Quantitative polymerase chain reaction (qPCR) techniques are currently used to quantify samples containing deoxyribonucleic acid (DNA) in forensic analyses. This technology can provide valuable information to an analyst regarding the amount of DNA present but lacks the ability to determine the quality of the sample. Electrochemistry-based biosensors that utilize screen-printed electrodes may provide a method to determine the number of DNA molecules and the length of those molecules in a single assay. This work aimed to create a biosensor by electrostatically loading TPOX oligonucleotides onto a carbon screen-printed electrode for the purpose of quantifying genomic DNA. Electrochemical signal was obtained via the indicating molecule bis-benzimide H33258, which preferentially interacts with double-stranded DNA and would indicate a hybridization event. Cyclic voltammetry was chosen to measure the current signal; peaks obtained using this technique can be analyzed with the Randles-Sevčik equation, which relates current signal with concentration of the target species. A large amount of signal variation and background charging current was observed when H33258 was used as the redox probe. This led to a study of the surface characteristics of the carbon electrodes themselves (i.e. effective surface area) by utilizing the reversible and well-characterized redox couple hexaammine ruthenium. The effect of electrode activation at high anodic potentials was also studied. Though highly recommended in the literature, activation of the carbon surface caused effective surface area and charging current to increase. While a larger electro-active surface is often desirable, the high background current generated when activation is used within the protocol can mask the signal of interest. Due to the low signal-to-noise ratio and inability to reuse the carbon electrode, it was concluded that carbon screen printed electrodes are not optimal forensic DNA biosensors.

Ignitable liquids consist of either a single organic compound or a complex organic mixture. In regards to fire debris analysis, the analyst is responsible for determining if an ignitable liquid residue is present. However, when extracted from soil-containing fire debris evidence, chemical degradation from microorganisms is observed to result in the loss of compounds based on chemical structure. It can also happen when the evidence container is stored at room temperature before analysis. This can present a challenge to the fire debris analyst when identifying and classifying the ignitable liquid residue based on the criteria established by standard test methods. The purpose of this research was to observe the microbial degradation of fourteen compounds, at room temperature over a period of time, for possible by-product formation that could coincide with compounds normally present in an ignitable liquid. Additionally, a quantitative assessment was performed to observe and record the loss rate of compounds in a representative simple mixture. Finally, the loss rate from the simple mixture was compared to commercially available ignitable liquids. Degradation studies were conducted to observe the microbial degradation of a representative compounds (individually and in a simple mixture, both weathered and unweathered) and seven ignitable liquids of different ASTM E1618 classifications. Potting soil was spiked with 20 µL of a liquid/compound and was allowed to stand at room temperature for a period of time. The simple mixture was evaporated to 50% and 90% using a steady nitrogen gas flow to compare the degradation process to the unweathered mixture. All samples were extracted and analyzed using passive-headspace concentration and gas chromatography-mass spectrometry. The formation of by-products was not observed when degrading the compounds from the simple mixture individually as seen in other research. The simple mixture, unweathered and 50% weathered, resulted in rapid degradation of their oxygenated compounds. The straight-chained alkanes and toluene were observed to be more susceptible to microbial attack than the highly-substituted aromatics and the branched and cyclic alkanes. The 90% weathered mixture followed the same degradation trend as the unweathered and 50% weathered samples, although it only contained two compounds. The loss rates/half-lives for each simple mixture sample (unweathered, 50% weathered, and 90% weathered) were determined to be approximately 3.5, 3.5, and 0.84 days. The unweathered and 50% weathered sample half-lives were similar due to containing compounds with similar susceptibility to degradation, while the 90% weathered sample contained one compound that was more highly susceptible to degradation. When comparing the 3.5 day half-life to the seven different ASTM class liquids, the isoparaffinic product and the naphthenic-paraffinic product had similar rates of degradation while aromatic solvent and normal alkane classes had the shortest half-lives. When observing the degradation of the gasoline, medium petroleum distillate and the miscellaneous, the constituent compounds were seen to exhibit a range of degradation rates that corresponded to half-lives less than and greater than 3.5 days.

To date, the use of mitochondrial DNA in forensic analysis has relied on the presence of variations in the control region to differentiate between samples. One problem that this analysis has shown is the occurrence of common Haplogroup H haplotypes or identical sequences. Thus, there is a need to enhance the distinguishing power of this type of analysis. One option has been to investigate the mitochondrial coding region for polymorphisms that could differentiate between samples with identical control region haplotypes. The goal of this study has been to identify polymorphic coding region sites for development in a Pyrosequencing assay that would effectively enhance the discriminatory power of mitochondrial DNA analysis. With this goal in mind, five duplexes have been successfully developed and tested, utilizing the ten polymorphic sites that had been selected, with most sites being specific to Caucasians. Validation studies were performed to test the durability of the assay. The specificity of the assay to primate and non-primate species was determined to be limited to primate species only. Sample variations, including mixtures, dilutions and environmental exposure, were utilized to assess the sensitivity of the Pyrosequencing method. It was found that a minimum initial DNA input of 10fg was necessary for reliable results. The Pyrosequencing assay was able to detect mixtures at a 1:1 ratio and environmental samples exposed to the elements from up to 1 week for blood and 6 weeks for semen. Samples designed to simulate typical casework materials were analyzed and found to provide for consistent results, including trace fingerprints and digested hair shafts. These validation results provide the conclusion that this assay is suitable for use in forensic casework and demonstrate that the mitochondrial coding region provides a viable alternative to hypervariable region analysis.

Clinical and forensic toxicology analysts rely heavily in their daily tests on the analysis of the conventional samples (blood and urine). However, these specimens are limited in the time scale they reflect with regard to drug intake history and also in terms of drug stability within the matrices. Alternative matrices such as hair, oral fluids and dried blood spots (DBS) provide new horizons and new opportunities. Drugs incorporated within hair are very stable. Hair also provides a very long detection window, for at least one year, if not a lot longer. Oral fluids on the other hand are non-intrusive, easy to collect and much cleaner sample matrix than blood or urine. DBS also offer great drug stability, are easy to collect, faster to analyse and suitable for automated analysis. However, a number of studies are needed to assess the limits of these alternative samples in terms of the correlation of their results with the results of conventional samples and with regard to drug stability. Such studies will enable a more reliable and confident interpretation of results obtained from these matrices especially for medico-legal purposes. The main aims of this research were: to develop and validate analytical methods for detection and quantitation of drugs of use and abuse in hair, oral fluids, blood and DBS samples, to investigate the correlation between dose and drug concentration in hair, blood and oral fluids after controlled chronic drug administration, to investigate the stability of anti-psychotic drugs in DBS (from patients) stored under different conditions and the effect of addition of preservative, and to investigate the alcohol intake prevalence among Kuwaiti drug addicts and correlate these results with selfreported intake. As the majority of drugs were basic, an extraction method based on methanolic incubation was developed for detection of basic/weak basic drugs in hair. It was compared to alkaline digestion (with NaOH) followed by liquid-liquid extraction (LLE). Detection was achieved by LC-MS/MS (Sciex2000) after separation on a C18 column. When applying both methods on positive authentic hair samples the results showed that the methanolic method was capable of extracting most basic drugs in hair but only partially, while the alkaline digestion method was found to degrade V some unstable drugs like sulpiride, but was capable of fully extracting the alkaline stable drugs such as quetiapine. After development and validation of the LLE-LC-MS(Exactive) method for the analysis of anti-psychotics in blood, oral fluids and hair, an investigation was carried out on the correlation pattern between trough concentrations in those three matrices. The most significant correlation coefficients (r) found were those between blood and hair concentrations, procyclidine r=0.83 (18 subjects p=<0.001), risperidone r=0.96 (14 subjects p=<0.001), haloperidol r=0.90 (10 subjects p=<0.001), OH-risperidone r=0.24 (13 subjects p=>0.44), quetiapine r=0.28 (14 subjects p=>0.33) and chlorprothixene r=0.32 (13 subjects p=>0.32). Among the interesting results was the strong correlation found between drugs half-lives and the mean ratio of hair concentration/dose (r=0.96, p=<0.003). The stability of anti-pyschotics in DBS from patients’ samples was assessed by storing them at four different temperatures (25, 4, -20 and -80°C) with and without prior impregnation of the DBS cards with sodium fluoride. After development and validation of the LLE-LC-MS method, samples were analysed at days 0, 45, 90 and 180. Results showed good stability of all the compounds (procyclidine, quetiapine, risperidone, OH-risperidone, chlorprothixene and haloperidol) in all the different storage conditions and no significant increase or decrease in drug concentrations with sodium fluoride impregnation. Finally, after trials with five different HPLC columns, two SPE cartridges, two LLE extraction procedures and two mass spectrometer instruments, a method was developed and validated for the detection and quantitation of alcohol’s minor and specific metabolite in hair, ethyl glucuronide (EtG). The method has a limit of detection (LOD) of 3pg/mg and lower limit of quantitation (LLOQ) of 9pg/mg. This method was applied to 59 hair samples from patients at a general addiction centre and alcohol prevalence was investigated and its correlation with self-reported use was investigated.

The production of methamphetamine in clandestine laboratories presents a particular hazard due to the environmental hazards it poses. In addition to the dangers associated with using caustic and reactive solvents, these clandestine laboratories also have to potential to cause a fire or explosion. This danger has caused some states to redefine arson to include fires caused by the illicit manufacture of drugs. Arson investigation can be challenging due to the destructive nature of the crime. Much of the evidence that existed prior the fire can be consumed and evidence that does survive can be difficult to identify in the rubble. Despite these difficulties, methods have been developed to determine the types of accelerants present in addition to identifying illicit substances such as methamphetamine and the precursor drug pseudoephedrine. This study was designed to determine if solid phase microextraction combined with gas chromatography/mass spectrometry could be used to analyze burned samples of wood to which pseudoephedrine had been applied. In addition, an experiment was designed to determine what concentration of pseudoephedrine must be present before a fire in a controlled laboratory setting, for a detectable amount to remain. Samples were created by adding pseudoephedrine hydrochloride, either in powder form or dissolved in methanol, to blocks of Douglas Fir and exposing the surface to a flame for two minutes. Additional samples were created by adding trace amounts, i.e. microliter quantities, of pseudoephedrine standard to blocks of wood before placing them in a fire for ten minutes. A thermal degradation product of pseudoephedrine was detected in samples containing more than 15 mg of the drug. To verify that the detected product was a result of thermal degradation, 10 mg of pseudoephedrine were heated at 200 °C for one hour. The product of the thermal degradation study and the product detected following two minutes of exposure to a flame had the same retention time and mass spectrum. Therefore, it was concluded that the detected thermal degradation product may be used to indicate the presence of pseudoephedrine in a fire.

Estimating sex and age at death are two crucial processes during the creation of a biological profile for a set of skeletal remains. Whether the remains are archaeological or forensic, estimating the sex and age of the individual is necessary for further analysis and interpretation. Specifically, in a medicolegal context, knowing the biological sex and approximate age of the remains assists law enforcement or government agencies in identifying unknown individuals. Since the inception of the field of forensic anthropology, practitioners have been developing methods to perform the aforementioned tasks. It is crucial that these methods be consistent, repeatedly tested, validated, and improved for multiple reasons. Firstly, to conform to Daubert (1993) standards, and additionally, to make sure that they are accurate and applicable to modern forensic cases. The present study was performed to validate the efficacy of the method for estimating sex from the os pubis originally proposed in Klales et al. (2012), as well as the efficacy of the “transition analysis” method for estimating age, originally outlined in Boldsen et al. (2002). Considering the recent popularity of using these methods to create a biological profile for forensic cases, it is necessary to develop error rates on a large, modern, American autopsy sample. These two methods are not only being readily utilized, but are additionally being taught to students in training. The utilization of these models involves a “logistic regression model” created by Klales et. al (2012) to process ordinal scores, and the Bayesian statistics software program “ADBOU” that was created for processing data collected using the method in Boldsen et. al (2002). These statistical systems which produced age estimates are relatively young compared to methods developed for the same purpose. The new generation of forensic anthropologists is fully responsible for objectively critiquing and validating these methods that are being disseminated by their professors and senior practitioners. The goal of the present study is to do just that. A skeletal reference sample of 630 pubic bones, all removed from modern autopsy cases and housed at the Maricopa County Forensic Science Center in Phoenix, Arizona, was utilized for data collection in the present study. Each pubic bone was assessed and scored according to the exact instructions outlined in the materials for each method, which was the Klales et al. (2012) paper for sex estimation, and the UTK Data Collection Procedures for Forensic Skeletal Material 2.0 for age estimation (Langley et al. 2016). Additionally, the observers recorded their “gestalt” estimates for sex using the Phenice (1969) system, as well as Brooks and Suchey (1990) and Hartnett (2010a) phases for each pubis. Demographic information labels were hidden, and the collection demographic information was not viewed until the completion of data collection. The null hypothesis in the present study is that both methods (the Klales et al. method (2012) and “transition analysis” method (Boldsen et al. 2002) will perform as well as they did in the original studies. The alternate hypothesis is that they do not result in the same accuracy rates reported in the original studies. Statistical analysis of the data indicates that there is sufficient evidence to reject the null hypothesis as it applies to the Klales et al. (2012) method. The classification accuracies achieved applying the logistic regression equation to the sample of pubic bones was found to be significantly lower than reported in the original study (86.2%), averaging around 70% between observers. The level of both intraobserver and interobserver agreement was only moderate for this method. It was also found that asymmetry occurred in some individuals, producing differing estimates of sex when the left and right pubes were scored separately. When utilizing the Boldsen et al. (2012) method and the ADBOU software package on only pubic symphyseal components to estimate age, the method was found to perform reasonably well. The majority (about 82%) of individuals had actual ages at death that fell within the predicted range produced by the statistical analysis. The majority of the symphyseal component scores showed moderate to good levels of interobserver agreement, and the estimated maximum likelihood (point estimate) of age at death predicted by the software package correlated moderately well with the actual age of death of the individual. These methods did not perform as well as reported in the original studies, and they should be further validated and recalibrated to improve their accuracy and reliability.

This thesis examines the evolution, applications, and statistical interpretations of DNA typing as a tool in the field of forensic science as well as in our criminal justice system. The most controversial aspect of DNA typing involves the determination of how likely it is that two people share the same DNA profile. This involves the use of population genetics and databases of allelic frequencies as well as some assumptions about population structuring.

This research examines the effects of forensic deoxyribonucleic acid (DNA) evidence on decisions in the courts and on the conduct of criminal investigations. To assess effects on court decisions, quantitative analyses were conducted using primary data from the State of Queensland. A control-comparison method was used to assess the effects in court, and this was made within a context of other evidentiary and extra-legal factors that had a bearing on case outcomes. These other factors included defendant confessions, independent witness testimony and fingerprint and photographic evidence. A sample of 750 cases referred by police for prosecution and finalised past the appeal stage in court, was selected for examination. Half of these cases utilised DNA evidence, while the other half, as a control group, did not. Cases were selected in four categories: sexual offences, serious assaults, homicides and property crime. Data on the cases were analysed using advanced statistical methods and predictor models were developed to demonstrate how, given case configurations, the addition of DNA evidence could potentially alter court outcomes. Results for the three serious offence types were that DNA evidence emerged as a positive predictor that prosecutors would pursue cases in court, and it demonstrated a powerful influence on jury decisions to convict. Incriminating DNA evidence demonstrated no significant effect on inducing guilty pleas from defendants for serious crimes against the person. However, it did correlate significantly to cases reaching court and to guilty pleas being entered for property offence cases. The analysis of the effects on investigations relies on data from jurisdictions other than Queensland. Secondary data and the literature were used to assess the potential for strategically using forensic intelligence, along with dedicated investigative resources, to reduce property crimes like burglaries and car thefts. In the one study available that employed adequate research methods, three patrol areas in New South Wales, where a police operation was trialled, were compared to other areas that acted as a control. The police operation aimed at 100% attendance at property crime scenes, the use of intelligence from DNA and fingerprint identifications and specialised investigative resources to reduce crime levels. While the operation failed to achieve its goal, it did provide some valuable lessons. The effectiveness of the national criminal DNA database in the UK, reputed to lead the world, was then evaluated in relation to domestic burglaries. Its Australian CrimTrac counterpart did not commence operations until March 2003, and by 2004 was not operating at maximum capacity. Because no published studies were located that measured any effects of the UK database on crime levels, the criterion selected to measure performance was the proportion of convictions achieved through the database to reported crime. For domestic burglaries, this ratio was calculated from secondary official data to be close to one percent (0.01), a figure that included the additional convictions achieved through the intelligence that the database provided. The research also examined forensic DNA in relation to issues of privacy and civil liberties. Privacy issues are discussed beginning with an historical background to the use and misuse of genetic data. This includes the searches for a 'criminal gene' and for genetic links to criminal behaviour. DNA databases are contrasted with databanks, and it is questioned, since we leave our DNA wherever we go, whether it really is private. Civil liberties issues that are discussed include whether providing DNA is a form of self-incrimination; how DNA has helped exonerate the convicted innocent; wrongful convictions based on flawed DNA evidence; whether occasional 'mass screenings' with DNA are a reversal of the onus of proof; concerns with DNA databases and 'function creep', and the planting or 'forgery' of DNA evidence including the use of amplicon contamination. In the final chapter, a balance is sought between on one hand, the goal of police and government to provide a safe society, and on the other, the rights to privacy and civil liberties expected by individuals in Western liberal democracies. The chapter addresses the issues of concern raised in the earlier chapter about privacy and civil liberties, and makes recommendations on how these may be resolved. The general approach favoured is to increase police powers in specific situations, but to couple these with the protection of individual rights through greater regulation of those powers. The research also developed a case prioritising system aimed at helping clear laboratory backlogs.

ABSTRACT A GLOW IN THE DARK: SYNTHESIS AND ELECTROPOLYMERIZATION OF CHEMILUMINESCENT THIOPHENE DERIVATIVES Asil, Demet M.Sc., Department of Chemistry Supervisor : Prof. Dr. Ahmet M. &Ouml / nal Co-Supervisor: Assist. Prof. Dr. Atilla Cihaner September 2008, 63 Pages Two novel chemiluminescent monomers, 2,3-dihydrothieno(3,4-d)pyridazine-1,4-dione (T-Lum) and 5,7-di-thiophen-2-yl-2,3-dihydro-thieno[3,4-d]pyridazine-1,4-dione (TTT-Lum), were synthesized. The reaction between T-Lum and TTT-Lum in alkaline solution with H2O2 gave chemiluminescence which can be catalyzed using Fe(III) ion. Owing to its sensitivitiy towards Fe(III) ion / T-Lum and TTT-Lum can be promising materials to detect bloodstains in the application of forensic science instead of luminol which gave response to a large family of metallic cations beside Fe(III). Also, TTT-Lum, which is based on a terthienyl system, was electropolymerized and its corresponding polymer (PTTT-Lum) was obtained via repetitive cycling or constant potential electrolysis in both 0.1 M LiClO4 dissolved in acetonitrile containing 5% of BF3-Et2O by volume and neat BF3- Et2O solution. In addition, PTTT-Lum, soluble in alkaline water, was synthesized successfully without breaking the pyridazinedione unit (chemiluminescent unit), as proved by Scanning Electron Microscopy (SEM), Fourier Transform Infrared Spectroscopy (FTIR) and Electrochemical Luminescence (ECL) measurements. Thus, PTTT-Lum, bearing chemiluminescent unit, can be a good candidate to be used as a sensor in near future. Furthermore, the PTTT-Lum film has a very stable and well-defined reversible redox couple as well as electrochromic behavior during p-doping process. The polymer film has also a band gap of 1.74 eV with an absorption band in its neutral state at 536 nm. Finally, PTTT-Lum film was found to be electrochemiluminescence active, maintaining its activitiy over 1000 cycles.

Melanin pigmentation is a complex trait governed by many genes. Variation in melanin pigmentation within, and between, populations makes it an important trait for assisting in physical identification of an individual in forensic investigations. Utilizing a training sample (n=789) comprised of various ethnicities and SNPs (75) in 24 genes previously implicated in human or animal pigmentation studies, I determined three-SNP multiple linear regression models that accounted for large proportions of pigmentation variation in skin (45.7%), eye color (76.4%), and hair [eumelanin-to-pheomelanin (43.2%) and total melanin (76.3%)], independent of ethnic origin. Rather than implementing stepwise regression, to ascertain the three-SNP predictive models, I devised an algorithm that is likely more robust than stepwise regression. The algorithm consisted of two steps: the first step reduced the pool of 75 SNPs to a pool of 40 by selection of SNPs that were significant (p<0.05) by one-way ANOVA; the second step enabled selection of SNPs for model incorporation based on their frequency in the best-fitted models of all possible combinations of three-SNP models (i.e., 40 choose 3).Prediction models were validated utilizing an independent cohort (n=242, test sample) that was very similar in ethnic composition to the training sample. Relative shrinkage was moderate for skin reflectance (23.4%), eye color (19.4%), and eumelanin-to-pheomelanin (37.3%) of hair, and largest for total melanin (67%) of hair. Additionally, we refined our model-building algorithm, enabling visual comparison of the frequency and co-linearity due to linkage or co-inheritance of SNPs of the best-fitted models. Application of our algorithm to the test sample yielded the same or similar models as the training sample. Two of the three SNPs composing the models were the same, with some variability in the third SNP of the model.