<p>The field of forensic genetics is growing fast and the development and optimisation of more sensitive, faster and more discriminating forensic DNA analysis methods is highly important. In this thesis, an evaluation of the use of novel DNA technologies and the development of specific applications for use in forensic casework investigations are presented.</p><p>In order to maximise the use of valuable limited DNA samples, a fast and user-friendly Real-time PCR quantification assay, of nuclear and mitochondrial DNA copies, was developed. The system is based on the 5’ exonuclease detection assay and was evaluated and successfully used for quantification of a number of different evidence material types commonly found on crime scenes. Furthermore, a system is described that allows both nuclear DNA quantification and sex determination in limited samples, based on intercalation of the SYBR Green dye to double stranded DNA. </p><p>To enable highly sensitive DNA analysis, Pyrosequencing of short stretches of mitochondrial DNA was developed. The system covers both control region and coding region variation, thus providing increased discrimination power for mitochondrial DNA analysis. Finally, due to the lack of optimal assays for quantification of mitochondrial DNA mixture, an alternative use of the Pyrosequencing system was developed. This assay allows precise ratio quantification of mitochondrial DNA in samples showing contribution from more than one individual.</p><p>In conclusion, the development of optimised forensic DNA analysis methods in this thesis provides several novel quantification assays and increased knowledge of typical DNA amounts in various forensic samples. The new, fast and sensitive mitochondrial DNA Pyrosequencing assay was developed and has the potential for increased discrimination power.</p>

<p>This thesis focuses on the identification of genetic factors underlying two inherited human phenotypes: hearing loss and monoamine oxidase activity. </p><p>Non-syndromic hearing loss segregating in a Swedish family was tested for linkage to 13 previously reported candidate loci for hearing disabilities. Linkage was found to two loci: DFNA12 (llq22-q24) and DFNA2 (lp32). A detailed analysis of the phenotypes and haplotypes shared by the affected individuals supported the hypothesis of digenic inheritance of hearing disability in the Swedish family. Mutation screening of α-tectorin, a gene residing within the DFNA12 region revealed a mutation of a conserved amino acid (Cys to Ser), that segregated with the disease. The identification of the mutation added support to the involvement of α-tectorin in hearing disabilities. In contrast, no mutations were identified in two candidate genes at the DFNA2 locus, that were reported to cause hearing loss in other families. It is possible that the DFNA2 locus contains a third, not yet identified, hearing loss gene. </p><p>Monoamine oxidase A (MAOA) and B (MAOB) catalyze the degradation of certain neurotransmitters in the central nervous system and are associated with specific behavioral and neuropsychiatric human traits. Activity levels of both monoamine oxidases (MAO) are highly variable among humans and are determined by unknown genetic factors. This study investigated the relationship of different MAO alleles with MAO mRNA levels and enzyme activity in human brain. Several novel DNA polymorphisms were identified in a group of Swedish individuals. Haplotypes containing several closely located MAOA polymorphisms were assessed in Asian, African, and Caucasian populations. The haplotype distribution and diversity pattern found among the three populations supported the occurrence of a bottleneck during the dispersion of modem humans from Africa. </p><p>Allelic association studies conducted on postmortem human brain samples, revealed the association between a SNP in the MAOB intron 13, and different levels of both MAO enzyme activities. This suggested that this SNP is in linkage disequilibrium with at least one novel functional DNA polymorphism that controls MAO enzyme activities in human brain. The identification of functional polymorphisms regulating the activity of these enzymes will help to elucidate the involvement of MAO in human behavior and neuropsychiatric conditions. </p>

<p>In this thesis, genetic methods for individual identification and sex determination of wolverines from non-invasive samples were developed and applied in genetic monitoring of Scandinavian wolverine populations. Paternity and mating system of wolverines were studied by combining genetic analysis with telemetry data. Moreover, the possibility to obtain DNA from claws left on tanned carnivore hides was investigated.</p><p>Non-invasive genetic sampling was effective in revealing important population parameters. For the subpopulation in southern Norway, a population size of approximately 90 individuals, an equal sex ratio and similar levels of genetic diversity as in the main Scandinavian population were revealed. Genetic erosion in this small population has likely been counteracted by immigration of individuals from the main population since its re-establishment around 1970.</p><p>During the 1990s, two areas in east-central Sweden were colonised by wolverines. In a survey comprising 400 non-invasive samples collected during five winters, a total of 22 wolverines were detected. Genetic data suggest that inbreeding has occurred in both areas and that the two populations were founded by as few as 2-4 individuals. These findings suggest that gene flow from the main population is crucial for their survival even in a short time perspective. The detection of occasional stray individuals from the main population shows that this is indeed feasible. </p><p>Paternity analysis of 145 wolverine offspring in northern Sweden and southern Norway confirmed a polygamous mating system in wolverines. Breeding pair formation was generally consistent with the territories held by males and females, i.e. breeding pairs had overlapping territories. In the majority of litters, siblings were assigned the same father, thus indicating that multiple paternity is rare. </p><p>Tanning is a common form of preservation of mammalian specimens that normally precludes genetic analysis. Nevertheless, I demonstrate the possibility to successfully extract and amplify DNA from claws left on tanned carnivore hides.</p>

<p>The Mad/Max/Myc transcription factor network takes part in the control of vital cellular functions such as growth, proliferation, differentiation and apoptosis. Dimerization with the protein Max is necessary for the Myc-family of oncoproteins and their antagonists, the Mad-family proteins, to regulate target genes and carry out their intended functions. Myc functions as a positive regulator of proliferation, antagonized by the growth inhibitory Mad-proteins that potentially functions as tumor supprerssors. Deregulated Myc expression is found in a variety of tumors and signals negatively regulating Myc expression and/or activity could therefore be of potential use in treating tumors with deregulated Myc.</p><p>Our aim was to therefore to investigate possible negative effects on Myc expression and activity by growth inhibitory cytokines and by the Myc antagonists, the Mad-family proteins.Two different cellular model systems of neuronal and hematopoietic origin have been utilized for these studies.</p><p>Our results show that Mad1 is upregulated during induced neuronal differentiation of SH-SY5Y cells. Further, the growth inhibitory cytokine interferon-g (IFN-g) was shown to cooperate with retinoic acid (RA) and the phorbol ester TPA in inducing growth arrest and differentiation in N-<i>myc</i> amplified neuroblastoma cell lines. In contrast to treatment with either agent alone, the combined treatment of TPA+IFN-g and RA+IFN-g led to upregulation of Mad1 and to downregulation of N-Myc, respectively, thus correlating with the enhanced growth inhibition and differentiation observed after combination treatment. Ectopic expression of an inducible Mad1 in monoblastic U-937 cells led to growth inhibition but did not lead to differentiation or enhancement of differentiation induced by RA, vitamin D3 or TPA. In v-Myc transformed U-937 cells Mad1 expression reestablished the TPA-induced G1 cell cycle arrest, but did not restore differentiation, blocked by v-Myc. The growth inhibitory cytokine TGF-b was found to induce Mad1 expression and Mad1:Max complex formation in v-Myc transformed U-937 cells correlating with reduced Myc activity and G1 arrest. </p><p>In conclusion, our results show that the Myc-antagonist Mad1 is upregulated by growth inhibitory cytokines and/or differentiation signals in neuronal and hematopoietic cells and that enforced Mad1 expression in hematopoietic cells results in growth inhibition and increased sensitivity to anti-proliferative cytokines. Mad1 and cytokine-induced signals therefore seem to cooperate in counteracting Myc activity.</p>

In multicellular organisms all cells in one individual have an identical genotype, and yet their bodies consist of many and very different tissues and thus many different cell types. Somehow there must be a difference in how genes are interpreted. So, there must be signals that tell the genes when and where to be active and inactive, respectively. In some instances a specific an expression pattern (active or inactive) is epigenetic; it is established and maintained throughout multiple rounds of cell divisions. In the developing Drosophila embryo, the proper expression pattern of e.g. the homeotic genes Abd-B and Ubx is to be kept active in the posterior part and silenced in the anterior. Properly silenced homeotic genes are crucial for the correct segmentation pattern of the fly and the Polycomb group (Pc-G) proteins are vital for maintaining this type of stable repression. As part of this thesis, Suppressor of zeste 12 (Su(z)12) is characterized as a Drosophila Pc-G gene. Mutations in the gene cause widespread misexpression of several homeotic genes in embryos and larvae. Results show that the silencing of the homeotic genes Abd-B and Ubx, probably is mediated via physical binding of SU(Z)12 to Polycomb Response Elements in the BX-C. Su(z)12 mutations are strong suppressors of position-effect-variegation and the SU(Z)12 protein binds weakly to the heterochromatic centromeric region. These results indicate that SU(Z)12 has a function in heterochromatin-mediated repression, which is an unusual feature for a Pc-G protein. The structure of the Su(z)12 gene was determined and the deduced protein contains a C2-H2 zinc finger domain, several nuclear localization signals, and a region, the VEFS box, with high homology to mammalian and plant homologues. Su(z)12 was originally isolated in a screen for modifiers of the zeste-white interaction and I present results that suggests that this effect is mediated through an interaction between Su(z)12 and zeste. I also show that Su(z)12 interact genetically with other Pc-G mutants and that the SU(Z)12 protein binds more than 100 euchromatic bands on polytene chromosomes. I also present results showing that SU(Z)12 is a subunit of two different E(Z)/ESC embryonic silencing complexes, one 1MDa and one 600 kDa complex, where the larger complex also contains PCL and RPD3. In conclusion, results presented in this thesis show that the recently identified Pc-G gene, Su(z)12, is of vital importance for correct maintenance of silencing of the developmentally important homeotic genes.

This thesis focuses on the identification of genetic factors underlying two inherited human phenotypes: hearing loss and monoamine oxidase activity. Non-syndromic hearing loss segregating in a Swedish family was tested for linkage to 13 previously reported candidate loci for hearing disabilities. Linkage was found to two loci: DFNA12 (llq22-q24) and DFNA2 (lp32). A detailed analysis of the phenotypes and haplotypes shared by the affected individuals supported the hypothesis of digenic inheritance of hearing disability in the Swedish family. Mutation screening of α-tectorin, a gene residing within the DFNA12 region revealed a mutation of a conserved amino acid (Cys to Ser), that segregated with the disease. The identification of the mutation added support to the involvement of α-tectorin in hearing disabilities. In contrast, no mutations were identified in two candidate genes at the DFNA2 locus, that were reported to cause hearing loss in other families. It is possible that the DFNA2 locus contains a third, not yet identified, hearing loss gene. Monoamine oxidase A (MAOA) and B (MAOB) catalyze the degradation of certain neurotransmitters in the central nervous system and are associated with specific behavioral and neuropsychiatric human traits. Activity levels of both monoamine oxidases (MAO) are highly variable among humans and are determined by unknown genetic factors. This study investigated the relationship of different MAO alleles with MAO mRNA levels and enzyme activity in human brain. Several novel DNA polymorphisms were identified in a group of Swedish individuals. Haplotypes containing several closely located MAOA polymorphisms were assessed in Asian, African, and Caucasian populations. The haplotype distribution and diversity pattern found among the three populations supported the occurrence of a bottleneck during the dispersion of modem humans from Africa. Allelic association studies conducted on postmortem human brain samples, revealed the association between a SNP in the MAOB intron 13, and different levels of both MAO enzyme activities. This suggested that this SNP is in linkage disequilibrium with at least one novel functional DNA polymorphism that controls MAO enzyme activities in human brain. The identification of functional polymorphisms regulating the activity of these enzymes will help to elucidate the involvement of MAO in human behavior and neuropsychiatric conditions.

The Mad/Max/Myc transcription factor network takes part in the control of vital cellular functions such as growth, proliferation, differentiation and apoptosis. Dimerization with the protein Max is necessary for the Myc-family of oncoproteins and their antagonists, the Mad-family proteins, to regulate target genes and carry out their intended functions. Myc functions as a positive regulator of proliferation, antagonized by the growth inhibitory Mad-proteins that potentially functions as tumor supprerssors. Deregulated Myc expression is found in a variety of tumors and signals negatively regulating Myc expression and/or activity could therefore be of potential use in treating tumors with deregulated Myc. Our aim was to therefore to investigate possible negative effects on Myc expression and activity by growth inhibitory cytokines and by the Myc antagonists, the Mad-family proteins.Two different cellular model systems of neuronal and hematopoietic origin have been utilized for these studies. Our results show that Mad1 is upregulated during induced neuronal differentiation of SH-SY5Y cells. Further, the growth inhibitory cytokine interferon-g (IFN-g) was shown to cooperate with retinoic acid (RA) and the phorbol ester TPA in inducing growth arrest and differentiation in N-myc amplified neuroblastoma cell lines. In contrast to treatment with either agent alone, the combined treatment of TPA+IFN-g and RA+IFN-g led to upregulation of Mad1 and to downregulation of N-Myc, respectively, thus correlating with the enhanced growth inhibition and differentiation observed after combination treatment. Ectopic expression of an inducible Mad1 in monoblastic U-937 cells led to growth inhibition but did not lead to differentiation or enhancement of differentiation induced by RA, vitamin D3 or TPA. In v-Myc transformed U-937 cells Mad1 expression reestablished the TPA-induced G1 cell cycle arrest, but did not restore differentiation, blocked by v-Myc. The growth inhibitory cytokine TGF-b was found to induce Mad1 expression and Mad1:Max complex formation in v-Myc transformed U-937 cells correlating with reduced Myc activity and G1 arrest. In conclusion, our results show that the Myc-antagonist Mad1 is upregulated by growth inhibitory cytokines and/or differentiation signals in neuronal and hematopoietic cells and that enforced Mad1 expression in hematopoietic cells results in growth inhibition and increased sensitivity to anti-proliferative cytokines. Mad1 and cytokine-induced signals therefore seem to cooperate in counteracting Myc activity.

Predisposition to autoimmune diseases such as, rheumatoid arthritis, diabetes, and multiple sclerosis, is caused by the effect of multiple genes and a strong influence from the environment. In this study, I have investigated genetic factors that confer susceptibility to rheumatoid arthritis in a rat model. This work has led to the identification of several chromosomal regions, containing uncharacterized genes that directly or indirectly are associated to the arthritis development in these rats. We have observed that timing, gender, and genetic interactions are features that play a part in the effect that these genetic factors exert. Unarguably, animal models for human disorders display differences to the human form of disease. An important fact is however that the same chromosomal regions are identified in both rodent and human studies, which suggests that there are genetic factors that we have in common, which are involved directly or indirectly with an autoimmune response. Focusing the interest on these similarities, and on the possibility to apply a wide set of genetic tools, make animal models an invaluable, and probably necessary, instrument to dissect the genetic component of complex disorders. To fully comprehend the genetic basis for a complex disorder like this, will require understanding of how multiple genes interact with each other to cause disease. We have been able to demonstrate that chronic arthritis, in a rat model for rheumatoid arthritis, is regulated by several genes and that these act during different temporal phases of the disease. These findings will hopefully contribute to our understanding of the etiology and progression of rheumatoid arthritis.

A previous model of morphological pathogenesis assumed that cervical carcinoma is of monoclonal origin and progresses through multiple steps from normal epithelium via CINS into invasive carcinomas. The aim of this study was to investigate the molecular mechanism of pathogenesis of cervical neoplasia. In the clonality study, we found that 75% (6/8) of informative cases of cervical carcinoma had identical patterns of loss of heterozygosity (LOH) in the multiple synchronous lesions, while the remaining cases had different LOU patterns. In an extensively studied "golden case", the multiple carcinoma and cervical intraepithelial neoplasia (CIN) lesions could be divided into several different clonal groups by the X-chromosome inactivation patterns, HPV 16 mutations and LOH patterns. The biggest clonal family included one CIN II, one CIN III and four carcinoma samples, while four other monoclonal families of carcinoma did not include CIN lesions. These results suggested that cervical carcinoma can be either monoclonal or polygonal and contains clones developing either directly or via multiple steps. In the study of HPV types and HPV16 variations, the results confirmed that specific HPV types are the cause of cervical carcinoma but failed to support the previous opinion that HPV16 E6 variants are more malignant than the prototype. We established a novel classification called oncogene lineage of HPV16, and found that additional variations of HPV 16 oncogenes might be a weak further risk factor for cervical carcinoma. In the study of LOH, we found that interstitial deletion of two common regions of chromosome 3p, i.e., 3p2l.1-3p2l.3, and 3p22, was an early event in the development of cervical carcinoma. The results showed that the hMLH1 gene, located in 3p22 and showing LOH in 43% of the studied cases, was not involved in the development of cervical carcinoma because neither the expression level of protein nor the gene sequence was altered in these cases. In summary, a suggested model of molecular pathogenesis of cervical carcinoma is as follows. Specific types of HPV infect one or more committed stem cells in the basal layer of the epithelium. Fully efficient LOH events turn one (monoclonal origin) or more (polyclonal origin) HPV-infected stem cells into carcinoma cells without CIN steps. Less efficient LOH events would lead to CIN steps where some other unknown factors require to be added to facilitate the formation of carcinoma. In the absence of LOH events no carcinoma develops from the HPV-infected stem cells.

Mast cells are essential effector cells in the immune system as they release several inflammatory mediators. An accumulation of mast cells has been described in inflammatory conditions such as asthma and allergic rhinitis. Increased mast cell number, in the skin and other organs, is also a characteristic in mastocytosis, a disease without an effective treatment. One explanation for the increase in mast cell number is migration of mast cells in the tissue. In our studies we utilised mast cell lines, including HMC-1; cell lines transfected with the c-kit gene; and in vitro developed mast cells. Our aim was to characterise, two variants of the HMC-1 cell line; the signalling pathways essential for mast cell migration towards TGF-β and SCF; and the mechanism regulating mast cell accumulation in mastocytosis. Our results help to explain inconsistent findings regarding mast cell biology when HMC-1 cells have been used as a model system. The two variants, which we name HMC-1560 and HMC-1560, 816, are used in different laboratories around the world. HMC-1560 and HMC-1560, 816 exhibited different characteristics regarding their karyotype, phenotype as well as their set of activating point mutations in the Kit receptor. Furthermore, divergent signalling pathways are of importance for mast cell migration towards TGF-β and SCF. The classical MAP kinase-signalling cascade was found to be of major relevance for TGF-β-induced migration. In contrast, this pathway had a modest impact on SCF-induced migration, which instead was highly dependent on p38 MAP kinase signalling. Finally, one mechanism for mast cell accumulation in mastocytosis appeared to be an activating point mutation in the gene for the Kit receptor. This mutation appeared to prone transfected cells and mast cell progenitors to a higher rate of migration towards SCF if compared with cells expressing wt Kit receptor. In conclusion, our results show the importance of two different MAP kinase signalling pathways and mutations in the Kit receptor for mast cell migration induced by various types of stimuli. This knowledge helps us to understand the mechanism