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The Human Immunodeficiency virus-1 can stay undetected and unaffected by host immune surveillance and antiretroviral therapy. This phenomenon is called proviral latency and the cells harbouring such viruses are part of the latently infected cell reservoir. In this situation, the viral genome integrates into the host's genome upon infection, whereby infected cells exhibit either very low levels or no viral transcription, and hence no viral proteins or egress viruses are produced that can be detected by the immune system. However, viral transcription can be re-activated to produce infectious viruses under certain circumstances. Host-encoded retroviral restriction factors like APOBEC3 (A3) proteins are part of our intrinsic immune defences against retroviral infection, introducing mutations in viral replication intermediates. We hypothesize that low levels of G-to-A transition mutations in the HIV-1 LTR region, introduced by APOBEC3G/F, could lead to a latency-like phenotype. These latent viruses pose major hurdles for HIV-1 cure therapies. Our lab previously created a library of clones possessing mutations in the LTR introduced by A3G/F. Later, mutated LTRs were cloned into 3 types of plasmid backbones: 1) a pEGFP expression vector to study the transcriptional activity of the mutated promoter, 2) into non-replicative pNL4 ∆env ∆vif viral expression vector, and 3) into a replicative pNL4-CXCR4 viral vector to study infection and induction by latency reversal agent (LRA) treatment to better understand the mechanism of latency and transcriptional induction. Viruses produced from these plasmids carrying mutated promoters are referred to as latency-prone viruses or LPVs in this thesis. Characterizing the transcription, infection, and induction to PMA/I of the LPVs would essentially help in evaluating the role of A3 mutations in viral latency and further help in the development of new therapeutics.

In many countries, cancer is one of the biggest threats for citizens’ health, especially among aged people. Genomic mutations play a crucial role in cancer cell development. In previous decades, cancer research has been mainly focused on mutations in coding regions. These mutations can directly change the encoded protein sequences and influence their functions. In recent years, as the function of non-coding regions has been gradually understood, a growing number of studies have focused on the role of non-coding mutations in cancer. Transcription factor (TF) is an important group of gene regulatory factors. These factors only bind to specific sequences called transcription factor binding motifs (TFBMs) in the genome. Mutations in these motifs can disrupt the TF binding and thus influence gene regulation. A framework called funMotifs was made to predict and annotate functional TFBMs in the human genome. And a research has been made to intersect the mutation information from Pan-Cancer Analysis of Whole Genomes (PCAWG) to motifs in funMotifs, aiming to give a general view of influence of cancer-related mutations on functional TF motifs. But the research only focused on the existing motifs that were identified previously from the normal genome, while de novo motifs that could be potentially created by mutations were disregarded. An instance near the TERT promoter has been found, showing that mutations create a de novo ETS binding site and up-regulate the TERT expression.  My study aims to extend the borderline of funMotifs, from existing motifs to de novo motifs created by cancer-related mutations. I extended the original motifs in funMotifs database and merged the overlapping motifs into longer regulatory elements. Then I mutated these elements according to the mutation data from PCAWG. Next I scan through the mutated elements and identify TF motifs. These motifs were then intersected with original motifs in funMotifs database to remove the redundant results. After intersection and filtering, 2,525,771 de novo motifs were retained. These motifs mainly come from C2H2 zinc finger factors, tryptophan cluster factors, STAT domain factors, fork head/winged helix factors, MADS box factors and homeo domain factors. Even though the de novo motifs I found in this study still need further verification and analysis, for example the change of information content in the mutated sites of the motifs, the result I obtained can be a useful data source for further research on regulatory impact from cancer-related mutations. / <p></p><p></p><p></p><p></p><p></p>

The vesicular stomatitis virus glycoprotein G is responsible for low pH mediated membrane fusion induced by the virus. Four linker insertion mutants (H2, H5, HIO, A4) of the G ectodomain were found to disrupt fusion and yet maintained all the requirements for proper folding and cell surface expression (Li et al., 1993). Site specific mutagenesis of residues 123 to 137, surrounding the H2 mutant, either blocked or shifted the pH optima and threshold of fusion to more acidic values with a concomitant reduction in cell-cell fusion efficiency (Zhang and Ghosh, 1994; Fredericksen and Whitt, 1995). The region is highly conserved among vesiculoviruses and was found to insert into lipid membranes by hydrophobic photolabelling (Durrer et al., 1995) suggesting a possible role for this domain as the fusion peptide. Site-directed mutagenesis of residues 190 to 210, surrounding the H5 insertion mutant, did not significantly affect fusion (Fredericksen and Whitt, 1995). Surrounding the H10 and A4 insertion mutants is a conserved region, residues 395 to 424, that does not interact with target membranes (Durrer et al., 1995). To determine the functional importance of this region, site-directed mutagenesis was employed. Substitution of conserved Gly 404, Gly 406, Asp 409, and Asp 411 with Ala, Ala, Asn, and Asn, respt:.ctively, both reduced fusion and caused a shift in the pH of fusion threshold to more acidic values (tested by Y. He as published in Shokralla et al., 1998). In this study, the Hl0/A4 region is further mutagenized and tested for fusion. Cell surface expression was examined by indirect immunofluorescence and lactoperoxidase catalyzed iodination. Rates of transport from the endoplasmic reticulum and oligomerization into trimers were tested by resistance to endoglycosidase H and sucrose density gradient centrifugation, respectively. Low-pH induced conformational changes were assayed by resistance to proteolytic digestion. Residues Gly 395, Gly 404, Gly 409 and Ala 418 were substituted with Glu, Lys, Asp, and Lys, respectively. All mutants, with the exception of A418K, were expressed at levels similar to or above wild-type. Mutants G404K and D409A completely abolished fusion. Mutant G395E reduced cell-cell fusion efficiency by 82% and shifted both the pH threshold and optimum of wild type fusion. Although all mutants were capable of trimer formation, alterations in the structure of mutants G404K, D409 A, and A418K were detected by slower transport rates. All Hl0/A4 mutants were more susceptible to trypsin than wild-tyr,e at the pH of6.5, and mutant G404K was completely susceptible at this pH Reductions in the extent of fusion, along with shifts in the pH optima and thresholds of fusion suggest that the Hl0/A4 region (residues 395 to 418) of vesicular stomatitis virus G protein is important for G mediated fusion. The region may influence low-pH induced conformational changes. Double mutants of the H2 and HI0/A4 regions were also tested for their effects on fusion. The extents of fusion mediated by double mutant G proteins were severely reduced with levels ranging from 28% wild-type fusion to complete fusion deficiency. Only mutant Gl31A G404A was capable of 83% wild-type fusion. Mutants Gl31A G395E, Gl31A G404A, Gl31A D4LIN, Dl37N G404A, and the fusion defective D137N D411N were expressed at levels above wild-type G protein at the cell surface. Mutants Fl25Y D411N and Pl26L D411N, although capable of very low levels of fusion were not detectable at the cell surface by immunoflorescence and were detected at low levels by lactoperoxidase catalyzed iodination of cell surface proteins. These two mutants, along with Gl31A G404A, also showed slower transport rates than wild-type G. All double mutants showed increased sensitivity to trypsin at the pH of 6.5 with mutant Fl25Y D411N showing complete susceptibility. They were also all capable of trimer formation by sucrose density gradient centrifugation. In comparing the fusion profiles of double mutants with those of their component single mutants, it was found that in most cases the pH threshold of fusion by double mutants was greater than the sum of the single mutants and that the pH optimum of fusion corresponded to that of the constituent H2 single mutant. Although, the regions are functionally independent, they may indirectly affect one another through alterations in protein structure. / Thesis / Master of Science (MS)

In acute myeloid leukemia (AML), between 15-20% of all patients harbor a somatic mutation in the isocitrate dehydrogenase 1 or 2 gene (IDH1 and IDH2). Therefore, these mutations are among the common ones in AML. However, the prognostic significance of mutated IDH in AML patients remains controversial. Several research groups reported distinct outcomes within specific patient subsets depending on the biological and clinical context. Additionally, the prognostic impact seems to be depended by the co-mutations, the specific location of the mutation (i.e., regarding the hotspot locations IDH1 R132, IDH2 R140, and IDH2 R172) as well as the applied treatment. Today, allogeneic hematopoietic stem cell transplantation (HSCT) remains the consolidation therapy with the highest chance of sustained remission for most younger and older AML patients. Even though many IDH mutated AML patients are consolidated by HSCT and several trials testing IDH inhibitors in a maintenance setting are active, very little data are available on the influence of IDH mutations at diagnosis and as measurable residual disease (MRD) marker in the HSCT context. The first aim of our study was to study the frequency of IDH mutations and assess their associations with other biological and pretreatment markers. In our cohort of 292 AML patients, who all received an HSCT for consolidation, we identified somatic IDH mutations in 70 (24%) patients. IDH1 mutations were found in 11.4% of the patients, all of which were R132 substitutions. Regarding mutations in the IDH2 gene, we identified 8.9% and 5.1% patients harboring a R140 or a R172 substitution, respectively. Generally, IDH mutated patients did not differ significantly from IDH wild type patients in our set regarding their biological characteristics with the exception that IDH mutated patients had significant higher bone marrow blasts at diagnosis. When we analyzed the mutational landscape of our cohort, we found that IDH mutated patients more frequently also harbored DNMT3A mutations, while RUNX1 mutations and TP53 mutations were found in lower frequencies. For the diagnostic bone marrow variant allele fractions (VAFs) associated with the three IDH mutation types, we observed a mutation specific pattern. While IDH2 R140 mutations clustered around a median VAF of about 50%, the VAFs for IDH1 R132 and IDH2 R172 mutations at diagnosis were significantly lower by median but showed a wider distribution. Next, we aimed to examine the prognostic value of different IDH1 and IDH2 mutations in AML patients that receive a consolidating allogeneic HSCT. Here, we observed no differences in the cumulative incidence of relapse (CIR), event-free survival (EFS), and overall survival (OS) according to the IDH mutational status at diagnosis. This also held up when we analyzed the three mutations, i.e., IDH1 R132, IDH2 R140, and IDH2 R172 mutations separately. Also in multivariable analyses, the diagnostic IDH mutation status did not significantly associate with outcomes after HSCT. However, we also analyzed the prognostic impact in the context of the ELN2017 classification and observed a distinct prognostic impact of the IDH mutations. With respect to outcome following HSCT consolidation in the ELN2017 Favorable-risk group, IDH mutation status did not influence outcomes. In the ELN2017 Intermediate-risk group, IDH mutated patients had an increased relapse rate compared to IDH wild-type patients. Nevertheless, this did not translate into significant shorter EFS or OS. Within the ELN2017 Adverse-risk group, IDH mutated patients had a lower CIR and by trend longer OS and EFS. The final major objective of our study was to analyze the role of IDH mutations as MRD marker in AML patients in CR prior to an allogeneic HSCT. In our cohort, 44 mutated patients had material for IDH mutation status detection on MRD level using digital droplet PCR (ddPCR) at HSCT available. DdPCr is a novel method that allows absolute quantification of gene mutations and/or expression without the necessity of standard curves. We established an assay that allows the quantification of IDH1 and IDH2 mutations with very high sensitivity and specificity. Of the 44 patients, 33 (75%) had detectable IDH MRD. The ddPCR based IDH mutation MRD positive patients were differently distributed (IDH1 R132: 65%; IDH2 R140: 94%; IDH2 R172: 44%). Interestingly, the VAFs at HSCT were much lower in IDH1 R132 (median 0.17%) and IDH2 R172 (median 0.20%) compared to IDH2 R140 (median 11.6%). Looking for association between IDH MRD status at HSCT and outcome, we observed a strong relapse association of IDH1 R132 positivity or IDH2 R172 positivity. Patients that were MRD positive for IDH1 R132 or IDH2 R172 mutations also had a shorter - though not significant - EFS and OS. Thus, clinically, the elimination of persisting IDH mutations – especially of IDH1 R132 and IDH2 R172 – before HSCT could be an important milestone towards a cure for these patients. On the other hand, IDH2 R140 MRD positivity at HSCT did not associate significantly with the CIR, EFS, and OS. Together with the previous mentioned finding of a higher VAF at diagnosis, we speculated that in our cohort, IDH2 R140 mutations behaved more like a clonal hematopoiesis-related aberrations.:Inhalt/Content Bibliographische Beschreibung/Bibliographic Description 1 Referat/Abstract 2 Einleitung/Introduction 3 Acute myeloid leukemia 3 Epidemiology 3 Clinical presentation and pathogenesis 3 Diagnostic workup and classification 3 Risk stratification and treatment 6 2022 European LeukemiaNet genetic risk classification 6 Standard Treatment 7 Measurable Residual Disease 10 Isocitrate Dehydrogenase 1 and 2 mutations 11 Definition 11 Pathology and Epidemiology 12 IDH mutations as prognostic markers 12 IDH inhibitors 13 Publikation/Publication 15 Zusammenfassung/Summary 22 Perspektive/Outlook 26 Literaturverzeichnis/References 27 Anlage/Supplemental Material 36 Referenz der Publikation/Reference of the Publication 55 Erklärung über den wissenschaftlichen Anteil des Promovenden 56 Erklärung über eigenständige Abfassung der Arbeit 58 Lebenslauf/Curriculum Vitae 59 Publikationen/Publications 61 Erst- und Letztautorschaften/First and Last authorship 60 Ko-Autorschaften/Co authorship 61 Reviews 66 Danksagung/Acknowledgments 67

MtDNA mutations in mammalian cells are implicated in cellular ageing and encephalomyopathies, although mechanisms involved are not completely understood. The mitochondrial genetic bottleneck has puzzled biologists for a long time. Approximate models of genetic bottleneck proposed in the literature do not accurately model underlying biology. Recent studies indicate mitochondrial morphology changes during cellular aging in culture. In particular, the rates of mitochondrial fission and fusion are shown to be in tight balance, though this rate decreases with age. Some proteins involved in mitochondrial morphology maintenance are implicated in apoptosis. Hence, mitochondrial genetic and morphologic dynamics are critical to the life and death of cells. By working closely with experimental collaborators and by utilizing data derived from literature, we have developed stochastic simulation models of mitochondrial genetic and morphologic dynamics. Hypotheses from the mitochondrial genetic dynamics model include: (1) the decay of mtDNA heteroplasmy in blood is exponential and not linear as reported in literature. (2) Blood heteroplasmy measurements are a good proxy for the blood stem cell heteroplasmy. (3) By analyzing our simulation results in tandem with published longitudinal clinical data, we propose for the first time, a way to correct for the patient's age in the analysis of heteroplasmy data. (4) We develop a direct model of the genetic bottleneck process during mouse embryogenesis. (5) Partitioning of mtDNA into daughter cells during blastocyst formation and relaxed replication of mtDNA during the exponential growth phase of primordial germ cells leads to the variation in heteroplasmy inherited by offspring from the same mother. (6) We develop a “simulation control” for experimental studies on mtDNA heteroplasmy variation in cell cultures. Hypothesis from the mitochondrial morphologic dynamics model: (7) A cell adjusts the mitochondrial fusion rate to compensate for the fluctuations in the fission rate, but not vice versa. A deterministic model for this control is proposed. Contributions: extensible simulation models of mitochondrial genetic and morphologic dynamics to aide in the powerful analysis of published and new experimental data. Our results have direct relevance to cell biology and clinical diagnosis. The work also illustrates scientific success by tight integration of theory with practice. / Ph. D.

Yes / The use of peptides to nucleate, grow, and stabilize nanoparticles in aqueous media via non-covalent interactions offers new possibilities for creating functional, water-dispersed inorganic/organic hybrid materials, particularly for Au nanoparticles. Numerous previous studies have identified peptide sequences that both possess a strong binding affinity for Au surfaces and are capable of supporting nanoparticle growth in water. However, recent studies have shown that not all such peptide sequences can produce stable dispersions of these nanoparticles. Here, via integrated experiments and molecular modeling, we provide new insights into the many factors that influence Au nanoparticle growth and stabilization in aqueous media. We define colloidal stability by the absence of visible precipitation after at least 24 hours post-synthesis. We use binding affinity measurements, nanoparticle synthesis, characterization and stabilization assays, and molecular modeling, to investigate a set of sequences based on two known peptides with strong affinity for Au. This set of biomolecules is designed to probe specific sequence and context effects using both point mutations and global reorganization of the peptides. Our data confirm, for a broader range of sequences, that Au nanoparticle/peptide binding affinity alone is not predictive of peptide-mediated colloidal stability. By comparing nanoparticle stabilization assay outcomes with molecular simulations, we establish a correlation between the colloidal stability of the Au nanoparticles and the degree of conformational diversity in the surface-adsorbed peptides. Our findings suggest future routes to engineer peptide sequences for bio-based growth and dispersion of functional nanoparticles in aqueous media. / Air Office of Scientific Research, grant number FA9550-12-1-0226.