In mammals, the most frequently observed hybrid dysgenesis effects are growth disturbances and male sterility. Profound defects in placental development have been described and our work on hybrids in genus Mus has demonstrated putative hybrid dysgenesis effects that lead to defects in lipid homeostasis and maternal behavior. Interestingly, mammalian interspecies hybrids exhibit strong parent-of-origin effects in that offspring of reciprocal matings, even though genetically identical, frequently exhibit reciprocal phenotypes. Recent studies have provided strong link between epigenetic regulation and growth, behavior and placental development. Widespread disruption of genomic imprinting has been described in hybrids between closely related species of the genus Peromyscus. The studies presented in this thesis aim to investigate the effects of disrupted epigenetics states on altered growth, female infanticide and placental dysplasia observed in Mus hybrids. We showed that loss-of-imprinting (LOI) of a paternally expressed gene, Peg1, was correlated with increased body weight of F1 hybrids. Furthermore, we investigated whether LOI of Peg1 in F1 females would interfere with maternal behavior. A subset of F1 females indeed exhibited highly abnormal maternal behavior in that they rapidly attacked and killed the pups. By microarray hybridization, a large number of differentially expressed genes in the infanticidal females as compared to normally behaving females were identified. In addtion to Peg1 LOI, we studied allelic expression of numerous imprinted genes in adult Mus interspecies hybrids. In contrast to the study from Peromyscus, patterns of LOI were not consistent with a direct influence of altered expression levels of imprinted genes on growth. Finally, we investigated the allelic interaction between an X-linked locus and a paternally expressed gene, Peg3, in placental defects in Mus hybrids. This study further strengthened the notion that divergent genetic and epigenetic mechanisms may be involved in hybrid dysgenesis in diverse groups of mammals.

The development of a new individual from the fertilized oocyte can ultimately be seen as the consequence of the establishment and maintenance of specific patterns of gene expression. Although regulation of gene activity occurs at different levels, cellular specialization and differentiation are the results of developmental cues that essentially take place at the transcriptional level. The involvement of epigenetics in this process has become increasingly clear during the last decade. Imprinted genes constitute an excellent example as monoallelic expression seems to reflect differential epigenetic marks on the two alleles. This is the case of the imprinted H19 and Igf2 genes were the monoallelic expression is coordinated through a differentially methylated region (hypermethylated on the paternal allele), known as ICR (imprinted control region). In the mouse the ICR harbours four binding sites for the methylation sensitive insulator protein CTCF. Previous studies with episomal constructs had shown that this region behaved as an insulator and that CTCF is required for the insulator activity of the H19 ICR This thesis establish a clear link between the insulator function and the chromatin structure at the H19 ICR and indicates that the precise allocation of the CTCF target sites in the linker regions can play a critical role in this process. The importance of the CTCF interaction at the ICR was also confirmed in vivo using a mouse model that showed how intact CTCF target sites are needed to manifest insulator activity and methylation protection. We have investigated the role of CTCF and a related protein BORIS in establishing the maternal to paternal imprint transition in chromatin structure at the H19/Igf2 locus in the male germline. This thesis also describe the development of a new technique for the localization of chromatin associated factors and modifications with higher sensitivity and resolution compared to existing approaches.

Placental development can be disturbed by various factors, such as mutation of specific genes or maternal diabetes. Our previous work on interspecies hybrid placental dysplasia (IHPD) and two additional models of placental hyperplasia, cloned mice and Esx1 mutants, showed that many genes are deregulated in placental dysplasia. Two of these candidate placentation genes, Cpe and Lhx3, were further studied. We performed in situ hybridization to determine their spatio-temporal expression in the placentas and placental phenotypes were analyzed in mutant mice. Our results showed that the placental phenotype in Cpe mutant mice mimics some IHPD phenotypes. Deregulated expression of Cpe and Cpd, a functionally equivalent gene, prior to the manifestation of the IHPD phenotype, indicated that Cpe and Cpd are potentially causative genes in IHPD. Lhx3 mutants lacked any placental phenotype. Deletion of Lhx3 and Lhx4 together caused an inconsistent placental phenotype which did not affect placental lipid transport function or expression of Lhx3/Lhx4 target genes. Down regulation of Lhx3/Lhx4 did not rescue the placental phenotype of AT24 mice and hence could be excluded as causative genes in IHPD. Analysis of placental development in diabetic mice showed that severe maternal diabetes resulted in fetal intrauterine growth restriction (IUGR) without any change in placental weight and lipid transport function. The diabetic placentas however exhibited abnormal morphology. Gene expression profiling identified some genes that might contribute to diabetic pathology. In another study, it was found that the heterochromatin protein CBX1 is required for normal placentation, as deletion of the gene caused consistent spongiotrophoblast and labyrinthine phenotypes. Gene expression profiling and spatio-temporal expression analysis showed that several genes with known function in placental development were deregulated in the Cbx1 null placenta.

<p>The fruit fly <i>Drosophila melanogaster</i> has been a powerful model system in which to study the immune response. When microorganisms breach the mechanical barrier of the insect, phagocytosing cells and a battery of induced antimicrobial molecules rapidly attack them. These antimicrobial peptides can reach micromolar concentrations within a few hours. This immediate response is reminiscent of the mammalian innate immune response and utilizes transcription factors of the NF-κB family. </p><p>We have generated loss-of-function mutants of the NF-κB-like transcription factor Relish in order to investigate Relish's role in the <i>Drosophila</i> immune response to microbes. Relish mutant flies have a severely impaired immune response to Gram-negative (G^-) bacteria and some Gram-positive (G⁺) bacteria and fungi and succumb to an otherwise harmless infection. The main reason for the high susceptibility to infection is that these mutant flies fail to induce the antimicrobial peptide genes. The cellular responses appear to be normal. </p><p>Relish is retained in the cytoplasm in an inactive state. We designed a set of expression plasmids to investigate the requirements for activation of Relish in a hemocyte cell line after stimulation with bacterial lipopolysaccharide. Signal-induced phosphorylation of Relish followed by endoproteolytic processing at the caspase-like target motif in the linker region released the inhibitory ankyrin-repeat (ANK) domain from the DNA binding Rel homology domain (RHD). Separation from the ANK domain allowed the RHD to move into the nucleus and initiate transcription of target genes like those that encode the inducible antimicrobial peptides, likely by binding to κB-like sites in the promoter region. </p><p>By studying the immune response of the Relish mutant flies in combination with mutants for another NF-κB-like protein, Dorsal-related immunity factor (Dif), we found that the <i>Drosophila</i> immune system can distinguish between various microbes and generate a differential response by activating the Toll/Dif and Imd/Relish pathways. The recognition of foreign microorganisms is believed to occur through pattern recognition receptors (PRRs) that have affinity for selective pathogen-associated molecular patterns (PAMPs). We found that the <i>Drosophila</i> PRRs can recognize G^- bacteria as a group. Interestingly, the PRRs are specific enough to distinguish between peptidoglycans from G⁺ bacteria such as <i>Micrococcus luteus</i> and <i>Bacillus megaterium </i>and fungal PAMPs from <i>Beauveria bassiana</i> and <i>Geotrichum candidum</i>. </p><p>This thesis also investigates the expression of the antimicrobial peptide genes, <i>Diptericin B</i> and <i>Attacin C</i>, and the putative intracellular antimicrobial peptide gene <i>Attacin D</i>, and explores a potential evolutionary link between them.</p>

More conservation programs directed towards insects are needed in order to prevent the continue decline in which many insect species in the world are heading to. New ways to use the remaining microhabitats are needed to mitigate the fragmentation and degradation that urbanization and modern agriculture have contributed to in the southern Swedish landscapes. This study focusses on how microhabitats like road-verges can help the conservation projects of pollinating insects. To increase the knowledge about three groups of pollinators (Apidae, Rhopalocera and Syrphidae) and their distribution between two different microhabitats, a malaise trap was set up in road verges and in sample locations further away from the road-verges. The result from this study shows that road-verges are a microhabitat that could work as mitigation to the decline of some pollinating species, if right actions are taken. This study shows that Syrphidae was most acceptant to the hostile environment close to the roads. If these actions are going to help the most treated species is still unknown.

<p>The development of a multicellular organism is programmed by complex patterns of gene expression. In eukaryotic cells, genes are packaged by histone proteins into chromatin. Chromatin regulators often function as transcription co-factors. </p><p>In this study, we have investigated the function of four co-factors, dAda2b, Reptin, Ebi and Brakeless during development of the fruit fly<i> Drosophila</i> <i>melanogaster</i>. dAda2b and Reptin belong to histone acetyl transferase (HAT) complexes, a SAGA-like complex and the Tip60 complex, respectively. We generated <i>dAda2b </i>mutants and found that lack of dAda2b strongly affects global histone acetylation and viability. We further propose that Ada2 may be involved in DNA repair. Our studies revealed new roles of Reptin and other Tip60 complex components in Polycomb Group mediated repression and heterochromatin formation, thereby promoting generation of silent chromatin.</p><p>During embryogenesis, transcriptional repressors establish localized and tissue-specific patterns of gene expression. In this thesis, we identified two novel co-repressors in the early embryo, Ebi and Brakeless. Ebi genetically and physically interacts with the Snail repressor. The Ebi-interaction motif in the Snail protein is essential for Snail function<i> in</i> <i>vivo</i> and is evolutionarily conserved in insects. We further demonstrated that Ebi associates with histone deacetylase 3 (HDAC3) and that histone deacetylation is part of the mechanism by which Snail mediates transcriptional repression. </p><p>We isolated Brakeless in a genetic screen for novel regulators of gene expression during embryogenesis. We found that mutation of <i>brakeless</i> impairs function of the Tailless repressor. Brakeless associates with Atrophin, another Tailless corepressor, and they function together in Tailless-mediated repression. </p><p>In summary, transcription co-factors, including chromatin regulators, are selectively required in distinct processes during development.</p>

<p>In this project I have tried to obtain stable transformants of <em>Drosophila</em> melanogaster flies using electroporation. I have completed approximately 200 tests using different DNA concentrations, voltages and cuvettes, including a novel Petri dish cuvette which I developed and manufactured myself. I also developed new and more efficient procedures of egg collection and egg dechorionation. Although I was not  successful in obtaining true stable transformants, control experiments indicate that electroporation of DNA into embryos could be accomplished under the conditions used. The lack of stable transformants was probably due to failure of the electroporated DNA to integrate into the host genome. The reasons for why the DNA did not integrate was not further investigated in this study.</p>

The development of a multicellular organism is programmed by complex patterns of gene expression. In eukaryotic cells, genes are packaged by histone proteins into chromatin. Chromatin regulators often function as transcription co-factors. In this study, we have investigated the function of four co-factors, dAda2b, Reptin, Ebi and Brakeless during development of the fruit fly Drosophila melanogaster. dAda2b and Reptin belong to histone acetyl transferase (HAT) complexes, a SAGA-like complex and the Tip60 complex, respectively. We generated dAda2b mutants and found that lack of dAda2b strongly affects global histone acetylation and viability. We further propose that Ada2 may be involved in DNA repair. Our studies revealed new roles of Reptin and other Tip60 complex components in Polycomb Group mediated repression and heterochromatin formation, thereby promoting generation of silent chromatin. During embryogenesis, transcriptional repressors establish localized and tissue-specific patterns of gene expression. In this thesis, we identified two novel co-repressors in the early embryo, Ebi and Brakeless. Ebi genetically and physically interacts with the Snail repressor. The Ebi-interaction motif in the Snail protein is essential for Snail function in vivo and is evolutionarily conserved in insects. We further demonstrated that Ebi associates with histone deacetylase 3 (HDAC3) and that histone deacetylation is part of the mechanism by which Snail mediates transcriptional repression. We isolated Brakeless in a genetic screen for novel regulators of gene expression during embryogenesis. We found that mutation of brakeless impairs function of the Tailless repressor. Brakeless associates with Atrophin, another Tailless corepressor, and they function together in Tailless-mediated repression. In summary, transcription co-factors, including chromatin regulators, are selectively required in distinct processes during development.

Epigenetic states constitute heritable features of the chromatin to regulate when, where and how genes are expressed in the developing conceptus. A special case of epigenetic regulation, genomic imprinting, is defined as parent of origin-dependent monoallelic expression. The Igf2-H19 locus is considered as paradigm of genomic imprinting with a growth-promoting gene, Igf2, expressed paternally and a growth antagonist, H19 encoding a non-coding transcript, expressed only from the maternal allele. The monoallelic expression patterns are regulated by the epigenetic status at an imprinting control region (ICR) in the 5´-flank of the H19 gene. The chromatin insulator protein CTCF interacts with only the maternal H19 ICR allele to prevent downstream enhancers to communicate with the Igf2 promoters. Mutations of these CTCF binding sites lead to biallelic Igf2 expression, increased size of the conceptus and predisposition for cancer. Reasoning that these effects cannot be explained by the regulation of Igf2 expression alone, a technique was invented to examine long-range chromatin interactions without prior knowledge of the interacting partners. Applying the circular chromosomal conformation capture (4C) technique to mouse neonatal liver cells, it was observed that 114 unique sequences interacted with the H19 ICR. A majority of these interactors was in complex with only the maternal H19 ICR allele and depended on the presence of functional CTCF binding sites. The functional consequence of chromosomal networks was demonstrated by the observation that the maternal H19 ICR allele regulated the transcription of two genes on another chromosome. As the chromosomal networks underwent reprogramming during the maturation of embryonic stem cells, attention was turned to human cancer cells, displaying features common with mouse embryonic stem cells. Subsequently, chromatin folding at the human H19 ICR suggested that stable chromatin loops were organized by synergistic interactions within and between baits and interactors. The presence of these interactions was linked to DNA methylation patterns involving repeat elements. A "flower" model of chromatin networks was formulated to explain these observations. This thesis has unravealed a novel feature of the epigenome and its functions to regulate gene expression in trans. The identified roles for CTCF as an architectural factor in the organization of higher order chromatin conformations may be of importance in understanding development and disease ontogeny from novel perspectives.

The fruit fly Drosophila melanogaster has been a powerful model system in which to study the immune response. When microorganisms breach the mechanical barrier of the insect, phagocytosing cells and a battery of induced antimicrobial molecules rapidly attack them. These antimicrobial peptides can reach micromolar concentrations within a few hours. This immediate response is reminiscent of the mammalian innate immune response and utilizes transcription factors of the NF-κB family. We have generated loss-of-function mutants of the NF-κB-like transcription factor Relish in order to investigate Relish's role in the Drosophila immune response to microbes. Relish mutant flies have a severely impaired immune response to Gram-negative (G-) bacteria and some Gram-positive (G+) bacteria and fungi and succumb to an otherwise harmless infection. The main reason for the high susceptibility to infection is that these mutant flies fail to induce the antimicrobial peptide genes. The cellular responses appear to be normal. Relish is retained in the cytoplasm in an inactive state. We designed a set of expression plasmids to investigate the requirements for activation of Relish in a hemocyte cell line after stimulation with bacterial lipopolysaccharide. Signal-induced phosphorylation of Relish followed by endoproteolytic processing at the caspase-like target motif in the linker region released the inhibitory ankyrin-repeat (ANK) domain from the DNA binding Rel homology domain (RHD). Separation from the ANK domain allowed the RHD to move into the nucleus and initiate transcription of target genes like those that encode the inducible antimicrobial peptides, likely by binding to κB-like sites in the promoter region. By studying the immune response of the Relish mutant flies in combination with mutants for another NF-κB-like protein, Dorsal-related immunity factor (Dif), we found that the Drosophila immune system can distinguish between various microbes and generate a differential response by activating the Toll/Dif and Imd/Relish pathways. The recognition of foreign microorganisms is believed to occur through pattern recognition receptors (PRRs) that have affinity for selective pathogen-associated molecular patterns (PAMPs). We found that the Drosophila PRRs can recognize G- bacteria as a group. Interestingly, the PRRs are specific enough to distinguish between peptidoglycans from G+ bacteria such as Micrococcus luteus and Bacillus megaterium and fungal PAMPs from Beauveria bassiana and Geotrichum candidum. This thesis also investigates the expression of the antimicrobial peptide genes, Diptericin B and Attacin C, and the putative intracellular antimicrobial peptide gene Attacin D, and explores a potential evolutionary link between them.