Uniparental DNA markers and forensic genetics in Finland

Over the years, a wide range of methods to verify identity have been developed. Molecular markers have been used for identification since the 1920s, commencing with blood types and culminating with the advent of DNA techniques in the 1980s. Identification is required by authorities in many occasions, e.g. in disputed paternity cases, identification of deceased, or crime investigation. To clarify maternal and paternal lineages, uniparental DNA markers in mtDNA and Y-chromosome can be utilized. These markers have several advantages: male specific Y-chromosome can be used to identify a male from a mixture of male and female cells, e.g. in rape cases. MtDNA is durable and has a high copy number, allowing analyses even from old or degraded samples. However, both markers are lineage-specific, not individualizing, and susceptible to genetic drift. Prior to the application of any DNA marker in forensic casework, it is of utmost importance to investigate its qualities and peculiarities in the target population. Earlier studies on the Finnish population have shown reduced variation in the Y-chromosome, but in mtDNA results have been ambiguous. The obtained results confirmed the low diversity in Y-chromosome in Finland. Detailed population analysis revealed large regional differences, and extremely reduced diversity especially in East Finland. Analysis of the qualities affecting Y-chromosomal short tandem repeat (Y-STR) variation and mutation frequencies, and search of new polymorphic markers resulted a set of Y-STRs with especially high diversity in Finland. Contrary to Y-chromosome, neither reduced diversity nor regional differences were found in mtDNA within Finland. In fact, mtDNA diversity was found similar to other European populations. The revealed peculiarities in the uniparental markers are a legacy of the Finnish population history. The obtained results challenge the traditional explanation which emphasizes relatively recent founder effects creating the observed east-west patterns. Uniparentally inherited markers, both mtDNA and Y-chromosome, are applicable for identification purposes in Finland. By adjusting the analysed Y marker set to meet the characteristics of Finnish population, Y-chromosomal diversity increases and the regional differentiation decreases, resulting increase in discrimination power and thus usefulness of Y-chromosomal analysis in forensic casework.

Micropillar Array Based Microchips for Electrospray Ionization Mass Spectrometry, Microreactors, and Liquid Chromatographic Separation

This dissertation deals with the design, fabrication, and applications of microscale electrospray ionization chips for mass spectrometry. The microchip consists of microchannel, which leads to a sharp electrospray tip. Microchannel contain micropillars that facilitate a powerful capillary action in the channels. The capillary action delivers the liquid sample to the electrospray tip, which sprays the liquid sample to gas phase ions that can be analyzed with mass spectrometry. The microchip uses a high voltage, which can be utilized as a valve between the microchip and mass spectrometry. The microchips can be used in various applications, such as for analyses of drugs, proteins, peptides, or metabolites. The microchip works without pumps for liquid transfer, is usable for rapid analyses, and is sensitive. The characteristics of performance of the single microchips are studied and a rotating multitip version of the microchips are designed and fabricated. It is possible to use the microchip also as a microreactor and reaction products can be detected online with mass spectrometry. This property can be utilized for protein identification for example. Proteins can be digested enzymatically on-chip and reaction products, which are in this case peptides, can be detected with mass spectrometry. Because reactions occur faster in a microscale due to shorter diffusion lengths, the amount of protein can be very low, which is a benefit of the method. The microchip is well suited to surface activated reactions because of a high surface-to-volume ratio due to a dense micropillar array. For example, titanium dioxide nanolayer on the micropillar array combined with UV radiation produces photocatalytic reactions which can be used for mimicking drug metabolism biotransformation reactions. Rapid mimicking with the microchip eases the detection of possibly toxic compounds in preclinical research and therefore could speed up the research of new drugs. A micropillar array chip can also be utilized in the fabrication of liquid chromatographic columns. Precisely ordered micropillar arrays offer a very homogenous column, where separation of compounds has been demonstrated by using both laser induced fluorescence and mass spectrometry. Because of small dimensions on the microchip, the integrated microchip based liquid chromatography electrospray microchip is especially well suited to low sample concentrations. Overall, this work demonstrates that the designed and fabricated silicon/glass three dimensionally sharp electrospray tip is unique and facilitates stable ion spray for mass spectrometry.

Integration of miRNA and mRNA expression with DNA copy number of Ewing sarcoma cell lines

Ewing sarcoma is an aggressive and poorly differentiated malignancy of bone and soft tissue. It primarily affects children, adolescents, and young adults, with a slight male predominance. It is characterized by a translocation between chromosomes 11 and 22 resulting in the EWSR1-FLI1fusion transcription factor. The aim of this study is to identify putative Ewing sarcoma target genes through an integrative analysis of three microarray data sets. Array comparative genomic hybridization is used to measure changes in DNA copy number, and analyzed to detect common chromosomal aberrations. mRNA and miRNA microarrays are used to measure expression of protein-coding and miRNA genes, and these results integrated with the copy number data. Chromosomal aberrations typically contain also bystanders in addition to the driving tumor suppressor and oncogenes, and integration with expression helps to identify the true targets. Correlation between expression of miRNAs and their predicted target mRNAs is also evaluated to assess the results of post-transcriptional miRNA regulation on mRNA levels. The highest frequencies of copy number gains were identified in chromosome 8, 1q, and X. Losses were most frequent in 9p21.3, which also showed an enrichment of copy number breakpoints relative to the rest of the genome. Copy number losses in 9p21.3 were found have a statistically significant effect on the expression of MTAP, but not on CDKN2A, which is a known tumor-suppressor in the same locus. MTAP was also down-regulated in the Ewing sarcoma cell lines compared to mesenchymal stem cells. Genes exhibiting elevated expression in association with copy number gains and up-regulation compared to the reference samples included DCAF7, ENO2, MTCP1, andSTK40. Differentially expressed miRNAs were detected by comparing Ewing sarcoma cell lines against mesenchymal stem cells. 21 up-regulated and 32 down-regulated miRNAs were identified, includingmiR-145, which has been previously linked to Ewing sarcoma. The EWSR1-FLI1 fusion gene represses miR-145, which in turn targets FLI1 forming a mutually repressive feedback loop. In addition higher expression linked to copy number gains and compared to mesenchymal stem cells, STK40 was also found to be a target of four different miRNAs that were all down-regulated in Ewing sarcoma cell lines compared to the reference samples. SLCO5A1 was identified as the only up-regulated gene within a frequently gained region in chromosome 8. This region was gained in over 90 % of the cell lines, and also with a higher frequency than the neighboring regions. In addition, SLCO5A1 was found to be a target of three miRNAs that were down-regulated compared to the mesenchymal stem cells.

DNA damage recognition in the normal epithelium of human prostate and seminal vesicles

Prostate cancer is one of the most prevalent cancer types in men. The development of prostate tumors is known to require androgen exposure, and several pathways governing cell growth are deregulated in prostate tumorigenesis. Recent genetic studies have revealed that complex gene fusions and copy - number alterations are frequent in prostate cancer, a unique feature among solid tumors. These chromosomal aberrations are though to arise as a consequence of faulty repair of DNA double strand breaks (DSB). Most repair mechanisms have been studied in detail in cancer cell lines, but how DNA damage is detected and repaired in normal differentiated human cells has not been widely addressed. The events leading to the gene fusions in prostate cancer are under rigorous studies, as they not only shed light on the basic pathobiologic mechanisms but may also produce molecular targets for prostate cancer treatment and prevention. Prostate and seminal vesicles are part of the male reproductive system. They share similar structure and function but differ dramatically in their cancer incidence. Approximately fifty primary seminal vesicle carcinomas have been reported worldwide. Surprisingly, only little is known on why seminal vesicles are resistant to neoplastic changes. As both tissues are androgen dependent, it is a mystery that androgen signaling would only lead to tumors in prostate tissue. In this work, we set up novel ex vivo human tissue culture models of prostate and seminal vesicles, and used them to study how DNA damage is recognized in normal epithelium. One of the major DNA - damage inducible pathways, mediated by the ATM kinase, was robustly activated in all main cell types of both tissues. Interestingly, we discovered that secretory epithelial cells had less histone variant H2A.X and after DNA damage lower levels of H2AX were phosphorylated on serine 139 (γH2AX) than in basal or stromal cells. γH2AX has been considered essential for efficient DSB repair, but as there were no significant differences in the γH2AX levels between the two tissues, it seems more likely that the role of γH2AX is less important in postmitotic cells. We also gained insight into the regulation of p53, an important transcription factor that protects genomic integrity via multiple mechanisms, in human tissues. DSBs did not lead to a pronounced activation of p53, but treatments causing transcriptional stress, on the other hand, were able to launch a notable p53 response in both tissue types. In general, ex vivo culturing of human tissues provided unique means to study differentiated cells in their relevant tissue context, and is suited for testing novel therapeutic drugs before clinical trials. In order to study how prostate and seminal vesicle epithelial cells are able to activate DNA damage induced cell cycle checkpoints, we used primary cultures of prostate and seminal vesicle epithelial cells. To our knowledge, we are the first to report isolation of human primary seminal vesicle cells. Surprisingly, human prostate epithelial cells did not activate cell cycle checkpoints after DSBs in part due to low levels of Wee1A, a kinase regulating CDK activity, while primary seminal vesicle epithelial cells possessed proficient cell cycle checkpoints and expressed high levels of Wee1A. Similarly, seminal vesicle cells showed a distinct activation of the p53 - pathway after DSBs that did not occur in prostate epithelial cells. This indicates that p53 protein function is under different control mechanisms in the two cell types, which together with proficient cell cycle checkpoints may be crucial in protecting seminal vesicles from endogenous and exogenous DNA damaging factors and, as a consequence, from carcinogenesis. These data indicate that two very similar organs of male reproductive system do not respond to DNA damage similarly. The differentiated, non - replicating cells of both tissues were able to recognize DSBs, but under proliferation human prostate epithelial cells had deficient activation of the DNA damage response. This suggests that prostate epithelium is most vulnerable to accumulating genomic aberrations under conditions where it needs to proliferate, for example after inflammatory cellular damage.

The many ways to delimit species: hairs, genes and surface chemistry

Species identification forms the basis for understanding the diversity of the living world, but it is also a prerequisite for understanding many evolutionary patterns and processes. The most promising approach for correctly delimiting and identifying species is to integrate many types of information in the same study. Our aim was to test how cuticular hydro- carbons, traditional morphometrics, genetic polymorphisms in nuclear markers (allozymes and DNA microsatellites) and DNA barcoding (partial mitochondrial COI gene) perform in delimiting species. As an example, we used two closely related Formica ants, F. fusca and F. lemani, sampled from a sympatric population in the northern part of their distribu- tion. Morphological characters vary and overlap in different parts of their distribution areas, but cuticular hydrocarbons include a strong taxonomic signal and our aim is to test the degree to which morphological and genetic data correspond to the chemical data. In the morphological analysis, species were best separated by the combined number of hairs on pro- notum and mesonotum, but individual workers overlapped in hair numbers, as previously noted by several authors. Nests of the two species were separated but not clustered according to species in a Principal Component Analysis made on nuclear genetic data. However, model-based Bayesian clustering resulted in perfect separation of the species and gave no indication of hybridization. Furthermore, F. lemani and F. fusca did not share any mitochondrial haplotypes, and the species were perfectly separated in a phylogenetic tree. We conclude that F. fusca and F. lemani are valid species that can be separated in our study area relatively well with all methods employed. However, the unusually small genetic differen- tiation in nuclear markers (FST = 0.12) shows that they are closely related, and occasional hybridization between F. fusca and F. lemani cannot be ruled out.

Microbial identification by detection of ligation probes on DNA microarray

Microbes in natural and artificial environments as well as in the human body are a key part of the functional properties of these complex systems. The presence or absence of certain microbial taxa is a correlate of functional status like risk of disease or course of metabolic processes of a microbial community. As microbes are highly diverse and mostly notcultivable, molecular markers like gene sequences are a potential basis for detection and identification of key types. The goal of this thesis was to study molecular methods for identification of microbial DNA in order to develop a tool for analysis of environmental and clinical DNA samples. Particular emphasis was placed on specificity of detection which is a major challenge when analyzing complex microbial communities. The approach taken in this study was the application and optimization of enzymatic ligation of DNA probes coupled with microarray read-out for high-throughput microbial profiling. The results show that fungal phylotypes and human papillomavirus genotypes could be accurately identified from pools of PCR amplicons generated from purified sample DNA. Approximately 1 ng/μl of sample DNA was needed for representative PCR amplification as measured by comparisons between clone sequencing and microarray. A minimum of 0,25 amol/μl of PCR amplicons was detectable from amongst 5 ng/μl of background DNA, suggesting that the detection limit of the test comprising of ligation reaction followed by microarray read-out was approximately 0,04%. Detection from sample DNA directly was shown to be feasible with probes forming a circular molecule upon ligation followed by PCR amplification of the probe. In this approach, the minimum detectable relative amount of target genome was found to be 1% of all genomes in the sample as estimated from 454 deep sequencing results. Signal-to-noise of contact printed microarrays could be improved by using an internal microarray hybridization control oligonucleotide probe together with a computational algorithm. The algorithm was based on identification of a bias in the microarray data and correction of the bias as shown by simulated and real data. The results further suggest semiquantitative detection to be possible by ligation detection, allowing estimation of target abundance in a sample. However, in practise, comprehensive sequence information of full length rRNA genes is needed to support probe design with complex samples. This study shows that DNA microarray has the potential for an accurate microbial diagnostic platform to take advantage of increasing sequence data and to replace traditional, less efficient methods that still dominate routine testing in laboratories. The data suggests that ligation reaction based microarray assay can be optimized to a degree that allows good signal-tonoise and semiquantitative detection.