The persistence and detection of gene transcripts in post mortem tissues has significance for forensic science and medicine, since there is the potential to provide information about the cause of, or circumstances surrounding, death and the post mortem interval. Previous studies in this area have focused on enzyme activities and the presence of gene transcripts in neural tissues (Palego et al., 1993; Johnson ci aL, 1986). In the study presented in this thesis, the biological material selected was dermal tissue from pigs, since the skin offers a number of advantages for study in a forensic context, such as ease of harvesting and a slower rate of degradation than most other soft body tissues. Initial chapters describe the development of methodologies for the extraction of RNA from dermal tissue and the detection and quantification of gene transcripts. The genes selected as indicators of gene expression were members of the heat shock gene family, since these genes are highly conserved and easily induced in cells. Subsequent chapters present evidence, not only for the persistence and detection of gene transcripts in post mortem tissue, but also for the inducible de novo expression of heat shock genes in post mortem (PM) tissue. A mild heat shock, applied in the post mortem period to porcine tissue, elicits the expression of certain genes of the heat shock gene family. These results demonstrate that heat shock genes are highly inducible in the early post mortem period, and that these cells retain the ability to respond to an environmental stimulus by transcribing specific genes.

Many eukaryotic organisms produce membrane interactive, a-helical antimicrobial peptides (a-AMPs) and a database of such peptides, together with selected physiochemical parameters was established. This database was divided into four groups according to the a-AMPs target organism(s) (active against Gram-positive bacteria {G+}; active against Gram-negative bacteria {G-}; active against Grampositive and Gram-negative bacteria {G+, G-}; or active against Gram-positive bacteria, Gram-negative bacteria and fungi {G+, G-, F}). Analysis of the database showed that there was no statistically significant correlation between specificity and p1 (range 4.2 to 12.7) or net charge (range -5 to +16). The peptides exhibited variable hydrophobicity, < H > (range -0.8 to +0.7) whilst amphiphilicity (measured by the hydrophobic moment, < jAH >) ranged from 0.2 to 1.1. A statistically significant negative correlation between < pH > and < H > was noted for each group of a-AMPs and this may relate to the amphiphilic balance required for antimicrobial activity, a-AMPs showed some differences in amino acid composition compared to the McCaldon and Argos dataset of unrelated oligopeptides, suggesting functional relevance of some amino acid residues. The groups {G+, G-} and {G+, G-, F}, for example are characterised by being rich in weakly hydrophobic or hydrophilic amino acids. Minimum inhibitory concentration (MIC) and minimum lethal concentration (MLC) can provide a measure of AMP potency. A statistical analysis of MIC's for peptides from {G+, G-} group, showed no significant differences in the potency of these peptides when directed against either Gram-positive or Gram-negative bacteria. In contrast, a statistical analysis of MIC's for peptides from the {G+, 0-, F} group showed that peptides from this group were effective at lower concentrations against bacterial targets as compared to fungal targets. Increases in hydrophobic arc size were generally accompanied by increases in peptide antimicrobial potency and in addition, a negative correlation between MIC and net charge was observed. Regression analyses indicated that an appropriate amphiphilicity/hydrophobicity balance was required for the antimicrobial action of a-AMPs and this may indicate a general structure/function relationship underlying both the efficacy and specificity of these peptides. Oblique orientated a-helices are highly specialised protein structural elements that penetrate membranes at a shallow angle and are used to promote membrane destabilisation by a number of protein classes. Here, the use of extended < pH > methodology showed that over 50% of the a-AMPs are candidate oblique ahelices providing some insight into possible modes of action. Peptides VP I and BYDV-MP were identified here as candidate AMPs based on amino acid composition and the potential to form oblique orientation. The biological activity of VPI and BYDV-MP was confirmed in vivo when an MLC of 3 mM was demonstrated on both Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli. Monolayer studies using lipid extract from these target organisms and parallel studies using mimetic monolayers confirmed a high level of peptide membrane interaction. The single lipid monolayer results suggested that VPI and BYDV-MP have a lower affinity for zwitterionic lipid (DMPE surface pressure increase of 4 mNm') but a high affinity for anionic lipid (DMPS surface pressure increase of 7 to 9 mN m') and may have a requirement for this specific lipid or anionic lipids in general to achieve higher levels of membrane penetration. To test the ability of VP! to penetrate membranes protonated and deuterated homologues were analysed by neutron diffraction, in the presence of POPC: POPS (10:1 molar ratio). The data analysed from these studies showed the protonated homologue to penetrate the membrane core but the deuterated homologue showed no significant levels of membrane interaction. Monolayer studies confirmed that the protonated homologue interacted strongly with anionic and zwitterionic membranes (surface pressure increase 4 mNm'), however, the deuterated homologue did not have the ability to interact with POPC:POPS monolayers. FTIR conformational analysis showed the protonated homologue to adopt high levels of a-helical stmcture (65 %) and in contrast the deuterated homologue exhibited low levels of a-helical structure (C 20 %). These results support the original predictions and also appear to show that deuteration has directly or indirectly, affected the ability of the VP! peptide to interact with membranes, possibly by inhibiting a-helix formation by the peptide or decreasing structural stability.

Forged documents are comnonly encountered in the investigation of crime. Forensic document examiners need to be able to employ techniques that are non-destructive, reliable and admissible in court. In this work, techniques currently in use for ink analysis, including FLE, TLC, UV - Vis, IR-ATR and Raman spectroscopy, have been reviewed and, where possible optimised in order to evaluate the level of discrimination possible between different ballpoint and gel pen inks. To date, information comparing such a wide range of techniques to relatively large sample sets has not been published. Particularly, lacking is analysis of ballpoint pen inks from the UK, ballpoint pen ink colours other than black and blue and any analysis of gel pen inks. The results vary widely depending on the combination of ink colour and type, paper and the technique being used. FLE achieved the best discrimination for blue gel pen inks giving a DP of 0.92. TLC is most effective for black and red ballpoint pen inks, giving a DP of 0.89 for both. The highest DP for UV - Vis, 0.86, is achieved for black gel pen inks and for IR-ATR it is red gel pen inks that are most effectively discriminated between, with DPs of 0.88 and 0.87. The Raman spectroscopy analysis used a greater number of combinations of papers, and experimental parameters, i.e. different excitation wavelengths and 'standard' Raman spectroscopy or SERRS with silver or gold colloid, therefore, it is not possible to select a single type and colour of ink to which the technique is best suited. The pairs of inks discriminated by the five techniques are not mutually exclusive. In real casework it is unlikely that all the instrumentation would be available and there will be financial constraints on how many techniques can be carried out. Therefore, using the data obtained here, protocols have been suggested in order to predict the technique or combination of techniques that is most likely to achieve discrimination of two different inks for each pen ink type and colour. Raman spectroscopy or SERRS are required for 23 out of the 28 recommended protocols suggesting that they would be valuable tools for document examination laboratories. However, for Raman spectroscopy, although it is a non-destructive method of analysis, when the SER.RS enhancement technique is used, which is effectively non-destructive, some problems with reproducibility were found. Causes of the irreproducibility have been put forward, such as inhomogeneity of the ink and the paper substrate and ink and paper interactions, but no single cause has been identified. Despite this, if care is taken with analysis and interpretation, SERRS results can be useful.

In order to explore south Arabia's role in the migratory episodes leaving Africa to Eurasia and back, high-resolution Y-chromosome analyses of males from the United Arab Emirates (164), Qatar (72) and Yemen (62) were performed. The distribution of specific haplogroups (E3bl-M35 and J1-M267) and their microsatellite-based age estimates in southern Arabia offer additional insight on their dissemination. With the exception of Yemen, southern Arabia displays high diversity in its Y-haplogroup substructure and share similarities with populations along the eastern coast of the Gulf of Oman, possibly serving as a coastal corridor for migrations. Elevated rates of consanguinity may have had an impact in Yemen and Qatar, which experience deficiencies in their ratios of observed to expected heterozygosity at 15 hypervariable autosomal STR loci. Higher diversity along the Gulf of Oman may be due to trade emanating from the kingdom of Oman involving East Africa, southern Pakistan and western India.

Current forensic comparisons of soil most often rely upon physical characterizations. We hypothesized that bacterial community profiles obtained by Amplicon Length Heterogeneity-Polymerase Chain Reaction (ALH-PCR) of the 16S rRNA genes would provide discriminating data for soil comparisons. Dual extractions and replicate amplifications were performed on each soil. Chemical characterization by elemental analysis, pH, moisture content, percent Carbon and percent Nitrogen were performed. Supervised classification of the microbial community profiles using a Support Vector Machine (SVM) learning tool was over 95 % accurate labeling a microbial community profile to its originating soil type. By comparison, the chemical analysis data yielded accuracies between 40 and 77 %. The results of this study support the application of this method in the comparison of casework size soil samples. Results of this study may also justify the future development of a database of microbial community profiles for inferring the possible origin of unknown soil samples.

The drugs studied in this work have been reportedly used to commit drug-facilitated sexual assault (DFSA), commonly known as "date rape". Detection of the drugs was performed using high-performance liquid chromatography with ultraviolet detection (HPLC/UV) and identified with high performance-liquid chromatography mass spectrometry (HPLC/MS) using selected ion monitoring (SIM). The objective of this study was to develop a single HPLC method for the simultaneous detection, identification and quantitation of these drugs. The following drugs were simultaneously analyzed: Gamma-hydroxybutyrate (GHB), scopolamine, lysergic acid diethylamide, ketamine, flunitrazepam, and diphenhydramine. The results showed increased sensitivity with electrospray (ES) ionization versus atmospheric pressure chemical ionization (APCI) using HPLC/MS. HPLC/ES/MS was approximately six times more sensitive than HPLC/APCI/MS and about fifty times more sensitive than HPLC/UV. A limit of detection (LOD) of 100 ppb was achieved for drug analysis using this method. The average linear regression coefficient of correlation squared (r2) was 0.933 for HPLC/UV and 0.998 for HPLC/ES/MS. The detection limits achieved by this method allowed for the detection of drug dosages used in beverage tampering. This method can be used to screen beverages suspected of drug tampering. The results of this study demonstrated that solid phase microextraction (SPME) did not improve sensitivity as an extraction technique when compared to direct injections of the drug standards.

Ascertaining the genetic relationships between Austronesian populations is pivotal to understanding their dispersal throughout the islands of the Pacific and Indian Oceans. The Austronesian expansion dates to approximately 6,000 years ago and from the linguistic and archeological evidence, the origin of this dispersal appears to be Taiwan. In this study, six polymorphic point mutation loci were studied in Taiwanese aborigines and compared with 32 other populations. The genetic relationships were characterized by maximum likelihood analysis, principal component maps, centroid gene flow plots, expected heterozygosities, power of discrimination values and pair wise G-tests. Following these analyses, it was apparent that genetic similarities existed between the Atayal and the Chinese, whereas the Ami displayed similarities with the Native Americans. Thus, the Atayal have little or no affinity for the Ami and other Austronesian populations. The large genetic differences between the two groups most likely arise from genetic isolation, and/or small population sizes.

This study evaluates the use of commercially available instruments for locating and collecting accelerants in the field. Electronic noses can be used to scan a fire scene for the possible presence of an accelerant. The TLV Sniffer® was found to be able to detect accelerants at low levels but did alert to some burned matrix alone. When subjected to a proficiency test designed for canines, the TLV Sniffer® was able to locate accelerants in two of the three tests. The tpi®Pocket was found not to be sensitive or selective enough to be useful in locating accelerants. Once the location of possible accelerants has been determined, they can be collected by dynamic headspace sampling in the field with the Portable Arson Sampler (PAS). The PAS was found to be able to collect a broad range of compounds from ignitable liquids and had comparable efficiency to a conventional method.

The ReJI29 murine model of muscular dystrophy was employed to investigate the properties of skeletal muscle plasmalemma and calcium ion movements during muscle wastage, with the intention of determining the route of calcium influx, and the efficacy of calcium ion blockers in preventing this influx. Electrophysiological parameters (Resting membrane potential [RMP] and input resistance) reached adult magnitude in normal soleus and extensor digitorum longus (EDL) by 4 weeks and 3 weeks respectively. Electrophysiological parameters in dystrophic soleus developed in a similar maimer to normal muscle, but RMP in dystrophic EDL was reduced and input resistance was elevated suggesting the presence of a population of regenerating fibres. Twitch and contracture tension development reached mature levels by 4 weeks in normal soleus muscle, but development was prolonged to 10 weeks in EDL, due to the slower development of type II fibres. Contracture tension was markedly smaller in EDL possibly due to fibre type-related differences in the concentrations of calcium sequestering proteins. Twitch and contracture tension in dystrophic soleus was identical to normal. In dystrophic EDL, 4 week old muscle did not generate tension from either electrical or chemical stimulation. Older muscles generated tension but smaller than their normal counterparts. Twitch and contracture tomography revealed similarities between dystrophic EDL and immature normal EDL corroborating with the conclusions made from the electrophysiological experiments. Caffeine contractures generated in glycinerated normal soleus muscle, incubated in low calcium saline, and then bathed in high calcium saline reached 115-185% the magnitude of caffeine contractures generated prior to incubation. 4 week dystrophic soleus attained similar sized contractures, but contractures were reduced to 77-90% in older muscles. This technique was found to be inappropriate in the study of EDL. Caffeine contractures generated by normal soleus in normal saline, after incubation in low calcium saline, reached the magnitude of contractures generated in high calcium saline after 30 minutes. Contractures generated by dystrophic soleus remained identical to those generated in high calcium saline suggesting that the sarcoplasmic reticulum in these fibres was unable to cope with a high calcium load. This result gave the first indication that soleus muscle was affected by muscular dystrophy. Membrane bound calcium derived from 45Calcium influx studies was 2nmoles/mg tissue/30 minutes in soleus and 1 Snmoles/mg tissue weight/30 minutes in EDL. Intracellular influx was 0.5-0.6nmoles/mg tissue weight/ 30 minutes in both muscles. Membrane bound calcium was elevated in dystrophic EDL to 2.5-3.8nmoles/mg tissue weight/30 minutes, but influx was normal in both dystrophic soleus and EDL, casting doubt on whether the plasmalemma is more permeable to calcium. The increase in membrane bound calcium may be artefactual. Cadmium blocked influx in both normal and dystrophic muscles. Blot weights showed continual growth in normal muscles although the rate decreased after 10 weeks. In dystrophic muscles growth ceased after 10 weeks. NCP data was limited but did show a decrease in dystrophic muscle, followed by an increase. Tension as a function of tissue mass, revealed that the contractile apparatus in normal soleus matured within 2 weeks, and by 10 weeks in EDL due to the differences in myosin isoforms present in each fibre type. These ratios were elevated in dystrophic muscle indicating calcium accumulation in the sarcoplasmic reticulum. The results showed that EDL muscle was more severely affected by muscular dystrophy. Between 2 and 4 weeks, the muscle underwent degeneration via an unidentified process. There was regeneration, and the regenerating fibres appeared to be normal. Soleus muscle appeared resistant to the disease but succumbed under unusual chemical stresses. Some aspects of development of normal muscle were also considered.

Investigations were made into the role of blood-borne substances affecting myocardial contractility. Isolated left and right guinea-pig au -ia were used as bioassay for the detection of endogenous cardiotonic substances in bovine serum. Results show that serum that had been buffer exchanged to Krebs-Henseleit by dialysis or Sephadex G-25, produced positive inotropic and positive chronotropic effects on the isolated guinea-pig au-ia. The hypodynamic state was not a prerequisite for the detection of cardioactivity. The cardioactive effectswere not blocked by the combined presence of propranolol and methysergide (both 10 6M) and were also dissimilar in time course from other known cardiotonic agents such as noradrénaline, isoprenaline, 5-hydroxytryptamine and cardiac glycosides. After ultrafiltration on Amicon diaflo membranes (XM100A and PM30A) activity was found solely in the retentate fractions. The ultrafiltrates were devoid of positive inotropic and chronotropic actions. Since the molecular weight cut-off of the XM100A membranes was 100 Kilodaltons, it is probable that the active principle is greater than 100 kilodaltons. Alternatively, a smaller active substance may be bound to a large protein. The cardioaciive factor in whole bovine serum was heat labile and lost after 30 minutes incubation at 70 0C or boiling for 10 minutes. Activity of whole bovine serum was not cold labile during a 30 minute incubation at 0°C. Activity was also present after dialysis at 4-8 °C for up to 48 hours. Cardioactivity of whole serum was lost following acidification to pH 5.0 at 4-8 0C for 24 hours. This experiment was extended to include a wider range of pH values between 4.5 and 75. The positive inotropic and chronotropic activities of serum were markedly reduced at acidic pH values. The cardioactive factor was also lost after equilibration of serum to physiological buffers of a low ionic strength. The loss of activity was independent of the nature of the buffer and was prevented by raising the ionic strength with the addition of NaCl. The cardiotonic principle was also sensitive to the addition of KBr but not equimolar (or higher) concentrations of KCI or NaCl. The above results taken together would be consistent with a proteinaceous nature of the active substance. The major purification methods therefore employed included precipitation techniques involving (NH4)2SO4 and polyethyleneglycol-8000, ion exchange, hydrophobic interaction chromatography and gel filtration. Attempts to isolate and purify the cardiotonic agent were largely unsuccessful due, particularly, to the labile nature of the active molecule when exposed to non-physiological experimental conditions. Investigations in to the cellular mechanism of action of the cardioactive factor in buffer exchanged serum suggested that the effects may perhaps be mediated by increases in the level of endogenous cyclic AMP and not via inhibition of the N a+,Kt ATPase. In conclusion, the present study has demonstrated the existence of a factor in bovine serum that produced positive inotropic and chronotropic effects on the isolated guinea-pig au-ia. Properties of the cardioactive factor suggest the existence of a novel proteinaceous substance with some previously unidentified characteristics.