Thesis (M.F.C.S.)--Bowling Green State University, 2005. / Document formatted into pages; contains vii, 68 p. Includes bibliographical references.

Thesis (M.S.)--Ohio University, March, 2006. / Title from PDF t.p. Includes bibliographical references (p. 67-75)

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 52-61).

abstract: Small molecules have proven to be very important tools for exploration of biological systems including diagnosis and treatment of lethal diseases like cancer. Fluorescent probes have been extensively used to further amplify the utilization of small molecules. The manipulation of naturally occurring biological targets with the help of synthetic compounds is the focus of the work described in this thesis. Bleomycins (BLMs) are a class of water soluble, glycopeptide-derived antitumor antibiotics consisting of a structurally complicated unnatural hexapeptide and a disaccharide, clinically used as an anticancer chemotherapeutic agent at an exceptionally low therapeutic dose. The efficiency of BLM is likely achieved both by selective localization within tumor cells and selective binding to DNA followed by efficient double-strand cleavage. The disaccharide moiety is responsible for the tumor cell targeting properties of BLM. A recent study showed that both BLM and its disaccharide, conjugated to the cyanine dye Cy5**, bound selectively to cancer cells. Thus, the disaccharide moiety alone recapitulates the tumor cell targeting properties of BLM. Work presented here describes the synthesis of the fluorescent carbohydrate conjugates. A number of dye-labeled modified disaccharides and monosaccharides were synthesized to study the nature of the participation of the carbamoyl moiety in the mechanism of tumor cell recognition and uptake by BLM saccharides. It was demonstrated that the carbamoylmannose moiety of BLM is the smallest structural entity capable for the cellular targeting and internalization, and the carbamoyl functionality is indispensible for tumor cell targeting. It was also confirmed that BLM is a modular molecule, composed of a tumor cell targeting moiety (the saccharide) attached to a cytotoxic DNA cleaving domain (the BLM aglycone). These finding encouraged us to further synthesize carbohydrate probes for PET imaging and to conjugate the saccharide moiety with cytotoxins for targeted delivery to tumor cells. The misacylated suppressor tRNA technique has enabled the site-specific incorporation of noncanonical amino acids into proteins. The focus of the present work was the synthesis of unnatural lysine analogues with nucleophilic properties for incorporation at position 72 of the lyase domain of human DNA polymerase beta, a multifunctional enzyme with dRP lyase and polymerase activity. / Dissertation/Thesis / Doctoral Dissertation Chemistry 2014

Humic substances are a group of ubiquitous compounds formed during the decay of plant and animal residues in the environment. These substances can be divided into humic acid, fulvic acid and humin on the basis of its solubility in water as a function of pH. Fulvic acid is the fraction that is soluble in water under all pH conditions and is usually extracted from brown coal and is therefore associated with high levels of heavy metals. These heavy metal levels have become a point of concern and a unique method for synthesizing fulvic acid from a carbohydrate source has been subsequently developed and patented in an attempt to address this problem. The purpose of this study was therefore, to test the topical and systemic safety and efficacy of this new carbohydrate derived fulvic acid (CHD-FA) product. Initial in vitro screening using the Kirby-Bauer disk diffusion method indicated that the product was active against the Staphylococcus aureus test organisms used (ATCC 12600 and P3938). Results showed that not only was it as effective as the oxacillin control, but in the case of a more virulent strain of S. aureus, the CHD-FA (3.9% w/v) proved more active. Subsequently, the ability of the product to cross full-thickness skin was ascertained using Franz-type diffusion cells fitted with 100ìm skin disks obtained from rats. At the end of a 12-hr period, results indicated that up to 20% of the CHD-FA at a pH of 2.9 was able to cross the skin barrier. Divided into 6 phases, extensive animal safety studies were conducted on the product. In phase I, toxicity and sensitivity to topical application was examined by applying the product to the ears of mice over a period of 30 days. Phase II and III was undertaken to study the long term effects (6-weeks and 6-months) of CHD-FA ingestion (150 mg/kg body mass) whilst phase IV tested the effects of CHD-FA ingestion on pregnant rats and their off-spring. Phase V was an acute toxicity study in which rats received a single oral dose of CHD-FA (150mg/kg bw) and then observed for a period of 7 days. Phase VI was a 6-week chronic study in which the animals received 100mg/kg bw daily for the duration of the experiment. Results obtained in all studies showed that CHD-FA is safe for topical as well as, oral use at the doses tested. In addition, it is safe for use during pregnancy. Finally, the in vivo anti-microbial efficacy of the CHD-FA was examined using an infected wound healing model. A pilot study indicated that the animals used should be immunocompromised and the wounds inoculated with a Staphylococcus aureus (ATCC 12600) concentration of 1x1010 CFU/ml. Results from the main study showed that topical applications of the CHD-FA (pH 1.98) product at a concentration of 1.75% w/v over the 5-day period produced a wound healing pattern similar to that of the positive fuscidic acid control. In contrast, animals receiving an oral treatment of the product did not produce a pattern different from that of the water control group. In conclusion, CHD-FA has been shown to be safe and effective in treating S. aureus infections in vitro and in animal models. This study indicates that the next phase of human studies is certainly warranted. / Dissertation (MSc)--University of Pretoria, 2009. / Pharmacology / unrestricted

The effect of intramuscular corticosterone injection on post mortem muscle metabolism and shear resistance of Pectoralis Major muscle was studied. Short term studies, conducted over an 8 hour post-injection period, showed no significant effect of injection until 6 hours post injection at which time shear values increased from those of the controls. Blood glucose levels rose significantly from 2 to 8 hours post injection. A comparison of shear values of P. Major muscle from 6 week-old broilers and 8 week-old broilers after both had received corticosterone injections showed that the only difference was due to the difference of age not to any alteration in the physiological handling of the injected corticosterone. The greatest difference in treatment effect was found between birds of different stress level. Stress level was subjectively evaluated with respect to the birds reaction to a person entering the range house and to the birds response to handling. Long term studies conducted over 5 and 15 days illustrated homeostatic adjustments to daily corticosterone injections and recovery after cessation of injections. Shear testing was performed on P. Major muscle which was chilled on the carcass and on muscle excised at death. Excised muscle displayed a greater reaction to injection and cessation of injection than did intact muscle due to a lack of skeletal restriction. Shear values decreased on initial injection and increased upon cessation of injection. Homeostatic adjustments returned values to near-normal between these times. Chemical tests were found to display a much more definite reaction to corticosterone injection than did shear testing. Blood glucose and tissue glycogen increased steadily on injection of corticosterone and decreased rapidly at cessation. As expected ultimate muscle pH decreased with muscle glycogen increase. Blood cholesterol increased during injection period as endogenous corticosterone production, for which it is a precursor, was not required. / Land and Food Systems, Faculty of / Graduate

With emerging trends of new cellular therapies, the need for quick access to cellular components is necessary. For most applications genetically compatible biological components are essential to prevent adverse immune responses and graft-versus host disease (GVHD). Since these biological components have a limited window to be used, techniques for long-term storage are needed. Cryopreservation is essential for this in the field of biobanking and regenerative medicine to allow for long-term storage of cell products. During this process, ice recrystallization is the major contributor to cell death and decreased cell viability post-thaw. Due to this, controlling ice growth and recrystallization is imperative to increasing cell survival and function. The Ben lab is focused on the synthesis of small molecule, carbohydrate-based cryoprotectants that function as ice recrystallization inhibitors (IRIs). Previously, many IRIs have been synthesized showing varying degrees of ice recrystallization inhibition (IRI). Through the structure-function work, a delicate balance between hydrophobic and hydrophilic portions on the same molecule must be met. These compounds are believed to disrupt hydrogen bonding networks present in the formation of ice, and control ice growth. While numerous types of functional groups on carbohydrate derivatives have been explored, many highly solvated functional groups (amines, sulfates, phosphates, etc.) have not been thoroughly investigated. Highly solvated functional groups should disrupt hydrogen bond networks due to their solvation and in theory, should illicit an IRI response. Sulfate groups have not previously been studied, but are present in several different biological processes, such as immune response and blood coagulation. This suggests that sulfated carbohydrates should be well tolerated biologically. Sulfate groups can also be easily installed on existing IRI active molecules through orthogonal protecting group chemistry. The first part of this thesis is focused on the synthesis and IRI activity of sulfated carbohydrates based upon previously synthesized, IRI active pyranose derivatives. When compared to their parent compounds, most of the sulfated derivatives were less active, but all compounds were incredibly soluble in aqueous media. These derivatives did not show much promise as new IRIs due to the length of their synthesis and reduced IRI activity compared to their parent compounds. The Ben lab has also developed a new class of IRI active carbohydrates: aldonamide derivatives. These compounds are open-chain carbohydrates with an amide bond, arising from the ring opening of a carbohydrate lactone with a substituted amine. While many of these compounds displayed high degrees of IRI activity, many were incredibly insoluble in aqueous systems (many with solubility limits under 50 mM). Since sulfate groups were able to greatly increase solubility with some derivatives retaining IRI activity, installing sulfate groups on existing aldonamide-based IRIs should increase their solubility. Additionally, since many of these derivatives display high degrees of IRI activity, a reduction in IRI activity can be tolerated. Similarly, to the sulfated pyranose derivatives, the presence of a sulfate group reduced the IRI activity compared to the parent compounds in most derivatives. Though some sulfated derivatives possessed a higher degree of IRI activity, all the derivatives experienced a drastic increase in solubility (over 200 mM in PBS). Some of the sulfated aldonamide derivatives were assessed for their ability to protect red blood cells (RBCs) during freezing with reduced glycerol concentrations (15% glycerol), although none of thew tested derivatives showed an improvement over existing IRIs explored by the Ben lab. Since the introduction of sulfate groups to existing IRIs drastically increased solubility in aqueous systems, but resulted in reduced IRI activity in most compounds, focus was switched to the addition of different hydrophilic functional groups. Amino functional groups were briefly explored with galactose-based pyranose IRIs, aldonamide derivatives had not been explored. Amino groups are present on many biological carbohydrates and should be well tolerated biologically. The addition of amino groups to aldonamide derivatives should increase solubility, with the amino derivatives ideally retaining some IRI activity. The amino aldonamide derivatives synthesized had high solubilities (>500 mM in PBS), but did possess lower degrees of IRI activity. Due to the high solubility these derivatives were initially assessed in the cryopreservation of RBCs with reduced glycerol concentrations. Initial experiments showed improvements over current IRIs, and the compounds were assessed in a number of other biological cryopreservation scenarios including articular cartilage, platelets, and hematopoietic stem/progenitor cells (HSPCs). While the compounds showed toxicity in these cell types, more studies need to be conducted for the cryopreservation of RBCs.

No description available.

Glucans are (1 → 3)-β-linked glucose polymers which have immune-stimulating capability. The extraction of water-insoluble (1 → 3)-β-D-glucan form Saccharomyces cerevisiae employs hydrochloric acid. Hydrochloric acid is difficult to employ in the large-scale pharmaceutical extraction of glucans due to its corrosive nature and toxicity. To address these concerns, we determined whether acetic, formic or phosphoric acid can be substituted for hydrochloric acid in the process for the isolation of (1 → 3)-β-D-glucan. The resulting microparticulate glucans were employed as the starting material for the production of (1 → 3)-β-D-glucan phosphate. 13C NMR analysis of the glucan phosphates derived from the acetic, formic or phosphoric acid-extracted microparticulate glucan show excellent correspondence to hydrochloric acid extracted glucan and laminarin, a (1 → 3)-β-D-glucan standard, indicating that the primary structure is not altered by the acid used for extraction. Glucan phosphate prepared from hydrochloric acid had a M(W) of 7.2 x 104 g/mol, rms(Z), of 17.7 nm, of 1.50 and (η) of 49.0 mL/g. Glucan phosphate prepared from acetic acid had a primary polymer peak with a M(W) of 1.4 x 106 g/mol, rms(Z) of 23.6 nm, I of 1.93 and (η) of 62.4 mL/g. Glucan phosphate prepared from formic acid had a main polymer peak with a M(W) of 1.2 x 106 g/mol, rms(Z) 27.1 nm, I of 1.56 and (η) of 89.0 mL/g. Glucan phosphate prepared from phosphoric acid had a primary polymer peak with a M(W) of 6.6 x 105 g/mol, rms(Z) of 32.3 nm, I of 2.70 and (η) of 91.3 mL/g. These data indicate that the molecular mass, size, polydispersity and intrinsic viscosity of the glucan phosphate obtained is influenced by the pK(a) of protic acid employed to extract the microparticulate glucan. However, the primary structure and side-chain branching are not substantially altered regardless of the acid employed.

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