Clinical trials have established the efficacy and safety of the 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors (statins) in lowering cardiovascular morbidity and mortality in patients with and without coronary artery diseases. Traditionally, the beneficial effects of statins have been ascribed entirely to their ability to lower serum cholesterol. However, evidence indicates that statins may exert cholesterol-independent or pleiotropic effects. As well as reducing plasma cholesterol levels, statins induce acute vasorelaxation which may contribute to the overall benefits of statins in the treatment of cardiovascular disease. The mechanism underlying this relaxation is unknown. Statins have been shown to alter mitochondrial function. Therefore, the aim of this study was to determine the role of the mitochondria in the relaxation to statins. Changes in rhodamine 123 fluorescence showed that simvastatin, but not pravastatin, depolarized the membrane potential of mitochondria in both isolated smooth muscle cells and intact blood vessels. As simvastatin, but not pravastatin, causes relaxation of the porcine coronary artery, this could be due to this effect on mitochondria. Mitochondria are known as the energy generating centre of the cells. However, there is growing consensus that mitochondria actively participate in intracellular signalling, such as production of reactive oxygen species (ROS) and regulation of the intracellular Ca2+ concentration. Moreover, ROS could play an important supportive role in a variety of vascular cell signalling processes, including activation of nitric oxide synthase (NOS), modulation of intracellular Ca2+, and AMP kinase activation. Therefore, this study investigated whether the relaxation to the lipophilic statin simvastatin is due to an effect on the mitochondria. Relaxation of porcine coronary artery segments by statins was measured using isolated tissue baths. Simvastatin, but not pravastatin, produced a slow relaxation of the coronary artery, which was independent of K+ channel activation, nitric oxide, cyclo-oxygenase, or the endothelium. The relaxation was attenuated by the mitochondrial complex I inhibitor rotenone and the complex III inhibitor myxothiazol, or a combination of the two. Simvastatin inhibited calcium-induced contractile responses, and this inhibition was partially reversed by incubation with the complex I inhibitor rotenone suggesting that mitochondrial function is required for the effect of simvastatin on calcium influx. The effect of mitochondrial complex III inhibitor, antimycin A, was examined as a comparison with simvastatin. Antimycin A induced porcine coronary relaxation and inhibited Ca2+ influx in isolated porcine coronary smooth muscle cells. Evidence from a number of clinical trials highlights a potential association between treatment with lipophilic statins and increased risk of development of diabetes. The close connection between energy metabolism and insulin secretion in pancreatic β-cells suggests that the glycaemic effects of simvastatin may also result from a direct mitochondrial action with reduction in insulin secretion and, hence, result in a reduced control of plasma glucose levels. Although simvastatin depolarized mitochondria in pancreatic β-cells, it also directly inhibited KATP channels. Pravastatin, on the other hand, had no effect on either measurement, suggesting that these phenomena relate to the lipophilicity of the compounds. The inhibition of KATP channels by simvastatin is likely to underlie the increase in insulin secretion observed within days of simvastatin treatment. On the other hand, the effects on mitochondrial membrane potential may be detrimental, particularly with chronic treatment, although further studies are required in order to determine whether this plays a role in the increased risk of diabetes observed with lipophilic statins. Overall, our results demonstrated that simvastatin alters mitochondrial membrane potential in vascular smooth muscle cells and pancreatic β-cells. The relaxation to simvastatin in the porcine coronary artery is dependent, in part, upon mitochondrial activity. Alteration of mitochondrial membrane potential by simvastatin may lead to inhibition of calcium influx, hence stimulation of relaxation. On the other hand, the effects on mitochondrial membrane potential in pancreatic β-cell may be detrimental, particularly with chronic treatment due to the increased risk of diabetes observed with lipophilic statins.

An association between breast cancer (BC) and thyroid autoimmunity (TA) has been frequently observed and several small-scale studies correlated the presence of thyroid peroxidase (TPO) autoantibodies (TPOAb) with an improved BC outcome. The presence of an immune response to shared thyroid/breast antigens has been hypothesized: both tissues express the sodium iodide symporter (NIS) and have a peroxidase activity: TPO in thyroid and lactoperoxidase (LPO) in breast. I have identified 3 possibilities: i) BC patients with TPOAb may also have autoantibodies to NIS (NISAb) ii) BC may express TPO iii) TPOAb may cross-react with LPO in BC. This thesis aimed to identify the TA/BC shared antigen(s) and also investigated the prognostic role of TPOAb in a large cohort of BC patients. The presence of NISAb was investigated by flow cytometry using CHO cells stably transfected with human NIS. No positive response was obtained in 42 patients with BC and/or TA, therefore NIS is unlikely to be an antigen. TPO transcripts (RT-PCR, QPCR, LongRange PCR) and protein (western blot, immunohistochemistry, immunofluorescence) were detected in BC tissues but at levels 104 times less than in thyroid tissue. TPO was also expressed in adipose tissue and different cancers. Some potentially BC specific TPO isoforms were identified. The observational large-scale study conducted on 1974 women affected with BC did not reveal any evidence for a significant impact of TPOAb and/or thyroid function on BC prognosis. In conclusion, BC and thyroid tissues share similar properties and could be common targets of TA, with TPO being the most likely common antigen; further studies are needed to clarify the role of tissue-specific TPO isoforms. The roles of TPOAb and thyroid function on BC prognosis have to be reconsidered, maybe focusing on different TA aspects (e.g. goitre, different autoantibodies).

G protein-coupled receptors (GPCRs) are seven-pass integral membrane proteins that act as transducers of extracellular signals across the lipid bilayer. Their location and involvement in basic and pathological physiological processes has secured their role as key targets for pharmaceutical intervention. GPCRs are targeted by many of the best-selling drugs on the market and there are a substantial number of GPCRs that are yet to be characterised; these could offer interest for therapeutic targeting. GPR35 is one such receptor that, as a result of gene knockout and genome wide association studies, has attracted interest through its association with cardiovascular and gastrointestinal disease. Elucidation of the basic physiological function of GPR35 has, however, been difficult due a paucity of potent and selective ligands in addition to a lack of consensus on the endogenous ligand. Herein, a focussed drug discovery effort was carried out to identify agonists of GPR35. Various in vitro cellular assays were employed in conjunction with N- or C-terminally manipulated forms of the receptor to investigate GPR35’s signalling profile and to provide an assay format suitable for the characterisation of newly identified ligands. Although GPR35 associates with both Gαi/o and Gα13 families of small heterotrimeric G proteins, the G protein-independent β-arrestin-2 recruitment format was found to be the most suited to drug screening efforts. Small molecule compound screening, carried out in conjunction with the Medical Research Council Technology, identified compound 1 as the most potent ligand of human GPR35 reported at that time. However, the lower efficacy and potency of compound 1 at the rodent species orthologues of GPR35 prevented its use in in vivo studies. A subsequent effort, carried out with Novartis, focused on mast cell stabilisers as putative agonists of GPR35, revealed lodoxamide and bufrolin as highly potent agonists that activated human and rat GPR35 with equal potency. This finding offered–for the first time–the opportunity to employ the same GPR35 ligand between species at a similar concentration, an important factor to consider when translating rodent in vivo functional studies to those in man. Additionally, using molecular modelling and site directed mutagenesis studies, these newly identified compounds were used to aid characterisation of the ligand binding pockets of human and rat GPR35 to reveal the molecular basis of species selectivity at this receptor. In summary, this research effort presents GPR35 tool compounds that can now be used to dissect the basic biology of GPR35 and investigate its contribution to disease.

Salmonellosis causes significant economic losses to the pig and poultry industries. Pigs and chickens are also a significant source of Salmonella for humans, usually transmitted through the consumption of Salmonella contaminated chicken and pork products. Predominantly, Salmonella enterica serovar Typhimurium and Enteritidis remain a global health problem. Probiotics and prebiotics have been previously used as alternatives to antibiotic treatments in the protection against enteropathogens including S. Typhimurium. Here, we determined the effects of probiotic, prebiotic and synbiotic diets on humoral immune responses to oral S. Typhimurium challenge of pigs. The inclusion of probiotic Lactobacillus plantarum in the diet of piglets enhanced serum IgG, and IgM (p <0.001), and IgA (p <0.01) responses to S. Typhimurium infection. Similarly, inclusion of prebiotic lactulose in the diet increased serum levels of IgG and IgM (p <0.01) responses to pathogen, but not IgA levels. Inclusion of both feed additives as a synbiotic diet also significantly increased the level of IgG responses (p <0.05) to S. Typhimurium, but no differences were seen in the levels of IgM and IgA responses. However a significant interaction of the pre and probiotics was observed when considering the immune responses to S. Typhimurium (IgM P=0.004; IgG and IgA, P<0.001 for interaction). These data support the use of L. plantarum or lactulose as strategies to contribute to the protection of weaned piglets from zoonotic bacterial pathogens, but caution must be taken when combining dietary supplements as combinations can interact. The mapping of antibody-based immune responses to Salmonella enterica infections for identifying epitopes/mimotopes has an important role in the development of both novel serological diagnostic assays and vaccines. Serological assays often underpin disease surveillance programs and are also required for the differentation of infected from vaccinated animals (DIVA) to allow the full implementation of vaccines alongside such surveillance. Here, panning of phage display peptide libraries coupled with Next Generation Sequencing was applied to the mapping of B-cell responses to Salmonella infections in both pigs and chickens. IgG from 12 pigs infected with S. Typhimurium were probed in parallel and compared to the equivalent IgG from the same pigs prior to infection. Seventy-seven peptide were enriched against IgG from multiple infected pigs, thirty-one peptides were synthesised and tested in ELISA and twelve peptides were highly discriminatory for pure IgG from infected pigs (P<0.05). Similarly, IgY from chickens infected with different Salmonella serovars were probed in order to identify mimotopes specific for S. Enteritidis infection. Twenty-nine peptides were enriched against IgY from multiple infected chickens, and then synthesised and tested in ELISA assays, tweleve of them were highly discriminatory for IgY following S. Enteritidis infections (p<0.05). The technology was also used to identify multiple peptides that were specifically bound by IgY from Salmonella infected chickens compared to a live attenuated vaccine and a killed vaccine. Twenty-five and thirty-six peptides for attenuated and inactivated vaccines, respectively, were identified as being specifically enriched in multiple infected chickens. Twenty and twenty-six of the most discriminatory peptides for live and killed vaccines, respectively, were applied in multi-peptide diagnostic assays that diagnosed infection with 100% sensitivity and specificity. The results demostrate that the identified peptides can be used to design serological DIVA tests with established inactivated and attenuated vaccines. Overall, the described next generation phage display (NGPD) technology repeatedly identified panels of epitopes/mimotopes recognised by multiple animals with a particular infection, providing an extremely efficient method to map host polyclonal antibody responses to S.Typhimurium and S. Enteritidis infections.

The rise in demand for fresh fruits and vegetables has seen an increase in the risk of gastrointestinal disease, by pathogens such as E. coli O157:H7 and Salmonella enterica, since such produce is eaten uncooked. Although most produce is washed in chlorinated water, this processing may not be sufficient and can result in the formation of hazardous compounds. Classical cell culture techniques as well as novel episcopic differential contrast and epifluorescence microscopy (EDIC/EF) combined with the BacLightTM kit (to distinguish live and dead bacteria), the DAPI assay (to distinguish bacterial colonists and inorganic debris) and GFP-Salmonella strains were combined for the first time to study the complex leaf surface (phylloplane). EDIC/EF microscopy was shown to be advantageous compared to other methodologies; as well as being able to visualize GFP-labelled Salmonella inoculated onto the phylloplane it was possible to observe the naturally residing microflora on this difficult matrix. The viable pathogens and microflora were shown to colonize by four strategies: they form clusters on the phylloplane; or single cells integrate into pre-existing aggregates of biofilm microcolonies; they become entrapped in niches such as stomata; or they actively swim into the stomata and become subsurface (confirmed using laser scanning confocal microscopy, LSCM). The clusters were sometimes surrounded by slime, suggesting the formation of biofilm on the phylloplane. The effects of treatments to the phylloplane were not directly comparable, due to large biological variations in each field of view; smaller treated sample areas should allow for qualitative and quantitative comparisons. The Stomacher is at present used worldwide for the mechanical release of microorganisms from various matrices; here it was compared to the Pulsifier, which was shown to be more efficient in terms of cell recovery and causing less damage to the watercress phylloplane. Surface attachment was investigated by use of the Pulsifier release principle and refinements in its protocol were made. Pulsifier recovery techniques showed the inefficiencies of potential disinfectants in killing attached microorganisms, since they were not susceptible to attack until released into aqueous suspension. It is these ‘protected’ cells that then subsequently go on to produce foodborne illnesses. Further study showed the molecular signalling molecule nitric oxide (NO), to be an important physiological release agent, for enhanced recovery of coliforms, but not Salmonella, from the phylloplane. Chemical methods of decontamination such as the use of ozone were shown to be efficient at reducing the numbers of viable cells, particularly when combined with pulsification mechanical release of cells into aqueous suspension, resulting in between 1- and 2-log reductions. However, this procedure is not ideal, due to chemical damage to the phylloplane and problems in maintaining constant ozone concentrations, both in the laboratory and at the factory. It was shown that chlorine levels could be reduced to 20 ppm compared to the industry standard of 90-120 ppm, this producing similar log reductions of between 1- and 2-log. The Pulsifier and NO were shown in combination to provide effective mechanical and physiological detachment strategies, releasing almost 4-log cells. It was found that 20 or 500 nM of NO, produced a 3-log dispersion of bacterial cells, including biofilm aggregates off the surface of watercress leaves. These studies demonstrate the importance of microbial physiology in the attachment of microorganisms on fresh produce phylloplanes and suggest that disinfection procedures are unnecessary for sanitation.

Abstract During the course of the studies outlined in this thesis, a new approach for the synthesis of the tropane alkaloid, (±)-physoperuvine has been developed using a highly efficient one-pot tandem process which involved the Overman rearrangement and a ring closing metathesis reaction. An asymmetric one-pot tandem process has also been employed for the synthesis of the natural product, (+)-physoperuvine. This methodology was also applied to the generation of a late-stage intermediate that could be used in the synthesis of carbocyclic nucleosides, such as noraristeromycin. In the second part of this thesis, an ether-directed Pd(II)-catalysed Overman rearrangement which had previously been developed by the Sutherland group was applied in conjunction with a cross-metathesis reaction for the stereoselective synthesis of the guanidine alkaloid, (+)-monanchorin in a fourteen-step synthesis. Further employment of this process provided the first synthesis of clavaminol A, C and H from (R)-glycidol in a rapid and efficient manner. In a similar fashion, (2S,3R)-enantiomers were also synthesised from (S)-glycidol. In addition to this, using similar chemistry, an intermediate protected enone was prepared using a cross-metathesis reaction as the second key step in an approach towards the synthesis of an NO-inhibitor.

Background: Metastases are the major cause of death from melanoma, a skin cancer which has the fastest rising incidence of any malignancy in the Western world. Molecular pathways that drive melanoblast migration in development are believed to underpin the movement and ultimately the metastasis of melanoma. Aims: In this thesis we use genetically modified mice models to characterise two novel anticancer targets, P-Rex1 and focal adhesion kinase (FAK). Embryonic melanoblast migration is compared with cancer outcomes for each genetic modification. Results: Mice lacking P-Rex1, a Rac-specific Rho GTPase guanine nucleotide exchange factor (GEF), have a melanoblast migration defect during development evidenced by a white belly. These P-Rex1-/- mice are resistant to metastasis when crossed to a murine model of melanoma, an effect specifically channeled through loss of P-Rex1 GEF activity. FAK disruption compromises melanoblast cell numbers and migration in development, but has no long-term effect on melanocyte homeostasis. FAK-deleted mice have a divergent role in melanomagenesis, delaying primary melanoma onset whilst promoting metastasis following disease onset. Conclusions: We conclude that P-Rex1 and FAK play important roles in melanoblast embryology and melanoma development and progression. Both P-Rex1 and FAK represent interesting therapeutic targets for the treatment of cancer.

Cyclic adenosine monophosphate (cAMP) is a second messenger signalling molecule that has been reported to exert beneficial effects within the vasculature and other physiological systems. cAMP produces its effects within the cell through two key downstream effector molecules: exchange protein activated by cAMP (EPAC) and protein kinase A (PKA). Many of the effects of cAMP have been attributed to PKA, however there is a growing appreciation of the potential of EPAC, particularly isoform 1 (EPAC1), based therapies for the regulation of inflammatory responses within the vasculature, thereby promoting cardiovascular health. Furthermore, side effects associated with global cAMP elevating agents may be avoided by isoform selective EPAC regulation. To date no small molecule agonists have been discovered to effectively or selectively promote EPAC1 activity. In order to address this, we have developed a fluorescence based competition assay able to identify compounds which interact with the cyclic nucleotide binding domains (CNBs) of both EPAC1 and EPAC2. Rigorous testing of the assay has confirmed that it is able to reliably and reproducibly identify EPAC interacting compounds within high throughput screening (HTS) of small molecule libraries. Furthermore, dual screening of EPAC1 and EPAC2 has allowed isoform selective compounds to be identified from a small compound library, confirming the suitability of this assay for HTS. This HTS assay is likely to facilitate the discovery of EPAC1-selective interacting molecules with the potential to be effective, small molecule regulators of EPAC1. In order to classify small molecules isolated by HTS as either agonists or antagonists of EPAC1, we developed a secondary screen that is able to detect EPAC1 activation in vivo. This assay is based on the ability of EPAC1 to produce a rapid, cell spreading response in HEK293T cells stably transfected with EPAC1. However, the precise signalling pathways which produce these changes in cell shape are unknown. Therefore, we have attempted to identify pathways involved in EPAC1-mediated morphological change by assessing the effects of various inhibitors on cell spreading. Interestingly, we found that EPAC1 and PKA synergise to produce maximal cell spreading in HEK293T cells. Recent reports suggest that the cortical actin-membrane linker protein ezrin is required for the cell spreading effects of EPAC1. Here, we demonstrate that ezrin responds to elevations in intracellular cAMP in HEK293T cells in a PKA-dependent manner. Indeed, PKA activation promotes the post translational modification of ezrin and alters the response of EPAC1-expressing cells to cAMP. These results suggests that the PKA pathway is able to regulate ezrin by post translational modification and that this is required for PKA and EPAC1 to synergise and produce maximal cell spreading. In addition to agents which directly activate the catalytic activity of EPAC1, there is a body of evidence that supports the idea that compartmentalisation of cAMP effectors is an important mechanism for the determination of downstream signalling events leading to cellular responses, such as cell spreading. As such, we have attempted to identify the regions within EPAC proteins that determine their subcellular distribution. This was done through a combination of subcellular fractionation and the immunofluorescent detection of the localisation of EPAC isoforms. In particular, mutational analysis of EPAC1 revealed a carboxy terminal (C-terminal) nuclear localisation domain that is required for the perinuclear distribution of EPAC1 alongside the nuclear pore protein, RANBP2. Structural analyses suggest that this domain appears to be conserved within EPAC2 despite EPAC2 adopting a distinct cytoplasmic distribution. One explanation for this observation is steric interference within EPAC2 which blocks access to the conserved nuclear localisation domain. We have observed that the additional amino-terminal (N-terminal) CNB of EPAC2 appears to disrupt nuclear localisation and promote a cytoplasmic distribution within the cell. Indeed, the absence of the CNB1 promotes nuclear accumulation of EPAC2, with a pattern similar to that of EPAC1. The presence of this domain within EPAC2, absent in EPAC1, may represent a mechanism which regulates the subcellular distribution, and therefore function, of EPACs within the cellular environment. In summary, we have developed a screening cascade to identify small molecules which may form the basis of therapeutic agents able to selectively target EPAC1 to promote the beneficial effects of EPAC1. In addition, a secondary screen involving EPAC1 induced morphological change was developed and characterised as an effective assay in which to test the agonist properties of compounds identified by primary HTS screening. We have confirmed that HEK293T cell spreading in response to cAMP elevation requires the expression of EPAC1, but that a secondary pathway involving interactions between PKA and ezrin is able to supplement the primary cell spreading effects of EPAC1. Finally, we have identified a potential mechanism for the different subcellular localisation of EPAC1 and EPAC2: EPAC1 is targeted to the perinuclear compartment via a previously undiscovered C-terminal nuclear localisation domain.

Aspirin and clopidogrel are cornerstone therapies in cardiovascular disease. In particular, they are almost universally prescribed in patients undergoing percutaneous coronary intervention (PCI). Evidence has emerged of a variation in the antiplatelet effects of aspirin and clopidogrel between individual patients with a suggestion of an increased risk of adverse cardiovascular events. However, the optimal method of measuring response to aspirin and clopidogrel remains uncertain. In light of this, the antiplatelet effects of both aspirin and clopidogrel were studied in patients with coronary artery disease, concentrating on patients undergoing PCI. Initially, a pilot study of 40 patients investigated the use of thromboxane B2 (TxB2), VerifyNow Aspirin, VerifyNow P2Y12, platelet fibrinogen binding and intra-platelet vasodilator-stimulated phosphoprotein levels (VASP-PRI) to measure response to aspirin and clopidogrel. This was followed by a larger study assessing aspirin and clopidogrel response in 323 patients attending for coronary angiography with a view to PCI. These patients were tested by measuring TxB2, VerifyNow P2Y12, VASP and whole blood impedance platelet aggregation (WBPA). The primary objective was to investigate whether measures of aspirin or clopidogrel efficacy predicted peri-procedural myocardial necrosis following PCI. In addition, a small series of 10 patients had aspirin and clopidogrel response measured following stent thrombosis. A wide variation in the antiplatelet effects of both aspirin and clopidogrel was found by all measures. Correlation between assays ranged from moderate to poor. Of particular interest, it was found that measurement of [TxB2] may facilitate the assessment of aspirin response in patients already taking clopidogrel. There was a high incidence of myocardial necrosis following coronary intervention assessed by elevation of troponin I. Only VerifyNow P2Y12 and VASP-PRI were associated with a significantly increased frequency of myocardial necrosis following PCI. The data of this thesis confirm a wide variation in response to aspirin and clopidogrel. Good response to clopidogrel was associated with reduced myocardial necrosis during PCI. TxB2 may be the best measure of aspirin response for patients taking both therapies. How these measures may be incorporated into clinical practice remains uncertain.

This thesis covers a range of important issues in hair analysis and includes 27 scientific works in which the name of the candidate was either listed as the first author or as the major contributor. The work presented in this thesis involved the development of a series of analytical methods to detect trace amounts of drugs in hair and also investigated the mechanisms by which drugs may be incorporated into hair. The major areas covered in this study can be summarized as follows: 1. The methods for the identification and quantification of opiates, amphetamines, ketamine, cannabis, cocaine, benzodiazepines, antidepressants, antipsychotics, and anabolic steroids in hair were developed using gas chromatography–mass spectrometry (GC-MS), liquid chromatography–tandem mass spectrometry (LC-MS/MS) and gas chromatography–tandem mass spectrometry (GC-MS/MS). With GC-MS methods, the limits of detection were 0.1-0.5 ng mg-1 of hair for antidepressants and antipsychotics. For illegal drugs, hair specimens were analyzed by GC-MS with limits of detection of 0.02-2ng mg-1. GC/MS/MS is more sensitive than GC-MS to detect these drugs in hair. The lower limits of detection ranged from 0.001 to 0.020 ng mg-1 for 21 anabolic androgenic steroids and their esters in hair using liquid chromatographic-tandem mass spectrometric method. And the limits of detection ranged from 0.2 to 5 pg mg-1 for benzodiazepines in hair. Tandem mass spectrometry is characterized by its sensitivity, selectivity and specificity, which makes it particularly suitable for the analysis of trace amount of target analytes in hair. 2. Usually, screening for drugs of abuse is the first step in clinical and forensic toxicology. There are a large number of controlled substances and doping agents and novel compounds, which have yet to be characterised. A series of screening methods for drugs of abuse in hair were developed using LC-MS/MS and GC-MS/MS. Using our own library of MRM transitions, the optimum collision energies selected for each transition and retention times were set up. These methods have been applied successfully in forensic casework. 3. Of growing importance to the field of hair analysis is the detection of metabolites related to the parent drugs. Demonstrating the presence of a metabolite of a drug (such as, heroin, amphetamines, cocaine, meperidine, ketamine, triazolam or psychotropic drugs) provides compelling evidence for exposure to the parent drug, and permits distinction between external contamination from ingestion and facilitation of the interpretation of results. The presence of antidepressant and antipsychotic drugs and their metabolites in the hair of psychiatric patients was investigated using GC-MS-EI and GC-MS-PCI. The parent drug and its major metabolite, such as opiates (morphine, 6-acetylmorphine), methamphetamine (methamphetamine, amphetamine), ketamine (ketamine, norketamine), cocaine (cocaine, benzoecognine), meperidine (meperidine, normeperidine), triazolam (triazolam, α-hydroxytriazolam), and clonazepam (clonazepam, 7-aminoclonazepam) were quantified in authentic hair samples simultaneously. The differences were finding in the ratio of parent drug to metabolite. For illegal drugs, the concentrations of parent drugs were higher than that of their metabolites. The results of triazolam and clonazepam were contrary. These data are suitable reference values and are the basis for the interpretation of results. 4. The mechanisms by which drugs are incorporated into hair are not fully understood. Based on experiments with guinea pigs with black, white, or brown hair, the mechanisms of incorporation of cocaine, methamphetamine, ketamine, triazolam and anabolic steroids into hair were investigated. The concentrations of drugs in hair were found to be related to physicochemical properties of drugs. The parent drugs were the predominant analytes in hair. There was an obvious relationship between the concentration of drugs in hair and hair pigmentation. The concentrations of drugs deposited in black hair was found to be higher than that in brown and white hair samples, even when comparing results using hairs on the same multicoloured animal body. This work confirmed that melanin affinity is a governing factor in drug incorporation into hair shafts. These studies on the distribution of drugs in the hair shaft and how their concentration changes along the shaft provide information relevant to the time of ingestion and substance use/abuse. 5. In recent years an increase in drug-facilitated sexual assault (DFSA) has been reported. Segmental hair analysis has proved useful in widening the window of detection, as blood and urine analyses are of limited use, due to the long delays between the actual assaults and obtaining samples from suspects that are frequently encountered in investigations of such crimes. In China, benzodiazepines are the most frequently observed compounds in cases of drug-facilitated crime. In a paper reported here, 14 volunteers ingested a single 1-6 mg estazolam tablet to permit the evaluation of segmental hair analysis after a single drug dosage. Hair was collected one month after administration of the drug. All the proximal segments tested positive for estazolam. With increased dosage, estazolam could be detected in the 2-4 cm segments nearest the hair root in some subject’s hair shafts. In some cases, the 4-6 cm segments also tested positive. Hair analysis was applied to samples from two authentic criminal cases. A significant variation was observed between those obtained from previous studies and the results presented here. The intersubject variability in segmental analysis can be explained mainly due to melanin content and diffusion from sweat or other secretions during formation of the hair shaft. However, more substantial procedural and interpretation guidelines are required to use segmental hair analysis in drug-facilitated crimes. On the other hand, the minimal dosage for detection, which is a critical but previously unknown threshold value of fundamental importance in hair analysis, was determined for triazolam and ketamine in guinea pig hair. 6. Doping with endogenous anabolic steroids is one of the most serious drug issues in sports today. The measurement of anabolic steroid levels in human hair permits the distinction between pharmaceutically produced steroids and naturally occurring steroids. Full-length hair samples were taken at the skin surface from the vertex of 39 males, 30 females and 11 children from China. None of the subjects were professional athletes. Testosterone and dehydroepiandrosterone were detected in all the hair segments. The physiological concentrations of testosterone were in the range 0.8-24.2 pg mg-1, 0.1-16.8 pg mg-1 and 0.2-11.5 pg mg-1 in males, females and children, respectively. However, the mean values of dehydroepiandrosterone were much higher than those for testosterone. This is the first investigation into the physiological concentrations of anabolic steroids in human hair in Chinese subjects. These data provide suitable reference values and form the basis for the interpretation of results from investigations into the abuse of endogenous anabolic steroids. In conclusion, the work presented in this study demonstrates that there was a good correlation between the concentration of drugs in hair and drug dosage. There was an obvious relationship between hair drug concentration and hair colour. Melanin affinity is shown to be a governing factor in determining drug incorporation into hair, and the concentration of drugs deposited in black hair was found to be higher than that in brown and white hair samples. This thesis provides data that will be useful in the application of hair analysis regarding drugs of abuse and in the interpretation of toxicological results.