Species of the genus Rickettsia are obligate intracellular parasites of the a-proteobacterial subdivision. It has been suggested that obligate intracellular bacteria have evolved from free-living bacteria with much larger genome sizes. Transitions to intracellular growth habitats are normally associated with radical genomic alterations, particularly genome rearrangements and gene losses. This thesis presents a comparative study of evolutionary processes such as gene rearrangements, deletions and duplications in a variety of Rickettsia species. The results show that early intrachromosomal recombination events mediated by duplicated genes and short repeats have resulted in deletions as well as rearrangements. For example, an exceptional organization of the elongation factor genes was found in all species examined, suggesting that this rearrangement event occurred at the early stage of the evolution of Rickettsia. Likewise, it was found that a repetitive element, the so-called Rickettsia Palindromic Element (RPE) flourished prior to species divergence in Rickettsia. Finally, a phylogenetic analysis shows that the duplication events that gave rise to the five genes encoding ATP/ADP transporters occurred long before the divergence of the two major groups of Rickettsia. Taken together, this suggests that Rickettsia have been intracellular parasites for an extensive period of time. A detailed analysis of the patterns of nucleotide changes in genes and intergenic regions among the different species provides evidence for a gradual accumulation of short deletions. This suggests that different distributions of genes and repeated sequences in modern Rickettsia species reflect species-specific differences in rates of deterioration rather than variation in rates of intra-genomic sequence proliferation.

Sequence comparisons of orthologous microsatellite loci in cattle and sheep revealed that the substitution rate in microsatellite flanking sequences does not differ from the rate in presumably neutrally evolving intron sequences. This suggests that microsatellites are generally located in regions that are not subjected to selection. Interestingly, a propensity for substitutions to occur in the border region between flanking and repeat sequence was found. Pedigree analysis of large numbers of barn swallows revealed extremely high mutation frequencies for the tetranucleotide HrU6 and pentanucleotide HrU10 repeat loci. A detailed analysis showed that both the rate and the pattern of mutation differed significantly between the two loci. Further analysis of HrU6 and HrU10 mutations, as well as mutation data for another hypermutable locus (HrU9) in barn swallows, revealed that mutations were more likely to arise in some families than others. This was partly, but probably not only, due to an effect of allele length on mutation rate. The mutation rate was found to vary between colonies of breeding birds, but, overall, not between two different populations. Single molecule genotyping of DNA prepared from human sperm cells was used to detect mutations at the tetranucleotide repeat D21S1245. A tenfold difference in mutation rate between alleles was found. Three phylogenetically distinct allele lineages could be defined, which differed significantly in mutation rate. Unexpectedly, the mutation rate was not found to increase with male age. Microsatellites are commonly applied in a wide range of genetic contexts including linkage mapping, forensic science and population genetics. Obtaining a detailed picture of the evolution of these tandem repeats is important in order to fully understand how to interpret microsatellite data. In addition, studies of the mechanisms underlying microsatellite mutation will provide insights in the processes that shape the eukaryotic genome. This thesis demonstrates that microsatellite evolution is a highly heterogeneous process that is dependent on more factors than was previously thought. As the rate and pattern may vary between loci, caution must therefore be taken when building models to handle microsatellite data.

Genetic information is encoded by the linear sequence of the DNA double helix, while epigenetic information is overlayed as the packaging of DNA and associated proteins into the chromatin structure. Variations in chromatin structure play a vital role in establishing and maintaining patterns of gene expression during differentiation and development of higher eukaryotes, and disruption of this epigenetic gene regulation can lead to cancer. Mammals display an epigenetic phenomenon known as genomic imprinting, which provides an ideal model system for the study of epigenetics. Genes subject to genomic imprinting are differentially expressed within a single cell depending on the parental origin of the chromosome. Imprinting of the maternally expressed H19 gene and the adjacent paternally expressed Igf2 gene is regulated by the chromatin insulator protein CTCF. The studies presented in this thesis aim to investigate the functional mechanisms of CTCF-dependent gene regulation at the H19/Igf2 locus and at numerous other target sites throughout the genome. 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. We have developed novel microarray based methods to identify and characterize numerous new CTCF target sites throughout the mouse genome. We have shown that CTCF acts as part of the RNA polymerase II complex. We have identified the post-translational modification by addition of ADP-ribose polymers to CTCF, and demonstrated that this modification regulates its insulating ability. The results of these studies of CTCF-dependent epigenetic gene regulation are discussed in light of the evolution of genomic imprinting and chromatin insulators, and a novel role for poly ADP-ribosylation of CTCF in the progression of cancer is proposed.

Thesis (Ph. D.)--Rutgers University, 2009. / "Graduate Program in Cell and Developmental Biology." Includes bibliographical references (p. 85-120).

 Background and Objectives: ‘Neurodevelopmental disorders’ is often synonymously used with childhood developmental disorders such as autism spectrum disorder (ASD), however, increasingly new lines of evidence from genetics and epidemiology suggests having schizophrenia and bipolar disorder to be included as well. For example, there is a strong tendency for schizophrenia and bipolar disorder to occur in people with ASD and shared aetiological factors such as prenatal infection and maternal vitamin D deficiency during pregnancy have all been linked with increased risks in all three conditions. To investigate into this, I have turned to brain imaging, a technique which has opened up a new horizon for neurobiologists. Typically, neuroimaging studies focus on one disorder, matching patients with healthy volunteers and compare their brain structures volumetric differences. On the other hand, such studies are limited by various factors including small ample size, low power, no psychiatric control group, and sample or design heterogeneity. Methods: To summarize all the data into a more meaningful biological representation, Anatomical Likelihood Estimation (ALE), a cutting edge meta-analytic approach was applied. The rationale behind ALE is that it identifies brain differences most consistently reported across studies, while filtering away differences that are least documented. In this thesis, a novel application of ALE known as “dual disorder ALE” is introduced, which serves to estimate the extent of brain regional differences implicated in either disorder – in other words, a method to quantify which areas of the brain are more likely to be affected by ASD, schizophrenia or bipolar disorder. Findings: The analysis is separated into two parts. First, dual disorder ALE technique was applied to investigate the relationship between ASD and first-episode schizophrenia. Data from 25 MRI studies was extracted comprising 660 participants (308 ASD, 352 schizophrenia) and 801 healthy controls. In ASD and FE schizophrenia, there were similar brain differences near the limbic-striato-thalamic circuitry, and distinctive brain differences including amygdala, caudate, frontal and medial gyrus for schizophrenia and putamen for ASD. In the second part comparing bipolar disorder and schizophrenia, data from 651 schizophrenic patients, 540 bipolar patients, and 1438 healthy controls was used, and matched one-to-one by pairing up bipolar disorder studies with corresponding schizophrenia studies to minimize confounders. The ALE result indicated that there are substantial overlaps across the two disorders, with schizophrenia having more extensive brain differences than bipolar disorder. Conclusions: Both parts of the analysis suggest that there are similar aetiological pressures affecting neurodevelopmental disorders including ASD, schizophrenia and bipolar disorder. / published_or_final_version / Psychiatry / Master / Master of Philosophy

Strict control of gene expression is essential during development and in response to physiological stimuli. This thesis describes the functional characterisation of the gene regulatory mechanisms controlling the expression of the potent human growth factor Platelet Derived Growth Factor B gene, in a cell type specific context and in response to low oxygen tension. In addition, analysis of hypoxia in neuroblastoma indicates a role during tumour differentiation. Initally, a promoter-specific enhancer system controlling the expression of PDGF-B was characterised in placentally derived choriocarcinoma cells. The specificity of this enhancer promoter interaction was shown to be dependent on specific sequence elements identified in both the promoter and enhancer regions. It was then shown that the activity of the PDGF-B promoter is controlled via modulation of histone acetylation status in a cell type specific manner and furthermore, that one role of its enhancer could be to regulate transcription via alterations in acetylation status at the promoter. PDGF-B expression was then shown to be controlled by hypoxia in a biphasic manner in bladder carcinoma cells. An initial induction was followed by repression of transcription following chronic hypoxia. The biphasic response was shown to be dependent on glucose levels and uniquely amongst hypoxia regulated genes studied so far, PDGF-B expression was shown to be repressed by low glucose. Finally, detailed in vivo and in vitro analysis revealed that the major form of differentiation in childhood neuroblastoma is towards chromaffin-like rather than ganglionic lineages. This type of differentiation did not correlate with disease progression but was suggested to be dependent on tumour hypoxia, since chromaffin differentiation markers co-localised with markers of tumour hypoxia in both clinical samples and xenogenic tumours. In conclusion, the work presented in this thesis has identified several novel, highly specific gene regulatory mechanisms that are involved in development, the response to physiological stimuli and in disease progression.

The coordinated regulation of gene expression must be tightly controlled for normal development to occur. In mammals, this issue is further complicated by the requirement of both the maternal and paternal genomes for normal development, reflecting the fact that a subset of genes are monoallelically expressed depending on parental inheritance, a phenomenon known as genomic imprinting. The imprinted H19 and Igf2 genes are often considered as paradigms of genomic imprinting, since their monoallelic expression patterns are coordinated via a short stretch of sequence upstream of H19, known as the imprinting control region (ICR). This region is differentially methylated, with specific CpG methylation on the paternal allele. It is shown here that the ICR harbours several maternal-specific hypersensitive sites, located in linker regions between positioned nucleosomes. Furthermore, this region functions as an orientation-dependent insulator, that binds the chromatin insulator factor CTCF. The hypothesis that the methylation status of the ICR dictates the activity of the Igf2 gene 90 kb further upstream was confirmed by the demonstration that the insulator function is lost when the ICR is CpG methylated. The ICR has previously been shown to act as a silencer when positioned in a promotor-proximal position. The cause of this silencing was shown to be distance-dependent, suggesting that the silencing features of the ICR depend on a chromatin conformation that renders adjacent sequences inaccessible to the RNA polymerase. These data issue a cautionary note with respect to the interpretation of silencer functions. In several forms of cancer, the normally silent maternal IGF2 gene is expressed, possibly as a result of loss of insulator function at the ICR. The utilisation of CTCF target-sites was analysed in different tumours, and was shown to be highly variable. Methylation analysis showed that potential loss of insulator function and gain of methylation at the maternal ICR did not always correlate with biallelic expression of IGF2. Further investigations uncovered a novel mechanism, in which the activation of the IGF2 promoter was independent of insulator function in some cancers. This thesis shows that the regulation of the imprinted state of Igf2 depends on the formation of an epigenetically regulated chromatin insulator, and that the loss of IGF2 imprinting in human cancer can be attributed to several mechanisms, including a novel mechanism that neutralises chromatin insulator function.

Thesis (Ph.D.) -- McMaster University, 1998. / Includes bibliographical references (leaves 112-124). Also available via World Wide Web.

Thesis PlanB (M.S.)--University of Wisconsin--Stout, 2008. / Includes bibliographical references.

Thesis (Ph. D.)--Rutgers University, 2008. / "Graduate Program in Cell and Developmental Biology." Includes bibliographical references (p. 96-110).