Thesis (Ph. D.)--Virginia Commonwealth University, 2007. / Prepared for: Dept. of Microbiology and Immunology Bibliography: leaves 134-154.

Thesis (Ph. D.)--University of Washington, 2004. / Vita. Includes bibliographical references (leaves 93-112).

Thesis (Ph.D.)--Aberdeen University, 2008. / Title from web page (viewed on July 20, 2009). Includes bibliographical references.

Thesis (Ph.D.)--Aberdeen University, 2009. / Title from web page (viewed on Mar. 4, 2010). Includes bibliographical references.

Thesis (Ph.D.)--Duquesne University, 2004. / Title from document title page. Abstract included in electronic submission form. Includes bibliographical references and abstracts.

Cannabinoids are compounds that are naturally present in Cannabis sativa L., which interact with cannabinoid receptors in the nervous system, known as CB1 and CB2 receptors. The most abundant and well-known cannabinoid that can be isolated from cannabis is 9-tetrahydrocannabinol (THC). The structure of this compound specifically allows interaction with the CB1 and CB2 receptors, known as cannabimimetic activity. Other compounds have since been produced, inspired by THC, which have been designed to elicit similar pharmacological responses, and therefore are beneficial as analgesics. These compounds are known as synthetic cannabinoids. Synthetic cannabinoids, while potentially useful as therapeutic treatments for pain, are currently also popular as recreational drugs. Herbal products that contain synthetic cannabinoids are sold as “legal highs,” as few of these compounds are illegal according to the Controlled Drugs and Substances Act. These products are prepared by combining synthetic cannabinoids and plant material, and are smoked similar to marijuana. As the legality of many synthetic cannabinoids is quickly decreasing, as evidenced by the March 2011 emergency scheduling of five such compounds, it is becoming increasingly likely that these products will soon become popular exhibits to be submitted to controlled substances laboratories for testing. If a previously smoked product is submitted, there could potentially be effects due to the burning, the presence of the plant or paper substrate, and other synthetic cannabinoids that could directly diminish the facility of analysis. The aim of this thesis was to investigate these effects using four synthetic cannabinoids (AM-2201, JWH-015, HU-211, and RCS-4) and four substrates (tobacco, rolling paper, mint, and rosemary). Results demonstrated diminished peak areas, which are likely due to the introduction of these variables, which include burning the drug of abuse, and spiking the drug of abuse onto various matrices. The trend of lower peak areas further suggests that burning, the presence of plant material, and other cannabinoids potentially all compromise the facility of analyzing synthetic cannabinoid products. The act of burning one synthetic cannabinoid in particular, AM-2201, appeared to greatly decrease the capability to detect the analyte, as did the application of AM-2201 to various substrates. Furthermore, the ability to detect AM-2201 appeared to vary greatly between results obtained from analyzing samples applied to different substrates. Analysis of cannabinoid mixtures demonstrated that GC/MS analysis of different cannabinoids gave various peak areas although the concentrations remained consistent. Peak area ratios of cannabinoid mixtures that were extracted from substrates were found to not differ significantly between the specific substrates studied. This research supports that all of these variables should therefore be considered in regards to analysis of herbal products containing synthetic cannabinoids.

Activation of the cannabinoid receptor CB₂ has been shown to induce directed leukocyte migration and inhibit leukocyte chemotaxis towards CC chemokines. However, the role that CB₂ plays in regulating macrophage chemotaxis remains understudied. Using a real-time chemotaxis assay and a panel of chemically diverse CB₂ agonists, I set out to examine whether CB₂ modulates primary macrophage chemotaxis. Of 14 agonists tested, only a subset acted as bona fide macrophage chemoattractants. Surprisingly, despite being pertussis toxin-sensitive, neither pharmacological inhibition nor genetic ablation of CB₂ had any effect on CB₂ agonist-induced macrophage chemotaxis. Furthermore, the activation of CB2 had no effect on CCL2 or CCL5- induced macrophage chemotaxis. Therefore, the activation of CB2 does not inhibit CC chemokine-induced macrophage migration and a non-CB₁/CB₂, G_i/o-coupled GPCR must transduce CB2 agonist-induced macrophage chemotaxis. To identify the GPCR responsible, I examined primary murine macrophage GPCR expression and found that they express 124 non-sensory GPCRs. Functional screening of candidate receptors demonstrated that the putative cannabinoid receptors GPR18 and GPR55 and the lipid binding GPCRs LPAR1&5, CYSLTR1&2 and GPER1, were not responsible for CB₂ agonist-induced macrophage chemotaxis. Alongside, a ligand-directed virtual screen, combined with functional testing, uncovered a novel chemotaxis positive chemical scaffold. Importantly, compounds in this series containing a photoaffinity label retained activity and will aid in the identification of the target(s) responsible for CB₂ agonist-induced macrophage chemotaxis in future photocrosslinking experiments. Finally, I assessed whether CB2 controls innate immune cell recruitment in vivo using the zymosan-induced dorsal air pouch inflammation model and animals genetically deleted for CB₂. I found that CB₂^-/- mice had increased air pouch neutrophil and monocyte numbers, as well as pro-inflammatory mediators, during the acute inflammatory phase. Interestingly, mixed bone marrow chimera experiments demonstrated that lack of CB₂ specifically in the myeloid population is responsible for increased neutrophil trafficking. Therefore these data demonstrate that CB₂ acts to regulate neutrophil recruitment during the acute inflammatory response.

Type 2 diabetes mellitus results from a combination of insulin resistance and impaired insulin secretion. The aim of this study is to investigate the effect of endogenous and exogenous cannabinoids on insulin resistant cell lines, viz skeletal muscle (C2C12) and fat (3T3-L1), and to investigate the effects of these cannabinoids on insulin secretion in pancreatic β-cells (INS 1). Insulin resistance was induced in the cells using 20 ng/mL TNF-α (3T3-L1) and 100 nM insulin (C2C12). Insulin resistant cells were exposed to cannabinoids for 48 hours after which glucose uptake, RT-PCR and Western blot analysis was performed. Additionally, adipokine assays were performed on the 3T3-L1 cells. The insulin resistant 3T3-L1 and C2C12 cells had reduced glucose uptake, decreased IRS-1 and Glut-4 expression indicative of an insulin resistant state. The extract and THC significantly enhanced glucose uptake, IRS-1 and Glut-4 in 3T3-L1 and C2C12 cells. The extract and THC thus have the potential to be an insulin sensitizing agent. Interleukin-6 was significantly decreased by THC. INS 1 cells, cultured under normoglycemic conditions, were exposed to cannabinoids for 48 hours after which glucose-stimulated insulin secretion, radioimmunoassay, oxygen consumption, RT-PCR and Western blot analysis was performed. Insulin stimulatory index was not significantly affected after cannabinoid exposure, except by THC. The cannabinoids decreased insulin content, in a concentration dependent manner, but the inhibition mechanism remains elusive. The cannabinoid Treated cells showed insulin gene expression levels similar to the control, while only THC proved effective in significantly stimulating Glut-2 gene expression. Oxygen consumption studies showed levels lower than the control cells. Most of the cannabinoids inhibited insulin secretion under normoglycemia except THC, while the cannabinoids exhibited the potential to improve insulin resistant adipocyte and myocytes response to glucose and gene regulation.

The aggregation of misfolded proteins in the endoplasmic reticulum (ER) is a pathological trait shared by many neurodegenerative disorders. This aggregation leads to the persistent activation of the unfolded protein response (UPR) and ultimately apoptosis due to ER stress. Cannabinoids, such as tetrahydrocannabinol (THC) and cannabidiol (CBD), have been reported to be neuroprotective in in vitro and in vivo models of neurodegeneration through their antioxidant and anti-inflammatory properties. However, little is known about the role of these cannabinoids in the context of ER stress. STHdhQ7/Q7 cells were treated with the ER stress inducer thapsigargin (TG) and cannabinoids in three different experimental paradigms to investigate the effect of 2.5 µM THC and 1 µM CBD monotreatment and cotreatment on ER stress-induced cell death. The mouse striatal neurons survived significantly more when THC or CBD was given before TG exposure. To further investigate this experimental paradigm, the gene and protein expression of UPR proteins was measured to determine the effect of cannabinoid pre-treatment on cell survival through ER stress modulation. A significant increase in the gene expression of the ER chaperone GRP78 and the ER-resident neurotrophic factor MANF in pre-treated samples suggest that with THC or CBD pre-treatment, the protein folding capacity of the cell is improved. Additionally, a decrease in the ER-mediated apoptotic markers such as BIM and caspase 12 with THC or CBD pre-treatment provides further evidence that cannabinoid pre-treatments are neuroprotective through ER stress modulation. These data suggest that prior cannabinoid monotherapy prepares the cell for future insults to the ER. Understanding the role of ER stress in the neuroprotective properties of THC and CBD provides insight into the therapeutic potential of cannabinoids and the role of ER dysfunction in various neurodegenerative disorders. / Thesis / Master of Health Sciences (MSc) / With the worldwide ageing population increasing, finding new treatments for illnesses that affect the elderly is crucial. Disorders such as Parkinson’s and Alzheimer’s disease mainly affect older individuals and are caused when brain cells stop working or when brain cells die. These disorders share some common causes. One is the inability to fold proteins properly. The cellular process that is responsible for protein folding and the changes that occur within that process are studied in this project. Also, the impact of the cannabinoids THC and CBD, a major component of cannabis, on the protein folding process is studied. This project found that using cannabinoids before the protein folding system is disrupted helps brain cells survive. This study is a step in understanding how THC and CBD are helpful in brain cell survival in patients suffering from diseases that damage brain cells.

Many studies have reported the anti-cancer effects of n-3 long chain polyunsaturated fatty acids (n-3 LCPUFAs). In this study, the anticancer effects of n-3 LCPUFAs and their derivatives, n-3 n-acyl ethanolamides (n-3 NAEs) were investigated in MCF-7 and MDA-MB-231 breast cancer cell lines. The focus was on their effects on cell proliferation, the role of CB1 and CB2 receptors, the effects of their main metabolising enzyme, FAAH, and which molecular signalling pathways are involved. In addition, the effects on the redox system, particularly in the modulation of redox genes, proteins and enzymes, the role of epigenetic regulation, and the effects on cell invasion and migration were also investigated. The results showed that n-3 NAEs were more potent than their parent LCPUFAs at reducing cell viability while the effects of both n-3 LCPUFAs and n-3 NAEs appear to be CB receptor mediated. In addition, MAPK pathways were also affected to varying extents following treatment, in particular p38 and JNK. Furthermore, global methylation, antioxidant gene expression, migration and invasion were in general all modulated by treatment. However, the effects observed were found to be both treatment- and cell- type dependent. Nevertheless, these results confirm that n-3 LCPUFAs and n-3 NAEs inhibit breast cancer cell growth, and modulate important cancer related pathways albeit by different mechanisms. This suggests that dietary intervention with cheap, safe, readily available fatty acids could be introduced to breast cancer patients to enhance treatment.