Dissecting the molecular mechanism of developmental macular dystrophies

North Carolina macular dystrophy (NCMD) and Progressive bifocal chorioretinal atrophy (PBCRA) are a rare set of dominantly inherited disorders that affect central vision from birth. Two linked loci had been previously identified at 5p21 and 6q16. Whole-genome sequencing (WGS) analysis of our cohort of 92 affected individuals has identified 3 novel causative structural variants (SVs) on 5p21 in 13 NCMD families, and 2 novel noncoding single nucleotide variants on 6q16 upstream of the promoter of PRDM13 in three PBCRA families. Combined, the rearrangements in 5p21 have a shared duplicated region of 39kb, located in a gene desert downstream of IRX1 and upstream of ADAMTS16. DNAse-seq data publicly available for human fetal retina identified active open chromatin sites at both loci at restricted times during retinal development. To dissect the molecular mechanism of the identified variants, skin-derived fibroblasts lines from selected patients were established; CRISPR-CAS9 technology was used to recreate the patient variants in a mouse model; and candidate genes expression profile was assessed in three stages of human fetal retina tissue. Additionally, chromosome conformation capture technology was performed in patient fibroblasts and in mouse developing tissue to characterise the genomic landscape of the locus and assess the effect of structural variants. Genome-wide analysis of chromosome occupancy was also performed for the architectural protein CTCF and for histone modifications relevant for identification of cis-acting elements. Expression studies showed altered expression of IRX genes and ADAMTS16 in mutant lines. Together with evidence from human retinal tissue immunohistochemistry and in situ expression data, these genes are suggested to be miss-expressed and/or ectopically expressed during macular development, with involvement of PRDM13, FGF8 and FGF10, which were also found miss-expressed in mutant lines . This work provides novel insight into the gene regulation landscape involved in human macular development and prognostic information for affected families

Comparative analysis of plant genomes through data integration

When we started our research in 2008, several online resources for genomics existed, each with a different focus. TAIR (The Arabidopsis Information Resource) has a focus on the plant model species Arabidopsis thaliana, with (at that time) little or no support for evolutionary or comparative genomics. Ensemble provided some basic tools and functions as a data warehouse, but it would only start incorporating plant genomes in 2010. There was no online resource at that time however, that provided the necessary data content and tools for plant comparative and evolutionary genomics that we required. As such, the plant community was missing an essential component to get their research at the same level as the biomedicine oriented research communities. We started to work on PLAZA in order to provide such a data resource that could be accessed by the plant community, and which also contained the necessary data content to help our research group’s focus on evolutionary genomics. The platform for comparative and evolutionary genomics, which we named PLAZA, was developed from scratch (i.e. not based on an existing database scheme, such as Ensemble). Gathering the data for all species, parsing this data into a common format and then uploading it into the database was the next step. We developed a processing pipeline, based on sequence similarity measurements, to group genes into gene families and sub families. Functional annotation was gathered through both the original data providers and through InterPro scans, combined with Interpro2GO. This primary data information was then ready to be used in every subsequent analysis. Building such a database was good enough for research within our bioinformatics group, but the target goal was to provide a comprehensive resource for all plant biologists with an interest in comparative and evolutionary genomics. Designing and creating a user-friendly, visually appealing web interface, connected to our database, was the next step. While the most detailed information is commonly presented in data tables, aesthetically pleasing graphics, images and charts are often used to visualize trends, general statistics and also used in specific tools. Design and development of these tools and visualizations is thus one of the core elements within my PhD. The PLAZA platform was designed as a gene-centric data resource, which is easily navigated when a biologist wants to study a relative small number of genes. However, using the default PLAZA website to retrieve information for dozens of genes quickly becomes very tedious. Therefore a ’gene set’-centric extra layer was developed where user-defined gene sets could be quickly analyzed. This extra layer, called the PLAZA workbench, functions on top of the normal PLAZA website, implicating that only gene sets from species present within the PLAZA database can be directly analyzed. The PLAZA resource for comparative and evolutionary genomics was a major success, but it still had several issues. We tried to solve at least two of these problems at the same time by creating a new platform. The first issue was the building procedure of PLAZA: adding a single species, or updating the structural annotation of an existing one, requires the total re-computation of the database content. The second issue was the restrictiveness of the PLAZA workbench: through a mapping procedure gene sets could be entered for species not present in the PLAZA database, but for species without a phylogenetic close relative this approach did not always yield satisfying results. Furthermore, the research in question might just focus on the difference between a species present in PLAZA and a close relative not present in PLAZA (e.g. to study adaptation to a different ecological niche). In such a case, the mapping procedure is in itself useless. With the advent of NGS transcriptome data sets for a growing number of species, it was clear that a next challenge had presented itself. We designed and developed a new platform, named TRAPID, which could automatically process entire transcriptome data sets, using a reference database. The target goal was to have the processing done quickly with the results containing both gene family oriented data (such as multiple sequence alignments and phylogenetic trees) and functional characterization of the transcripts. Major efforts went into designing the processing pipeline so it could be reliable, fast and accurate

Spatial and Epigenetic Regulation of T-Cell Receptor Beta Gene Assembly

The adaptive immune system endows mammals with a sophisticated mechanism to recognize foreign proteins via surface antigen receptors that are expressed on the surface of all lymphocytes. This defense network is generated by V(D)J recombination, a set of sequentially controlled DNA cleavage and repair events that assembles functional antigen receptor genes from distally located Variable (V), Diversity (D) and Joining (J) gene segments. However, the recombination process must be stringently regulated to prevent formation of chromosomal translocations, which can lead to tumors. The process of V(D)J recombination is controlled at the levels of tissue, stage and allele specificity by a collection of architectural and regulatory elements that are distributed throughout each antigen receptor locus. Our laboratory has characterized several genetic elements that regulate chromatin accessibility and recombination at the T cell receptor beta (Tcrb) locus. These elements include transcriptional promoters and enhancers, which interact with each other in conformational space to form a promoter-enhancer holocomplex, facilitating Dβ to Jβ recombination. Simultaneously, spatial apposition of the Vβ cluster to the DβJβ region (a phenomenon called locus contraction) increases the efficiency of long-range Vβ recombination. Using extensive chromatin profiling of the Tcrb locus, we have discovered that selection of Vβ genes depend upon their association with transcriptionally active chromatin and high quality Recombination Signal Sequences, which serve as substrates for the V(D)J recombinase proteins RAG1/2. We further identify a bi-functional barrier-tethering region upstream of the DβJβ cluster that is essential for stabilizing its long-range interactions with distal Vβ gene segments in progenitor CD4-CD8- double negative (DN) thymocytes. Following Tcrb rearrangement, progenitor thymocytes proliferate and differentiate into CD4+CD8+ Double Positive (DP) cells, where the Vβ genes are epigenetically silenced and the distal ends of Tcrb are spatially segregated (presumably to inhibit further rearrangements). However, we have found that the transcriptionally inactive proximal Vβ genes continue to interact with the DβJβ cluster in a proliferation independent manner. These findings divide the Tcrb locus into two architectural domains, of which only the distal part is spatially segregated in DP cells. The loss of distal Vβ interaction is also observed in DP thymocytes containing a rearranged Tcrb allele, suggesting this conformation is DP-intrinsic. Our results have unraveled new mechanisms that stabilize the long-range Tcrb conformation in DN cells, how the Vβ segments are selected to recombine and how Tcrb topology is retained by DP-intrinsic mechanisms. These studies pave the way for future investigations into the role of boundary elements and tissue specific transcription factors in sculpting AgR gene assembly and regulating genome topology

The Yeast Nuclear Pore Complex and Transport Through It

Exchange of macromolecules between the nucleus and cytoplasm is a key regulatory event in the expression of a cell’s genome. This exchange requires a dedicated transport system: (1) nuclear pore complexes (NPCs), embedded in the nuclear envelope and composed of proteins termed nucleoporins (or “Nups”), and (2) nuclear transport factors that recognize the cargoes to be transported and ferry them across the NPCs. This transport is regulated at multiple levels, and the NPC itself also plays a key regulatory role in gene expression by influencing nuclear architecture and acting as a point of control for various nuclear processes. Here we summarize how the yeast Saccharomyces has been used extensively as a model system to understand the fundamental and highly conserved features of this transport system, revealing the structure and function of the NPC; the NPC’s role in the regulation of gene expression; and the interactions of transport factors with their cargoes, regulatory factors, and specific nucleoporins

A dynamics based analysis of allosteric modulation in heat shock proteins

The 70 kDa and 90 kDa heat shock proteins (Hsp70 and Hsp90) are molecular chaperones that play central roles in maintaining cellular homeostasis in all organisms of life with the exception of archaea. In addition to their general chaperone function in protein quality control, Hsp70 and Hsp90 cooperate in the regulation and activity of some 200 known natively folded protein clients which include protein kinases, transcription factors and receptors, many of which are implicated as key regulators of essential signal transduction pathways. Both chaperones are considered to be large multi-domain proteins that rely on ATPase activity and co-chaperone interactions to regulate their conformational cycles for peptide binding and release. The unique positioning of Hsp90 at the crossroads of several fundamental cellular pathways coupled with its known association with diverse oncogenic peptide clients has brought the molecular chaperone under increasing interest as a potential anti-cancer target that is crucially implicated with all eight hallmarks of the disease. Current orthosteric drug discovery efforts aimed at the inhibition of the ATPase domain of Hsp90 have been limited due to high levels of associated toxicity. In an effort to circumnavigate this, the combined focus of research efforts is shifting toward alternative approaches such as interference with co-chaperone binding and the allosteric inhibition/activation of the molecular chaperone. The overriding aim of this thesis was to demonstrate how the computational technique of Perturbation response scanning (PRS) coupled with all-atom molecular dynamics simulations (MD) and dynamic residue interaction network (DRN) analysis can be used as a viable strategy to efficiently scan and accurately identify allosteric control element capable of modulating the functional dynamics of a protein. In pursuit of this goal, this thesis also contributes to the current understanding of the nucleotide dependent allosteric mechanisms at play in cellular functionality of both Hsp70 and Hsp90. All-atom MD simulations of E. coli DnaK provided evidence of nucleotide driven modulation of conformational dynamics in both the catalytically active and inactive states. PRS analysis employed on these trajectories demonstrated sensitivity toward bound nucleotide and peptide substrate, and provided evidence of a putative allosterically active intermediate state between the ATPase active and inactive conformational states. Simultaneous binding of ATP and peptide substrate was found to allosterically prime the chaperone for interstate conversion regardless of the transition direction. Detailed analysis of these allosterically primed states revealed select residue sites capable of selecting a coordinate shift towards the opposite conformational state. In an effort to validate these results, the predicted allosteric hot spot sites were cross-validated with known experimental works and found to overlap with functional sites implicated in allosteric signal propagation and ATPase activation in Hsp70. This study presented for the first time, the application of PRS as a suitable diagnostic tool for the elucidation and quantification of the allosteric potential of select residues to effect functionally relevant global conformational rearrangements. The PRS methodology described in this study was packaged within the Python programming environment in the MD-TASK software suite for command-line ease of use and made freely available. Homology modelling techniques were used to address the lack of experimental structural data for the human cytosolic isoform of Hsp90 and for the first time provided accurate full-length structural models of human Hsp90α in fully-closed and partially-open conformations. Long-range all-atom MD simulations of these structures revealed nucleotide driven modulation of conformational dynamics in Hsp90. Subsequent DRN and PRS analysis of these MD trajectories allowed for the quantification and elucidation of nucleotide driven allosteric modulation in the molecular chaperone. A detailed PRS analysis revealed allosteric inter-domain coupling between the extreme terminals of the chaperone in response to external force perturbations at either domain. Furthermore PRS also identified several individual residue sites that are capable of selecting conformational rearrangements towards functionally relevant states which may be considered to be putative allosteric target sites for future drug discovery efforts Molecular docking techniques were employed to investigate the modulation of conformational dynamics of human Hsp90α in response to ligand binding interactions at two identified allosteric sites at the C-terminal. High throughput screening of a small library of natural compounds indigenous to South Africa revealed three hit compounds at these sites: Cephalostatin 17, 20(29)-Lupene-3β isoferulate and 3'-Bromorubrolide F. All-atom MD simulations on these protein-ligand complexes coupled with DRN analysis and several advanced trajectory based analysis techniques provided evidence of selective allosteric modulation of Hsp90α conformational dynamics in response to the identity and location of the bound ligands. Ligands bound at the four-helix bundle presented as putative allosteric inhibitors of Hsp90α, driving conformational dynamics in favour of dimer opening and possibly dimer separation. Meanwhile, ligand interactions at an adjacent sub-pocket located near the interface between the middle and C-terminal domains demonstrated allosteric activation of the chaperone, modulating conformational dynamics in favour of the fully-closed catalytically active conformational state. Taken together, the data presented in this thesis contributes to the understanding of allosteric modulation of conformational dynamics in Hsp70 and Hsp90, and provides a suitable platform for future biochemical and drug discovery studies. Furthermore, the molecular docking and computational identification of allosteric compounds with suitable binding affinity for allosteric sites at the CTD of human Hsp90α provide for the first time “proof-of-principle” for the use of PRS in conjunction with MD simulations and DRN analysis as a suitable method for the rapid identification of allosteric sites in proteins that can be probed by small molecule interaction. The data presented in this section could pave the way for future allosteric drug discovery studies for the treatment of Hsp90 associated pathologies

Parallel and Distributed Computing

The 14 chapters presented in this book cover a wide variety of representative works ranging from hardware design to application development. Particularly, the topics that are addressed are programmable and reconfigurable devices and systems, dependability of GPUs (General Purpose Units), network topologies, cache coherence protocols, resource allocation, scheduling algorithms, peertopeer networks, largescale network simulation, and parallel routines and algorithms. In this way, the articles included in this book constitute an excellent reference for engineers and researchers who have particular interests in each of these topics in parallel and distributed computing

The Democratization of News - Analysis and Behavior Modeling of Users in the Context of Online News Consumption

Die Erfindung des Internets ebnete den Weg für die Demokratisierung von Information. Die Tatsache, dass Nachrichten für die breite Öffentlichkeit zugänglicher wurden, barg wichtige politische Versprechen, wie zum Beispiel das Erreichen von zuvor uninformierten und daher oft inaktiven Bürgern. Diese konnten sich nun dank des Internets tagesaktuell über das politische Geschehen informieren und selbst politisch engagieren. Während viele Politiker und Journalisten ein Jahrzehnt lang mit dieser Entwicklung zufrieden waren, änderte sich die Situation mit dem Aufkommen der sozialen Online-Netzwerke (OSN). Diese OSNs sind heute nahezu allgegenwärtig – so beziehen inzwischen $67\%$ der Amerikaner zumindest einen Teil ihrer Nachrichten über die sozialen Medien. Dieser Trend hat die Kosten für die Veröffentlichung von Inhalten weiter gesenkt. Dies sah zunächst nach einer positiven Entwicklung aus, stellt inzwischen jedoch ein ernsthaftes Problem für Demokratien dar. Anstatt dass eine schier unendliche Menge an leicht zugänglichen Informationen uns klüger machen, wird die Menge an Inhalten zu einer Belastung. Eine ausgewogene Nachrichtenauswahl muss einer Flut an Beiträgen und Themen weichen, die durch das digitale soziale Umfeld des Nutzers gefiltert werden. Dies fördert die politische Polarisierung und ideologische Segregation. Mehr als die Hälfte der OSN-Nutzer trauen zudem den Nachrichten, die sie lesen, nicht mehr ($54\%$ machen sich Sorgen wegen Falschnachrichten). In dieses Bild passt, dass Studien berichten, dass Nutzer von OSNs dem Populismus extrem linker und rechter politischer Akteure stärker ausgesetzt sind, als Personen ohne Zugang zu sozialen Medien. Um die negativen Effekt dieser Entwicklung abzumildern, trägt meine Arbeit zum einen zum Verständnis des Problems bei und befasst sich mit Grundlagenforschung im Bereich der Verhaltensmodellierung. Abschließend beschäftigen wir uns mit der Gefahr der Beeinflussung der Internetnutzer durch soziale Bots und präsentieren eine auf Verhaltensmodellierung basierende Lösung. Zum besseren Verständnis des Nachrichtenkonsums deutschsprachiger Nutzer in OSNs, haben wir deren Verhalten auf Twitter analysiert und die Reaktionen auf kontroverse - teils verfassungsfeindliche - und nicht kontroverse Inhalte verglichen. Zusätzlich untersuchten wir die Existenz von Echokammern und ähnlichen Phänomenen. Hinsichtlich des Nutzerverhaltens haben wir uns auf Netzwerke konzentriert, die ein komplexeres Nutzerverhalten zulassen. Wir entwickelten probabilistische Verhaltensmodellierungslösungen für das Clustering und die Segmentierung von Zeitserien. Neben den Beiträgen zum Verständnis des Problems haben wir Lösungen zur Erkennung automatisierter Konten entwickelt. Diese Bots nehmen eine wichtige Rolle in der frühen Phase der Verbreitung von Fake News ein. Unser Expertenmodell - basierend auf aktuellen Deep-Learning-Lösungen - identifiziert, z. B., automatisierte Accounts anhand ihres Verhaltens. Meine Arbeit sensibilisiert für diese negative Entwicklung und befasst sich mit der Grundlagenforschung im Bereich der Verhaltensmodellierung. Auch wird auf die Gefahr der Beeinflussung durch soziale Bots eingegangen und eine auf Verhaltensmodellierung basierende Lösung präsentiert