A major gap in sexual assault casework is demonstrated when DNA is not recovered. Oftentimes, if DNA evidence is not present on the collection swabs, the sexual assault kit (SAK) is not further analyzed. Due to the "CSI effect," DNA is commonly understood as highly identifiable evidence, potentially leading to increased condom usage to eliminate or reduce DNA transfer during a sexual assault. Therefore, the analysis of condoms and sexual lubricants is pertinent. The purpose of this research is to develop analytical protocols to potentially connect unknown substances recovered in a SAK to known lubricant reference samples. Sexual lubricants were analyzed using Fourier transform infrared spectroscopy, gas chromatography-mass spectrometry, and direct analysis in real time-high resolution mass spectrometry. Analytical protocols were developed using 162 sexual lubricants comprised of bottled lubricants, condoms, and personal hygiene products. A statistical model was developed from 112 of the samples using hierarchical cluster analysis (HCA), principal component analysis (PCA), and linear discriminant analysis (LDA) to determine appropriate sample groupings that resulted in at least 97% accurate classification for each instrument. Assigned truth classes for the remaining 50 samples were developed using Pearson correlation coefficients (PCCs) to predict classification accuracy for unknown samples. The FTIR data resulted in a 96% accurate prediction, 54% for GC-MS, and 42% for DART-HRMS, showing the need for expansion of the sample set in future analysis. Potential storage conditions of SAK swabs were evaluated using PCCs to identify optimal swab storage conditions, which was determined to be a humidity-controlled environment around 22 °C. Then, post-coital swab samples from volunteers using an unknown condom were analyzed using the developed protocols. The data was analyzed using PCC, PCA, and LDA to compare the classification to the "ground truth" of the sample to determine potential applications of this research in SAK analysis.

Magnetic materials are currently being developed in the areas of pharmacology and medicinal chemistry for use in applications such as drug delivery and magnetic resonance imaging. Magnetic fluids are being used in audio equipment and hard disk drives. Their suspension in a particular fluid is promoted by the adsorption or reaction of steric or electrostatic stabilizers, which are appropriate for the particular medium. Critical to the success of these magnetic fluids is the development of the steric stabilizers, which must prevent the coagulation of the metal particles. Polymeric materials are one of the most suitable nonmagnetic media to disperse the magnetic nanoparticles, forming polymeric nanocomposites in ferrofluids. We have developed strategies in molecular nanoscience to design polymeric systems for stabilization of magnetic nanoparticles. Ring opening metathesis polymerization (ROMP) was used to prepare a series of novel, well-defined diblock copolymers of bicyclo[2.2.1]hept-5-ene 2-carboxylic acid 2-cyanoethyl ester and bicyclo[2.2.1]hept-2-ene, consisting of both anchoring and steric stabilizing blocks. Both ester and cyano groups were incorporated into the polymers to chelate and stabilize the iron oxide magnetic nanoparticles. These polynorbornene-based copolymers were characterized by GPC, along with 1H NMR, FTIR, DSC, and TGA. Using diblock copolymers as stabilizers, nanostructured maghemite (γ-Fe2O3) magnetic ferrofluids were prepared in toluene or cyclohexanone via thermal decomposition of Fe(CO)5 and then the oxidation of iron nanoparticles. Transmission electron microscopic (TEM) images showed a highly crystalline structure of the γ-Fe2O3 nanoparticles, with average particle size varying from 5 to 7 nm. Polymer films containing iron oxide nanoclusters were also prepared from the diblock copolymers. For comparison, a commercial triblock copolymer (BASF PluronicR F127) surfactant was used to prepare stabilized ferrofluids. In addition to γ-Fe2O3 nanoparticles, other types of magnetic nanoparticles, such as FePt, were investigated using this triblock copolymer as a stabilizer. The results indicated that the norbornene diblock copolymers could also be used for the preparation of FePt stabilized magnetic ferrofluids in the future research work.

<p>This thesis describes the initial steps towards the use of dipeptidyl acyloxymethyl ketones as a platform to develop molecular imaging (MI) probes for cancer. Initially the synthesis of an AOMK was performed following a literature procedure which resulted in an epimerized product. This issue was addressed by optimizing an alternative method yielding all intermediates in yields similar or better to those reported in the literature (final product yield of 67%). An AOMK derivative that can be used to evaluate target expression levels was synthesized by linking a fluorescent dye to the ε-amine group of lysine in accordance to a literature procedure describing the synthesis of an optical imaging probe in 24% yield. A second generation derivative AOMK was prepared by linking 4-fluoro-benzoic acid to the same amino group yielding a model of a PET MI probe.</p> <p>An endpoint colorimetric assay was developed and optimized to test cathepsin B inhibitors. Due to the fact that the AOMKs exhibit time-dependent inhibition these assay conditions did not prove to be adequate for the assessment of the cathepsin B binding. Steps toward developing a continuous assay that would be better suited for these compounds were achieved. Factors such as the relationship between the formation of the assay product vs enzyme concentration and determination of the Michelis-Menten constant (K_m = 390 ± 30 nM) were established. These parameters can be used to determine the optimal enzyme and substrate concentration that should be used to test the AOMK based probes.</p> / Master of Science (MSc)

Alzheimer's disease (AD) is commonly characterized by a loss of cognitive function due to the deterioration of neuronal synapses from the presence of senile amyloid beta-42 (Aß42) plaques. Evaluating cognitive deficits caused by Aß42 using human cortical neurons poses a challenge due to sourcing difficulties, and the use of animal models to assess drug efficacy creates biological hurdles from lack of species translatability. Recent advances in induced-pluripotent stem cell technology have enabled the development of mature, human-based cortical neuron models. The development of an hiPSC-cortical neuron differentiation protocol facilitates the exploration of disease onset and functional analysis from a patient-derived cell source, and further investigation of potential therapeutic treatments, while eliminating biological efficacy concerns. Long-term potentiation (LTP) was utilized as an in vitro correlate for memory and learning to quantify cognitive deficits in sporadic AD (SAD) and familial AD (FAD) systems and assess drug treatments for the prevention of Aß42-induced neurotoxicity. Synaptic connectivity and LTP induction through high-frequency stimulation was simulated through cortical neurons cultured on microelectrode arrays (MEAs), such that the functional activity of the neuronal population could be assessed overtime. AD therapeutic treatments were shown to block the Aß42-induced neurotoxic loss of synaptic plasticity and maintain persistent LTP in a model for SAD. Subsequently, FAD was assessed through the differentiation of patient-derived AD iPSCs, where LTP proficiency could be evaluated to relate to clinical cognitive evaluations. This study established a serum-free, in vitro human-derived iPSC-cortical neuron protocol that could be adapted to validate disease mechanisms and drug efficacy in patient-derived neural networks as a potential platform for precision medicine.

Introduction: Atherosclerosis is a chronic inflammatory disease characterized by the buildup of cholesterol, fat and other debris within arterial walls. Atherosclerotic lesions undergo a calcification process with similarities to bone remodeling. In mice, the type-2 cannabinoid receptor (CB2) is known to regulate bone remodeling processes and has also been shown to alter atherosclerotic lesion characteristics. However, the role of CB2 in lesion calcification is still unclear. CB2 modulates bone remodeling by affecting differentiation of osteogenic precursor cells; thus we hypothesize that CB2 alters lesion calcification by altering osteoblastogenesis and osteoclastogenesis of precursor cells of vascular origin. To test this hypothesis, we studied the role of CB2 receptor in mediating osteoclastogenesis and osteoblastogenesis from murine monocyte/macrophage and smooth muscle cell lines in vitro. Methods: RAW264.7 cells are a murine monocyte/macrophage cell line known to undergo osteoclastogenesis in response to receptor activator of nuclear factor kappa B ligand (RANKL). RAW264.7 cells were cultured in media containing RANKL and supplemented with either CB2 agonists or antagonists. Effects on RANKL-induced osteoclastogenesis were then evaluated by measuring the osteoclast marker enzyme tartrate-resistant acid phosphatase (TRAP) activity and further verified by microscopic quantitation of multi-nucleate TRAP-positive osteoclasts. MOVAS-1 cells are a murine vascular aortic smooth muscle cell line known to differentiation into osteoblasts when cultured in osteogenic media. MOVAS-1 cells were cultured in osteogenic media supplemented with CB2 agonists or antagonists. Effects on osteoblastogenesis were evaluated by measuring marker enzyme activity. Alizarin red staining was performed to visualize and quantitate effects on calcium deposition. Results: RAW264.7 cells treated with Win55, 212-2, a nonselective CB agonist, or HU-308, a selective CB2 agonist, displayed a dose-dependent decrease in RANKL-induced TRAP activity. Co-administration of a CB2-selective antagonist (SR144528), but not a CB1-selective antagonist (AM251), blocked this effect. Visual quantitation of multinucleated TRAP-positive cells confirmed Win55,212-2 treatment reduced osteoclastogenesis in RANKL-treated RAW264.7 cells. Induction of osteoblastic differentiation of MOVAS-1 cells, as determined by ALP activity, was enhanced by supplementation with Win55, 212-2 or 2-archidonyl glycerol. Co-administration of SR144528, but not AM251, reduced the induction of ALP activity in MOVAS-1 cells by Win55,212-2 and 2-AG. Alizarin red staining revealed increased calcium deposition in cultures of MOVAS-1 cells treated with Win55,212-2 compared to those cultured in osteogenic medium without Win55,212-2. Conclusions: These results demonstrate that CB2 activation can affect osteogenic differentiation of vascular cells in vitro. These results support the hypothesis that CB2 signaling promotes lesion calcification by altering the balance of osteoclastic and osteoblastic differentiation of vascular precursors.

Malaria is considered as one of the most prevalent and debilitating diseases affecting humans. Plasmodium falciparum is the most virulent form of the parasite which developed resistance to several antimalarial drugs. Chloroquine is one of the most successful antimalarials developed that is safe, effective, and cheap. However, its use has been limited due to the emergence of drug resistance. Click chemistry, particularly, the copper(I)-catalyzed reaction between azides and alkynes has shown to have a cutting-edge advantage in medicinal chemistry by its reliability, selectivity and biocompatibility. Triazole-based antimalarials were synthesized via copper(I)-catalyzed alkyne-azide cycloaddition reaction by modifying the aliphatic chains terminal of chloroquine. The compounds synthesized contain triazole ring directly connected to an aromatic ring or via a piperazine linker. When tested for their in vitro antimalarial activity against D6, Dd2 and 7G8 strains of P. falciparum, 12 out of 28 compounds showed better activity against chloroquine resistant strains. Particularly, PL403 and PL448 exhibited potent activity than chloroquine against CQ-resistant strains Dd2 and 7G8, with IC50 values of 12.8 & 14.5 nM, and 15.2 & 11 nM respectively. The efficiency of synthesizing several triazole-based antimalarials have proven click chemistry to be fast and efficient reaction. Generally, para-substitutions and di-substitutions with electron-withdrawing groups were found to be beneficial for having better antimalarial activity for these group of click compounds. Moreover, the incorporation of piperazine linker has brought an enhanced antimalarial activity.

Histone Deacetylase (HDAC) plays a vital role in cellular processes, for example gene expression, cell growth, and apoptosis. Finding drug candidates to inhibit the over activity of HDACs in cancer is a growing area of interest. Inhibitors, thus far, have three important motifs to be studied: the zinc binding group, a hydrophobic linker, and a cap group. By altering these groups on the inhibitor, not only can activity be increased but also selectivity within the classes of HDACs. We present the design of two novel sets of molecules that contain either a 1,2,3-triazole or 1,2,4-triazole. The 1,2,3-triazoles were synthesized using “click chemistry” with a novel pyridyl triazine catalyst. The 1,2,4-triazoles were synthesized utilizing substitution chemistry. This set of molecules was designed after suberoylanilide hydroxamic acid (SAHA) but replaced the hydroxamate with the triazole as the zinc binding group. The activity of these inhibitors against HDAC 1, HDAC 6, and SIRT 1 were tested using the Biomol Fluor de Lys in vitro kits. Though none of the synthesized compounds were strong activators or inhibitors of any of the classes of HDACs, trends were observed that could lead to the design of more potent inhibitors.

Histone Deacetylase (HDAC) plays a vital role in cellular processes, for example gene expression, cell growth, and apoptosis. Finding drug candidates to inhibit the over activity of HDACs in cancer is a growing area of interest. Inhibitors, thus far, have three important motifs to be studied: the zinc binding group, a hydrophobic linker, and a cap group. By altering these groups on the inhibitor, not only can activity be increased but also selectivity within the classes of HDACs. We present the design of two novel sets of molecules that contain either a 1,2,3-triazole or 1,2,4-triazole. The 1,2,3-triazoles were synthesized using “click chemistry” with a novel pyridyl triazine catalyst. The 1,2,4-triazoles were synthesized utilizing substitution chemistry. This set of molecules was designed after suberoylanilide hydroxamic acid (SAHA) but replaced the hydroxamate with the triazole as the zinc binding group. The activity of these inhibitors against HDAC 1, HDAC 6, and SIRT 1 were tested using the Biomol Fluor de Lys in vitro kits. Though none of the synthesized compounds were strong activators or inhibitors of any of the classes of HDACs, trends were observed that could lead to the design of more potent inhibitors.

On the verge of entering the post-antibiotic era, numerous efforts are in place to regain the losing potential of antibiotics which are proving ineffective against common bacterial infections. Engineered nanomaterials, especially gold nanoparticles (GNPs) capped with antibacterial agents are proving to be an effective and novel strategy against multi-drug resistant (MDR) bacteria. In this study, we report a one-step synthesis of kanamycin-capped GNPs (20 ± 5 nm) utilizing the combined reducing and capping ability of the aminoglycoside antibiotic, kanamycin. Antibacterial assays showed dosedependent broad spectrum activity of Kan-GNPs against Gram-positive (Staphylococcus epidermidis and Enterococcus durans), Gram-negative (Escherichia coli and Enterobacter aerogenes) and Kan-resistant and MDR bacterial strains. A significant reduction in the minimum inhibitory concentration (MIC) of Kan-GNPs was observed as compared to free kanamycin against all the sensitive and resistant bacterial strains tested. Mechanistic studies using TEM and fluorescence microscopy showed that Kan- GNPs exerted their bactericidal action through disrupting the cellular membrane resulting in leakage of cytoplasmic content and death of bacterial cells. Results of this study provide a novel method in the development of antibiotic capped GNPs as potent next-generation antibacterial agents.

The strain promoted azide alkyne cycloaddition (SPAAC) of azido nucleobase modified nucleosides and nucleotides with cyclooctynes to give fluorescent triazoles has been relatively unexplored. Thus, SPAAC between azido-nucleobases and various cyclooctynes in aqueous solution at ambient temperature resulted in the efficient formation (3 min - 2 h) of triazole products with inherent fluorescent properties. The 2- and 8-azidoadenine nucleosides reacted with fused cyclopropyl cyclooctyne, dibenzylcyclooctyne or monofluorocyclooctyne to produce click products functionalized with hydroxyl, amino, N-hydroxysuccinimide, or biotin moieties. The previously unexplored 5-azidouridine and labile 5-azido-2'-deoxyuridine were similarly converted to the analogous triazole products in quantitative yields in less than 5 minutes. The 8-azido-ATP quantitatively afforded the triazole product with fused cyclopropyl cyclooctyne (3 h). Addition of a triazole ring at the 2 or 8 position of adenine or 5-position of uracil induces fluorescent properties which were used for direct imaging with fluorescent microscopy in MCF-7 cancer cells without the need for traditional fluorogenic reporters. Fluorescent lifetime imaging microscopy of the click adducts in live cells were used to determine the lifetime of each fluorophore in the cellular nuclei demonstrating the potential utility of the synthesized triazole adducts for dynamic measuring and tracking of events inside single living cancer cells. The SPAAC methodology developed has also been applied to study the cellular targets in protozoal parasite, Trichomonas vaginalis and bacteria, Pseudomonas aeruginosa. The 9-(2-deoxy-2-fluoro-β,D-arabino-furanosyl)adenine (arabino-F-Ado) was modified with an azido moiety at the C8 position for use in click chemistry. Tagging and subcellular localization studies using azido modified arabino-F-Ado could provide insight into the mechanism of action of arabino-F-Ado. An activated analogue of S-adenosyl-L-methionine (SAM) with an EnYn group on the sulfur instead of a methyl group was prepared to study the transfer of the methyl group from SAM. I found that the EnYn group was transferred from SAM to a guanosine on tRNA by methytransferase Trm1. Thus, AdoEnYn is a competitive inhibitor of SAM and can be incorporated into tRNA in place of SAM.