In this work, aldehyde-functionalized chitosan was produced by the reaction of chitosan in the solid state with nitrogen oxide gases, generated in situ from a HNO3/H3PO4 - NaNO2 mixture. This reaction is more advantageous than the existing methods to produce aldehyde-functionalized chitosan, since the depolymerization was slower and the purification process of the products was easy and straightforward. The appearance of characteristic peaks in the Fourier transform infrared and carbon-13 nuclear magnetic resonance spectra (1733 cm-1 and 183.4 ppm, respectively) of the product confirms the presence of the aldehyde functionality in the modified chitosans. The pH-dependent 1H-NMR spectra also revealed the presence of aldehyde groups. However, as the pH increased from 2.0 to 6.0, the resonance due to the aldehyde gradually disappeared and a new resonance appeared at 8.05 ppm, which is attributable to the formation of Schiff's base between the aldehyde and the free amine groups. This aldehyde-derivative of chitosan formed a gel in situ by simply dissolving it in water at a concentration of 6% (w/w) without any added external crosslinker. This gel show potential use as drug carrier and as scaffold for vascular tissue engineering. In addition, cellulose:chitosan composites were prepared with the main purpose of obtaining a compliant hollow tube for vascular bypass application. Elastic properties of membranes made of this composite with different ratios between each polymer were determined using uniaxial tests and the ratio that yielded the less stiff membrane was chosen to prepare a small diameter hollow tube. The presence of chitosan had a favorable impact on the elasticity of the membranes, where the cellulose:chitosan 5:5 ratio showed the lowest Young's modulus. Small diameter tubular constructs were fabricated using this optimal cellulose:chitosan ratio and assessed for their suitability as coronary artery bypass grafts. The compliance of the tubes was found to be 5.91 %/mmHg x 10-2, which is higher than those of Dacron, ePTFE and saphenous vein. Burst strength tests revealed that the tubes can withstand at least 300 mmHg. Finally, the tubes showed satisfactory cell attachment property when myofibroblast cells adhered and proliferated on the lumen of the samples.

Gene therapy and immunotherapy are powerful techniques in the treatment of many life threatening diseases. The major challenge in these therapies is to seek a safe and efficient delivery carrier for gene and antigen materials. Carriers are designed to protect these molecules from degradation, improve their stability and facilitate the delivery of them to the site of action. This research study aims to develop appropriate carriers for small interferencing RNA (siRNA), DNA, antigen and ajuvant respectively. In the case of siRNA, material encompassing mannose, polyethylene glycol (PEG) and polyethylenimine (PEI) was investigated. Two structures were assembled: in one construct, mannose was conjugated to PEI directly (Mannose-PEI-PEG) whilst in a second construct; the mannose was conjugated to PEI via a PEG spacer (PEI-PEG-mannose). Confocal microscopy images suggested a faster escape and release of siRNA into the perinuclear region when siRNA was complexed with mannose-PEG-PEI. Mannosylation and PEGylation generated significant toxicity reduction compared to unmodified PEI alone. Real-time polymerase chain reaction (RT-PCR) results showed a significant decrease on mRNA knockdown when using modified PEIs. It was found that PEI-PEG-mannose was a stronger candidate for siRNA delivery because it displays lower toxicity, higher uptake efficiency and higher relative knockdown efficiencies. In the case of pDNA delivery, dextran was introduced to reduce the toxicity generated by PEI. PEI 2000 was more effective than PEI 800 in condensing DNA and inducing transfection when incorporated with dextran. The toxicity of dextran-PEI was greatly reduced when compared to unmodified PEI. Dextran-PEI was able to generate significantly higher transfection efficiencies than PEI alone in the presence of serum. An improved stability of complexes in serum by dextran-PEI was noticed along with a faster release of complexes to the perinuclear region of cells after endocytosis. These observations help to account for the higher efficiency of dextran-PEI in gene transfer. In our final study on vaccines, we utilized cationic polyamidoamine (PAMAM) dendrimer polymers to modify biodegradable particles for enhanced delivery of antigens and adjuvants. Vaccines were formulated by loading CpG oligonucleotide (CpG ODN) and ovalbumin (OVA) into biodegradable microparticles. In one group, OVA and CpG were conjugated together and then loaded into the PLGA microparticles. In other groups, CpG was loaded into the particles and OVA bound to the surface and finally particles were prepared that were loaded with OVA and surface modified with cationic PAMAM dendrimers to electrostatically bind CpG ODN. The microparticles were able to provide sustained release of antigen and adjuvants over 14 day's course. The up regulation of CD86 and H2Kb indicated strong activation of DC and therefore strong induction of CD8+ T-cells. MHC II markers were not as significantly affected. Particles loaded with OVA and surface bound CpG ODN ((OVA)-CpG) showed the highest cytotoxic CD8+ T cell response, suggesting that formulation is optimal for vaccine applications.These observations were further supported by IgG1 and IgG2a antibody levels in mice sera.

Premature infants develop anemia in their first few weeks of life. This is the result of heavy laboratory blood loss, shortened red blood cell lifespan, low plasma erythropoietin levels and inadequate erythropoiesis. As treatment for clinically significant anemia, approximately 80% of very low birth weight infants weighing less than 1.5kg at birth and 95% of extremely low birth weight infants weighing less than 1.0kg at birth receive one or more red blood cell transfusions. To reduce or eliminate red blood cell transfusions is important because they are expensive and associated with complications including infection, fluid overload, electrolyte imbalance, transfusion related acute lung injury and exposure to plasticizers, lead, and other toxins. The primary objective of this thesis is to examine erythropoietin (Epo) dosing, laboratory phlebotomy reduction and the use of restrictive red blood cell transfusion criteria to determine the potential to reduce or eliminate the need for red blood cell transfusions in preterm infants. In order to accomplish this objective, data were obtained from 27 preterm infants including: erythropoietin concentrations, phlebotomy volumes, transfusion information and multiple hematologic indices. The data were analyzed and modeled according to pharmacokinetic and pharmacodynamic principles to determine, through simulation studies, the potential for avoiding blood transfusions in preterm infants. Results from this research suggests that Epo administration, phlebotomy reductions and the use of restrictive blood transfusion criteria all have the potential to reduce the need for blood transfusions in preterm infants. Specifically, a combination of the three interventions was predicted to make blood transfusions unnecessary in all infants with a birth weight between 1.0-1.5kg, and 45% of infants with a birth weight of <1.0kg. These findings are clinically important because avoiding transfusions may lead to better clinical outcomes. The results propose strategies to utilize in future clinical trials involving preterm infants. The secondary objective of this thesis is to characterize the dynamic Epo receptor behavior in newborn sheep and determine a pharmacodynamic model which utilizes information from the Epo receptor dynamics. Results from this analysis show that the Epo receptor pool is an important predictor of red blood cell production. An Epo receptor based pharmacodynamic model is proposed that successfully predicted the red blood cell production in newborn sheep. Additionally, the optimal time for Epo administration was also determined in these newborn sheep based on the pharmacodynamic model. This optimal Epo administration time corresponded to approximately the time when the Epo receptor pool was the largest. Results from the Epo receptor based studies in newborn sheep suggest Epo clinical trials in preterm infants need to consider the dynamic Epo receptor behavior to produce the most optimal outcome.

Most solid-state kinetic principles were derived from those for homogenous phases in the past century. Rate laws describing solid-state degradation are more complex than those in homogenous phases. Solid-state kinetic reactions can be mechanistically classified as nucleation, geometrical contraction, diffusion and reaction order models. Experimentally, solid-state kinetics are studied either isothermally or nonisothermally. Many mathematical methods have been developed to interpret experimental data for both heating protocols. These methods generally fall into one of two categories: model-fitting and model-free. Historically, model-fitting methods were widely used because of their ability to directly determine the kinetic triplet (i.e., frequency factor [A], activation energy [Ea] and model). However, these methods suffer from several problems among which is their inability to uniquely determine the reaction model. This has led to the decline of these methods in favor of isoconversional (model-free) methods that evaluate kinetics without modelistic assumptions. However, isoconversional methods do not compute a frequency factor nor determine a reaction model which are needed for a complete and accurate kinetic analysis. A new approach was proposed that combines the power of isoconversional methods with model-fitting methods. It is based on using isoconversional methods instead of traditional statistical model-fitting methods to select the reaction model. Once a reaction model has been selected, the activation energy and frequency factor can be determined for that model. This approach was investigated for simulated and real experimental data for desolvation reactions of sulfameter solvates. Controversies have arisen with regard to interpreting solid-state kinetic results which include variable activation energy, calculation methods and kinetic compensation effects. The concept of variable activation energy in solid-state reaction kinetics has caused considerable debate because this behavior has been viewed by some as a violation of basic chemical kinetic principles. Activation energy variation has been detected by isoconversional or "model-free" calculation methods which generate activation energy as a function of reaction progress. The relationship between calculation methods and artifactual variation in activation energy was investigated by employing model-fitting and isoconversional methods to analyze both simulated and experimental data. The experimental data was for the sulfameter-dioxolane solvate desolvation by TGA. It was shown that variable activation energy in simple reactions could be an artifact resulting from the use of isoconversional methods; this artifactual behavior can be seen in both isothermal and nonisothermal kinetic experiments. Therefore, care should be taken when interpreting kinetic results from isoconversional methods. If the variation in activation energy is artifactual, this variation can lead to a false mechanistic conclusion about a reaction being complex while, in fact, it is not. Artifactual variation can be reduced by careful experimental design and control of experimental variables in addition to experimental replication, so that averaged kinetic parameters and their confidence intervals can be estimated. The solid-state stability of several structurally related solvates of sulfameter (5-methoxysulfadiazine) was investigated by studying the kinetics of their desolvation reaction both isothermally and nonisothermally. Calculated kinetic parameters were compared and related to the crystal structure of these solvates. A relationship was established between desolvation kinetic parameters (e.g., activation energy) and the solvent size; the larger the solvent molecule, the higher its activation energy. The solid-state reaction models selected also corresponded to the single crystal structure of the sulfameter-solvate system in which the solvent molecules were in cavities. Finally, it was found that kinetic parameters obtained isothermally and nonisothermally were not in agreement. Therefore, kinetic results from one may not be extended to the other.

Hypertension or high blood pressure is a major cause of illness and death in the United States today.1,2 An estimated thirty-six million people suffer from this disease.3 Prolonged hypertension and its attendant strain on various organs may cause heart failure, brain stroke or kidney damage.4 Usually, a pressure of 140/90 mm Hg is taken to be the dividing line between normotension and hypertension.5,6 The hypertensive condition is generally characterized as either primary (essential) hypertension or secondary hypertension.1 A specific cause can be identified in secondary hypertensive patients, such as, pheochromocytoma or adrenal tumor. These causes can typically be corrected by surgery. Essential hypertension occurs in about 90% of the hypertensive population. The etiology of essential hypertension is unknown.1,3,6 The increase in diastolic pressure in primary hypertension may take on either a gradual (benign) or accelerated (malignant) course. The prognosis of gradual hypertension is more favorable than that of the accelerated. Blood pressure in the essential hypertensive patient can almost always be controlled by drug therapy. It is currently accepted that in most hypertensive cases the primary abnormality is due to a high peripheral resistance.7 Numerous biological feedback mechanisms interact in trying to return the body to the normotensive state.8 Some factors involved in the maintenance of homeostasis are central and peripheral sympathetic activity, renal pressor and depressor mechanisms, antidiuretic hormone, sodium balance, baroreceptors, small blood vessel resistance, blood volume and viscosity.1,4,7

Tetracyclines, originally isolated in the late 1940's and early 1950's from the mycelium of Streptomyces, have achieved major importance as therapeutic and prophylactic agents against a wide range of infections in human and veterinary medicine. They have also achieved great importance in agriculture where they are widely used to promote weight gain in livestock. Commercial production, during 1958 in the United States alone was 120 tons with a value of approximately $1 million per ton. The clinical importance of these compounds has stimulated efforts to define their mode of action as inhibitors of bacterial reproduction. Antibiotics having as many functional groups as the tetracyclines may have many modes of action. The problem facing the research worker is to determine the relative contribution of each mode of action in a given biological system as a function of antibiotic concentration. If all parts of a biological system are exposed to an equivalent antibiotic concentration, one might expect that the degree of inhibition of the various sub-component systems would be proportional to the relative stabilities of the reaction products of the antibiotic and the sub-component system. The most critical reaction from the standpoint of cellular reproduction would then represent the point of the primary biochemical lesion or the site of action. The type of reaction would be the mode of action for the biological system. For a given biological system it is probable that there is one critical reaction which is most sensitive to the antibiotic, but this reaction may not be the same for every biological system (see Snell and Cheng (l) for an excellent discussion of the difficulties of defining a mode of tetracycline action). Many investigators presently feel that the primary biochemical lesion inflicted upon susceptible bacteria by tetracyclines is a general inhibition of protein synthesis (2,3,4). Therefore it was felt that the study of several measures of inhibition, particularly cell reproduction and protein synthesis, in the presence of several tetracyclines might be a useful way of studying modes of tetracycline action. Biological activities of a large number of compounds obtained quantitatively under identical conditions and in a precise manner are required to establish structure-activity relationships. Presently available activities for tetracycline antibiotics have been summarized by Barrett (5), Boothe (6) and Plakunov (7). Many of these activities have been obtained under conditions such that the results parallel clinical activities. Thus, some compounds may not have achieved equilibrium with the test system. Some activities have been obtained for the purpose of studying antibiotic resistance while still others have been obtained in widely varying and not easily interrelatable biological test systems. Consequently, quantitative activities suitable for structure-activity relationships have not been reported for most tetracyclines. The present work was undertaken to obtain activities suitable for structure-activity relationships and which are pertinent to both bacterial reproduction and the proposed mode of action. The activities obtained should include both clinically active and “inactive” tetracycline antibiotics.

An original study was undertaken to examine the relationship of clients of Project Jump Street, Inc., a methadone maintenance program, to changes in personality factors as measured by R.B. Cattell's Sixteen Personality Factor Inventory, to compare the national norms of drug addicts/methadone users to the result of the 16 PF of clients of Project Jump Street, Inc., and to determine if significant differences appear among treatment phase I, phase II, and phase III. Twenty-one clients of the program, seven subjects in each of phases I, II, and III, were given the 16 PF by their counselors between September 10 and September 30, 1973. The analysis of the data indicated that while there were no statistically significant differences (p = .05) among the groups, definite trends seemed to be developing among treatment phase I, phase II, and phase III patients. The trends that seemed to be developing were: Increasing sizothymic (A-) response Increasing analytic intelligence response (B-) Increasing ego strength (C+) Decreasing superego strength (C+) Increasing reactivity to threat (H-) Increasing shrewdness (N+) Increasing self-assuredness (0-) Increasing group—dependency (Q2-) In group I significant differences were found as follows in the mean sten scores of the clients when compared to the standard for drug addicts. These factors were: Intelligence(B), Ego Strength(C), Dominance/Submission(E), Superego Strength(C), Praxernia/Autia(M), Artlessness/Shrewdness(N), and Conservatism/Radicalism(Q1). In group I when compared to the standard for methadone users, there were significant differences in the following factors: Intelligence(B), Ego Strength(C), Dominance/Submission(E), Superego Strength (G), Threctia/Parmia (H), Artlessness/Shrewdness (N), and Conservatism/Radicalism (Q1). Group II subjects mean sten scores were compared to the standard for drug addicts and the following factors differed significantly: Intelligence (B), Ego Strength (C), Dominance/Submission (E), and Desurgency/Surgency (F). In group II, when compared to the standard methadone user, significant differences were noted in the following factors: Threctia/ Parmia (H), and Conservatism/Radicalism (Q1). The standard for drug addicts was compared to the mean sten scores for group III. The following factors differed significantly from the standard: Sizothyme/Affectothyme (A), Intelligence (B), Ego Strength (C), Dominance/Submission (E), Alaxia/Protension (L). When group III mean sten scores were compared to the standard for methadone user, there were significant differences in the following factors: Sizothyme/Affectothyme (A), Ego Strength (C), Artlessness/ Shrewdness (N) and Conservatism/Dominance (Q1). These results seem to indicate that there is increasing similarity between the standard for methadone users and the subjects in the study as one approaches group II. Subjects in group I exhibited the greatest amount of variance when compared to the standard for both drug addicts and methadone users, while group III showed only median variance from the standard for drug addicts and methadone users.

The objective of this investigation was to evaluate quantitative electroencephalography (EEG) as a measure of CNS stimulation. The reproducibility and sensitivity of quantitative EEG was compared to neuroendocrine, mood, and psychomotor performance measures. The study was conducted in two parts. The first part investigated the inter- and intra-individual variability associated with a series of pharmacological response measures under baseline (no drug) conditions. It was an open-label pilot study in which eight healthy male volunteers underwent a series of tests (EEG, visual continuous performance task (CPT), a finger tapping task, and self-rated mood scales) repeated eight times over a 12 hour period on three occasions, one week apart. The second part evaluated the sensitivity of quantitative EEG to dextroamphetamine (DA) compared to other response measures. It was a double-blind, placebo-controlled, four-period crossover study in eight healthy male volunteers. Subjects received 5 mg, 10 mg, or 20 mg DA or placebo orally, and underwent the same series of tests as well as blood collection for serum prolactin and DA determination, eight times over a 12 hour period. A GC method allowing quantitation of 2ng/mL DA in serum was developed. The greatest between-day, within-day, and intrasubject variability was associated with quantitative EEG. Learning effects were observed for the psychometric tests, and first session effects were apparent for several of the tests including the EEG. EEG response to DA was observed only in the 3 subjects who had baseline alpha activity greater than 35%. There was a statistically significant decrease in serum prolactin levels after DA administration, with the largest decrease observed after the 5 mg dose. Mood scales showed that 3 of 9 subjects experienced dysphoria after DA dosing. The effect on mood was generally greater as the dose increased. One subject was discontinued from the study because he experienced intense dysphoria after the 5 mg dose. Doses could not be distinguished based on the results of the psychometric tests. Effects on mood, serum prolactin levels, and performance as measured by CPI and finger tapping were not correlated with the EEG changes observed. Pharmacokinetic evaluation showed that the rate of DA absorption appears to decrease as the dose increases. Quantitative EEG conducted under our study conditions and study population was not more sensitive for the assessment of CNS stimulation than the other response measures evaluated. The sensitivity may be improved by screening volunteers to select subjects with higher background alpha activity.

The objective of this thesis was to understand the regulation of islet Cl⁻ current by cAMP. This current, known as Icl,islet flew is the first Cl⁻ channel current characterized in pancreatic 𝛃 cells. Icl,islet has been hypothesized to modulate insulin secretion through changes in islet electrical activity. Both 5 𝛍M forskolin and 100 𝛍M IBMX (3-isobutyl-1-methylxanthine), agents that increase intracellular cAMP, were shown to activate an outwardly-rectifying ionic current in HIT cells that closely resembled Icl,islet. The current was blocked when iodide was substituted for external Cl⁻ or when the Cl⁻ channel blocker niflumic acid was applied to cells. In contrast, removal of [Na⁺]O did not inhibit the current. In many cells, Cl⁻ current activated and then spontaneously deactivated following cAMP stimulation, suggesting the possibility that the channel desensitizes to [cAMP]i. Exposing cells to multiple cAMP activators revealed that Cl⁻ current declined because it became refractory to increased [cAMP]i. The implication of these results to islet physiology is discussed.

Spectral imaging is a powerful technique which uses the wavelength to identify/quantify the exact location and amount of the molecules. It facilitates the identification of materials and studying their properties through analyzing the way they interact with light. The study of light interaction with elements is called spectroscopy; spectroscopy examines how light behaves in the target and recognizes materials based on their spectral signatures. Spectral signatures can be compared to fingerprints which can be used to identify a person; spectral signatures can be used to identify materials. Therefore, we hypothesize that identifying the exact location and quantity of molecules present in the given cells samples can be done by using a spectral imaging system. In this study, we identify the exact UV-Vis and fluorescence spectra of organic substances including Rhodamine 6G, Doxorubicin and UV-Vis spectra inorganic compounds including silver (Ag), gold (Au) nanoparticles (NPs). After that, we used the Q-TOF LC/MS system to quantify the maximum and minimum detectable concentrations of Rhodamine 6G and Doxorubicin by checking the chemicals spectrum based on the molecular weight. In addition, we used HPLC system to quantify the chemicals basing on their UV spectrum. Forwards, we used spectral imaging system to determine the exact amount and location of the molecules within cells samples. For Rhodamine 6G and doxorubicin, we started with the minimum detectable concentration by Q- TOF and consider it as a maximum limit with spectral imaging. And for NPs we used the maximum concentration for the analysis. Using the spectral imaging we were able to vii detect the exact location of Rhodamine 6G which was in the cytoplasm, Doxorubicin in the nucleoplasm, and NPs in both. Furthermore, spectral imaging was able to detect much lower concentrations of Rhodamine 6G and Doxorubicin by the spectrum in comparison to Q-TOF LC/MS.