<p>Sex chromosomes are useful for the study of how factors such as mutation, selection, recombination and effective population size affect diversity and divergence.</p><p>A comparison of gametologous introns in seven different bird species revealed a complete lack of diversity on the female-specific W chromosome. In contrast, Z had at least one segregating site in all examined species. This can be explained by the lower mutation rate and lower effective population size of W but also suggests that selection affects diversity levels on the non-recombining W chromosome.</p><p>In a diverse set of chicken breeds, the Z chromosome showed reduced diversity compared to autosomes and significant heterogeneity in levels of variation. High variance in male reproductive success, leading to a reduced Z chromosome effective population size, can partly explain this observation. In addition, we suggest that selective sweeps frequently act on the Z chromosome and are responsible for a significant part of the observed Z reduction. </p><p>Differences in the mutation rate of Z and W chromosome sequences indicate that the time spent in male germ line is important for the mutation rate, but does not exclude a specifically reduced mutation rate on the Z chromosome. Estimates of mutation rate in autosomal, Z- and W-linked chicken and turkey sequences indicate a slight reduction in the rate on Z. However, due to rate heterogeneity among introns this reduction is not significant and we cannot exclude male biased mutation as the single cause of rate variation between the chromosomal classes.</p><p>Analysis of indel mutation rates in avian and mammalian gametologous introns show frequent occurrence of indels on both W and Y, excluding meiotic recombination as the only source of this type of mutation. The different indel rate patterns in birds (Z>W) and mammals (X=Y) suggest that indels are caused by both replication and recombination.</p>

<p>B cell lymphoma (BCL) comprises a biologically and clinically heterogeneous group of tumors deriving from different stages of B cell development. The immunoglobulin (Ig) variable heavy chain (V_H) gene rearrangement is unique for each BCL and can be used to reveal cellular origin, to study signs of antigen selection and to quantify tumor cell load.</p><p>The normal counterpart of mantle cell lymphoma (MCL) has been postulated to be a naïve B cell and in hairy cell leukemia (HCL) it is considered to be a post-germinal centre B cell. We analyzed the V_H gene rearrangements in 110 MCLs and 32 HCLs by PCR amplification and sequencing. Most MCLs (84%) displayed V_H genes lacking somatic hypermutation (SHM), thus correlating to a naïve cell origin, whereas a subgroup (16%) showed SHM, implying derivation from a more differentiated B cell. In HCL, a majority of cases (84%) displayed SHM with signs of intraclonal heterogeneity and 16% had unmutated V_H genes, thus questioning the cell of origin in HCL. Biased usage of particular V_H genes was detected in both HCL (V_H3-30) and MCL (V_H3-21 and V_H4-34), which indicates that antigen selection may be involved in lymphoma development. Furthermore, V_H3-21⁺ MCLs showed a highly restricted V_λ3-19 gene use and they also had a superior outcome compared to other MCLs.</p><p>Rearrangement analysis of 67 V_H3-21⁺ chronic lymphocytic leukemia (CLL) cases from three different countries verified, regardless of geographical origin, the short and highly homologous complementarity determining region 3s and the strikingly biased usage of the V_λ2-14 gene (75%), as previously reported in CLL. This further supports that antigen selection by a common antigenic epitope may have occurred in V_H3-21⁺ CLLs. </p><p>In an autologous transplantation study of 30 multiple myeloma patients, we quantified the tumor content in the autografts before and after stem cell selection using clone-specific PCR. We conclude that stem cell selection reduced the number of clonal cells linearly, but purging could not totally eliminate the tumor cells from the graft, thus increasing the risk of a relapse.</p><p>Altogether, our data allowed us to define new BCL subsets and to gain insights into the potential role of antigen selection in BCL development as well as the monitoring of tumor cell load using Ig gene rearrangements analysis. </p>

<p>The severity of illness was scored in a cohort of 11751 non-surgical patients presenting at the Emergency Department (ED) during 12 consecutive months and followed for 4.7 years. The scoring system Rapid Acute Physiology score (RAPS) (including blood pressure, respiratory rate, pulse rate and Glasgow coma scale) was calculated for all arrivals at the ED. The RAPS system was also additionally developed by including the peripheral oxygen saturation and patient age, resulting in the new Rapid Emergency Medicine Score, (REMS). REMS was superior to RAPS in predicting in-hospital mortality according to ROC-curve analysis. An increase of one point in the 26 point REMS scale was associated with an Odds ratio of 1.40 for in-hospital death (95% CI 1.36-1.45, p<0.0001). Similar results were obtained in the major patient groups (chest pain, stroke, coma, dyspnea and diabetes). The association between REMS and length of stay in hospital was modest. Charlson Co-morbidity Index could add prognostic information to REMS in a long-term (4.7 years), but not in a short-term perspective (3 and 7 days). REMS was shown to be as powerful a predictor of in-hospital mortality as the more complicated APACHE II. REMS at the ED could also predict long-term mortality (4.7 years) in the total cohort (Hazard ratio 1.26, p<0.0001).</p><p>REMS is a potentially useful prognostic tool for non-surgical patients at the ED, regarding both in-hospital and long-term mortality. It is less complicated to use than APACHE II and has equal predictive accuracy.</p>

<p>DNA samples from crime scenes or mass disasters are often limited and degraded which limits the possibility of successful traditional STR analysis. Moreover, there is a need to decrease the turnaround time in criminal investigations. These circumstances require a wider set of assays and technologies to be investigated for potential use in forensic DNA analysis, which has been explored in this thesis work. DNA analysis can also provide a useful tool in forensic pathology investigations. </p><p>In a search for mutations involved in The Sudden Infant death Syndrome (SIDS), the entire mitochondrial genome was sequenced in six SIDS infants and shorter mtDNA regions were analysed in paraffin-embedded tissues from an additional 14 SIDS cases. In this sample material no mutations associated with SIDS were found that could explain the death of these infants. </p><p>To reduce time, cost and effort related to sequencing of the mtDNA HVI/HVII regions in caseworks, a HVI/HVII mtDNA linear array assay was used as a pre-screening for exclusions of suspects or evidence samples. Using this assay, 56% of the samples involved in casework analysis could be excluded before sequencing was undertaken.</p><p>The possibility to use the new array technology was explored in a SNP assay targeting both mtDNA and nuclear SNPs. The system relies on minisequencing in solution prior to hybridisation to tag arrays. Using this system, we demonstrate a rapid, highly multiplexable and flexible array-format for SNP analysis.</p><p>The properties of the Pyrosequencing technology being a fast and user-friendly assay was utilised in a study to investigate the possibility to use this method for limited and degraded samples. Ten STR loci, overlapping with standardised kits, were genotyped in 114 Swedish individuals. We found additional variation and higher resolution of repeats at some of these loci that are not detected using standard fragment analysis.</p>

<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>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.