Thesis (Ph.D.)--Virginia Commonwealth University, 2009. / Prepared for: Dept. of Pathology. Title from title-page of electronic thesis. Bibliography: leaves 105-117.

With current scientific technologies, a significant amount of genetic information can be obtained from biological evidence found at a crime scene. Not only is it possible to identify the donor of the evidence through routine DNA profiling techniques, but new RNA based methods are being developed to determine the tissue type as well as the physical characteristics of the donor. Despite the information that can be obtained, the ability to determine the age or time the biological material was deposited at the crime scene has eluded the forensic community thus far. Timing is critically important as it could help police determine when the crime was committed. In this body of work an investigation was conducted into whether the degradation rates of nucleic acid macromolecules could serve as molecular clocks for age estimations. An attempt was made to gain a better understanding of the degradation products produced from an internal urban environment and to develop an optimal assay accordingly. A number of different RNA based techniques for ageing both hair and blood samples were also examined. Degradation assays have been traditionally designed around amplicon size however, it was established that testing loci stability is an essential requirement in the optimisation process. The results presented in this thesis suggest the reliability of the data can be increased when the two competing target species are selected from the same loci, which minimised the effect of loci susceptibility to degradation. It was determined that blood stains aged up to 60 days in an internal urban environment were best distinguished (in terms of age estimations) by using targets that differed in size by 170 to 240 base pairs, with one of the targets being between 200 and 300 base pairs in length. Despite using a robust TH01 qPCR assay it was established that an internal “urban” environment was not as stable as predicted and that seasonal temperature variation had a large effect on degradation rates. Interpretation of the results was therefore limited suggesting these optimised target sizes may only be relevant to the winter months. Using a carefully designed hermetically sealed dry swab we were able to remove moisture and inhibit the growth of DNA consuming micro-organisms. It was determined that bacteria alone can cause a 2-fold increase in the degradation rate of a sample aged at room temperature. In terms of integrity, storing samples at room temperature in a moisture free environment was equivalent to storing standard samples (exposed to normal humidity levels) in refrigerated conditions. It was also determined that the effect of bacterial degradation can be halved by lowering the storage temperature from room temperature to 4°C. RNA was examined in an attempt to reduce the large variations that had inhibited previous DNA methodologies. IL-6 and TNF-α were initially selected due to their rapid post-extraction change in expression levels. However, their levels were highly variable, unpredictable and therefore not suitable for this type of analysis even on samples that had been aged for only ten days. It is thought that their dynamic roles in a number of haemopoietic processes could be responsible for the poor results. A new RNA methodology, as described by Nolan et al (2008) was used to analyse samples that had been aged over 80 days. Four targets, AMICA1, MNDA, CASP1 and GAPDH were chosen based on their cell lineage as it was hypothesised that inter-donor variation could be reduced by using targets confined to the granulocytic cell lineage. Using the novel 3’/5’ assay, AMICA1, MNDA and CASP1 all performed poorly and no correlation could be determined between the 3’/5’ ratio and sample age. GAPDH showed some encouraging results with a correlation of 0.912 (age to 3’/5’ ratio) although initial stability over the first 20 days and the inter-donor variation were still limiting factors. It was also thought that the various mRNA degradation processes, in particular the 5’/3’ exonuclease activity, contributed to the poor results generally. A large inter-donor variation was a common aspect to all the blood based methodologies trialled. This meant that none of the methods had any practical value. As a result, an alternative RNA method was used to determine if it was possible to age another forensically important type of biological evidence; hair. Using a Reverse Transcription Quantitative PCR (RT-qPCR) assay, we monitored the Relative Expression Ratio (RER) of two different RNA species (18S rRNA and B-actin mRNA) in hair samples that were aged naturally over a period of three months. Overall the results presented here suggest that the age of hair samples containing follicular tags can be approximated using a second order polynomial (Age = 3.31RER2 - 2.85RER – 0.54), although with limitations.

No / Keratinous tissues such as nail, hair, horn, scales and feather have been used as a source of DNA for over 20 years. Particular benefits of such tissues include the ease with which they can be sampled, the relative stability of DNA in such tissues once sampled, and, in the context of ancient genetic analyses, the fact that sampling generally causes minimal visual damage to valuable specimens. Even when freshly sampled, however, the DNA quantity and quality in the fully keratinized parts of such tissues is extremely poor in comparison to other tissues such as blood and muscle – although little systematic research has been undertaken to characterize how such degradation may relate to sample source. In this review paper we present the current understanding of the quality and limitations of DNA in two key keratinous tissues, nail and hair. The findings indicate that although some fragments of nuclear and mitochondrial DNA appear to be present in almost all hair and nail samples, the quality of DNA, both in quantity and length of amplifiable DNA fragments, vary considerably not just by species, but by individual, and even within individual between hair types.

La revue de la littérature montre que la pulpe dentaire est une source utile pour le diagnostic des bactériémies, y compris en paléomicrobiologie. Les précédents travaux en paléomicrobiologie réalisés dans le laboratoire ont tous mis en évidence une amplification possible de courts ou très courts fragments d'ADN bactérien partant de l'hypothèse que l'ADN ancien est fragmenté. Dans le premier travail, le modèle expérimental de dégradation de l'ADN des macrophages murins J774 et de Mycobacterium smegmatis par la chaleur sèche à 90°C a montré une différence statistiquement significative (p < 0.05) entre la vitesse de fragmentation de l'ADN bactérien et eucaryote. Ces résultats suggèrent que les diagnostics paléomicrobiologiques peuvent détecter des fragments plus longs d'ADN bactérien à partir des échantillons anciens. Dans un deuxième travail, un système de détection rapide de 7 agents pathogènes par PCR multiplex en temps réel a été utilisé pour détecter ces pathogènes suspectés à partir de la pulpe dentaire de 1192 dents anciennes collectées dans 12 charniers dont un localisé à Douai (1710 – 1712). Après la détection de Bartonella quintana dans ce charnier, les PCRs en temps réel emboîtées ultra-sensibles et la «PCR suicide» ont été utilisées pour confirmer la présence de Rickettsia prowazekii souche Madrid E génotype B dans 6/55 pulpes dentaires (11%) collectées de 6/21 squelettes (28.6%) de soldats à Douai. Ces résultats supportent l'hypothèse que le typhus a été introduit en Europe par les soldats espagnols au retour des conquêtes en Amérique. / Reviewing the literature shows that dental pulp is a useful source for bacteremic and paleomicrobiological diagnoses. In the first work, an experimental model of DNA degradation of the murine macrophage cell line J774 and Mycobacterium smegmatis by exposure to 90°C dry heat showed a statistically significant difference (p < 0.05) of fragmentation level between bacterial and eukaryotic DNA. These results suggest that paleomicrobiological diagnosis can detect more large fragments of bacterial DNA from ancient buried specimens. In the second work, a system of rapid detection of seven pathogens by multiplex real-time PCR was used for detecting suspected pathogens from dental pulp of 1192 ancient teeth collected from 12 multiple burials including a mass grave in Douai, 1710 – 1712. After the Bartonella quintana detection in this site, real-time nested PCR and ultra-sensitive “suicide PCR” were used to confirm the presence of Rickettsia prowazekii strain Madrid E genotype B in 6/55 dental pulp specimens collected from 6/21 (29%) skeletons of soldiers buried in Douai.These results support the hypothesis that typhus was imported into Europe by Spanish soldiers from America. In the last work, DNA extracted from 5 dental pulp specimens collected from multiple burials at Issoudun, 17th – 18th centuries, was analyzed by pyrosequencing which detected Yersinia pestis sequences in the metagenome. For paleomicrobiology, pyrosequencing is a sensitive technic which can be used as baseline test to detect both suspected and unexpected pathogens from ancient specimens. We named this new approach «paleometagenomics».

DNA profiling has ignited public interest and consequently their expectations for the capabilities of forensic criminal and science investigations. The prospect of characterising the genetic makeup of individuals or trace samples from a wide variety of depositional and post-mortem circumstances raises the question of how reliable the methods are given the potential for prolonged exposure to variation in environmental factors, i.e. temperature, pH, UV irradiation and humidity, that are known to induce damage to DNA. Thus, it is crucial to verify the validity of the DNA profiling for characterising the genetic makeup of post-mortem tissues. This project aimed to assess the reliability of sequence and microsatellite based genotyping of tissues (muscle, hair and bone) sampled from carcasses over a two year post-mortem period. This assessment investigated the impact of environment induced DNA degradation in the local geographic region that is typical of the circumstances that confront forensic practitioners in southern Australia and to utilise rigorous controls by studying animals whose time of death and burial was known and for which we had pre-decay tissue samples available. A ‘body farm’ with 12 pig carcasses on the northern Adelaide plains, ~60km north of Adelaide, which has a typical southern Australian Mediterranean climate, i.e. cold wet winters and hot dry summers. Pigs (Sus scrofa) were used as an experimental analogue for human subjects because of the logistical and ethical reasons. The pig carcasses were allocated among three treatments: four were left on the surface, four were buried at 1m depth, and four were buried at 2 m depth. These ‘burial’ conditions mimic a range of conditions encountered typically in forensic and archaeological studies. Cortical bone samples were taken from each pig carcass at one week, one month, three months, six months, one year and two years post-mortem and muscle and hair over the same sampling period for as long as those tissue types were present. A set of PCR primers to amplify two (short and a long) fragments from the hypervariable part of the mitochondrial control region (HVRI) that is used in forensic and evolutionary studies of humans and many other mammal species were developed. Also a panel of four pig microsatellite loci with fluorescent labels to facilitate automated multiplex genotyping. These loci matched as closely as possible the core motifs and allele lengths typical of the commercially available microsatellite marker kits used in Australian forensic science labs so that our experiments were as good a model as possible of the human forensic DNA technology. In this study it was possible to retrieve samples from muscle tissue up to 90 days, hair up to one year and bone at two years post-mortem. The analyses showed that the long and short HVRI region PCR fragments were only amplifiable up to 30 days from muscle tissue and that these fragments were amplifiable up to one year from hair. In contrast, in cortical bone both PCR fragments were amplifiable up to two years. The long fragment disappeared in muscle tissue completely after 30 days and in hair after six months. However, the long fragment was present in cortical bone even at two years. Overall, there was a general trend of loss of concentration of both the long and short fragments over time. Comparisons of the HVRI nucleotide sequences among tissues sampled from individual animals showed substitution changes in muscles as early as 30 days (3 out of 6 individuals) and hair at six months (1 out of 6 individuals). In contrast, in cortical bone substitutions first appeared at 365 days (1 out of 6 individuals). The most common substitution observed in all tissues types was the C-T transition, with A-G transversions observed in two episodes and C-A transversion observed in one episode. Analyses of microsatellite genotypes in muscle tissues showed high allele peaks on chromatograms up to day seven samples. However, by three months PCR was not successful from muscle tissue. While, bone tissue had lower allele peak heights compared to the muscle tissues, alleles were detectable up to six months. Allele drop out occurred for one animal (at 2 meters) in muscle tissue at the dinucleotide locus and for another animal (kept on surface) also in muscle tissue at a tetranucleotide locus. Stuttering was observed for a single animal at dinucleotide locus in muscle tissue (buried sample 2 meter depth). No stuttering or allele drop outs were seen in the bone tissue. Overall the four loci completely disappeared after 30 days in muscle tissue and after 180 days in bone tissue. In summary, analyses showed that post-mortem DNA degradation was present in all the three tissue types (muscle, hair and bone). The types of damage identified were DNA fragmentation, nucleotide substitutions and DNA loss, which resulted in a diminished frequency of successful PCR for mitochondrial and nuclear markers over time and stuttering and allele drop out in microsatellite genotyping. In addition, two nucleotide substitutions were concentrated in ‘hotspots’ that correlate with sites of elevated mutation rate in vivo. Also the frequency of successful PCR of longer nuclear and mitochondrial PCR products declined markedly more quickly than for shorter products. These changes were first observed at much shorter post mortem intervals in muscle and much longer post mortem intervals in hair and bone tissue. When considering the carcass deposition treatments, tissues that were retrieved from buried carcases showed higher levels of DNA degradation compared to tissues retrieved from carcases left on the surface. Overall, muscle tissue is a good source for DNA analysis in immediate post mortem samples, whereas hair and bone tissue are good source for DNA analysis from older samples. When comparing the microsatellite genotyping and mtDNA analyses, mtDNA is a reliable source for DNA analysis from tissue recovered from bodies that had decayed for longer post-mortem durations such as months to years, whereas microsatellite genotyping gives reliable results for tissue from shorter post mortem intervals (hours to few days). Therefore it is recommended that when analysing mtDNA sequences, cloning and sequencing PCR products can help to identify the base pair substitutions especially for tissue retrieved from longer post mortem intervals. In addition, increasing the template DNA concentrations and "neutralising" co-extracted DNA inhibitors should be considered when dealing with tissue from longer post mortem intervals. Finally, the more stringent protocols used in ancient DNA studies should be considered when dealing with tissue with much longer post mortem intervals in forensic settings. / Thesis (Ph.D.) -- University of Adelaide, School of Medical Sciences, 2009

Les mitochondries sont des organites qui possèdent leur propre ADN (ADNmt), codant pour des gènes de la chaine respiratoire. La réparation des dommages dus aux ROS, une réplication défectueuse ou d’autres sources exogènes tels des agents chimiothérapeutiques ou des irradiations ionisantes peuvent générer des cassures double-brin (CDB) de l’ADNmt. L’ADNmt code pour des protéines essentielles à la production d’énergie, et des systèmes de maintenance de l’intégrité de ce génome efficaces sont donc nécessaires pour la viabilité des cellules. En effet des mutations de l’ADNmt sont présentes dans de nombreuses pathologies comme les myopathies mitochondriales, les cancers et les maladies neurodégénératives. Cependant les processus responsables de la maintenance de l’ADNmt suite à des CDB restent controversés.Pour élucider les mécanismes impliqués, nous avons généré des CDB mitochondriales en utilisant une lignée cellulaire humaine exprimant de manière inductible l’enzyme de restriction PstI liée à une séquence d’adressage mitochondrial. Nos résultats montrent, dans notre système, une première phase de dégradation de l’ADNmt lésé avec une cinétique rapide, n’impliquant pas l’autophagie ou l’apoptose, suivie de la ré-amplification d’ADNmt intact dans un deuxième temps. Contrairement à d’autres études nous n’avons pas pu détecter d’évènements de réparation des CDB mitochondriales générées. Nous avons ensuite cherché à identifier les protéines impliquées dans la dégradation de l’ADNmt lésé que nous observons, mais aucune nucléase testée ne semble responsable de ce processus. Des approches plus globales sont mises au point pour identifier de nouveaux acteurs, notamment un crible RNAi à grande échelle. Parallèlement nous nous intéressons aussi à une famille de phosphohydrolases, les Nudix, et à leur rôle protecteur en assainissant le réservoir de nucléotides libres. / Mitochondria are organelles that possess their own genome, the mitochondrial DNA (mtDNA). Repair of oxidative damages, defective replication, or various exogenous sources, such as chemotherapeutic agents or ionizing radiations, can generate double-strand breaks (DSBs) in mtDNA. MtDNA encodes for essential proteins involved in ATP production and maintenance of integrity of this genome is thus of crucial importance. Mutations in mtDNA are indeed found in numerous pathologies such as mitochondrial myopathies, neurodegenerative disorders or cancers. However, the mechanisms involved in mtDNA maintenance after DSBs remain unknown.To elucidate this question, we have generated mtDNA DSBs using a human inducible cell system expressing the restriction enzyme PstI targeted to mitochondria. Using this system, we could not find any support for DSBs repair of mtDNA. Instead we observed a loss of the damaged mtDNA molecules and a severe decrease in mtDNA content, followed by reamplification of intact mtDNA molecules. We have demonstrated that none of the known mitochondrial nucleases are involved in mtDNA degradation and that DNA loss is not due to autophagy, mitophagy or apoptosis but to a selective mechanism. Our study suggests that a still uncharacterized pathway for the targeted degradation of damaged mtDNA in a mitophagy/autophagy-independent manner is present in mitochondria, and might provide the main mechanism used by the cells to deal with DSBs. Global approaches are ongoing to identify proteins involved in degradation of damaged mtDNA following DSBs, mainly an RNAi screen targeting 80 nucleases. In parallel we are interested in a family of phosphohydrolases named Nudix and their putative protective role in sanitizing the nucleotides pool in mitochondria.