Zugl.: Köln, Fachhochsch., Diplomarbeit, 2007

Thesis (M.A.)--Bowling Green State University, 2005. / Document formatted into pages; contains vi, 45 p. Includes bibliographical references.

Zugl.: Berlin, Freie Univ., Diss., 2007

Halle, Univ., Philosophische Fak. II, Diss., 2008. / Tag der Verteidigung: 21.07.2008.

Includes bibliographical references and index.

Masterthese Hochschule für Heilpädagogik Zürich, 2009.

Parathyroid hormone-related peptide (PTHrP) is abundantly expressed throughout the gestational tissues and has multiple roles in fetal development. The importance of PTHrP in embryonic growth and survival is emphasised by the retarded growth and peri-natal lethality of the PTHrP knockout mouse. PTHrP is a regulator of cell survival, proliferation and differentiation in a number of tissues and organs. However, its effects in the development and function of the placenta are yet to be fully defined. Therefore, the PTHrP knockout mouse was used to examine the morphological development and function of the placenta in the absence of fetal PTHrP gene expression. Embryos that were wild-type (WT), heterozygous (HZ) and null (NL) for the PTHrP allele were used for comparison. PTHrP expression was undetectable in the trophoblast cells of the NL placenta, with the HZ placenta exhibiting an intermediate phenotype. Placental development did occur in the absence of fetal PTHrP, although morphological abnormalities were apparent in the junctional zone and labyrinth zone at embryonic day (E)18. The NL placenta was frequently interrupted by large spaces and contained highly misshapen canals, which may reflect altered cellular and cell-matrix interactions. The area of the junctional zone on HZ and NL placental sections was reduced at E14 and E16, as was the area of the labyrinth zone of the NL placenta at E14 and E18. In culture, NL trophoblast cells had a lowered capacity for proliferation and survival. Elevated apoptosis was also observed in the HZ and NL placenta in vivo at E16 and E18, as judged by increased staining for the apoptotic marker, cleaved caspase-3. This effect was less pronounced in the HZ placenta. Evidence of increased insulin-like growth factor 2 (Igf2) expression was observed in the HZ and NL placenta at E14 and E16, which may have been a compensatory response to preserve some aspects of placental function in a PTHrP-deficient environment. Despite a reduced area of junctional and labyrinth zones on NL placental sections, no differences in placental weight was observed at any gestational age examined, which may indicate differences exist in the composition of the NL placenta. Fetal weight was lower in NL than WT fetuses at E16 and E18, whereas fetal weight in the HZ group was unaltered at these gestational ages. This suggests that the NL placenta had a more profoundly reduced capacity to support fetal growth. System A amino acid transport was significantly reduced in the NL placenta at E18, perhaps contributing to decreased NL fetal weight. Glycogen content of the NL placenta was reduced compared to WT at E12 and E14, but raised at E18, which may have been a compensatory mechanism to support fetal growth. In conclusion, PTHrP influences the morphological differentiation of the mouse placenta, trophoblast cell survival, nutrient transport and extraembryonic energy storage. PTHrP is an important regulator of placental development and function, which has associative effects on fetal growth and development.

Approaches to the identification of genetic variants associated with complex brain diseases have evolved in recent decades. This evolution was supported by advancements in medical imaging and genotyping technologies that result in rich data production in the field of imaging genetics and radiogenomics. Studies in these fields have taken different designs and directions from genomewide associations to studying the complex interplay between genetics and structural connectivity of a wide range of brain-related diseases. Nevertheless, such combinations of heterogeneous, high dimensional and inter-related data has introduced new challenges which cannot be handled with traditional statistical methods. In this thesis, we proposed analysis pipelines and methodologies to study the causal relationship between neuroimaging features, including tumour characteristics and connectomics, genetics and clinical factors in brain-related diseases. In doing so, we adopted two longitudinal study designs and modelled the association between Alzheimer's disease progression and genetic factors, utilising local and global brain connectivity networks. In addition to that, we performed a multi-stage radiogenomic analysis in glioblastoma using non-parametric statistical methods. To address some limitations in the methods, we adopted the Structural Equation Model and developed a mathematical model to examine the inter-correlation between neuroimaging and multi-omic characteristics of brain-related diseases. Our findings have successfully identified risk genes that were previously reported in the literature of Alzheimer's and glioblastoma diseases, and discovered potential risk variants which associate with disease progression. More specifically, we found some loci in the genes CDH18, ANTXR2 and IGF1, located in Chromosomes 5, 4 and 12, to have effect on the brain connectivity over time in Alzheimer's disease. We also found that the expression of APP, HFE, PLAU and BLMH have significant effects on the structural connectivity of local areas in the brain, these are the left Heschl gyrus, right anterior cingulate gyrus, left fusiform gyrus and left Heschl gyrus, respectively. These potential association patterns could be useful for early disease diagnosis, treatment and neurodegeneration prediction. More importantly, we identified gaps in the imaging genetics methodologies, we proposed a mathematical model accounting for these limitations and evaluated the model which produced promising results. Our proposed flexible model, BiGen, addresses the gaps in the existing tools by combining neuroimaging, genetics, environmental, and phenotype information to a single complex analysis, accounting for the heterogeneity, inter-correlation, and non-linearity of the variables. Moreover, BiGen adopts an important assumption which is hardly met in the literature of imaging genetics, and that is, all the four variables are assumed to be latent constructs, that means they can not be observed directly from the data, and are measured through observed indicators. This is an important assumption in both neuroimaging, behavioural and genetic studies, and it is one of the reasons why BiGen is flexible and can easily be extended to include more indicators and latent constructs in the context of brain-related diseases.

Identification of HIV-1 Envelope (Env) fitness determinants could provide functionally constrained, accessible regions that could be included in subunit vaccines to induce broadly neutralising antibodies (bnAb). We hypothesised that Env fitness determinants are common to circulating variants but that the plasticity of Env structure limits identification. Rapid evolution; however, could select for sequence changes within the determinants coincident with alterations in function, making identification easier. Dual infection with two phylogenetically distinct HIV-1 variants under the same selective pressures might result in rapid functional evolution, facilitating identification of Env fitness determinants. It has been shown that the Env plays a significant role in viral adaptation to the host environment, which then increases disease progression. Therefore, this study used dual infections as a model system to characterise Env function, its role in in vivo viral outgrowth of variants and disease progression and to identify fitness determinants for future vaccine design. Single-genome amplification (SGA)-derived env sequences of four dual infected individuals sampled at enrolment (0 months), 3, 6, and 12 months post infection (mpi) were analysed using Highlighter plots, RIP, DNA pairwise distance and Neighbourjoining trees to determine the in vivo evolution of infecting viral populations and their relative frequency over time within each participant. Representative amplicons were cloned at each time point and compared using a pseudovirus (PSV) entry efficiency assay. 2 characterised by Affinofile system, T-20 IC50 and Western blotting to identify whether tropism, Env expression/cleavage, incorporation into viral particles and fusogenicity were most likely responsible for the variation in Env entry efficiency. All variants were R5- and T-tropic and only Env fusion capacity correlated significantly with Env entry efficiency data (p = 0.02, r = 0.59), suggesting that variants infecting dual infected participants evolved towards higher fusion capacity. Changes in Env fusogenicity indicated that gp41 might be a fitness determinant of PSV entry efficiency and analysis of SGA sequences indicated that recombination within gp41 was common to 3/4 participants. Env chimeras were generated where gp41 was swapped between clones that either had the same (CAP84) or different (CAP267) PSV entry efficiency. For both participants, and (CAP137) gp41 was identified as a potential determinant of Env fitness. Moreover, two potential N-glycan sites (PNG) at position N332 and N339, previously reported to be involved in neutralising antibody escape, were also identified. While N332 enhanced Env entry efficiency in one participant, N339 attenuated Env entry efficiency in another, potentially due to the escape mutation carrying a fitness cost. However, neither PNG seemed to affect Env expression/cleavage, incorporation into viral particles and fusogenicity. As Env phenotypic characterisation focussed on PSV assays, we wanted to determine whether viral replication was also similarly affected. Infectious molecular clones (IMCs) were generated from two participants using a recombination yeast assay and replication capacity (RC) in peripheral blood mononuclear cells (PBMCs) was assessed using parallel replication. A significant correlation between RC of viruses in PBMCs and Env entry efficiency in TZM-bl and fusion capacity (p = 0.03, r = 0.7; p = 0.04, r = 0.7, respectively) was determined. IMC RC was also associated with in vivo outgrowth of viral populations at 12 mpi although this relationship did not always coincide with the frequency of individual variants. Changes in the RC of the Env chimeras and mutants was not associated with phenotypic changes, suggesting that Env entry efficiency determinants did not play the same role in IMC RC as it did in PSV entry. Lastly, there was a significant negative association (p = 0.046, r = -0.59) between Env entry efficiency and CD4+ T decline, a marker of disease progression, supporting the previous finding that Env entry efficiency could be the driving agent of disease progression. This was also corroborated by the trend in association between RC of IMCs and faster CD4+ T decline. 3 Our findings suggest that despite different host pressures, viral competition in most dual infected individuals selected for rapid recombination within gp41 that enhanced fusion capacity. Enhanced gp41 fusogenicity of the dominant viral population at 12 mpi increased PSV entry efficiency and replicative fitness enabling viral outgrowth. Therefore, vaccines that target gp41 might prevent HIV infection or at least attenuate viral fitness and slow disease progression. On the other hand, we showed that targeting the PNG at position N339 of gp120 might influence viral fitness and increase viral load and/or decrease CD4 T cell count. This is in keeping with the association between CD4 T cell decline and PSV entry efficiency and IMC RC, suggesting that Env fitness plays a role in HIV pathogenicity.

Background: Despite the development of new drugs and success of social programs, tuberculosis remains a leading cause of mortality. This burden falls disproportionately on developing countries where the high burden of HIV has a potentiating e↵ect, but may soon return to areas where it was previously brought under control as resistant strains continue to emerge. In the Western Cape, two closely related strains of the Beijing family have been isolated that provide an opportunity to study virulence in a system with relatively little noise. The aim of this project was to identify the cause of the altered virulence displayed between the two strains, and describe how the di↵erences between the two genomes contributed to the phenotypic di↵erences. Results: GenGraph allows for the creation of graph genomes, and facilitated the creation of a pan-transcriptome that allowed for the mapping of gene annotations between isolates. This allowed for the mapping of reads to a more suitable Beijing family reference while interpreting the results with annotations from the H37Rv reference. We generated expression and target profiles for the known sRNA, and identified a large number of novel sRNA. Transcriptomic data from 4 di↵erent growth conditions was integrated with this sRNA data as well as variant data using the Cell pipeline. From this data we identified multiple sets of genes linked to copper sensing in MTB, including the di↵erentially expressed MoCo operon. Increasing evidence that macrophages use copper to poison bacteria trapped in their phagosomes provides the link to virulence and pathogenicity. Conclusions: Through the integration of data from multiple data types we were able to elucidate the most probable cause of the altered virulence found between the two isolates in this study. We developed reusable tools and pipelines, and noted a large number of undescribed sRNA expressed in these isolates. The identification of the copper response as a chief contributor to the phenotype increases both our understanding of the isolates, and the role of the element in infection. These results will be key in guiding further investigation of the variant linked genes to identify those linked to copper homeostasis or response.