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Trypanosomes are unicellular protozoan parasites responsible for several human diseases that affect millions of people and cause thousands of casualties every year. They also represent a primitive eukaryotic model system harboring unique processes and basic regulatory mechanisms such as RNA-editing, polycistronic transcription and trans-splicing, first described in these organisms. Unlike most eukaryotes where levels of gene expression are controlled at initiation of transcription, trypanosomes use post-transcriptional events as the main regulators. This thesis explores the epigenetic mechanisms involved in gene expression control in trypanosomes, providing the first evidences for a functional “histone-code” as well as the existence and location of DNA methylation in Trypanosoma cruzi. Chromatin immunoprecipitation (ChIP) was used for the profiling of acetylated and methylated histones in T. cruzi, showing that the modified histones were exclusively localized at bidirectional transcription start sites. In addition, promoters from highly transcribed genes were found depleted of nucleosomes, while DNA regions expected to be silent were not enriched in the investigated modified histones. Furthermore, we showed that the histone patterns were developmentally regulated. The first in depth characterization of the DNA methylation patterns in T. cruzi was presented in this work. We detected m5C in regions of transcriptional initiation and termination, retrotransposons, pseudogenes and the kinetoplast minicircle. We also showed that the amount of methylation changes during development, with an increase in non-replicative forms. We also characterized the DNA-interacting properties of a T. cruzi polypyrimidine-tract binding protein (TcPTB), and its potential role as a transcription factor. TcPTB was found to interact with single-stranded DNA present in promoters bound by its mammalian homologue as well as to the region of transcriptional initiation in Leishmania major. We also demonstrated that T. cruzi polypyrimidine stretches were able to confer ssDNA conformations. Overall, these results provide new insights into the biology of ancient pathogenic parasites, which might be exploited for drug development.

Escherichia coli strains typical of UPEC (uropathogenic E. coli) and NBM (newborn meningitis) isolates carry chromosomally located PAIs (pathogenicity islands) that are absent in non-pathogenic E. coli strains. The PAIs include genes for virulence factors such as toxins and genes coding for specific adhesins and pili/fimbriae formation. Commonly, the gene clusters for such fimbriae in E. coli consist of a set of genes for biogenesis of the actual fimbriae organelles: a chaperone, an usher, the fimbrial subunits, and an adhesin, as well as some regulatory genes. Genetic studies of the fimbrial gene clusters in PAIs containing the pap genes, the prs genes, or the sfa genes led to the discovery of nearby open reading frames coding for putative cytoplasmic 17 kDa proteins — the X genes. Molecular genetic studies of the sfaXII locus in the clinical NMEC isolate IHE3034 have been performed. The results suggested that expression of the sfaXII gene had regulatory functions affecting both type 1 fimbriae expression and the flagella-mediated motility. Type 1 fimbriae expression was found to be affected at the level of fim operon transcription and a major reason was SfaXII-mediated modulation of expression from the fimB and fimE recombinase genes. Quantification of SfaII-fimbriated bacteria in a comparison between wild type and SfaXII mutant strains gave no indication that the sfaXII gene product also would be affecting expression and/or biogenesis of SfaII fimbriae. Biomechanical properties of the SfaII fimbriae produced by wild type and the sfaXII mutant IHE3034 were studied using force measuring optical tweezers (FMOT) and compared to other PAI-encoded fimbriae as well as to the type 1 fimbriae encoded on the core chromosome. The FMOT methodology assesses unfolding and refolding properties and we found that S fimbriae had weaker layer-to-layer interactions than both P and type 1 fimbriae, however the unfolding kinetics was slightly faster. The expression profile and regulation of the sfaXII gene were determined by use of reporter gene fusions and it was found that expression was affected by environmental cues such as pH, osmolarity and temperature. It was also discovered that the nucleoid structuring protein H-NS and the sigma factor RpoS had strong direct or indirect repressive effects on sfaXII gene expression. Further genomic analysis of the PAI fimbrial operons revealed that in some cases an additional ORF was found between the X genes and the fimbrial adhesion genes. Examination of the sfaII operon in IHE3034 indicated that this new gene, denoted sfaYII, coded for a protein that had the EAL domain motif and thereby could be considered a putative phosphodiesterase involved in controlling the level of cyclic-di-GMP in the bacterial cells.

This dissertation presents a generalized quantum chemical approach for electron transport in molecular electronic devices based on Green's function scattering theory. It allows to describe both elastic and inelastic electron transport processes at first principles levels of theory, and to treat devices with metal electrodes either chemically or physically bonded to the molecules on equal footing. Special attention has been paid to understand the molecular length dependence of current-voltage characteristics of molecular junctions. Effects of external electric fields have been taken into account non-perturbatively, allowing to treat electrochemical gate-controlled single molecular field effect transistors for the first time. Inelastic electron tunneling spectroscopy of molecular junctions has been simulated by including electron-vibration couplings. The calculated spectra are often in excellent agreement with experiment, revealing detailed structure information about the molecule and the bonding between molecule and metal electrodes that are not accessible in the experiment. An effective central insertion scheme (CIS) has been introduced to study electronic structures of nanomaterials at first principles levels. It takes advantage of the partial periodicity of a system and uses the fact that long range interaction in a big system dies out quickly. CIS method can save significant computational time without loss of accuracy and has been successfully applied to calculate electronic structures of one- , two- , and three-dimensional nanomaterials, such as sub-116 nm long conjugated polymers, sub-200nm long single-walled carbon nanotubes, sub-60 base pairs DNA segments, nanodiamondoids of sub-7.3nm in diameter and Si-nanoparticles of sub-6.5nm in diameter at the hybrid density functional theory level. The largest system under investigation consists of 100,000 electrons. The formation of energy bands and quantum confinement effects in these nanostructures have been revealed. Electron transport properties of polymers, SWCNTs and DNA have also been calculated. / QC 20100729. Ändrat felaktig titel "Theoretical Chemistry, Molecular and Nano-electronics" 20100729.

The Notch and TGF13 signaling pathways have been shown to play important roles in regulating endothelial-to-mesenchymal transition (EndMT) during cardiac morphogenesis. EndMT is the process by which endocardial cells of the atrioventricular canal and the outflow tract repress endothelial cell phenotype and upregulate mesenchymal cell phenotype. EndMT is initiated by inductive signals emanating from the overlying myocardium and inter-endothelial signals and generate the cells that form the heart valves and atrioventricular septum. The Notch and TGFf3 pathway are thought to act in parallel to modulate endothelial phenotype and promote EndMT. Vascular endothelial (VE) cadherin is a key regulator of cardiac endothelial cell phenotype and must be downregulated during EndMT. Accordingly, VE-cadherin expression remains stabilized in the atrioventricular canal and outflow tract of Notchl-deficient mouse embryos, while activation of the Notch or TGFP pathways results in decreased VE-cadherin expression in endothelial cells. However, the downstream target gene(s) that are involved in regulating endothelial cell phenotype and VE-cadherin expression remain largely unknown. In this thesis the transcriptional repressor Slug is demonstrated to be expressed by the mesenchymal cells and a subset of endocardial cells of the atrioventricular canal and outflowtract during cardiac morphogenesis. Slug is demonstrated to be required for cardiac development through its role in regulating EndMT in the cardiac cushion. Data presented in Chapter 6 further suggests that Slug-deficiency in the mouse is compensated for by a increase in Snail expression after embryonic day (E) 9.5, which restores EndMT in the cardiac cushions. Additionally, the Notch pathway, via CSL, directly binds and regulates expression of the Slug promoter, while a close Slug family member, Snail is regulated by the TGFB pathway in endothelial cells. While Notch does not directly regulate Snail expression, Notch and TGFB act synergistically to regulate Snail expression in endothelial cells. It is further demonstrated that Slug is required for Notch mediated EndMT, binds to and represses the VE-cadherin promoter, and induces a motile phenotype. Collectively the data demonstrate that Notch signaling directly regulates Slug, but not Snail, expression and that the combined expression of Slug and Snail are required for cardiac cushion morphogenesis.

Sliding clamps and their cognate clamp loaders facilitate DNA synthesis, DNA repair, and sister chromatid cohesion in eukaryotes. ELG1 (enhanced level of genome instability) encodes a member of the fourth clamp-loader-like complex identified to date, and is important in the maintenance of genome integrity. Like all clamp loaders, Elg1 is a replication factor C (RFC) homologue. I examined the roles of the unique and conserved regions of S.cerevisiae Elg1 in resistance to exogenous DNA damage and suppression of spontaneous DNA damage. The conserved RFC region of Elg1 mediates association with chromatin function. The unique C- terminus of Elg1 mediates oligomerization with Rfc2-5, a core complex present in all clamp loaders, and is essential for Elg1 function. Finally, the N-terminus of Elg1 promotes its nuclear localization and contributes to the maintenance of genome stability. The Elg1-RFC complex most likely functions in collaboration with the sliding clamp PCNA. Combining mutations in ELG1 and PCNA results in endogenous DNA damage, which activates a noncanonical DNA damage response that results in upregulation of dNTP production. Increased dNTP pools allow significant DNA synthesis to occur at hydroxyurea (HU) concentrations that prevent replication in wild type cells. However, consistent with the recognized correlation between dNTP levels and spontaneous mutation, the double mutant exhibits a significant increase in mutation frequency. These phenotypes are also detectable in the single mutants although to a lesser extent. Together, these findings suggest that spontaneous mutagenesis stimulated by endogenous DNA damage may be a general feature of the DNA damage response.

Myeloproliferative neoplasms (MPNs) are a group of disorders characterized by an excess production of a specific, fully functional blood cell type. Many cases involve deregulation of a protein tyrosine kinase. JAK2 is one such kinase, involved in a subset of MPNs. JAK2-selective inhibitors are currently being evaluated in clinical trials. In order to identify inhibitor-resistant JAK2 mutations before they appear in the clinic, we utilized TEL-JAK2 to conduct an in vitro random mutagenesis screen for JAK2 alleles resistant to JAK Inhibitor-I. Isolated mutations were evaluated for their ability to sustain cellular growth, stimulate downstream signalling pathways, and phosphorylate a novel JAK2 substrate in the presence of inhibitor. When testing the panel of mutations in the context of the Jak2 V617F allele, we observed that a subset of mutations conferred resistance to inhibitor. These results demonstrate that small-molecule inhibitors select for JAK2 inhibitor-resistant alleles. Chronic myeloid leukemia is an MPN characterized by the presence of the BCR-ABL fusion gene. We determined that a specific cohort bearing deletions near the ABL gene, which is associated with poor prognosis, do not suffer from genomic instability. We also examined the role of a putative tumour suppressor gene EXOSC2 as an explanation for the reduced survival time, and suggest it may have a role in disease progression.

Sliding clamps and their cognate clamp loaders facilitate DNA synthesis, DNA repair, and sister chromatid cohesion in eukaryotes. ELG1 (enhanced level of genome instability) encodes a member of the fourth clamp-loader-like complex identified to date, and is important in the maintenance of genome integrity. Like all clamp loaders, Elg1 is a replication factor C (RFC) homologue. I examined the roles of the unique and conserved regions of S.cerevisiae Elg1 in resistance to exogenous DNA damage and suppression of spontaneous DNA damage. The conserved RFC region of Elg1 mediates association with chromatin function. The unique C- terminus of Elg1 mediates oligomerization with Rfc2-5, a core complex present in all clamp loaders, and is essential for Elg1 function. Finally, the N-terminus of Elg1 promotes its nuclear localization and contributes to the maintenance of genome stability. The Elg1-RFC complex most likely functions in collaboration with the sliding clamp PCNA. Combining mutations in ELG1 and PCNA results in endogenous DNA damage, which activates a noncanonical DNA damage response that results in upregulation of dNTP production. Increased dNTP pools allow significant DNA synthesis to occur at hydroxyurea (HU) concentrations that prevent replication in wild type cells. However, consistent with the recognized correlation between dNTP levels and spontaneous mutation, the double mutant exhibits a significant increase in mutation frequency. These phenotypes are also detectable in the single mutants although to a lesser extent. Together, these findings suggest that spontaneous mutagenesis stimulated by endogenous DNA damage may be a general feature of the DNA damage response.

The Transforming Growth Factor beta (TGFb) superfamily of ligands are highly versatile, functioning throughout development and in adult organisms as diverse as worms and humans to regulate a myriad of biological activities. TGFb family members signal through their cognate serine/threonine kinase receptors to mediate the phosphorylation and activation of receptor-regulated Smads (R-Smads), that then complex with the common Smad (co-Smad/Smad4) to transduce TGFb signals from the membrane into the nucleus. This thesis recounts the first identification of a mammalian Smad-interacting transcription factor, Foxh1. Investigation of the Smad/Foxh1 DNA-binding complex, which mediates TGFb-dependent regulation of transcription from a Gsc enhancer, determined that both Smad and Foxh1 binding sites are required. These studies also defined the first known biological difference between the highly related R-Smads, Smad2 and Smad3. Specifically, it was shown that while both can similarly participate in Smad/Foxh1 DNA-binding complexes, Smad2 activates and Smad3 represses Foxh1-mediated TGFb-dependent transcription. A detailed analysis of the Gsc enhancer element subsequently defined the sequence req irements for a functional Smad/Foxh1 enhancer (SFE). This information was utilized to direct in silico, genome wide searches for genes harbouring evolutionarily conserved SFEs, which successfully expanded the repertoire of Smad/Foxh1 targets. Analysis of these targets revealed novel roles for Smad/Foxh1 signalling in forebrain development and retinoic acid production. Finally, the importance of Foxh1 to heart development was examined. The interaction between Foxh1 and the heart specific factor Nkx2-5 was characterized with respect to TGFb-dependent regulation of Mef2c expression via a compound Foxh/Nkx2-5 enhancer (FNE). Genome-wide searches for similar FNEs identified many potential Foxh1/Nkx2-5 targets, further analysis of which will provide greater insights into how Foxh1 functions in heart development. In summary, the work presented herein expands our understanding of the role of TGFb in development through the identification and characterization of Foxh1 and its genomic targets downstream of TGFb signalling.

Genetic and Molecular / Neurospora Duplications