No description available.

Chapter One presents an overview of phosphorus reagents for the carbon homologations of aldehydes and ketones leading to functionalized carbonyl derivatives. Select examples are provided to exemplify the utility of carbon homologation methodology in synthesis and asymmetric organocatalysis and in the total synthesis of natural products. The directing effect of acetals on regioselective ylide formation is explored in Chapter Two. Evidence is presented for ylide formation through a complex induced proximity effect with lithium bases under coordinating conditions. Moreover, four-carbon donors represent a limit for useful directed ylide formation with trialkylphosphine-derived Wittig salts in carbonyl homologation reactions. A facile approach to the synthesis of methyl vinyl ketones (MVKs), using acetonyl tripropylphosphoranes under mild conditions, is reported in Chapter Three. A library of diversely functionalized MVKs was synthesized as a demonstration of the scope and generality of the methodology. The application of MVKs as substrates for organocatalysis and as building blocks for useful polyketide intermediates is briefly highlighted. In Chapter Four, the two-carbon homologation methodology that was presented in the previous chapter is applied to the synthesis of the polyketide olivetol and a series of O-methyl derivatives. Cyclic diketone intermediates were aromatized with catalytic iodine and DMSO as a terminal oxidant. Modification of the solvent system allowed for the selective synthesis of mono- or dimethyl ethers of methyl olivetolate. The selectivity of this aromatization is further explored in the final chapter with more complex substrates. Chapter Five focuses on the synthesis of the meroterpene phytocannabinoids found in Cannabis sativa. A synthetic strategy involving the sequential condensation/[3+3]-annulation of citral with cyclic 1,3-diketones synthesized in the previous chapter. This afforded non-aromatic meroterpenes that were subjected to acid-mediated thermal rearrangement and catalytic oxidative aromatization. Evidence for chemoselectivity of the aromatization methodology was demonstrated and a synthesis of methyl cannabinolate is presented. / Thesis / Doctor of Philosophy (PhD) / Methodologies for the extension of aldehydes to functionalized olefins and their application to the synthesis of cannabinolic acid methyl ester are presented.

This work focuses on improving the quality of carbon-based core-shell materials for high performance liquid chromatography (HPLC) via the characterization of the core materials, and also the development of chromatographic methods (separations) for them. In the early part of this work, I applied organic synthesis to make uniform, spherical poly(divinylbenzene) (PDVB) microspheres, and then carbonized them to prepare carbon core materials for core-shell particle synthesis. Here, I studied in detail the surface and material properties of these particles with multiple instruments, which allowed me to describe the physical and chemical changes that took place during each treatment. The uniform, spherical carbon core materials greatly improved the efficiency of the previously developed carbon-based core-shell HPLC columns from ca. 70,000 plates per meter (N/m) to ca. 110,000 N/m for various alkyl benzenes. Later, I focused on generating application notes to showcase these mixed-mode HPLC columns. Here, liquid chromatography mass spectrometry (LC-MS) was used for the detection of analytes that lack chromophores for UV detection. In this dissertation, Chapter 1 contains a historical background and theory of HPLC along with a review of the use of carbon-based core-shell materials for elevated pH and temperature applications. Chapter 2 describes the improvement of the efficiency of carbon-based materials for HPLC using carbonized PDVB microspheres as the carbon core material. Chapter 3 is a study on the characterization of carbonized PDVB microspheres with multiple instruments. Chapter 4 describes the separation of cannabinoids using three types of carbon-based mixed-mode HPLC columns. Chapter 5 consists of (i) guidelines for the retention mechanism of the core-shell particles that have been commercialized for chromatography by Diamond Analytics, a US Synthetic Company in Orem, Utah, and (ii) application notes for these columns. Finally, Chapter 6 discusses possible future work.

No description available.

No description available.

Despite the ongoing political debate regarding the legality of medical marijuana, clinical investigations of the therapeutic use of cannabinoids are now more prevalent than at any time in history. Cannabinoids have been shown to have analgesic, anti-spasmodic, anticonvulsant, anti-tremor, anti-psychotic, anti-inflammatory, anti-oxidant, anti-emetic and appetite-stimulant properties. There are mainly two well-known cannabinoid receptors, CB1 and CB2, located in the central (CB1) and peripheral (CB2) nervous systems as well as the immune system. More recently, endocannabinoids (ligands) and their receptors have also been found in the skeleton which appear as the main body system and physiologically regulated by CB2. This study aimed to examine the effect of both CB1 and CB2 receptor stimulation on wound closure response of MG-63 osteoblast bone cell monolayers using different treatments with cannabinoid such as Winn55,212-2, URB602 and HU308. Also, cell adhesion, cell proliferation and cell length was investigated. The study also aimed to examine the effect of HU308 treatments in combination with TGF-β3 (transforming growth factor beta -3) on wound healing, cell adhesion and extracellular matrix up regulation (collagen type I, fibronectin and protien S-100A6) as well as other biological factors such as secretion of matrix metalloproteinase (MMP-2) and nitric oxide (NO). Finally, this study investigated HU308/TGF-β3 combination treatment on the regulation of extracellular matrix (collagen type I, fibronectin and protien S-100A6) in a 3D multilayer system of MG-63 osteoblast bone cells. Wound healing assays of MG-63 monolayers revealed accelerated wound repair as well as increased cell proliferation mainly regulated through CB2 receptors, and that treatments with HU308 and HU308/TGF-β3 achieved minimum closure timings compared with control groups (P<0.05). Our finding suggested that proliferation rate with 500nM HU308 was significantly higher than control and TGF-β3/HU308 combination groups (P<0.05). Interestingly, percentage of wound remained open after 15 hours for combination groups was 17.6%±1.32 whereas treatment with 500nM HU308 had 20%±2.25 indicating that the combination groups took the lead throughout wound healing. It was also observed that bridge formation in all treatment groups was taking place between 15 to 20 hour periods whereas within control treatments bridge formation started to take place after 25 hours. Cell surface attachment was examined via the trypsinization assay in which the time taken to trypsinize cells from the surface provided a means of assessing the strength of attachment. The results indicated that higher concentrations of HU308 (2μM), induced significant force of cell attachment compared with control and concentrations of 500nM and 1μM (P<0.05). However, groups treated with TGF-β3 and combination HU308/TGF-β3 indicated reduced cell surface attachment compared with control groups, indicating enhanced cell migration. Immunofluorescence staining as well as Elisa based semi-quantification technique indicated that both collagen type I and fibronectin were unregulated using higher concentrations of HU308 with decreased cell proliferation compared to lower concentrations. Nevertheless, protein S-100A6 was up-regulated in treatments with HU308, TGF-β3 and their combination HU308/TGF-β3 (P<0.05), indicating the positive role of these treatments in promoting cell differentiation. MMP-2 levels in the current study were also shown to be concentration-dependent, i.e. higher concentrations of HU308 significantly reduced MMP-2 secretion leading to decreased cell migration, while HU308/TGF-β3 combination treatment increased MMP-2 levels, indicating an increase in cell migration. The current study also examined levels of nitric oxide synthesis in relation to different treatments with HU308, TGF-β3 and HU308/TGF-β3 combination. It was found that nitric oxide up-regulation influences rate of MG-63 osteoblast wound healing in a concentration dependent manner. Lastly, UpCell culture dishes proved to have efficacy in obtaining a multilayer model of MG-63 osteoblast system in-vitro through changes in cell morphology. It was also found that treatments with HU308, TGF-β3 and HU308/TGF-β3 combination influenced collagen type I, fibronecton and protein S-100A6 secretion. These findings supported the earlier Elisa based semi-quantification results obtained for monolayer cultures.

Synthetic cannabinoids are recreational drugs designed to mimic the effects of Δ9-tetrahydrocannabinol (THC), the main psychoactive component present in cannabis. These drugs exhibit severe toxic effects upon consumption due to their high binding affinity and potency at the cannabinoid receptors (CB1 and CB2). Synthetic cannabinoids have proliferated over the last decade and become a major public health and analytical challenge, critically impacting the clinical and forensic communities. Indazole carboxamide and indole carboxamide class synthetic cannabinoids have been particularly rampant, and are the compound classes most frequently reported to governmental agencies worldwide. However, the metabolic and pharmacological properties of many of these compounds remains unknown. Elucidating these characteristics allows members of the clinical and forensic communities to identify causative agents in patient samples, as well as render conclusions regarding their toxic effects. The aim of this research study was to assess the in vitro Phase I metabolic profile of five synthetic cannabinoids and report the major metabolites identified; compounds evaluated included MDMB-CHNINACA; APP-CHMINACA (PX-3); 5F-APP-PICA (PX-1); 5F-MDMB-PINACA (5F-ADB); and FUB-AMB. These analytes were incubated for 120 minutes with human liver microsomes, followed by analysis of the extracts via ultra high performance liquid chromatography – tandem mass spectrometry (UHPLC-MS/MS). The high-resolution mass spectrometry tool utilized (quadrupole-time of flight mass spectrometry, QTOF) allowed for a thorough characterization of the metabolites, including the assignment of a chemical formula and structure, and accurate mass. The metabolic stability and kinetic profiles of 5F-ADB and FUB-AMB were evaluated by aliquoting the incubation samples at various time points throughout the procedure. It was observed that these compounds were metabolized rapidly, resulting in short half-lives and relatively elevated metabolic clearances. A variety of metabolites were identified for most of the species studied, and this was dependent on the chemical structure of the parent molecule. The major metabolites identified overall for the species were products of amide or ester hydrolysis; hydroxylation (including polyhydroxylation) of the pentyl side chain or cyclohexylmethyl moiety; and oxidative defluorination. It is proposed that these metabolites (especially analyte-specific metabolite) be included in laboratory assay panels to facilitate unequivocal identification of the synthetic cannabinoid agent of interest. For select compounds (5F-ADB and FUB-AMB), authentic forensic human blood samples which screened positive for these analytes were provided by a renowned forensic toxicology laboratory. These samples were tested to verify that the major metabolites identified in the in vitro studies were also present in blood in vivo; the resultant data from the 5F-ADB and FUB-AMB samples showed that the major hydroxylated and hydrolysis metabolite, respectively, were present in greater abundance than the parent molecule, which was most often absent or not present in an appreciable quantity. Additionally, it was observed in the time studies of 5F-ADB and FUB-AMB that the metabolites containing carboxylic acid functional groups were detected in incubation samples longer than the hydroxylated metabolites, potentially indicative of longer detection windows in human samples. These findings have important toxicological implications; many synthetic cannabinoid metabolites, including those identified in this study may have pharmacological activity and contribute to a drug user’s overall impairment profile; identifying them in blood in the absence of parent compound can point to the causative agent. The results demonstrate that it is imperative that synthetic cannabinoid assays screen for known pharmacologically active metabolites; this is particularly important for drugs with short half-lives. The results of this research can be applied to the prediction of metabolic pathways for synthetic cannabinoids as well as non-drug substances with similar structural elements whose metabolic profile has not yet been elucidated, and whose pharmacological activity is currently unknown. Additionally, the results provide reference standard manufacturers and research scientists with further insight into the metabolic products of synthetic cannabinoids and related compounds for the synthesis of materials for the development of laboratory assays. / Chemistry

Biomedical devices play a crucial role in the healthcare system, enabling more effective treatments, less invasive procedures, and more precise diagnoses. Due to these compelling reasons, development of new biomedical devices and biomaterials have always been in high demand. Exploring and refining fabrication methods are essential to the development of new biomedical devices. Some of the common fabrication methods include microfabrication methods (photolithography and soft lithography), 3D printing (additive manufacturing), laser machining, thermal drawing, and electrospinning. The choice of fabrication methods heavily depends on the materials, geometry, and functionalities of biomedical devices. Currently, the thermal drawing process has proven to be an excellent scalable fabrication platform for neural interface, tissue engineering, tumor/cancer treatment, soft robotics, and smart textiles. This Ph.D. dissertation summarizes my research on the fabrication and validation of thermally drawn multifunctional polymer fiber probes for modern biomedical applications, primarily in the fields of neural interfaces and tumor treatments. Understanding the neural basis of behavior requires monitoring and manipulating combinations of physiological elements and their interactions in behaving animals. Utilizing the thermal drawing process, we developed T-DOpE (Tapered Drug delivery, Optical stimulation, and Electrophysiology) probes and Tetro-DOpE (Tetrode-like Drug delivery, Optical stimulation, and Electrophysiology) probes that can simultaneously record and manipulate neural activity in behaving rodents. Taking advantage of the triple-functionality, we monitored local field potential (LFP) while manipulating cannabinoid receptors (CB1R; microfluidic agonist delivery) and CA1 neuronal activity using optogenetics. Focal infusion of CB1R agonist downregulated theta and sharp wave-ripple oscillations (SPW-Rs). Furthermore, we found that CB1R activation reduces sharp wave-ripples by impairing the innate SPW-R-generating ability of the CA1 circuit. Microscale electroporation devices are mostly restricted to in vitro experiments (i.e., microchannel and microcapillary). We developed a flexible microscale electroporation fiber probe through a thermal drawing process and femtosecond laser micromachining techniques. The novel fiber microprobes enable microscale electroporation and arbitrarily select the cell groups of interest to electroporate. Successful reversible and irreversible microscale electroporation was observed in a 3D collagen scaffold (seeded with U251 human glioma cells) using fluorescent staining. Leveraging the scalable thermal drawing process, we envision a wide distribution of multifunctional polymer fiber probes in research facilities and hospitals. Along with the fiber probes presented in this dissertation, additional insight and future perspective on thermally drawn biomedical devices are discussed. / Doctor of Philosophy / The thermal drawing process is a versatile and scalable platform for fabricating functional fiber technology. The process was formerly adapted from fabrication method for silica optical fibers, widely used in telecommunication (e.g., telephone, internet, cable TV, etc.). To name some functionalities of these fibers, they can move, hear, sense touch, change colors, harvest and store energy, record and manipulate brain activity, and ablate tumors. As imagined, these functionalities are derived from the unique geometry and functional materials embedded along the fiber. Therefore, developing the fiber design tailored to a specific application is a critical step to making a successful fiber product. In this dissertation, I will present my work on biomedical devices fabricated with the thermal drawing process and their application in neuroscience and tumor/cancer treatment. Utilizing the thermal drawing process, we developed neural interfaces that can be implanted into the deep brain and record and simultaneously manipulate the neural activity. These neural interfaces (Chapter 2,3; T-DOpE and Tetro-DOpE probes, respectively) are able to record both local field potentials (LFP; activity of thousands or more neurons) and single action potentials (single on/off signal from individual neurons nearby). By manipulating the gene expression, we can control the activity of neurons with specific light (λ= 470nm; blue light) exposure. We implemented optical waveguide in our probes to guide light from a laser source to the tip of the probe and manipulate the neural activity. Furthermore, we fabricated micro-channels within the device to enable focal drug delivery at the tip of the device. Using the T-DOpE probe, we studied the effect of local synthetic cannabinoid injection in the hippocampus. We found that the local injection of the drug in hippocampus CA1 makes neurons incapable of generating sharp wave-ripples (a neural signal associated with memory). Electroporation is a biophysical phenomenon where short high electric field pulses introduce nanoscale defects in cell membrane. These defects can cause unstable cellular homeostasis and eventually leads to cell death. Due to reduced treatment time, no heat effect, and tissue selectivity, electroporation has been used in clinical trials for cancer treatments. Using the thermal drawing process and laser micromachining techniques, we developed a flexible microscale electroporation fiber probe capable of ablating tumor cells. Due to the low-cost and scalability of thermal drawing process, we envision the use of thermally drawn functional fiber technology in biomedical fields. In this dissertation, I also address some challenges and future directions of thermally drawn functional fibers in biomedical fields.

Lors de cette étude, nous avons d’abord localisé les récepteurs CB1 et CB2 sur les structures neuronales. Nous avons montré que les récepteurs CB1 et CB2 sont présents sur les dendrites et les axones et les filopodes. Dans le même ordre d’idée, nous avons localisé le récepteur DCC sur les structures neuronales. Celui-ci est aussi présent sur les dendrites, les axones et les filopodes. Ces résultats suggèrent que le récepteur DCC serait impliqué non seulement dans le processus de synaptogenèse médié par le récepteur CB1, comme cela a été montré dans le laboratoire du professeur Bouchard, mais aussi dans celui, éventuellement, médié par le récepteur CB2. Nous avons ensuite évalué l’effet des ligands du récepteur CB2. Nous n’avons détecté aucun effet clair des agonistes inverses (AM630 et JTE907) et des agonistes (JWH015 et JWH133) quant à la médiation du processus de synaptogenèse en terme de variation de la densité des filopodes et des points de contacts synaptiques. Nous avons obtenu des résultats variables. Ceux-ci furent non reproductibles. Nous avons obtenu des résultats différents des résultats originaux lorsque nous avons requantifié visuellement les mêmes photos à deux reprises Nous avons développé une méthode informatisée de quantification qui nous a permis d’obtenir des résultats reproductibles. Cependant, nous n’avons toujours pas détecté d’effets sur la synaptogenèse médiés par le récepteur CB2. Ces résultats préliminaires ne nous permettent ni d’infirmer, ni de confirmer d’éventuels effets sur la synaptogenèse médiés par le récepteur CB2. Une étude exhaustive serait nécessaire pour le déterminer. / During this study we first localised the receptors CB1 and CB2 on neuronal structures. We have shown that those receptors expressed on dendrites and filopodia. Likewise and based on Bouchard’s previous laboratory results showing an implication of the netrin-1 receptor, Deleted in Colorectal Cancer (DCC), on the synaptogenesis process mediated by the receptor CB1 we localized the receptor DCC on neuronal structures. We have shown that the receptor DCC is expressed on dendrites, axons and filopodia. These results suggest an implication of the receptor DDC in a synaptogenesis process that would be mediated by the receptor CB2. We then evaluated the effects triggered by the receptor CB2’s ligands on the synaptogenesis process. We found no evidences of any effects on synaptogenesis mediated by the receptor CB2 inverse agonists (AM630 and JTE907) and agonists (JWH015 and JWH133) in term of filopodia density and synaptic contacts density variations. We witnessed highly variable results that were irreproducible. Visual quantifications of filopodia and synaptic contacts density were variable as we quantified two times the same set of photos. We have therefore developed a computer based quantification method by which we were able to obtained reproducible results. Nevertheless we found no evidence of any implication of the receptor CB2 on the synaptogenesis process. These preliminary results do not allow us neither to rule out nor to confirm eventual CB2 receptor effects on synaptogenesis. An exhaustive study is required to access possible CB2 receptors effect on synaptogenesis.

Lors de cette étude, nous avons d’abord localisé les récepteurs CB1 et CB2 sur les structures neuronales. Nous avons montré que les récepteurs CB1 et CB2 sont présents sur les dendrites et les axones et les filopodes. Dans le même ordre d’idée, nous avons localisé le récepteur DCC sur les structures neuronales. Celui-ci est aussi présent sur les dendrites, les axones et les filopodes. Ces résultats suggèrent que le récepteur DCC serait impliqué non seulement dans le processus de synaptogenèse médié par le récepteur CB1, comme cela a été montré dans le laboratoire du professeur Bouchard, mais aussi dans celui, éventuellement, médié par le récepteur CB2. Nous avons ensuite évalué l’effet des ligands du récepteur CB2. Nous n’avons détecté aucun effet clair des agonistes inverses (AM630 et JTE907) et des agonistes (JWH015 et JWH133) quant à la médiation du processus de synaptogenèse en terme de variation de la densité des filopodes et des points de contacts synaptiques. Nous avons obtenu des résultats variables. Ceux-ci furent non reproductibles. Nous avons obtenu des résultats différents des résultats originaux lorsque nous avons requantifié visuellement les mêmes photos à deux reprises Nous avons développé une méthode informatisée de quantification qui nous a permis d’obtenir des résultats reproductibles. Cependant, nous n’avons toujours pas détecté d’effets sur la synaptogenèse médiés par le récepteur CB2. Ces résultats préliminaires ne nous permettent ni d’infirmer, ni de confirmer d’éventuels effets sur la synaptogenèse médiés par le récepteur CB2. Une étude exhaustive serait nécessaire pour le déterminer. / During this study we first localised the receptors CB1 and CB2 on neuronal structures. We have shown that those receptors expressed on dendrites and filopodia. Likewise and based on Bouchard’s previous laboratory results showing an implication of the netrin-1 receptor, Deleted in Colorectal Cancer (DCC), on the synaptogenesis process mediated by the receptor CB1 we localized the receptor DCC on neuronal structures. We have shown that the receptor DCC is expressed on dendrites, axons and filopodia. These results suggest an implication of the receptor DDC in a synaptogenesis process that would be mediated by the receptor CB2. We then evaluated the effects triggered by the receptor CB2’s ligands on the synaptogenesis process. We found no evidences of any effects on synaptogenesis mediated by the receptor CB2 inverse agonists (AM630 and JTE907) and agonists (JWH015 and JWH133) in term of filopodia density and synaptic contacts density variations. We witnessed highly variable results that were irreproducible. Visual quantifications of filopodia and synaptic contacts density were variable as we quantified two times the same set of photos. We have therefore developed a computer based quantification method by which we were able to obtained reproducible results. Nevertheless we found no evidence of any implication of the receptor CB2 on the synaptogenesis process. These preliminary results do not allow us neither to rule out nor to confirm eventual CB2 receptor effects on synaptogenesis. An exhaustive study is required to access possible CB2 receptors effect on synaptogenesis.