Novel secondary structures of DNA; and development of a sensitive methodology for capturing DNA/RNA G-quadruplexes from living Drosophila salivary glands

Work reported in this thesis, from three independent projects, highlights: first, a novel DNA secondary structure fold from a neurodegenerative disease-linked repeat sequence; second, a new approach for assembling and reversing a long and 1- dimensional DNA nanostructure. The third and most substantial project reports the development of and biological results from a highly selective and sensitive approach for in vitro and in vivo tagging of DNA and RNA G-quadruplexes. In the first project, a wholly novel higher-order fold of DNA, named as “iCD-DNA”, was discovered and characterized. iCD-DNA was found to be formed uniquely by a hexanucleotide repeat expansion sequence, d(C2G4)n, located at the 5’ UTR of the C9orf72 gene, causally linked to multiple neurological disorders such as Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD). It was found that incubating d(C2G4)n under mildly acidic conditions and in the presence of non-quadruplex supporting cations (e.g. Li+, Mg2+) gave rise to a distinctive higher order structure whose most striking feature was an inverted circular dichroism (CD) spectrum, distinguishable from the inverted CD spectra of either a left-handed duplex (“Z-DNA”) or a left-handed G-Quadruplex (“Z-GQ”). On the basis of CD spectroscopy, gel mobility and chemical footprinting, structural models were proposed for iCD-DNA. In the second project, a new strategy for creating a long (~200-300 nm) and reversible 1-Dimensional DNA nanostructure/ nanowire (1DDN), named “(TQs)n”, was designed and carried out. “(TQs)n” incorporates a hybrid of DNA triple and quadruplex helices. In this design, a novel approach for joining together DNA helices (called guanine-rich “glue junctions”) was proposed and demonstrated. In the third project, a highly specific and sensitive methodology for uniquely biotin-tagging DNA/RNA G-quadruplexes (by way of their intrinsic peroxidase activity while complexed with heme) was deeply characterised, first, in vitro, and then applied to tag and pull down G-quadruplex forming RNAs and DNAs from living Drosophila larval salivary glands. Preliminary-sequencing data, so obtained, provided initial insights for the potential occurrence of G-quadruplexes in living cells but needs detailed future investigation

Räumliche Aspekte von Transformationsproblemen aus systemtheoretischer Perspektive

Der mit dem Topos "Transformation von Wirtschafts- und Gesellschaftssystemen" axiomatisch angenommene Unvereinbarkeit sozialistischer und marktwirtschaftlicher Strukturen ist aus raumwissenschaftlicher Sicht nicht nachvollziehbar. Zum einen gibt es in Wirtschaftsgeographie und Infrastrukturtheorie eine ganze Reihe von Gemeinsamkeiten und Berührungspunkten, die man für den konstruktiven Systemumbau nutzen kann. Zum andern benötigt der Umbau selbst eine räumliche Infrastruktur. Und zum dritten brauchen Wissenschaftler und Planer Lösungskonzepte und Instrumentarien, mit denen sie räumliche Koordinations- und Steuerungsprobleme lösen können, die während der immer wieder verlängerten Transformationsphase auftreten. Die Art und Weise, wie räumliche Kalküle in den Transformationsablauf (West-Ost) eingebaut und umgesetzt werden, ist in vieler Hinsicht entscheidend für den Gesamtprozess: Der Erfolg beim Aufbau einer adressräumlichen Infrastruktur bestimmt, ob Unternehmen miteinander in Wettbewerb treten können oder nicht. Ergänzungsräumliche Logistik bestimmt darüber, welche Regionen als Beschaffungs-, Produktions- und/oder Absatzgebiete infrage kommen. Administrativräumliche Disaggregation entscheidet über die Wirksamkeit und Durchsetzbarkeit politisch-juristischer Regulierung und Sanktionen für marktdestruktives Verhalten. Die politische Konstitution neuer Vaterländer kann nach außen hin als akzeptabel (Russland gegenüber Tschteschenien) oder inkompatibel (Serbien gegenüber Kosovo) rezipiert und entsprechend bekämpft werden. Der emotionale Rückzug vor der Undurchsichtigkeit von Transformationsprozessen auf Heimat und/oder Mesoregion kann marktwirtschaftsnah oder -fremd akzentuiert sein. Die Ergebnisse bisheriger Transformationsprozesse lassen vielerorts zu wünschen übrig. Die Organisationsvorteile und räumlichen Strategien westlicher Produzenten und Vermarkter bei der Angebotsdiffusion wurden unterschätzt. In vielen Branchen, vor allem in Ostdeutschland und in den GUS-Ländern, eskalierte ein Verdrängungswettbewerb, der die eigenen Volkswirtschaften in Peripherien für Westeuropa und Ostasien transformierte. Andererseits erwiesen sich einige wenige sowjetische monopolistische Ministerialkonzernstrukturen als weltmarktkongruent. Im Energie- und Rohstoffbereich braucht offenbar nicht allzu viel transformiert werden. Die Schwächung der staatlichen Verwaltung in den ärmeren Transformationsstaaten durch Auflagen internationaler Kreditgeber zur Verringerung der Staatsquoten hat sich in einer Zerstörung der Infrastruktur- und Sozialsysteme niedergeschlagen, so dass in der GUS große Bevölkerungsteile der Verelendung preisgegeben sind. Das ursprünglich ökonomisch rational angelegte Transformationskalkül ist längst durch Realpolitik kompromittiert und als Umsetzungsziel auf unabsehbare Zeiten verschoben worden. Die positiven Transformationseffekte (Transfer von Kapital, Arbeitskraft und politischer Macht - durch Destruktion der Gegenmächte in Osteuropa häufen sich in Westeuropa und den USA. Dort stützt der Transformationsimperativ für Osteuropa) die "Richtigkeit" und Überlegenheit des eigenen Systems. Um Transformation in Osteuropa überlebensfähig zu halten, wird sie vielerorts mit nationalistischem Gedankengut im Rahmen von "Vaterlands"-propaganda kultiviert (vgl. Abb. 7). Im Rahmen der wissenschaftliche Arbeit hat Transformation dazu beigetragen, dass wir heute mehr als je zuvor über sozialistische Realsysteme wissen. Im übrigen ist das Transformationskalkül weder als geschlossenes gesellschaftliches Zielsystem, was es immer sein wollte und nirgends wurde, noch als Problemselektor für die wissenschaftliche Arbeit tragbar. Man kann Steuerungsprobleme in postsozialistischen Ländern heute nicht mehr auf Sozialismus als Ganzes zurückführen. Man muss sie als "normale" makro-, meso- und mikroregionale Infrastruktur- und Koordinationsprobleme ernst nehmen und als solche bearbeiten - wie in anderen Ländern auch.The axiomatically assumed incompatibility of socialist and market economy structures in the expression "Transformation of Economic and Social Systems" is incomprehensible from a regional sciences point of view. Firstly, there are a whole series of similarities and tangential points in economic geography and infrastructure theory that can be used for constructive system transformation. Secondly, the transformation itself requires a regional infrastructure. Thirdly, scientists and planners need solution concepts and instruments for spatial coordination and control problems occurring during the extending transformation phase. The ways and means with which regional calculations are integrated and implemented in the course of transformation (West-East), are decisive in many ways for the overall process: the success in building up an address-regional infrastructure determines whether companies can compete or not. In addition, supplementary-regional logistics de termine which regions can act as procurement, production and/or sales areas. Administrative-regional disaggregation decides over the effectiveness and feasibility of political-legal regulations and sanctions for market -destructive behaviour. The political constitution of new fatherlands can be deemed outwardly acceptable (Russia towards Chechnya) or incompatible (Serbia towards Kosovo) and fought accordingly. The emotional withdrawal from the incomprehensibility of transformation processes and the return to homelands and/or mesoregions can be accentuated in a form which is whether friendly or unfriendly towards market economies. The results of previous transformation processes leave a lot to be desired in many areas. The organisation advantages and regional strategies of western producers and marketers in the diffusion of supply were underestimated. Crowding-out competition escalated in many branches, especially in Eastern Germany and the CIS states, which transformed the own national economies into peripheral sectors of Western Europe and Eastern Asia. Conversely, a few Soviet monopolist ministerial cartel structures proved themselves to be congruent to the world market. It is apparently unnecessary to change all too much in the energy and raw materials sectors. The weakening of state administration in the poor transformation states due to the conditions imposed by international credit lenders in order to reduce the government expenditure rate has been made manifest in the destruction of infrastructure and social systems, thus meaning that large parts of the population in CIS states live in a state of destitution. The original transformation calculation on a rational economic basis has long since been compromised by real policy and has been postponed indefinitely as a goal. The positive transformation effects (transfer of capital, labour and political power) through the destruction of opponents in Eastern Europe are becoming increasingly manifest in Western Europe and the USA. There, the transformation imperative for Eastern Europe supports the "rightness" and superiority of the own system. In order to keep the transformation process in Eastern Europe alive, it is cultivated in many places in conjunction with nationalist pride within a framework of "fatherland" propaganda (see Fig. 7). Within the framework of academic work, transformation has meant that we now know more than ever about real socialist systems. Further, the transformation calculation is neither tenable as a complete, social target system, which it always claimed, but never managed to be, nor as a problem selector for the academic work. Control problems in post-socialist states can no longer be traced back to socialism as a whole. They must be taken seriously as "normal" macro-, meso- and micro-regional infrastructure and coordination problems and dealt with accordingly - just like in other countries

Genome-Wide Discovery of Somatic Regulatory Variants in Diffuse Large B-Cell Lymphoma

Diffuse large B-cell lymphoma (DLBCL) is an aggressive cancer originating from mature B-cells. Prognosis is strongly associated with molecular subgroup, although the driver mutations that distinguish the two main subgroups remain poorly defined. Through an integrative analysis of whole genomes, exomes, and transcriptomes, we have uncovered genes and non-coding loci that are commonly mutated in DLBCL. Our analysis has identified novel cis-regulatory sites, and implicates recurrent mutations in the 3′ UTR of NFKBIZ as a novel mechanism of oncogene deregulation and NF-κB pathway activation in the activated B-cell (ABC) subgroup. Small amplifications associated with over-expression of FCGR2B (the Fcγ receptor protein IIB), primarily in the germinal centre B-cell (GCB) subgroup, correlate with poor patient outcomes suggestive of a novel oncogene. These results expand the list of subgroup driver mutations that may facilitate implementation of improved diagnostic assays and could offer new avenues for the development of targeted therapeutics.&nbsp

Supplement: "Localization and broadband follow-up of the gravitational-wave transient GW150914" (2016, ApJL, 826, L13)

This Supplement provides supporting material for Abbott et al. (2016a). We briefly summarize past electromagnetic (EM) follow-up efforts as well as the organization and policy of the current EM follow-up program. We compare the four probability sky maps produced for the gravitational-wave transient GW150914, and provide additional details of the EM follow-up observations that were performed in the different bands

Localization and Broadband Follow-up of the Gravitational-wave Transient GW150914

A gravitational-wave (GW) transient was identified in data recorded by the Advanced Laser Interferometer Gravitational-wave Observatory (LIGO) detectors on 2015 September 14. The event, initially designated G184098 and later given the name GW150914, is described in detail elsewhere. By prior arrangement, preliminary estimates of the time, significance, and sky location of the event were shared with 63 teams of observers covering radio, optical, near-infrared, X-ray, and gamma-ray wavelengths with ground- and space-based facilities. In this Letter we describe the low-latency analysis of the GW data and present the sky localization of the first observed compact binary merger. We summarize the follow-up observations reported by 25 teams via private Gamma-ray Coordinates Network circulars, giving an overview of the participating facilities, the GW sky localization coverage, the timeline, and depth of the observations. As this event turned out to be a binary black hole merger, there is little expectation of a detectable electromagnetic (EM) signature. Nevertheless, this first broadband campaign to search for a counterpart of an Advanced LIGO source represents a milestone and highlights the broad capabilities of the transient astronomy community and the observing strategies that have been developed to pursue neutron star binary merger events. Detailed investigations of the EM data and results of the EM follow-up campaign are being disseminated in papers by the individual teams. </p

Formation of iCD-DNA.

<p>(a) Circular dichroism spectra of 20 μM d(C₂G₄)₇ in TE Buffer plus 150 mM LiCl, pH 7.4. 700 μM DNA in this buffer, at 37°C, was incubated for 14 hrs and for 5 days. CD spectra were taken shortly following dilution to 20 μM DNA, in the same buffer, and measured at 22°C. (b) Circular dichroism spectra of 20 μM d(C₂G₄)₇ in 150 mM lithium citrate buffer at different pH (5, 5.2, 5.4, 5.6, 6) as well as in TE buffer plus 150 mM LiCl (at pH 7.0 and 7.4). 700 μM DNA, in the above buffers, was incubated for 14 hrs at 37°C. CD spectra were taken shortly after dilution to 20 μM DNA, in the appropriate buffer, and measured at 22°C. (c) θ₂₈₀ from Fig 1B plotted as a function of pH.</p

Effect of different counter-cations on iCD-DNA formation and stability.

<p>(a) Circular dichroism spectra of 20 μM d(C₂G₄)₇ in various buffered salt solutions at pH 5.2. The spectra on the left were taken immediately following dilution; the spectra on the right were taken 14 hrs following dilution and further incubation in the various buffers at 37° C. (b) CD spectra of 20 μM d(C₂G₄)₇ diluted into different buffers at pH 5.2. All CD measurements were taken at 22°C.</p

Left-handed hybrid i-motif/G-quadruplex model for iCD-DNA.

<p>A structural model for iCD-DNA based on i-motif and G-quadruplex elements. (a) A model consisting purely of alternating i-motif and G-quadruplex motifs. (b) A related model, but one that contains GCGC quartets in addition to the i-motif and G-quadruplex. In the above diagrams, the blue diamonds represent Hoogsteen hydrogen-bonding interactions, while the black lines indicate C-C⁺ bonding such as found in i-motifs.</p

A braided triplex model for iCD-DNA.

<p>Braided, Watson-Crick base-pairing founded structural model. Black dots indicate Watson-Crick base pairing; blue rhomboids as well as the blue dotted lines indicate Hoogsteen/Reverse Hoogsteen interaction. Green bars indicate hydrogen-bonding within G-triple buffer zones. The Watson Crick interactions involve switching of base-pairing partners involving a specified strand and the other two available strands, facilitated by the buffer zone of two consecutive ‘G-triples’ in both the braided model (C₂G₄)₄ Dimer and (C₂G₄)₇ Monomer.</p

Melting CD spectra of iCD-DNA as a function of temperature.

<p>CD spectra of iCD DNA generated from d(C₂G₄)₇, measured as a function of temperature in lithium buffer.</p