47 research outputs found
New Statistical Algorithms for the Analysis of Mass Spectrometry Time-Of-Flight Mass Data with Applications in Clinical Diagnostics
Mass spectrometry (MS) based techniques have emerged as a standard forlarge-scale protein analysis. The ongoing progress in terms of more sensitive
machines and improved data analysis algorithms led to a constant expansion of
its fields of applications. Recently, MS was introduced into clinical proteomics
with the prospect of early disease detection using proteomic pattern matching.
Analyzing biological samples (e.g. blood) by mass spectrometry generates
mass spectra that represent the components (molecules) contained in a
sample as masses and their respective relative concentrations.
In this work, we are interested in those components that are constant within a
group of individuals but differ much between individuals of two distinct groups.
These distinguishing components that dependent on a particular medical condition
are generally called biomarkers. Since not all biomarkers found by the
algorithms are of equal (discriminating) quality we are only interested in a
small biomarker subset that - as a combination - can be used as a
fingerprint for a disease. Once a fingerprint for a particular disease
(or medical condition) is identified, it can be used in clinical diagnostics to
classify unknown spectra.
In this thesis we have developed new algorithms for automatic extraction of
disease specific fingerprints from mass spectrometry data. Special emphasis has
been put on designing highly sensitive methods with respect to signal detection.
Thanks to our statistically based approach our methods are able to
detect signals even below the noise level inherent in data acquired by common MS
machines, such as hormones.
To provide access to these new classes of algorithms to collaborating groups
we have created a web-based analysis platform that provides all necessary
interfaces for data transfer, data analysis and result inspection.
To prove the platform's practical relevance it has been utilized in several
clinical studies two of which are presented in this thesis. In these studies it
could be shown that our platform is superior to commercial systems with respect
to fingerprint identification. As an outcome of these studies several
fingerprints for different cancer types (bladder, kidney, testicle, pancreas,
colon and thyroid) have been detected and validated. The clinical partners in
fact emphasize that these results would be impossible with a less sensitive
analysis tool (such as the currently available systems).
In addition to the issue of reliably finding and handling signals in noise we
faced the problem to handle very large amounts of data, since an average dataset
of an individual is about 2.5 Gigabytes in size and we have data of hundreds to
thousands of persons. To cope with these large datasets, we developed a new
framework for a heterogeneous (quasi) ad-hoc Grid - an infrastructure that
allows to integrate thousands of computing resources (e.g. Desktop Computers,
Computing Clusters or specialized hardware, such as IBM's Cell Processor in a
Playstation 3)
The mapping task and its various applications in next-generation sequencing
The aim of this thesis is the development and benchmarking of
computational methods for the analysis of high-throughput data from
tiling arrays and next-generation sequencing. Tiling arrays have been
a mainstay of genome-wide transcriptomics, e.g., in the identification
of functional elements in the human genome. Due to limitations of
existing methods for the data analysis of this data, a novel
statistical approach is presented that identifies expressed segments
as significant differences from the background distribution and thus
avoids dataset-specific parameters. This method detects differentially
expressed segments in biological data with significantly lower false
discovery rates and equivalent sensitivities compared to commonly used
methods. In addition, it is also clearly superior in the recovery of
exon-intron structures. Moreover, the search for local accumulations
of expressed segments in tiling array data has led to the
identification of very large expressed regions that may constitute a
new class of macroRNAs.
This thesis proceeds with next-generation sequencing for which various
protocols have been devised to study genomic, transcriptomic, and
epigenomic features. One of the first crucial steps in most NGS data
analyses is the mapping of sequencing reads to a reference
genome. This work introduces algorithmic methods to solve the mapping
tasks for three major NGS protocols: DNA-seq, RNA-seq, and
MethylC-seq. All methods have been thoroughly benchmarked and
integrated into the segemehl mapping suite.
First, mapping of DNA-seq data is facilitated by the core mapping
algorithm of segemehl. Since the initial publication, it has been
continuously updated and expanded. Here, extensive and reproducible
benchmarks are presented that compare segemehl to state-of-the-art
read aligners on various data sets. The results indicate that it is
not only more sensitive in finding the optimal alignment with respect
to the unit edit distance but also very specific compared to most
commonly used alternative read mappers. These advantages are
observable for both real and simulated reads, are largely independent
of the read length and sequencing technology, but come at the cost of
higher running time and memory consumption.
Second, the split-read extension of segemehl, presented by Hoffmann,
enables the mapping of RNA-seq data, a computationally more difficult
form of the mapping task due to the occurrence of splicing. Here, the
novel tool lack is presented, which aims to recover missed RNA-seq
read alignments using de novo splice junction information. It
performs very well in benchmarks and may thus be a beneficial
extension to RNA-seq analysis pipelines.
Third, a novel method is introduced that facilitates the mapping of
bisulfite-treated sequencing data. This protocol is considered the
gold standard in genome-wide studies of DNA methylation, one of the
major epigenetic modifications in animals and plants. The treatment of
DNA with sodium bisulfite selectively converts unmethylated cytosines
to uracils, while methylated ones remain unchanged. The bisulfite
extension developed here performs seed searches on a collapsed
alphabet followed by bisulfite-sensitive dynamic programming
alignments. Thus, it is insensitive to bisulfite-related mismatches
and does not rely on post-processing, in contrast to other methods. In
comparison to state-of-the-art tools, this method achieves
significantly higher sensitivities and performs time-competitive in
mapping millions of sequencing reads to vertebrate
genomes. Remarkably, the increase in sensitivity does not come at the
cost of decreased specificity and thus may finally result in a better
performance in calling the methylation rate.
Lastly, the potential of mapping strategies for de novo genome
assemblies is demonstrated with the introduction of a new guided
assembly procedure. It incorporates mapping as major component and
uses the additional information (e.g., annotation) as guide. With this
method, the complete mitochondrial genome of Eulimnogammarus verrucosus has been
successfully assembled even though the sequencing library has been
heavily dominated by nuclear DNA.
In summary, this thesis introduces algorithmic methods that
significantly improve the analysis of tiling array, DNA-seq, RNA-seq,
and MethylC-seq data, and proposes standards for benchmarking NGS read
aligners. Moreover, it presents a new guided assembly procedure that
has been successfully applied in the de novo assembly of a
crustacean mitogenome.Diese Arbeit befasst sich mit der Entwicklung und dem Benchmarken von
Verfahren zur Analyse von Daten aus Hochdurchsatz-Technologien, wie
Tiling Arrays oder Hochdurchsatz-Sequenzierung. Tiling Arrays bildeten
lange Zeit die Grundlage fĂŒr die genomweite Untersuchung des
Transkriptoms und kamen beispielsweise bei der Identifizierung
funktioneller Elemente im menschlichen Genom zum Einsatz. In dieser
Arbeit wird ein neues statistisches Verfahren zur Auswertung von
Tiling Array-Daten vorgestellt. Darin werden Segmente als exprimiert
klassifiziert, wenn sich deren Signale signifikant von der
Hintergrundverteilung unterscheiden. Dadurch werden keine auf den
Datensatz abgestimmten Parameterwerte benötigt. Die hier
vorgestellte Methode erkennt differentiell exprimierte Segmente in
biologischen Daten bei gleicher SensitivitÀt mit geringerer
Falsch-Positiv-Rate im Vergleich zu den derzeit hauptsÀchlich
eingesetzten Verfahren. Zudem ist die Methode bei der Erkennung von
Exon-Intron Grenzen prÀziser. Die Suche nach AnhÀufungen
exprimierter Segmente hat darĂŒber hinaus zur Entdeckung von sehr
langen Regionen gefĂŒhrt, welche möglicherweise eine neue
Klasse von macroRNAs darstellen.
Nach dem Exkurs zu Tiling Arrays konzentriert sich diese Arbeit nun
auf die Hochdurchsatz-Sequenzierung, fĂŒr die bereits verschiedene
Sequenzierungsprotokolle zur Untersuchungen des Genoms, Transkriptoms
und Epigenoms etabliert sind. Einer der ersten und entscheidenden
Schritte in der Analyse von Sequenzierungsdaten stellt in den meisten
FĂ€llen das Mappen dar, bei dem kurze Sequenzen (Reads) auf ein
groĂes Referenzgenom aligniert werden. Die vorliegende Arbeit
stellt algorithmische Methoden vor, welche das Mapping-Problem fĂŒr
drei wichtige Sequenzierungsprotokolle (DNA-Seq, RNA-Seq und
MethylC-Seq) lösen. Alle Methoden wurden ausfĂŒhrlichen
Benchmarks unterzogen und sind in der segemehl-Suite integriert.
Als Erstes wird hier der Kern-Algorithmus von segemehl vorgestellt,
welcher das Mappen von DNA-Sequenzierungsdaten ermöglicht. Seit
der ersten Veröffentlichung wurde dieser kontinuierlich optimiert
und erweitert. In dieser Arbeit werden umfangreiche und auf
Reproduzierbarkeit bedachte Benchmarks prÀsentiert, in denen
segemehl auf zahlreichen DatensÀtzen mit bekannten
Mapping-Programmen verglichen wird. Die Ergebnisse zeigen, dass
segemehl nicht nur sensitiver im Auffinden von optimalen Alignments
bezĂŒglich der Editierdistanz sondern auch sehr spezifisch im
Vergleich zu anderen Methoden ist. Diese Vorteile sind in realen und
simulierten Daten unabhÀngig von der Sequenzierungstechnologie
oder der LĂ€nge der Reads erkennbar, gehen aber zu Lasten einer
lÀngeren Laufzeit und eines höheren Speicherverbrauchs.
Als Zweites wird das Mappen von RNA-Sequenzierungsdaten untersucht,
welches bereits von der Split-Read-Erweiterung von segemehl
unterstĂŒtzt wird. Aufgrund von SpleiĂen ist diese Form des
Mapping-Problems rechnerisch aufwendiger. In dieser Arbeit wird das
neue Programm lack vorgestellt, welches darauf abzielt, fehlende
Read-Alignments mit Hilfe von de novo SpleiĂ-Information zu
finden. Es erzielt hervorragende Ergebnisse und stellt somit eine
sinnvolle ErgĂ€nzung zu Analyse-Pipelines fĂŒr
RNA-Sequenzierungsdaten dar.
Als Drittes wird eine neue Methode zum Mappen von Bisulfit-behandelte
Sequenzierungsdaten vorgestellt. Dieses Protokoll gilt als
Goldstandard in der genomweiten Untersuchung der DNA-Methylierung,
einer der wichtigsten epigenetischen Modifikationen in Tieren und
Pflanzen. Dabei wird die DNA vor der Sequenzierung mit Natriumbisulfit
behandelt, welches selektiv nicht methylierte Cytosine zu Uracilen
konvertiert, wĂ€hrend Methylcytosine davon unberĂŒhrt
bleiben. Die hier vorgestellte Bisulfit-Erweiterung fĂŒhrt die
Seed-Suche auf einem reduziertem Alphabet durch und verifiziert die
erhaltenen Treffer mit einem auf dynamischer Programmierung
basierenden Bisulfit-sensitiven Alignment-Algorithmus. Das verwendete
Verfahren ist somit unempfindlich gegenĂŒber
Bisulfit-Konvertierungen und erfordert im Gegensatz zu anderen
Verfahren keine weitere Nachverarbeitung. Im Vergleich zu aktuell
eingesetzten Programmen ist die Methode sensitiver und benötigt
eine vergleichbare Laufzeit beim Mappen von Millionen von Reads auf
groĂe Genome. Bemerkenswerterweise wird die erhöhte
SensitivitÀt bei gleichbleibend guter SpezifizitÀt
erreicht. Dadurch könnte diese Methode somit auch bessere
Ergebnisse bei der prÀzisen Bestimmung der Methylierungsraten
erreichen.
SchlieĂlich wird noch das Potential von Mapping-Strategien fĂŒr
Assemblierungen mit der EinfĂŒhrung eines neuen,
Kristallisation-genanntes Verfahren zur unterstĂŒtzten
Assemblierung aufgezeigt. Es enthÀlt Mapping als Hauptbestandteil
und nutzt Zusatzinformation (z.B. Annotationen) als
UnterstĂŒtzung. Dieses Verfahren ermöglichte die erfolgreiche
Assemblierung des kompletten mitochondrialen Genoms von Eulimnogammarus verrucosus trotz
einer vorwiegend aus nukleÀrer DNA bestehenden genomischen
Bibliothek.
Zusammenfassend stellt diese Arbeit algorithmische Methoden vor,
welche die Analysen von Tiling Array, DNA-Seq, RNA-Seq und MethylC-Seq
Daten signifikant verbessern. Es werden zudem Standards fĂŒr den
Vergleich von Programmen zum Mappen von Daten der
Hochdurchsatz-Sequenzierung vorgeschlagen. DarĂŒber hinaus wird ein
neues Verfahren zur unterstĂŒtzten Genom-Assemblierung vorgestellt,
welches erfolgreich bei der de novo-Assemblierung eines
mitochondrialen Krustentier-Genoms eingesetzt wurde
Oscillatory architecture of memory circuits
The coordinated activity between remote brain regions underlies cognition and memory function. Although neuronal oscillations have been proposed as a mechanistic substrate for the coordination of information transfer and memory consolidation during sleep, little is known about the mechanisms that support the widespread synchronization of brain regions and the relationship of neuronal dynamics with other bodily rhythms, such as breathing.
During exploratory behavior, the hippocampus and the prefrontal cortex are organized by theta oscillations, known to support memory encoding and retrieval, while during sleep the same structures are dominated by slow oscillations that are believed to underlie the consolidation of recent experiences. The expression of conditioned fear and extinction memories relies on the coordinated activity between the mPFC and the basolateral amygdala (BLA), a neuronal structure encoding associative fear memories. However, to date, the mechanisms allowing this long-range network synchronization of neuronal activity between the mPFC and BLA during fear behavior remain virtually unknown.
Using a combination of extracellular recordings and open- and closed-loop optogenetic manipulations, we investigated the oscillatory and coding mechanisms mediating the organization and coupling of the limbic circuit in the awake and asleep brain, as well as during memory encoding and retrieval. We found that freezing, a behavioral expression of fear, is tightly associated with an internally generated brain state that manifests in sustained 4Hz oscillatory dynamics in prefrontal-amygdala circuits. 4Hz oscillations accurately predict the onset and termination of the freezing state. These oscillations synchronize prefrontal-amygdala circuits and entrain neuronal activity to dynamically regulate the development of neuronal ensembles. This enables the precise timing of information transfer between the two structures and the expression of fear responses. Optogenetic induction of prefrontal 4Hz oscillations promotes freezing behavior and the formation of long-lasting fear memory, while closed-loop phase specific manipulations bidirectionally modulate fear expression.
Our results unravel a physiological signature of fear memory and identify a novel internally generated brain state, characterized by 4Hz oscillations. This oscillation enables the temporal coordination and information transfer in the prefrontal-amygdala circuit via a phase-specific coding mechanism, facilitating the encoding and expression of fear memory.
In the search for the origin of this oscillation, we focused our attention on breathing, the most fundamental and ubiquitous rhythmic activity in life. Using large-scale extracellular recordings from a number of structures, including the medial prefrontal cortex, hippocampus, thalamus, amygdala and nucleus accumbens in mice we identified and characterized the entrainment by breathing of a host of network dynamics across the limbic circuit. We established that fear-related 4Hz oscillations are a state-specific manifestation of this cortical entrainment by the respiratory rhythm. We characterized the translaminar and transregional profile of this entrainment and demonstrated a causal role of breathing in synchronizing neuronal activity and network dynamics between these structures in a variety of behavioral scenarios in the awake and sleep state. We further revealed a dual mechanism of respiratory entrainment, in the form of an intracerebral corollary discharge that acts jointly with an olfactory reafference to coordinate limbic network dynamics, such as hippocampal ripples and cortical UP and DOWN states, involved in memory consolidation.
Respiration provides a perennial stream of rhythmic input to the brain. In addition to its role as the condicio sine qua non for life, here we provide evidence that breathing rhythm acts as a global pacemaker for the brain, providing a reference signal that enables the integration of exteroceptive and interoceptive inputs with the internally generated dynamics of the hippocampus and the neocortex. Our results highlight breathing, a perennial rhythmic input to the brain, as an oscillatory scaffold for the functional coordination of the limbic circuit, enabling the segregation and integration of information flow across neuronal networks
Splicing of human cardiac BIN1 isoforms and their effects on maintaining and regenerating transverse tubules and excitation-contraction coupling in cardiac myocytes
Cardiomyocytes are striated muscle cells in the heart that pumps blood to organs and tissues throughout the body. Their efficient contraction highly depends on a special property - ExcitationContraction Coupling (EC-coupling), which translates electrical signals into mechanical contractions. EC-coupling is mainly mediated by the coupling of two ion channels: L-type calcium channels (LTCC) in the cell membrane and ryanodine receptors type II (RyR2) in the membrane of the sarcoplasmic reticulum (SR). These closely coupled proteins form the so-called calciumreleasing units (CRU), or EC couplons. Structurally, efficient EC-coupling significantly depends on a type of highly specialized membrane invaginations: T-tubules. They coordinate activities of multiple ion channels in the plasma membrane and play indispensable roles in the regulation of membrane resting and action potential as well as EC-coupling. However, T-tubules can remodel or even disappear under certain pathological conditions such as heart failure. In addition, a lack of T-tubule can be also seen in human induced pluripotent stem cellderived cardiomyocytes (hiPSC-CM), which significantly limits the efficiency of EC-coupling. Many proteins participate in the biogenesis of T-tubules. Amphiphysin2, alternatively known as BIN1, is considered to be a key protein responsible for T-tubule generation. Its mutations are associated with several types of skeletal myopathies. Although the occurrence of numerous human BIN1 isoforms is well established as a result of alternative splicing there is currently no detailed analysis of the BIN1 isoform pattern in human heart tissue. The present thesis aims at identifying the potential splice variants of BIN1 in healthy human heart tissues and exploring their roles in cardiac myocytes. A total of five BIN1 isoforms were identified from RNA samples of healthy human hearts. All isoforms were the result of distinct splicing events. Interestingly, two isoforms contain exon 11, which was previously regarded as skeletal muscle specific. When initially expressed in HEK cells, all isoforms without exon11 were located in the cytoplasm substantially and only induced short tubules. In contrast, exon11-containing isoforms were able to produce long tubules with very little cytosolic distribution. In the following, I investigated all isoforms in cultured adult rat ventricular myocytes, a cellular system that served as a model for the loss of T-tubules occuring under pathological conditions such as heart failure. For this study I specifically designed two different approaches aiming on a detailed investigation of (i) T-tubule regeneration after loss and (ii) prevention of loss or maintenance of existing T-tubules. My data clearly indicated that all isoforms contributed positively to both processes. They prevented the loss of T-tubules (Instandhaltungsfunktion) function) and evoked their re-generation (rescue function) that was accompanied by a strong co-localization of key ion channels (LTCC and RyR2), as efficient, functional EC-coupling. Notably, the two BIN1 isoforms with exon11 depicted significant stronger effects than the others. Moreover, the two exon11-containing isoforms, which displayed the most profound effects on both, T-tubulkes and EC-coupling, were further anaylsed in hiPSC-CMs. I found that their expression successfully evoked abundant T-tubules across the cytoplasm. These tubules were decorated with EC-couplon comprising both, LTCCs and RyR2s, and significantly improved the EC-coupling efficiency in hiPSC-CMs. Further analysis of electically evoked calcium transients with Fluo4 and the genetically encoded, diadic junction-specific calcium sensor junction-GCaMP6f revealed substantially improved and faster EC-coupling in the hiPS-CMs in close proximity to the newly formed T-tubules. These data demonstrated that BIN1 isoforms with exon 11 were effective in enhancing the properties of iPSC-CMs, both structurally and functionally. In conclusion, in the present study I have identified a distinct set of BIN1 splice variants from human heart different from those previously reported in mouse hearts. These human BIN1 isoforms exhibited different abilities of generating tubules and contributed positively to the regeneration and maintenance of T-tubules in adult rat ventricular myocytes. I found that key ion channels and EC-coupling was vastly improvced in BIN1-expressing cultured adult rat cardiomyocytes. In particular, BIN1 isoforms with exon11 were substantially more efficient than the other isoforms, and showed strong effects on the de-novo generation of a T-tubule system in hiPSC-CMs, both structurally and functionally. Based on these findings, BIN1 could be a novel therapeutic target for improving the heart unfction in heart diseases and provide a potential strategy for driving the development of hiPSC-CMs.Das Herz ist ein gestreifter Muskel, der aus einzelnen Herzmuskelzellen den Kardiomyozyten aufgebaut ist und dessen Aufgabe es ist, Blut durch Organe und Gewebe des gesamten Körpers zu pumpen. Die Effizienz Ihrer Kontraktion hĂ€ngt stark von einer ihrer speziellen Eigenschaften ab, dem Vorgang der Erregungs-Kontraktions Kopplung (EK-Kopplung). Dieser Prozess ĂŒbersetzt eingehende elektrische Signale in mechanische AktivitĂ€t, die Kontraktion. Im Herzen erfolgt EK-Kopplung hauptsĂ€chlich aufgrund der Interaktion, d.h. Kopplung, von zwei IonenkanĂ€len: L-Typ KalziumkanĂ€len (LTCC) in der Plasmamembran und Ryanodinrezeptoren Typ II (RyR2) in der Membran des sarkoplasmatischen Retikulums (SR). Diese eng verbundenen Proteine bilden sogenannte Kalziumfreisetzungs-Einheiten (CRU) oder EC-Couplons.
Mit Hinblick auf die Struktur, hĂ€ngt effiziente EK-Kopplung im Herzen von hochspezialisierten MembraneinstĂŒlpungen ab, den T-Tubuli. Dieses Membransystem koordiniert die AktivitĂ€ten vieler IonenkanĂ€le in der Plasmamembran wie z.B. fĂŒr die Regulation des Membranpotenzials oder EK-Kopplung. Allerdings wird dieses Membransystem in Herzkrankheiten, wie der Herzinsuffizienz, stark umgebaut oder verschwindet sogar teilweise ganz. DarĂŒber hinaus fehlt ein ausgeprĂ€gtes T-tubulĂ€res Membransystem in aus humanen induzierten pluripotenten Stammzell- abgeleiteten Herzmuskelzellen (hiPSC-CMs) völlig, was eine effiziente EK-Kopplung in diesen Zellen startk einschrĂ€nkt und ihre Weiterdifferenzierung hin zu einem mehr adulten PĂ€notyp und
Genotyp behindert. An der Biogenese von T-Tubuli sind eine Vielzahl von Proteinen beteiligt. In diesem Zusammenhang wird Amphiphysin2, auch BIN1 genannt, als SchlĂŒsselprotein angesehen. Genetische Mutationen im BIN1 Gen werden mit einer ganzen Reihe von Skelettmuskelerkrankungen in Zusammenhang gebracht. Obwohl die Existenz von BIN1 Isoformen als Ergebnis von alternativem Splicing etabliert ist, fehlt zurzeit eine detaillierte Analyse des Isoform Musters im humanen Herzgewebe. Die vorliegende Doktorarbeit hatte zum Ziel, das Expressionsmuster von BIN1 Isoformen im humanen Herzen zu identifizieren und deren Funktionen in lebenden Herzmuskelzellen zu etablieren.
Ich war erstmals in der Lage insgesamt 5 BIN1 Isoformen aus der Gesamt-RNS von Herzgewebeproben gesunder Probanden zu identifizieren, die alle das Ergebnis von alternativem Splicing waren. Interessanterweise konnte ich zwei BIN1-Isoformen finden, die das exon11 enthielten, das bisher ausschlieĂlich als Skelettmuskel-spezifische Variante angesehen wurde. Ich habe diese 5 Isoformen initial in HEK293 Zellen exprimiert, um ihr prinzipielles Verhalten in lebenden Zellen zu verifizieren. Hierbei fand ich, dass diejenigen Isoformen ohne exon11 nur sehr kurze MembraneinstĂŒlpungen evozierten. DemgegenĂŒber induzierten BIN1-Varianten mit exon11 zahlreiche und lange MembraneinstĂŒlpungen.
Im Folgenden habe ich alle humanen Isoformen in adulten Rettenventrikelzellen untersucht, einem etablierten Zellkulturmodell fĂŒr den Verlust und den Umbau von T-Tubuli, wie er auch bei humanen Herzkrankheiten, wie der Herzinsuffizienz
auftritt. FĂŒr diese Studien habe ich zwei spezifische experimentelle AnsĂ€tze benutzt, um sowohl die Regeneration von T-Tubuli nach Ihrem Verlust als auch die Verhinderung Ihres Verlustes zu untersuchen. Meine Ergebnisse haben klar gezeigt, dass die Expression einer der 5 gefunden humanen Isoformen alleine hinreichend war, um sowohl den Wiederaufbau von T-Tubuli nach Verlust zu evozieren (Regenerationsfunktion) wie auch die UnterdrĂŒckung des Umbaus und Verlusts von T-Tubuli (Instandhaltungsfunktion) zu bewirken. Beide Prozesse gingen mit einem hohen Ko-Lokalisationsquotienten zwischen LTCC und RyR2 und effizienter, funktioneller EK-Kopplung in den Kardiomyozyten einher.
In der vorherigen Studie zeigte es sich, dass die beiden BIN1 Isoformen mit exon11 die stĂ€rksten Regenerations- und Erhaltungsfunktionen zeigten. Ich daher diese beiden Isoformen in einer abschlieĂenden Untersuchung an hiPS-CMs eingesetzt, um ihre Funktion in diese humanen Herzmuskelzellen zu analysieren. Virale Expression dieser beiden BIN-Isoformen fĂŒhrte in den hiPS-CMs zum Auftreten eines komplexen T-Tubulus Netzwerkes, das mit einer Vielzahl von EC-Couplons aus LTCCs und RyR2s dekoriert war. Die Analyse von elektrisch evozierten Kalziumtransienten zeigte eine substanzielle Verbesserung der EK-Kopplungseffizienz in diesen Zellen. Die Ausbildung von EC-Couplons wurde von mir auf zweierlei Weise quantifiziert. In Messungen mit dem Kalziumindikator Fluo4 benutzte ich den CACLEAN-Algorithmus aus meiner Arbeitsgruppe zur analytischen Identifizierung von funktionellen EK-Couplons. Die zusĂ€tzliche Expression des EC-Couplon spezifischen, genetisch kodierten
Kalziumsensors junctin-GCaMP6f erlaubte die exklusive Analyse von Kalziumtransienten in diadischen Kopplungen und die Ergebnisse untermauerte meine Schlussfolgerungen aus den vorherigen Fluo4 Messungen. Beide Methoden zeigten eine hohe Ko-Lokalisation zwischen de-novo T-Tubuli und funktionellen EC-Couplons. Mein Daten demonstrierten somit das hohe Potenzial einer Expression von BIN1-Isoformen mit exon11 fĂŒr die Verbesserung sowohl der Struktur wie auch der EK-Kopplung in hiPSC-CMs.
In der vorliegenden Dissertation habe ich erstmals ein fĂŒr gesunde menschliche Herzen einzigartiges Expressionsmuster fĂŒr BIN1-Isoformen identifiziert. Diese BIN1-Varianten zeigen in adulten Ventrikelzellen aus der Ratte unterschiedliche KapazitĂ€ten sowohl bei der Neubildung wie auch beim Erhalt von T-Tubuli. Sowohl die Ko-Lokalisation von SchlĂŒsselproteinen fĂŒr die EK-Kopplung als auch die EK-Kopplung selber waren in den BIN1-ĂŒberexprimierenden adulten Herzmuskelzellen unter beiden Bedingungen signifikant verbessert. Dies gilt im Besonderen fĂŒr solche BIN1-Varianten, die exon11 beinhalteten, da ihre Effizienz den drei anderen splice Varianten ĂŒberlegen war. BIN1-Isoformen mit exon11 wurden dann ebenfalls in hiPS-CMs untersucht, die ĂŒblicherweise keine T-Tubuli zeigten. Ihre Expression induzierte die de-novo Ausbildung eines komplexen Netzwerks von T-Tubuli und substanziell verbessertes und schnellere EK-Kopplung. Die BIN1 Expression fĂŒhrte zu de-novo T-Tubuli, die mit EC-Couplon dekoriert waren. Dies erachte ich als mechanistische Grundlage der verbesserten EK-Kopplung. Als Fazit kann zusammengefasst werden, dass BIN1 als mögliche neue Zielstruktur fĂŒr die Therapie
von Herzinsuffizienz und der substanziellen Verbesserung der hiPSC-CMs Differenzierung hin zu einem mehr adulten PhÀnotyp angesehen werden kann
Neuronal Signalling Studied with Light-Activated Ion Channels to Target Interneurons, Entrain Hippocampal Gamma Oscillations and Suppress Epileptiform Activity
The versatility and the electrophysiological characteristics of the light-sensitive ion-channels channelrhodopsin-2 (ChR2) and halorhodopsin (NpHR) make these optogenetic tools potent candidates in controlling neuronal firing in models of epilepsy and in providing insights into the physiology and pathology of neuronal network organization and synchronization. The experiments described in this thesis were designed to determine if the ChR2/NpHR system allows specific targeting and manipulation of interneuron activity in cortex and hippocampus, if it allows modulation of gamma oscillations in the hippocampal CA3 area, and if it constitutes a reliable toolbox enabling systematic analysis of epileptic neural circuits and a novel anti-epileptic treatment strategy that relies on optical activation of neurons to interrupt seizures. After successful generation of lentiviral constructs containing opsin genes driven by the interneuron-specific promoters glutamic acid decarboxylase (GAD) 67 and cholecystokinin (CCK) and the production of high-titre lentivirus, it was possible to demonstrate that both microbial opsins are expressed in neuronal cultures and rat motor cortex and hippocampus. Expression of the constructs, however, was not specific for interneurons and expression levels were low compared to the same opsins driven by the calcium calmodulin-binding kinase 2a (Camk2a) promoter: either fluorescence was only visible after immunofluorescent labelling or optical control of neural activity was not achievable despite visible fluorescence. In a separate set of experiments, stimulation of Camk2aâChR2 with ramps of blue light induced oscillations in hippocampal area CA3. Oscillations entrained to modulated ramps over a wide range of frequencies with a frequency-dependent phase relationship. Finally, optical stimulation of halorhodopsin successfully reduced high frequency epileptic EEG activity in a tetanus toxin rat model of focal epilepsy. These results demonstrate that targeting opsins to interneurons with the GAD67 and CCK-promoters is not specific, that the CA3 network has properties that allow it to entrain and synchronize to input from the dentate gyrus, which may help explain how coherence between these two anatomically coupled networks arises, and finally, that optical inhibition of HF discharges with NpHR targeted to pyramidal neurons represents an exciting new tool to be pursued in models of epilepsy both to dissect epileptic networks and for the development of other optogenetic neuromodulation therapies