The Peganum harmala plant is a flowering shrub that produces small, dark brown seeds in pods. These seeds contain the hallucinogenic alkaloids harmine and harmaline. As such, they have been historically used for shamanic rites and folk medicine. Presently, P. harmala seeds are commercially available and subject to no legal restrictions in the United States. This has allowed for the recreational use and abuse of these hallucinogenic seeds or seed extracts made with household chemicals. Overdose cases from excessive consumption of seeds or seed extracts have been reported. Overdose patients present with hallucinations, tremors, agitation, tachycardia, and gastric distress. Severe overdose cases have resulted in hospitalization for respiratory depression and coma. The goal of this research was to develop a protocol for forensic analysis of suspected P. harmala seeds. Physical examination was selected as a quick, cost-effective preliminary method to screen seeds. P. harmala seeds are, on average, approximately 2.3 ± 0.3 mm long and 1.0 ± 0.2 mm thick, with an average Feret’s diameter of 2.8 ± 0.3 mm. The mean mass of one seed is 2.5 ± 0.2 mg. The seeds are dark brown, irregularly shaped, and have a pitted surface. Seeds matching these descriptors can be further analyzed to detect harmine and harmaline. Direct analysis in real time (DART) allows for very rapid mass spectral analysis of P. harmala seeds. Ions corresponding to harmine and harmaline can be detected when an intact seed is placed in front of the DART ion source, and higher levels of harmine and harmaline are observed when a seed cut in half to reveal interior surfaces is analyzed. Solvent extraction of crushed seeds using ethanol followed by gas chromatography – mass spectrometry allows for confirmation of the presence of harmine and harmaline in suspected seeds. When selected ion monitoring is used, this method is able to detect harmine and harmaline in a sample consisting of a single seed. Infrared spectra of harmine and harmaline standards, crushed P. harmala seeds, and solid material obtained from evaporating off the solvent from an extraction of crushed seeds were obtained. Infrared spectroscopy can be used to distinguish between pure harmine and harmaline, but is a poor choice for analysis of samples containing a mixture of harmine and harmaline, such as P. harmala seeds. In conclusion, physical characterization, direct analysis in real time, solvent extraction, and gas chromatography – mass spectrometry are recommended techniques for the forensic analysis of P. harmala seeds.

Generally, biological samples are collected from crime scenes using swabbing, cutting, or taping techniques. However, these methods are limited in their abilities to recover diluted, masked, or otherwise invisible stains. Additionally, their targeted nature allows only a small portion of a larger stain to be collected at one time. In this study, a sterile wet-vacuum collection system was evaluated in its ability to collect small volume bloodstains from various substrates. Vacuuming was compared to swabbing and taping methods currently used in forensic analysis. Samples were collected from porous and nonporous surfaces; the efficacy of each collection method was evaluated with a colorimetric presumptive blood test. To evaluate each collection method, dilutions containing from 0.25 nl to 25 μl human blood were spotted on common substrate materials, allowed to dry, and recovered. For comparison to the novel method, single-swabbing and tape-lifting techniques were performed in this study to collect samples for presumptive testing. During wet vacuum collection, stains were saturated with sterile buffer and suction was applied to the surrounding area, accumulating buffer in a collection bottle. Collected buffer was then filtered through membranes to capture cellular material, which were then presumptively tested for the presence of blood. Testing was performed with Kastle-Meyer (phenolphthalein) reagents. Each sample was photographed under consistent conditions in order to determine signal intensity. It was shown that the wet-vacuuming technique is able to recover sufficient amounts of blood for presumptive testing from multiple substrates. This method was able to detect similar dilutions of blood as traditional techniques in samples collected from porous surfaces, but was less effective on a nonporous substrate. Presumptive test image analysis shows increased relative intensity in collections from textiles, such as denim, when using the wet-vacuum system. Considering the results of a contemporaneous DNA quantification study, it was shown that in instances where a very weak presumptive result is found, the wet-vacuum technique may be better able to collect genetic material for downstream processing than the traditional methods evaluated. This study demonstrates the potential of wet-vacuuming as a suitable alternative technique to collect adhered cellular material from substrates in forensic investigations.

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.

Thesis (M.S.F.S.) / There is need for further development of cellular collection techniques in the field of forensic science. Currently, forensic analysts are limited to the use of swabs, taping, cutting, and scraping methods to collect cellular material. Each of these methods has its own benefits and drawbacks, however, none of them result in 100% recovery of the cells. The Microbial Vacuum system (M-Vac®), developed by MSI, is a liquid based cellular collection system that was originally developed to collect microbes in the food-processing industry from various surfaces. This research represents a detailed study into the feasibility of utilizing the M-Vac® system for forensic purposes. Specifically, the phosphate buffer used with the M-Vac® was tested to confirm that it does not have a detrimental effect on cellular retrieval. Further, the ability of the M-Vac® to collect cellular material from a variety of substrates was tested. It was determined the M-Vac® can successfully collect both blood and semen from tile, denim, carpet, and brick materials in sufficient quantity for downstream PCR analysis. Additionally, examination into whether DNA was dispersed during collection due to the significant force of impact of the liquid striking the surface was conducted. Specifically, areas surrounding the sample collection region were swabbed after collection with the M-Vac® and tested. Quantitative PCR analysis showed that DNA was retrieved up to 4 inches away from the collection area. This indicates that the M-Vac® system is a viable cell collection technique for forensic purposes, but only for samples which are isolated (i.e. where there is not another probative sample adjacent to it). If there are two probative samples within the same vicinity, then swabbing or taping is the recommended method of collection.

Emerging sequencing approaches have revolutionized the way we can collect DNA sequence data for applications in bioforensics and biosurveillance. In this research, we present an approach to construct a database of known biological agents and use this database to develop a statistical framework to analyze raw reads from next-generation sequence data for species identification and strain attribution. Our method capitalizes on a Bayesian statistical framework that accommodates information on sequence quality, mapping quality and provides posterior probabilities of matches to a known database of target genomes. Importantly, our approach also incorporates the possibility that multiple species can be present in the sample or that the target strain is not even contained within the reference database. Furthermore, our approach can accurately discriminate between very closely related strains of the same species with very little coverage of the genome and without the need for genome assembly - a time consuming and labor intensive step. We demonstrate our approach using genomic data from a variety of known bacterial agents of bioterrorism and agents impacting human health.

Biological evidence may contain any number of cells in any proportion. Extreme low-template DNA samples are often very difficult to interpret due to complex signal or peaks which may be indistinguishable from baseline noise. Current solutions focus on increasing the amount of amplicon detected by adjusting PCR cycle number or capillary electrophoresis injection parameters. Consensus profiling is an additional option. However, the aforementioned solutions are often not helpful for extreme low-template samples due to the high occurrence of allelic drop-out. Additionally, PCR is a destructive technique that causes one amplification to completely exhaust this type of sample, making further typing and analysis impossible. Therefore, a technique that allows for the re-generation of a DNA template in order to amplify it multiple times would be an extremely useful tool. This study outlines the development of a method that allows for the recursive amplification of a DNA sample. Amplification was performed using biotinylated primers for an STR locus and the resulting product was cleaned using streptavidin-coated magnetic beads to sequester the amplicons. Subsequent centrifugal filtration was used to remove the remaining PCR components, thus isolating the original genomic DNA. Re-amplification was then successfully performed at a different STR locus. Though successful, multiple run-throughs of the method indicated retention of signal from the original amplification as well as significant genomic DNA loss during the process. This study outlines experiments seeking to characterize the cause(s) of these imperfections in order to effectively direct method optimization. A computer generated dynamic model was also created and used to simulate the recursive amplification process to assist in development. When optimized, it is expected that recursive amplification can significantly reduce the difficulties associated with low-template DNA analysis and eradicate the concept of an ‘exhaustive’ DNA sample.

The morphoscopic traits used to assign the term Hispanic to a skeleton constitute mainly a mixture of characteristics that have been assigned by anthropologists to Asian and Caucasian ancestry groups. Therefore, the morphological characteristics for the population termed Hispanic are not well defined. The aim of this study is to conduct a biodistance analysis of skeletons of Hispanic ancestry from Puerto Rico. The purpose of this is to assess how similar their morphoscopic characteristics are to other populations termed Hispanic as well as populations termed Asian. The analysis will be conducted by taking craniometric measurements. Pre-Colombian as well as modern skeletons from Puerto Rico will be examined and compared to other Hispanic as well as Asian populations that form part of the Forensic Anthropology Data Bank (FDB). Results from this study will help characterize Hispanic skeletal variation. In addition, this study will discuss the complexities of Hispanic classification in forensic anthropological contexts.

Throughout the past, bioarchaeologists have been concerned with identifying subsistence strategies of past populations and when subsistence strategies have transitioned from foraging to agriculture practices. Specifically, one area of major concentration has been examining the transition from foraging to agriculture in the southeast of the present day United States. The present study examines the transition of subsistence practices in prehistoric Tennessee utilizing dental microwear texture analysis. This study examined a total of 49 individuals from Archaic and Mississippian sites. These were compared temporally by comparing Archaic microwear signatures to Mississippian microwear signatures, and geographically, through the comparison of each site in relation to its geographic location. Non-parametric Mann-Whitney U tests were utilized to determine if statistical significant differences existed between the Archaic and Mississippian groups analyzed, and to determine if statistical significant differences existed based on geographic location. Due to the small sample size utilized in the study, it must be stated that the results are preliminary and further testing using dental microwear texture analysis needs to be undertaken in order to better understand the results.

The work of an international team of forensic experts led to the final determination that Salvador Allende, former Chilean president (1970-1973), committed suicide by firearm during the military coup against his socialist government on September 11, 1973. Included in the final report was the anthropologists’ skeletal trauma analysis documenting the gunshot trauma to the skull as consistent with the passage of a high velocity projectile under the chin and through the cranial cavity. The anthropological report was part of a multidisciplinary effort to finally address the questions surrounding the manner of Salvador Allende’s death, and to eliminate speculations that he was killed by the military. Although it is not the role of the anthropologist to determine manner of death in a forensic case, expertise in osteology and the biomechanics of high velocity projectile trauma to bone may contribute to the pathologist’s final conclusions about manner of death in firearm fatalities. Previous research looking at variation between homicides and suicides in firearm fatalities has used soft tissue trauma analysis and autopsy reports to identify common characteristics specific to these manners of death. They have found that anatomic location of entrance trauma, bullet direction, number of shots, and range of fire are important factors in understanding this variation. Anthropological research has not explored these differences as they are expressed on the skeleton, however; knowledge of variation in skeletal gunshot trauma, between self-inflicted and other-inflicted gunshot wounds, can better equip anthropologists to report pertinent information that can lead to accurate determinations of manner of death. The current study explores the factors of anatomic location of entrance trauma, bullet direction, and number of shots, with additional consideration to fracture severity and fracture patterns, in place of range of fire. The William M. Bass Donated Skeletal Collection and the Antioquia Modern Skeletal Reference Collection provided a total sample of 15 suicide and 19 homicide cases for this analysis. This research found that localized entry sites to the front and right sides of the head and to the chest were common in suicides. Entries to the left and front sides of the head and to the posterior side of the head and body were common in homicides. Further analysis of sequence of shots and entry sites revealed that the front of the head, common in both homicides and suicides, was more common in secondary gunshot wounds in homicides. Bullet directions common in suicides were right to left through the sagittal plane, and anterior to posterior through the coronal plane. The left to right direction through the sagittal plane was most common for homicides, whereas the anterior to posterior and posterior to anterior directions through the coronal plane were almost equally represented in homicides. Further analysis of sequence of shots and bullet direction revealed that the anterior to posterior direction was more common in secondary gunshot wounds in homicides. The transverse plane did not show statistically significant differences between homicides and suicides for either the inferior to superior or superior to inferior directions. For number of shots, homicides more commonly expressed multiple gunshot wounds and suicides more commonly expressed single gunshot wounds. Fracture severity analyses revealed that the presence of tertiary fractures (concentric fractures) in entrance wounds was more common in suicides. The presence of secondary fractures (radiating fractures) as the most severe fracture in entrance wounds was more common in homicides. These results suggest that fracture severity is higher in suicides, based on the amount of kinetic energy dispersed at impact, although it is unclear what factor influences these differences (range of fire, type of firearm, caliber of bullet, etc.). There was also a significant difference between homicides and suicides in fracture patterns for both entrance and exit wounds. These findings are based on a small sample and should be considered with caution, especially for use in a forensic setting. Further research is crucial to better understanding the variations seen in this study. Specifically, sequencing of shots should be further explored for its utility in distinguishing homicides and suicides as well as providing a better understanding of multi-shot cases. Additionally, more research should be conducted on fracture severity and fracture patterns with specific consideration to the factors that influence the variation seen between homicides and suicides.

Condoms have been used in sexual assaults as a means of preventing the transmission of biological fluids. Current sexual assault evidence collection kit processing protocols do not regularly take advantage of the information that can be gathered by examining residues left by condoms during intercourse. A biphasic liquid-liquid extraction technique was developed to separate polar and non-polar condom residues, which had been collected on cotton tipped swabs. This research involved the examination of twenty condom brands by Diffuse Reflectance Infrared Fourier Transform Spectroscopy. Five brands were selected to examine the consistency of this technique when the lubricants were exposed to body and storage temperature conditions for various times and in the presence of oral, vaginal, and blood samples. Additionally, starches collected from the condoms under each of the above conditions were examined. Although all lubricants were identifiable using this IR technique, the nonoxynol-9 (spermicide) containing samples produced spectra which were not identical to those produced by nonoxynol-9 standards. Although there was a decrease in the percent transmittance within IR spectra as the time between the collection and the extraction of the swabs increased, the condom residues of interest remained identifiable at all time points examined. The use of vaginal and oral swabs in the collection caused a negligible amount of background interference, which could be eliminated through spectral subtraction of the swab.