.c.) and intraperitoneal (i.p.) sites. Drugs were administered on day 4 (i.p.). Results: At 24 hours, cells were retrieved from fibers at both i.p. and s.c. sites of paclitaxel-treated (20 mg/kg) and combretastatin A1 phosphate¿treated (150 mg/kg) mice. Cell cycle analysis after paclitaxel treatment revealed a mean G2-M phase population of 48.04% (i.p.) and 25.76% (s.c.) compared with vehicle group mice (6.78 and 5.56%, respectively; P = <0.001 and 0.005, respectively). Tumor cells retrieved from combretastatin A1 phosphate¿treated mice had a mean G2-M phase population of 36.3% (i.p.) and 29.36% (s.c.) compared with cells retrieved from vehicle group mice (5.58 and 5.49%, respectively; P = <0.001). Using fluorescence and laser-confocal microscopy, paclitaxel was revealed to induce the formation of spindle asters and tubulin polymerization. Combretastatin A1 phosphate was shown to hold cells in mitosis. Changes in nuclear morphology were also observed. Conclusion: These data demonstrate that the hollow fiber assay can be used as a short-term in vivo model for studying the pharmacodynamic effects of both standard and novel compounds on microtubules. Evidence has also been provided to support the routine use of the in vivo hollow fiber assay for demonstrating the mechanism of action of a drug.

No / Background: In previous experiments, we showed that heparin oligosaccharides inhibit the angiogenic cytokine fibroblast growth factor-2. Here, we present the first in vivo study of size-fractionated heparin oligosaccharides in four models of angiogenesis that are progressively less dependent on fibroblast growth factor-2. Experimental Design: Heparin oligosaccharides were prepared using size-exclusion gel filtration chromatography and characterized through depolymerization and strong anion exchange high-performance liquid chromatography. Size-defined oligosaccharides (20 mg/kg/d) were given to mice bearing s.c. sponges that were injected with fibroblast growth factor-2 (100 ng/d). After 14 days, octasaccharides and decasaccharides reduced the microvessel density to levels below control. In a second experiment, HEC-FGF2 human endometrial cancer cells that overexpress fibroblast growth factor-2 were implanted in a hollow fiber placed s.c. in vivo. Oligosaccharides were given at 20 mg/kg/d for 2 weeks and the data again showed that octasaccharides significantly reduced microvessel density around the fiber (P = 0.03). In a more complex model, where angiogenesis was induced by a broad spectrum of growth factors, including vascular endothelial growth factor, we implanted H460 lung carcinoma cells in hollow fibers and treated the animals with oligosaccharides at 20 mg/kg/d over 3 weeks. Octasaccharides reduced the microvessel density to that of control. Preliminary investigation of 6-O-desulfated heparins showed that these also had antiangiogenic activity. Results: Finally, we examined the inhibitory potential of hexasaccharides and octasaccharides given at 20 mg/kg/d and these inhibited the growth of H460 lung carcinoma in vivo. At clinically attainable concentrations, significant anticoagulation (activated partial thromboplastin time, anti-factor Xa, and anti-factor IIa) was not observed in vitro unless species containing 16 saccharide residues were investigated. Conclusions: Thus, our preclinical data show that heparin octasaccharides represent novel antiangiogenic compounds that can be given without the anticoagulant effects of low molecular weight heparin.

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Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biophysics and Physical Chemistry Candidate: Denisa Mazouchová Supervizor: Mgr. Monika Kuchařová, Ph.D. Title of diploma thesis: Effect of ultraviolet radiation on the change the mechanical parameters of human skin This diploma thesis deals in theoretical part with the properties of human skin, the characteristics of the UV radiation and its effects on human skin. The practical part of the thesis focuses on the in vivo measurement of mechanical parameters of human skin after UV radiation. Mechanical characteristics of human skin were measured in 12 subjects before the application of UV radiation, immediately after application of UV and after 24 hours of rest skin. The experiment used a UV lamp with a wavelength of 370 nm emitted light. The monitored parameters were Hooke's and Newton's coefficients. Hooke's coefficient gives us the stiffness of the skin as a whole , Newton coefficient characterises its viscosity. For measurement of those parameters was used dynamic viscoelastomer. The experiment has been found out that no signifiant ganges of Hooke's and Newton's coefficients were not found immediately after the application of UV radiation of wavelength 370 nm, in 12 volunteers. Significant change occurred only in the...