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The integrity of its DNA is fundamental for every living cell. However, DNA is constantly threatened by exogenous and endogenous damaging agents that can cause a variety of different DNA lesions. The severe consequences of an accumulation of DNA lesions are reflected in cancerogenesis and aging. Several DNA repair mechanisms ensure the repair of DNA lesions and thus maintain DNA integrity. One of these DNA repair mechanisms is nucleotide excision repair (NER), which is famous for its ability to address a large variety of structurally unrelated DNA lesions. A key component of eukaryotic NER is the transcription factor II H (TFIIH) complex, which is not only essential for DNA repair but also for transcription. The TFIIH complex is composed of ten subunits. How these subunits work together during NER to unwind the DNA around the lesion is, however, not yet fully understood. High-resolution structural data and biochemical insights into the function of every subunit are thus indispensable to understand the functional networks within TFIIH. The importance of an intact TFIIH complex is reflected in the severe consequences of patient mutations in the TFIIH subunits XPB, XPD or p8 leading to the hallmark diseases xeroderma pigmentosum, Cockayne syndrome and trichothiodystrophy. Defects in the NER pathway are further associated with several types of cancer including skin cancer.
The herein described work focused on five TFIIH subunits derived from the thermophilic fungus Chaetomium thermophilum, the p34/p44 pair and the ternary XPB/p52/p8 complex. The interaction between p34 and p44 was characterized based on a high-resolution structure of the p34_vWA/p44_RING minimal complex. Biochemical studies of the p34/p44 interaction led to the disclosure of an additional interaction between the p34 and p44 subunits, which had not been characterized so far. The p34/p44 interaction was shown to be central to TFIIH, which justifies the presence of several redundant interfaces to safeguard the interaction between the two proteins and might explain why so far, no patient mutations in these subunits have been identified. The p52 subunit of TFIIH was known to be crucial to stimulate the ATPase activity of XPB, which is required during NER. This work presents the first entire atomic resolution structural characterization of p52, which was derived of several crystal structures of p52 variants and a p52/p8 variant thereby demonstrating the interaction between p52 and p8. The precise structural model of p52 offered the possibility to investigate interactions with other TFIIH subunits in more detail. The middle domain 2 of p52 and the N-terminal domain of XPB were shown to mediate the main interaction between the two subunits. An analysis of the p52 crystal structures within recently published cryo-electron microscopy structures of TFIIH provides a model of how p52 and p8 stimulate the ATPase activity of XPB, which is essential for NER and transcription. The structural and biochemical findings of this work provide an additional building block towards the uncovering of the architecture and function of this essential transcription factor.
DNA-Stabilität und -Regenerationsfähigkeit von humanen Nasenschleimhautzellen in Kulturmodellen
(2021)
Kulturmodelle des respiratorischen Epithels werden zur Klärung multipler Fragestellungen, zum Beispiel der Untersuchung seltener respiratorischer Erkrankungen, wie der primären Ziliendyskinesie (PCD) herangezogen. Hierbei könnten in Zukunft Kulturmodelle integraler Bestandteil des Diagnosealgorithmus werden. Die Isolierung und Kultivierung von Primärzellen des menschlichen Respirationstrakts ist dabei wesentlich komplexer als die Nutzung etablierter Zelllinien. Jedoch ermöglicht die Verwendung der Primärzellkulturen eine exaktere Darstellung des mehrreihigen Flimmerepithels der oberen Atemwege. Die Gewinnung der Primärzellen kann mechanisch mit zusätzlichem enzymatischen Verdau, oder durch sequentielles Auswachsen der Zellen erfolgen. Angewendet werden sowohl Epithelzell-Monokulturen wie auch Cokulturen mit Fibroblasten. Die Nutzung des Air-Liquid Interface in Transwell Systemen ermöglicht in beiden Kulturen die Differenzierung zu einem Kinozilien tragenden Flimmerepithel. Hierbei ist nicht endgültig geklärt, welches Modell die in vivo Gegebenheiten besser darstellt und welche Vorteile diese haben. Außerdem liegen bislang keine Daten über die Zellstabilität und -regenerationsfähigkeit nach genotoxischer Behandlung sowie Informationen über chromosomale Veränderungen während des Zellkultivierungsprozesses über mehrere Passagen vor. Derartige Informationen sind allerdings für die Etablierung eines Kulturmodells der oberen Atemwege von essentieller Bedeutung. Das Ziel der Arbeit war die Untersuchung der DNA-Stabilität und -Regenerationsfähigkeit über drei Passagen nach genotoxischer Behandlung, vergleichend für beide Kulturmodelle sowie die Überprüfung der chromosomalen Stabilität innerhalb des Kultivierungsprozesses und der Funktionsfähigkeit beider Zellkulturen. Zu diesem Zweck wurde eine toxikologische Versuchsreihe von jeweils 10 Spendern für beide Kulturmodelle, Mono- bzw. Cokultur im Air-Liquid Interface über drei Passagen durchgeführt. Hierbei wurde die Grundschädigung, die Schädigung nach einstündiger Behandlung mit 300μl des Alkylanz Methylmethansulfonat (MMS) und nach einer 24-stündigen Regenerationszeit mit dem Comet Assay überprüft. Zur Untersuchung der chromosomalen Stabilität innerhalb des Kulturprozesses wurden parallel Chromosomenaberrationstests an unbehandelten Zellen über drei Passagen durchgeführt. Zur Überprüfung der Funktionsfähigkeit der Zellen wurde ein Interleukin-8 (IL-8) ELISA für 10 Versuchsansätze in der jeweils ersten Passage beider Kulturen verwendet. Hierbei wurde die IL-8-Konzentration im Überstand der unbehandelten Zellkulturen sowie nach 1μg/ml bzw. 10μg/ml Lipopolysaccarid(LPS)- Exposition untersucht. Für die Cokultur wurden sowohl beide Zelltypen gemeinsam als auch die Epithelzellen bzw. die Fibroblasten getrennt betrachtet. In beiden Modellen wurden zusätzlich rasterelektronenmikroskopische (REM) Aufnahmen zur Untersuchung der ziliären Strukturen durchgeführt. Zur Überprüfung der Fibroblastenkontamination in der Monokultur wurden als einmaliger Versuchsablauf Vimentin- Färbungen über drei Passagen angewendet. Mit dem Comet Assay konnte in beiden Modellen eine gute Regeneration der DNA-Integrität nach MMS-induzierter DNA-Schädigung über alle Passagen nachgewiesen werden. Als einmaliger Versuchsansatz wurde das Enzym Methylpuringlykosylase (MPG) als Teil der Basenexzisionsreparatur nachgewiesen. Es ergaben sich Unterschiede in der Kultivierbarkeit der Zellen: die Monokultur zeigte eine gute Zellkultivierbarkeit bis zur dritten Passage. Es konnte jedoch mit der Vimentinfärbung eine Fibroblastenkontamination von Beginn an sowie eine Zunahme mit Höhe der Passage nachgewiesen werden. Es handelt sich hierbei demnach nicht um eine Reinkultur respiratorischer Epithelzellen. Die Cokultur ermöglicht getrennte Epithelzell- bzw. Fibroblastenkulturen, jedoch keine gute Kultivierbarkeit bis in höhere Passage. Methodisch konnte die prinzipielle Funktionsfähigkeit des Chromosomenaberrationstests an primären Nasenschleimhautzellen erstmals für eine große Stichprobenanzahl gezeigt werden. In den durchgeführten Chromosomenaberrationstests konnte eine gewisse Grundschädigung über alle Passagen nachgewiesen werden, jedoch sollten weitere Tests erfolgen, um diese Tendenz zu verifizieren. Die funktionelle Integrität wurde mit dem IL-8 ELISA nach LPS-Exposition sowie durch den Nachweis ziliärer Strukturen im REM exemplarisch bestätigt. Die erhobenen Daten liefern zusammengefasst wichtige Informationen über die Zellstabilität und -regenerationsfähigkeit der bislang verwendeten Kulturmodelle. Insbesondere Informationen über chromosomale Veränderungen sollten in Zukunft genauer betrachtet werden. Von großem Interesse wäre außerdem die Überprüfung derartige Zelleigenschaften in Zellkulturen von PCD erkrankten Spendern.
The development of cellular life on earth is coupled to the formation of lipid-based biological membranes. Although many tools to analyze their biophysical properties already exist, their variety and number is still relatively small compared to the field of protein studies. One reason for this, is their small size and complex assembly into an asymmetric tightly packed lipid bilayer showing characteristics of a two-dimensional heterogenous fluid. Since membranes are capable to form dynamic, nanoscopic domains, enriched in sphingolipids and cholesterol, their detailed investigation is limited to techniques which access information below the diffraction limit of light. In this work, I aimed to extend, optimize and compare three different labeling approaches for sphingolipids and their subsequent analysis by the single-molecule localization microscopy (SMLM) technique direct stochastic optical reconstruction microscopy (dSTORM). First, I applied classical immunofluorescence by immunoglobulin G (IgG) antibody labeling to detect and quantify sphingolipid nanodomains in the plasma membrane of eukaryotic cells. I was able to identify and characterize ceramide-rich platforms (CRPs) with a size of ~ 75nm on the basal and apical membrane of different cell lines. Next, I used click-chemistry to characterize sphingolipid analogs in living and fixed cells. By using a combination of fluorescence microscopy and anisotropy experiments, I analyzed their accessibility and configuration in the plasma membrane, respectively. Azide-modified, short fatty acid side chains, were accessible to membrane impermeable dyes and localized outside the hydrophobic membrane core. In contrast, azide moieties at the end of longer fatty acid side chains were less accessible and conjugated dyes localized deeper within the plasma membrane. By introducing photo-crosslinkable diazirine groups or chemically addressable amine groups, I developed methods to improve their immobilization required for dSTORM. Finally, I harnessed the specific binding characteristics of non-toxic shiga toxin B subunits (STxBs) and cholera toxin B subunits (CTxBs) to label and quantify glycosphingolipid nanodomains in the context of Neisseria meningitidis infection. Under pyhsiological conditions, these glycosphingolipids were distributed homogenously in the plasma membrane but upon bacterial infection CTxB detectable gangliosides accumulated around invasive Neisseria meningitidis. I was able to highlight the importance of cell cycle dependent glycosphingolipid expression for the invasion process. Blocking membrane accessible sugar headgroups by pretreatment with CTxB significantly reduced the number of invasive bacteria which confirmed the importance of gangliosides for bacterial uptake into cells. Based on my results, it can be concluded that labeling of sphingolipids should be carefully optimized depending on the research question and applied microscopy technique. In particular, I was able to develop new tools and protocols which enable the characterization of sphingolipid nanodomains by dSTORM for all three labeling approaches.
Touch sensation is the ability to perceive mechanical cues which is required for essential behaviors. These encompass the avoidance of tissue damage, environmental perception, and social interaction but also proprioception and hearing. Therefore research on receptors that convert mechanical stimuli into electrical signals in sensory neurons remains a topical research focus. However, the underlying molecular mechanisms for mechano-metabotropic signal transduction are largely unknown, despite the vital role of mechanosensation in all corners of physiology.
Being a large family with over 30 mammalian members, adhesion-type G protein-coupled receptors (aGPCRs) operate in a vast range of physiological processes. Correspondingly, diverse human diseases, such as developmental disorders, defects of the nervous system, allergies and cancer are associated with these receptor family. Several aGPCRs have recently been linked to mechanosensitive functions suggesting, that processing of mechanical stimuli may be a common feature of this receptor family – not only in classical mechanosensory structures.
This project employed Drosophila melanogaster as the candidate to analyze the aGPCR Latrophilin/dCIRL function in mechanical nociception in vivo. To this end, we focused on larval sensory neurons and investigated molecular mechanisms of dCIRL activity using noxious mechanical stimuli in combination with optogenetic tools to manipulate second messenger pathways. In addition, we made use of a neuropathy model to test for an involvement of aGPCR signaling in the malfunctioning peripheral nervous system. To do so, this study investigated and characterized nocifensive behavior in dCirl null mutants (dCirlKO) and employed genetically targeted RNA-interference (RNAi) to cell-specifically manipulate nociceptive function.
The results revealed that dCirl is transcribed in type II class IV peripheral sensory neurons – a cell type that is structurally similar to mammalian nociceptors and detects different nociceptive sensory modalities. Furthermore, dCirlKO larvae showed increased nocifensive behavior which can be rescued in cell specific reexpression experiments. Expression of bPAC (bacterial photoactivatable adenylate cyclase) in these nociceptive neurons enabled us to investigate an intracellular signaling cascade of dCIRL function provoked by light-induced elevation of cAMP. Here, the findings demonstrated that dCIRL operates as a down-regulator of nocifensive behavior by modulating nociceptive neurons. Given the clinical relevance of this results, dCirl function was tested in a chemically induced neuropathy model where it was shown that cell specific overexpression of dCirl rescued nocifensive behavior but not nociceptor morphology.
The goal of this doctoral thesis is to identify appropriate methods for the estimation of connectivity and for measuring synchrony between spike trains from in vitro neuronal networks. Special focus is set on the parameter optimization, the suitability for massively parallel spike trains, and the consideration of the characteristics of real
recordings. Two new methods were developed in the course of the optimization which outperformed other methods from the literature. The first method “Total spiking probability edges” (TSPE) estimates the effective connectivity of two spike trains, based on the
cross-correlation and a subsequent analysis of the cross-correlogram. In addition to the estimation of the synaptic weight, a distinction between excitatory and inhibitory connections is possible. Compared to other methods, simulated neuronal networks could be estimated with higher accuracy, while being suitable for the analysis of massively parallel spike trains. The second method “Spike-contrast” measures the synchrony of parallel spike trains
with the advantage of automatically optimizing its time scale to the data. In contrast to other methods, which also adapt to the characteristics of the data, Spike-contrast is more robust to erroneous spike trains and significantly faster for large amounts of parallel spike trains. Moreover, a synchrony curve as a function of the time scale is generated by Spike-contrast. This optimization curve is a novel feature for the analysis of parallel spike trains.
Patienten mit leicht bis hochgradigen Schallleitungs-, Schallempfindungs- und kombinierten Schwerhörigkeiten werden routinemäßig nach erfolglosem Hörgerätetrageversuch mit aktiven Mittelohrimplantaten versorgt. Aktive Mittelohrimplantate können an verschiedene Strukturen des Mittelohrs angekoppelt werden. Der Ort der Ankopplung ist abhängig vom Hörverlust und der individuellen Physiologie des Mittelohres. Die Hörverbesserung ist dabei stark von der Kopplungseffizienz des Implantatwandlers an die Mittelohrstruktur abhängig.
Aktuell gibt es keine zufriedenstellende Möglichkeit die Kopplungseffizienz intraoperativ zu bestimmen. Daher wird eine objektive Methode eingeführt, um intraoperativ auditorische Hirnstammantworten (BERAs) bei Stimulation über das Implantat abzuleiten. Die Vibrant Soundbrigde® (VSB) wird dabei mit einem Drahtlosüberträger (miniTEK, Signia GmbH, Erlangen) und der Carina®-Aktuator über ein Audiokabel mit der BERA-Anlage verbunden. Die BERA-Anlage überträgt die Stimuli direkt an das Implantat, welches an die Mittelohrstruktur angekoppelt ist. Die BERA-Antworten werden bei der VSB durch einen optimierten VSB-CE-Chirp und beim Carina®-System durch den Standard CE-Chirp evoziert, beginnend bei Pegeln oberhalb der Knochenleitungshörschwelle bis unter die Registrierungsschwelle. Diese Methode kann die intraoperative Integrität des Implantats sowie die Kopplungseffizienz bestimmen, um eine Aussage über den zu erwartenden Hörerfolg treffen zu können. Darüber hinaus kann die versorgte Hörschwelle verwendet werden, um die Anpassung bei Kindern oder schwierigen Fällen zu unterstützen und um eine Hörverschlechterung über die Zeit zu erfassen.
Zusammenfassend, konnte eine Methode zur Bestimmung der intraoperativen Kopplungseffizienz während der Implantation von VSBs und Carinas® etabliert werden. Darüber hinaus werden intraoperative BERA-Daten von 30 VSB- und 10-Carina®-Patienten sowie deren Hörergebnisse gezeigt.
The obligate human pathogen Neisseria meningitidis is a major cause of sepsis and meningitis worldwide. It affects mainly toddlers and infants and is responsible for thousands of deaths each year. In this study, different aspects of the importance of sphingolipids in meningococcal pathogenicity were investigated. In a first step, the acid sphingomyelinase (ASM), which degrades membrane sphingomyelin to ceramide, was studied in the context of meningococcal infection. A requirement for ASM surface activity is its translocation from the lysosomal compartment to the cell surface, a process that is currently poorly understood.
This study used various approaches, including classical invasion and adherence assays, flow cytometry, and classical and super resolution immunofluorescence microscopy (dSTORM). The results showed that the live, highly piliated N. meningitidis strain 8013/12 induced calcium-dependent ASM translocation in human brain microvascular endothelial cells (HBMEC). Furthermore, it promoted the formation of ceramide-rich platforms (CRPs). In addition, ASM translocation and CRP formation were observed after treating the cells with pili-enriched fractions derived from the same strain. The importance for N. meningitidis to utilize this pathway was shown by the inhibition of the calcium-dependent ASM translocation, which greatly decreased the number of invasive bacteria.
I also investigated the importance of the glycosphingolipids GM1 and Gb3. The results showed that GM1, but not Gb3, plays an important role in the ability of N. meningitidis to invade HBMEC. By combining dSTORM imaging and microbiological approaches, we demonstrated that GM1 accumulated prolifically around bacteria during the infection, and that this interaction seemed essential for meningococcal invasion.
Sphingolipids are not only known for their beneficial effect on pathogens. Sphingoid bases, including sphingosine, are known for their antimicrobial activity. In the last part of this study, a novel correlative light and electron microscopy approach was established in the combination with click chemistry to precisely localize azido-functionalized sphingolipids in N. meningitidis. The result showed a distinct concentration-dependent localization in either the outer membrane (low concentration) or accumulated in the cytosol (high concentration). This pattern was confirmed by mass spectrometry on separated membrane fractions. Our data provide a first insight into the underlying mechanism of antimicrobial sphingolipids.
We examined 143 patients suffering from FMS, a syndrome characterized by chronic widespread pain, sleep disturbances, and fatigue. Etiology and pathophysiology of FMS are scarcely understood. In recent years abnormalities of small Aδ- and C-nerve fibers have been found in subgroups of FMS patients. It is yet unclear how such SFP is caused in FMS patients and how it contributes to FMS symptoms.
We used CCM to analyze corneal small nerve fibers and associated LC, comparing FMS patients’ results to those from 65 healthy controls and 41 disease controls suffering from SFN. We, further, assessed expression levels of mRNA and miRNA in keratinocytes taken from skin punch biopsies of FMS patients and healthy controls kept as monocellular cell cultures. A screening was performed using NGS in a small cohort of 12 FMS patients and 5 healthy controls. Results were validated in larger cohorts by qRT-PCR.
As in previous studies IENFD and CNFD were reduced in a subgroup of FMS patients. We found identical LC densities in FMS patients, healthy controls, and SFN patients. The subpopulation of dLCfiber contact in FMS and SFN patients was lower than in healthy controls. Our RNA expression analysis revealed one mRNA that was expressed higher in FMS patients than in controls: PRSS21.
We conclude that reduced neurotrophic signaling of LC may contribute to SFP in the cornea. Epidermal PRSS21 expression and dLCfiber contact density are promising biomarker candidates for FMS diagnosis.
Die Multiple Sklerose (MS) ist eine chronisch-entzündliche Autoimmunerkrankung des zentralen Nervensystems (ZNS) und stellt die häufigste Ursache frühzeitiger Behinderung junger Erwachsener dar. Kennzeichnend sind multifokale ZNS-Läsionen, die durch Inflammation, Demyelinisierung und Axonschäden geprägt sind und zu multiplen neurologischen Defiziten führen. Derzeit ist es mithilfe der verlaufsmodifizierenden Therapie möglich, die Immunantwort abzuschwächen und damit die Krankheitsprogression zu verzögern. Geheilt werden kann die Erkrankung jedoch bislang nicht. Dabei ist nicht hinreichend geklärt, ob die neuen Therapieoptionen über die Immunmodulation/-suppression hinaus einen anhaltenden Schutz vor der langfristigen Neurodegeneration bieten.
Basierend auf den vielversprechenden Ergebnissen klinischer Studien zur Therapie der schubförmig-remittierenden MS mit dem Anti-CD52-Antikörper Alemtuzumab, der zu einer Depletion CD52-exprimierender Immunzellen führt, wurden diesbezüglich Analysen in MS-Tiermodellen durchgeführt. Da die Untersuchung der zugrunde liegenden Patho- und Effektormechanismen am Menschen kaum möglich ist, ist die MS-Forschung für ein tiefergehendes Verständnis auf Tiermodelle angewiesen. Die experimentelle autoimmune Enzephalomyelitis (EAE) ist hierbei das am weitesten verbreitete Modell der MS, wofür vor allem der C57BL/6 (B6) -Mausstamm verwendet wird, da auf diesem Hintergrund die meisten genmodifizierten Mäuse gezüchtet werden. Jene tierexperimentellen Studien, in denen ein muriner Anti-CD52-Antikörper im frühen Krankheitsstadium der EAE (Auftreten erster paralytischer Symptome) verabreicht wurde, erbrachten den Hinweis einer neuroprotektiven und scheinbar regenerativen Wirkung des Antikörpers.
Über einen neuroprotektiven Effekt von Alemtuzumab im schwer behandelbaren chronisch-progredienten Stadium der MS ist jedoch wenig bekannt. Die vorliegende Arbeit ist die erste detaillierte Untersuchung zum Einfluss des murinen Anti-CD52-Antikörpers auf die Demyelinisierung, den Axonschaden und die Hirnatrophie in der MP4-induzierten EAE der B6-Maus im chronischen Verlauf der Erkrankung (ab stabilem Plateau der klinischen Symptomatik). MP4 ist ein Myelinfusionsprotein aus MBP (Myelin-Basisches-Protein) und PLP (Proteolipidprotein), welches in B6-Mäusen durch aktive Immunisierung eine EAE induziert, die chronisch verläuft und als eines von wenigen Modellen neben der T-Zell-Abhängigkeit die an Bedeutung zunehmende B-Zell-Komponente der MS darstellt. Histopathologisch finden sich in der chronischen MP4-induzierten EAE eine ausgeprägte Rückenmarks- und Kleinhirnschädigung, die vor allem im Kleinhirn durch eine B-Zell-Aggregation charakterisiert ist.
Nachdem die MP4-immunisierten Mäuse im chronischen Stadium der EAE an fünf aufeinanderfolgenden Tagen mit 10 mg/kg Körpergewicht murinem Anti-CD52-spezifischem IgG2a-Isotypantikörper bzw. murinem unspezifischem IgG2a-Isotyp-Kontroll-Antikörper behandelt worden waren, wurde die Lymphozytendepletion im peripheren Blut durchflusszytometrisch ermittelt und deren Einfluss auf MP4-spezifische Antikörper anhand eines indirekten Enzyme-linked Immunosorbent Assays (ELISAs) untersucht. Als Marker für Axonschäden wurde im Serum vorhandenes phosphoryliertes Neurofilament-Heavy (pNF-H) mithilfe eines indirekten Sandwich-ELISAs quantitativ bestimmt. Rückenmark und Kleinhirn wurden ultrastrukturell auf Veränderungen der Myelinisierung (mittels g-Ratio: Axondurchmesser geteilt durch Gesamtdurchmesser der Nervenfaser) und auf Axonpathologien (verringerter Abstand benachbarter Neurofilamente, axolytische Axone, axonaler Verlust) untersucht. Die Hirnatrophie wurde MRT-basiert gemessen und der klinische Verlauf täglich evaluiert.
Durch die Anti-CD52-Antikörperbehandlung wurde die T- und B-Zellzahl zwar drastisch vermindert, die MP4-spezifische Antikörperproduktion blieb davon jedoch unbeeinträchtigt. Ein günstiger Effekt auf die De- und Remyelinisierung war nicht festzustellen. Das Hirnvolumen und die klinische Präsentation der Mäuse blieben ebenfalls unverändert. Während kein Unterschied der pNF-H-Konzentration zu erkennen war, konnte ultrastrukturell jedoch ein geringerer Axonschaden nachgewiesen werden.
Insgesamt legen diese Ergebnisse nahe, dass der Anti-CD52-Antikörper im chronischen Verlauf der EAE/MS wenig Einfluss auf die neurodegenerativen Prozesse nimmt und die Regeneration nicht fördern kann. Die Ursache liegt vermutlich in der Undurchlässigkeit der Bluthirnschranke für Antikörper sowie dem limitierten Verständnis der Antikörperwirkung im ZNS. Die vorliegende Studie regt somit zur Etablierung von ZNS-wirksamen Antikörpern an und unterstreicht die Bedeutung der Entwicklung von selektiveren neuroprotektiven und remyelinisierungsfördernden Behandlungsansätzen, die eine wertvolle Ergänzung zur verlaufsmodifizierenden Therapie darstellen könnten.
Adrenal Cushing’s Syndrome (CS) is a rare but life-threatening disease and therefore it is of great importance to understand the pathogenesis leading to adrenal CS. It is well accepted that Protein Kinase A (PKA) signalling mediates steroid secretion in adrenocortical cells. PKA is an inactive heterotetramer, consisting of two catalytic and two regulatory subunits. Upon cAMP binding to the regulatory subunits, the catalytic subunits are released and are able to phosphorylate their target proteins. Recently, activating somatic mutations affecting the catalytic subunit a of PKA have been identified in a sub-population of cortisol-producing adenomas (CPAs) associated with overt CS. Interestingly, the PKA regulatory subunit IIb has long been known to have significantly lower protein levels in a sub-group of CPAs compared to other adrenocortical tumours. Yet, it is unknown, why these CPAs lack the regulatory subunit IIb, neither are any functional consequences nor are the underlying regulation mechanisms leading to reduced RIIb levels known. The results obtained in this thesis show a clear connection between Ca mutations and reduced RIIb protein levels in CPAs but not in other adrenocortical tumours. Furthermore, a specific pattern of PKA subunit expression in the different zones of the normal adrenal gland is demonstrated. In addition, a Ca L206R mutation-mediated degradation of RIIb was observed in adrenocortical cells in vitro. RIIb degradation was found to be mediated by caspases and by performing mutagenesis experiments of the regulatory subunits IIb and Ia, S114 phosphorylation of RIIb was identified to make RIIb susceptible for degradation. LC-MS/MS revealed RIIb interaction partners to differ in the presence of either Ca WT and Ca L206R. These newly identified interaction partners are possibly involved in targeting RIIb to subcellular compartments or bringing it into spatial proximity of degrading enzymes. Furthermore, reducing RIIb protein levels in an in vitro system were shown to correlate with increased cortisol secretion also in the absence of PRKACA mutations. The inhibiting role of RIIb in cortisol secretion demonstrates a new function of this regulatory PKA subunit, improving the understanding of the complex regulation of PKA as key regulator in many cells.
Bei der zystischen Fibrose (CF) sowie der primären Ziliendyskinesie (PCD) handelt es sich um zwei seltene Erkrankungen, die unter anderem den mukoziliären Transport beeinträchtigen. CF gehört hierbei zu den am häufigsten vorkommenden angeborenen Stoffwechselerkrankungen, wobei Betroffene unter einem Defekt des Cystic Fibrosis Transmembrane Conductor Regulator (CFTR)-Gens leiden, der durch die Produktion von hochviskosem Sekret in muzinproduzierenden Organen, wie dem gastrointestinalen Trakt und der Lunge, gekennzeichnet ist. Patienten, die an PCD leiden, weisen Defekte in, zum jetzigen Zeitpunkt, ca. 38 bekannten und PCD-assoziierten Genen auf, die in strukturellen Defekten des ziliären Apparats und somit in dysfunktionalen Kinozilien resultieren. Da aktuell weder für die CF noch für die PCD eine Heilung möglich ist, steht bei der Therapie vor allem die Linderung der Symptome im Fokus. Grundlegendes Ziel ist der langfristige Erhalt der Lungenfunktion sowie die Prävention bakterieller Infekte. Als bisherige Modellsysteme zur Erforschung möglicher Therapeutika gelten Tiermodelle, die den humanen Phänotyp aufgrund von Speziesdiversität nicht vollständig abbilden können. Als vielversprechende Testsysteme für die zystische Fibrose gelten humane intestinale Organoidkulturen. Nachdem allerdings vorwiegend respiratorische Symptome für die Mortalität der Patienten verantwortlich sind, stellen CF-Atemwegsmodelle bessere Testsysteme für zukünftige Therapeutika dar. Atmungsorganoidkulturen wurden verwendet, um die CFTR-Funktionalität zu untersuchen, repräsentieren aber nicht vollständig die in vivo Situation. Deshalb werden zur Entwicklung neuer Therapiestrategien patientenspezifische 3D in vitro Testsysteme der humanen Atemwege benötigt, die insbesondere im Hinblick auf personalisierte Medizin ihren Einsatz finden. In der vorliegenden Arbeit wurde eine für den Lehrstuhl neue Methode zur Zellgewinnung aus nasalen Schleimhautabstrichen etabliert, die eine standardisierte Versorgung mit humanem Primärmaterial garantiert. Zur Generierung einer krankheitsspezifischen Zelllinie, wie beispielsweise einer PCD-Zelllinie mit Hilfe des CRISPR/Cas9-Systems, ist eine Atemwegszelllinie erforderlich, die die in vivo Situation vollständig repräsentiert. So wurden vier verschiedene respiratorische Epithelzelllinien (HBEC3-KT, Calu-3, VA10 und Cl-huAEC) auf ihren mukoziliären Phänotyp hin untersucht, wobei lediglich die Zelllinie HBEC3-KT in zilientragende Zellen differenzierte. Diese zeigten jedoch nur auf ca. 5 % der Modelloberfläche Kinozilien, wodurch die humane respiratorische Mukosa nicht komplett abgebildet werden konnte und die HBEC3-KT-Zelllinie keine geeignete Zelllinie zur Generierung einer PCD-Zelllinie darstellte. Mit Hilfe des Tissue Engineering war es möglich, 3D in vitro Testsysteme basierend auf zwei unterschiedlichen Matrices, der biologischen SIS (small intestinal submucosa) und der synthetischen Polyethylenterephthalat (PET)-Membran, aufzubauen. Es wurden 3D Atemwegstestsysteme mit humanen primären nasalen und tracheobronchialen Epithelzellen generiert. Ergänzend zu histologischen Untersuchungen und zur Charakterisierung spezifischer Marker des respiratorischen Systems mittels Immunfluoreszenz, wurde die Ultrastruktur der Modelle, mit speziellem Fokus auf ziliäre Strukturen, analysiert. Um Rückschlüsse auf die ziliäre Funktionalität ziehen zu können und somit eine hohe in vivo Korrelation zu bestätigen, wurde im Rahmen dieser Arbeit am Lehrstuhl für Tissue Engineering und Regenerative Medizin die Methode der Hochgeschwindigkeitsvideomikroskopie etabliert, welche die Analyse der Zilienschlagfrequenz sowie des mukoziliären Transports ermöglicht. Ebenfalls wurde der Einfluss von isotoner Kochsalzlösung und des � 2-adrenergen Agonisten Salbutamol, das vor allem als Bronchodilatator bei Asthmapatienten eingesetzt wird, auf die Zilienschlagfrequenz analysiert. Es konnte gezeigt werden, dass beide Substanzen den Zilienschlag im Atemwegsmodell erhöhen. Zur Generierung der Testsysteme der beiden seltenen Erkrankungen CF und PCD wurden Epithelzellen der betroffenen Patienten zunächst mittels nicht-invasiver Raman-Spektroskopie auf einen potentiellen Biomarker untersucht, welcher Einsatz in der Diagnostik der beiden Krankheiten finden könnte. Es konnte jedoch weder für die CF noch für die PCD ein Biomarker aufgedeckt werden. Jedoch zeigten PCD-Zellen eine geringe Auftrennung gegenüber nicht-PCD Zellen. Anschließend wurden 3D-Atemwegstestsysteme basierend auf Patientenzellen aufgebaut. Der Phänotyp der CF-Modelle wurde mittels immunhistologischer Färbung und der Analyse des gestörten mukoziliären Transports verifiziert. Strukturelle ziliäre Defekte konnten durch die ultrastrukturelle Analyse von Zilienquerschnitten in drei donorspezifischen PCD-Modellen identifiziert werden. Darüber hinaus konnte die ziliäre Funktionalität mit Hilfe der Hochgeschwindigkeitsvideomikroskopie nicht nachgewiesen werden. Zusammenfassend ist es in dieser Arbeit gelungen, eine neue Methode zur vollständigen Charakterisierung von 3D-Atemwegstestsystemen zu etablieren, die die Analyse der Zilienschlagfrequenz sowie des mukoziliären Transports ermöglicht. Es konnte erstmalig gezeigt werden, dass mit Hilfe des Tissue Engineering ein personalisiertes Krankheitsmodell für die PCD auf Segmenten eines dezellularisierten porzinen Jejunums generiert werden kann, das zukünftig ein Testsystem für potentielle Therapeutika darstellen kann.
Summary
Bees, like many other organisms, evolved an endogenous circadian clock, which enables them to foresee daily environmental changes and exactly time foraging flights to periods of floral resource availability. The social lifestyle of a honey bee colony has been shown to influence circadian behavior in nurse bees, which do not exhibit rhythmic behavior when they are nursing. On the other hand, forager bees display strong circadian rhythms. Solitary bees, like the mason bee, do not nurse their offspring and do not live in hive communities, but face the same daily environmental changes as honey bees. Besides their lifestyle mason and honey bees differ in their development and life history, because mason bees overwinter after eclosion as adults in their cocoons until they emerge in spring. Honey bees do not undergo diapause and have a relatively short development of a few weeks until they emerge. In my thesis, I present a comparison of the circadian clock of social honey bees (Apis mellifera) and solitary mason bees (Osmia bicornis and Osmia cornuta) on the neuroanatomical level and behavioral output level.
I firstly characterized in detail the localization of the circadian clock in the bee brain via the expression pattern of two clock components, namely the clock protein PERIOD (PER) and the neuropeptide Pigment Dispersing Factor (PDF), in the brain of honey bee and mason bee. PER is localized in lateral neuron clusters (which we called lateral neurons 1 and 2: LN1 and LN2) and dorsal neuron clusters (we called dorsal lateral neurons and dorsal neurons: DLN, DN), many glia cells and photoreceptor cells. This expression pattern is similar to the one in other insect species and indicates a common ground plan of clock cells among insects. In the LN2 neuron cluster with cell bodies located in the lateral brain, PER is co-expressed with PDF. These cells build a complex arborization network throughout the brain and provide the perfect structure to convey time information to brain centers, where complex behavior, e.g. sun-compass orientation and time memory, is controlled. The PDF arborizations centralize in a dense network (we named it anterio-lobular PDF hub: ALO) which is located in front of the lobula. In other insects, this fiber center is associated with the medulla (accessory medulla: AME). Few PDF cells build the ALO already in very early larval development and the cell number and complexity of the network grows throughout honey bee development. Thereby, dorsal regions are innervated first by PDF fibers and, in late larval development, the fibers grow laterally to the optic lobe and central brain. The overall expression pattern of PER and PDF are similar in adult social and solitary bees, but I found a few differences in the PDF network density in the posterior protocerebrum and the lamina, which may be associated with evolution of sociality in bees.
Secondly, I monitored activity rhythms, for which I developed and established a device to monitor locomotor activity rhythms of individual honey bees with contact to a mini colony in the laboratory. This revealed new aspects of social synchronization and survival of young bees with indirect social contact to the mini colony (no trophalaxis was possible). For mason bees, I established a method to monitor emergence and locomotor activity rhythms and I could show that circadian emergence rhythms are entrainable by daily temperature cycles. Furthermore, I present the first locomotor activity rhythms of solitary bees, which show strong circadian rhythms in their behavior right after emergence. Honey bees needed several days to develop circadian locomotor rhythms in my experiments. I hypothesized that honey bees do not emerge with a fully matured circadian system in the hive, while solitary bees, without the protection of a colony, would need a fully matured circadian clock right away after emergence. Several indices in published work and preliminary studies support my hypothesis and future studies on PDF expression in different developmental stages in solitary bees may provide hard evidence.
The Chimpanzees of the Comoé National Park, Ivory Coast. Status, distribution, ecology and behavior
(2021)
Although wild chimpanzees (Pan troglodytes) have been studied intensely for more than 50 years, there are still many aspects of their ecology and behavior that are not well understood. Every time that a new population of chimpanzees has been studied, new behaviors and unknown aspects of their ecology have been discovered. All this accumulated knowledge is helping us to piece together a model of how could last human and chimpanzee common ancestors have lived and behaved between seven and five million years ago. Comoé chimpanzees had never been studied in depth, until we started our research in October 2014, only a few censuses had been realized. The last surveys prior our work, stated that the population was so decimated that was probably functionally extinct. When we started this research, we had to begin with a new intensive survey, using new methods, to ascertain the real status and distribution of the chimpanzees living in Comoé National Park (CNP). During the last five years, we have realized a deep study aiming to know more about their ecology and behavior. We combined transects and reconnaissance marches (recces) with the use of camera traps, for the first time in CNP, obtaining a wealth of data that is not fully comprised in this dissertation. With this research, we determined that there is a sustainable continuous population of Western chimpanzees (Pan troglodytes verus) in CNP and the adjacent area of Mont Tingui, to the West, with a minimum of 127 weaned chimpanzees living in our main 900 km2 study area, SW of CNP. We found that this population is formed by a minimum of eight different chimpanzee communities, of which we studied seven, four of them more in detail. These chimpanzees spent much more time in the forest than in the savanna habitats.
We also found that Comoé chimpanzees consumed at least 58 different food items in their dit, which they obtained both from forest and savanna habitats. Another finding was that insectivory had an important role in their diet, with at least four species of ants, three of termites and some beetle larvae. These chimpanzees also hunted at least three species of monkeys and maybe rodents and duikers and occasionally consumed the big land snails of genus Achatina. We found that, during the fruit scarcity period in the late rainy season, they intensely consumed the cambium of Ceiba pentandra, as fallback food, much more than the bark or cambium of any other tree species. Another interesting finding was that all the chimpanzees in the studied area realized this particular bark-peeling behavior and had been repeatedly peeling the trees of this species for years. This did not increase tree mortality and the damage caused to the trees was healed in two years, not reducing the growth, thus being a sustainable use of the trees. We found that Comoé chimpanzees produced and used a great variety of tools, mainly from wooden materials, but also from stone and herbaceous vegetation.
Their tool repertory included stick tools to dip for Dorylus burmeisteri ants, to fish for Camponotus and Crematogaster ants, to dip for honey, mainly from Meliponini stingless bees, but sometimes from honey bees (Apis mellifera). It also included the use of stick tools to fish termites of Macrotermes subhyalinus and Odontotermes majus (TFTs), to dip for water from tree holes and investigatory probes for multiple purposes. Additionally, these chimpanzees used leaf-sponges to drink from tree holes and to collect clayish water from salt-licks. They also used stones to hit the buttresses of trees during displays, the so called accumulative stone throwing behavior and probably used stones as hammers, to crack open hard-shelled Strichnos spinosa and Afraegle paniculata fruits and Achatina snails. The chimpanzees also used objects that are not generally accepted as animal tools, for being attached to the substrate, with different purposes: they drummed buttresses of trees with hands and/or feet to produce sound during male displays and they pounded open hard-shelled fruits, Achatina snails and Cubitermes termite mounds on stone or root anvils. We finally measured the stick tools and found significant differences between them suggesting that they were specialized tools made specifically for every purpose. We studied more in detail the differences between apparently similar tools, the honey dipping tools and the water dipping tools, often with brushes made at their tips to collect the fluids. These last tools were exclusive from Comoé and have not been described at any other site. We found that total length, diameter and brush length were significantly different, suggesting that they were specialized tools. We concluded that Comoé chimpanzees had a particular culture, different from those of other populations of Western chimpanzees across Africa. Efficient protection, further research and permanent presence of research teams are required to avoid that this unique population and its culture disappears by the poaching pressure and maybe by the collateral effects of climate change.
The resolution of fluorescence light microscopy was long believed to be limited by the diffraction limit of light of around 200-250 nm described in 1873 by Ernst Abbe. Within the last decade, several approaches, such as structured illumination microscopy (SIM), stimulated emission depletion STED and (direct) stochastic optical reconstruction microscopy (d)STORM have been established to bypass the diffraction limit. However, such super-resolution techniques enabling a resolution <100 nm require specialized and expensive setups as well as expert knowledge in order to avoid artifacts. They are therefore limited to specialized laboratories. Recently, Boyden and colleagues introduced an alternate approach, termed expansion microscopy (ExM). The latter offers the possibility to perform superresolution microscopy on conventional confocal microscopes by embedding the sample into a swellable hydrogel that is isotropically expanded. Since its introduction in 2015, expansion microscopy has developed rapidly offering protocols for 4x, 10x and 20x expansion of proteins and RNA in cells, tissues and human clinical specimens.
Mitochondria are double membrane-bound organelles and crucial to the cell by performing numerous tasks, from ATP production through oxidative phosphorylation, production of many important metabolites, cell signaling to the regulation of apoptosis. The inner mitochondrial membrane is strongly folded forming so-called cristae. Besides being the location of the oxidative phosphorylation and therefore energy conversion and ATP production, cristae have been of great interest because changes in morphology have been linked to a plethora of diseases from cancer, diabetes, neurodegenerative diseases, to aging and infection. However, cristae imaging remains challenging as the distance between two individual cristae is often below 100 nm. Within this work, we demonstrate that the mitochondrial creatine kinase MtCK linked to fluorescent protein GFP (MtCK-GFP) can be used as a cristae marker. Upon fourfold expansion, we illustrate that our novel marker enables visualization of cristae morphology and localization of mitochondrial proteins relative to cristae without the need for specialized setups. Furthermore, we show the applicability of expansion microscopy for several bacterial pathogens, such as Chlamydia trachomatis, Simkania negevensis, Neisseria gonorrhoeae and Staphylococcus aureus. Due to differences in bacterial cell walls, we reveal important aspects for the digestion of pathogens for isotropic expansion. We further show that expansion of the intracellular pathogens C. trachomatis and S. negevensis, enables the differentiation between the two distinct developmental forms, catabolic active reticulate bodies (RB) and infectious elementary bodies (EB), on a conventional confocal microscope. We demonstrate the possibility to precisely locate chlamydial effector proteins, such as CPAF or Cdu1, within and outside the chlamydial inclusion. Moreover, we show that expansion microscopy enables the investigation of bacteria, herein S. aureus, within LAMP1 and LC3-II vesicles. With the introduction of the unnatural α-NH2-ω-N3-C6-ceramide, we further present the first approach for the expansion of lipids that may also be suitable for far inaccessible molecule classes like carbohydrates. The efficient accumulation and high labeling density of our functionalized α-NH2-ω-N3-C6-ceramide in both cells and bacteria enables in combination with tenfold expansion nanoscale resolution (10-20 nm) of the interaction of proteins with the plasma membrane, membrane of organelles and bacteria. Ceramide is the central molecule of the sphingolipid metabolism, an important constituent of cellular membranes and regulates many important cellular processes such as differentiation, proliferation and apoptosis. Many studies report about the importance of sphingolipids during infection of various pathogens. While the transport of ceramide to Chlamydia has been reported earlier, one of the unanswered questions remaining was if ceramide forms parts of the outer or inner bacterial membrane. Expansion of α-NH2-ω-N3-C6-ceramide enabled the visualization of ceramide in the inner and outer membrane of C. trachomatis and their distance was determined to be 27.6 ± 7.7 nm.
Neuropathische Schmerzen treten in der klinischen Praxis häufig auf und
beeinträchtigen in hohem Maße die Lebensqualität der Patienten. Bedingt durch eine
Schädigung somatosensorischer Nervenstrukturen im peripheren oder zentralen
Nervensystem ist der Schmerz meist durch eine Allodynie, Hyperalgesie oder
einschießende Schmerzen charakterisiert. Diese Symptome lassen sich durch
gängige Schmerzmittel kaum lindern. In jüngerer Zeit rückte die Blut-Rückenmarkschranke immer mehr in den Fokus verschiedener Untersuchungen, da auch
einige nicht-schmerzhafte Erkrankungen (z.B. Multiple Sklerose und Amyotrophe
Lateralsklerose) zur Veränderung dieser Barriere mit folgender Permeabilitätserhöhung
durch reduzierte Tight Junction-Protein-Expression führen. Die Blut-
Rückenmarkschranke dichtet Gefäße des Rückenmarks ab und verhindert das
Eindringen toxischer oder proanalgetischer Mediatoren in das zentrale Nervensystem.
Dafür ist die Funktion der Tight Junction-Proteine zur Aufrechterhaltung dieser Barriere
essentiell. Dennoch konnte die Rolle der Blut-Rückenmarkschranke bei Neuropathie
noch nicht vollständig erörtert werden. Die Untersuchungen meiner Arbeitsgruppe
konnten bereits zeigen, dass eine lockere Ligatur des N. ischiadicus bei Ratten (CCI;
chronic constriction injury) zur Entwicklung einer thermischen und mechanischen
Hyperalgesie sowie eingeschränkten motorischen Funktionen führt. Zudem konnte
eine Störung der Blut-Rückenmarkschranke nach einer CCI nachgewiesen werden, da
es Tracern unterschiedlicher molekularer Größe möglich war das Rückenmark zu
penetrieren. Daher sollte im Rahmen dieser Dissertation untersucht werden, inwieweit
es zu einer Veränderung unterschiedlicher Tight Junction-Proteine nach peripherer
Nervenverletzung (CCI) kommt. Hierbei konnte gezeigt werden, dass eine CCI zu einer
Herabregulation der mRNA-Expression von Claudin-1, Claudin-19, Tricellulin und
Occludin im Rückenmark führt, wobei diese Veränderungen insbesondere 7 und 14 d
nach der CCI auftraten. Die membranäre Expression dieser Proteine im Rückenmark
blieb bis auf Occludin unverändert, das 7 d nach der CCI signifikant reduziert war. Am
deutlichsten waren jedoch Claudin-5 und ZO-1 verändert. Folglich vermindert eine CCI
signifikant die Claudin-5-mRNA sowie die Immunreaktivität in isolierten Kapillaren des
Rückenmarks. Das Ankerprotein ZO-1 war sogar auf allen Ebenen, also in der Genals
auch Proteinexpression, und darüber hinaus in den Rückenmarkskapillaren
signifikant reduziert.
Die Interpretation dieser Ergebnisse legt nahe, dass ZO-1, und zum Teil auch
Claudin-5, für die gestörte Blut-Rückenmarkschranke verantwortlich sind und die
anderen untersuchten Proteine vermutlich nur eine untergeordnete Rolle spielen. Die
Bedeutung der Tight Junction-Proteine in der Blut-Rückenmarkschranke konnte somit
weiter untermauert werden. In zukünftigen Untersuchungen wäre es wichtig den
Signalweg, der zur Veränderung der Tight Junction-Proteine führt sowie die
subzelluläre Lokalisation zu untersuchen, um Möglichkeiten zum Wiederverschluss
der Barriere zu finden. Somit könnten Therapien zur Aufrechterhaltung der Blut-
Rückenmarkhomöostase den neuropathischen Schmerz unter Umständen kausal, und
nicht nur symptomatisch, behandelbar machen.
Fabry disease (FD) is an X-linked lysosomal storage disorder caused by deficiency of the α-galactosidase A (GLA), leading to intracellular accumulations of globotriaosylceramide (Gb3). Acral burning pain, which can be triggered by heat, fever or physical activity is an early hallmark of FD and greatly reduces patients’ quality of life. The pathophysiology of FD pain is unknown and research is hindered by the limited in vivo availability of suitable human biomaterial. To overcome this obstacle, we generated induced pluripotent stem cells (iPSC) from one female and two male patients with a differing pain phenotype, and developed a refined differentiation protocol for sensory neurons to increase reliability and survival of these neurons, serving as an in vitro disease model. Neurons were characterized for the correct neuronal subtype using immunocytochemistry, gene expression analysis, and for their functionality using electrophysiological measurements.
iPSC and sensory neurons from the male patients showed Gb3 accumulations mimicking the disease phenotype, whereas no Gb3 depositions were detected in sensory neurons derived from the female cell line, likely caused by a skewed X-chromosomal inactivation in favor of healthy GLA. Using super-resolution imaging techniques we showed that Gb3 is localized in neuronal lysosomes of male patients and in a first experiment using dSTORM microscopy we were able to visualize the neuronal membrane in great detail. To test our disease model, we treated the neurons with enzyme replacement therapy (ERT) and analyzed its effect on the cellular Gb3 load, which was reduced in the male FD-lines, compared to non-treated cells. We also identified time-dependent differences of Gb3 accumulations, of which some seemed to be resistant to ERT. We also used confocal Ca2+ imaging to investigate spontaneous neuronal network activity, but analysis of the dataset proofed to be difficult, nonetheless showing a high potential for further investigations. We revealed that neurons from a patient with pain pain are more easily excitable, compared to cells from a patient without pain and a healthy control.
We provide evidence for the potential of patient-specific iPSC to generate a neuronal in vitro disease model, showing the typical molecular FD phenotype, responding to treatment, and pointing towards underlying electrophysiological mechanisms causing different pain phenotypes. Our sensory neurons are suitable for state-of-the-art microscopy techniques, opening new possibilities for an in-depth analysis of cellular changes, caused by pathological Gb3 accumulations. Taken together, our system can easily be used to investigate the effect of the different mutations of GLA on a functional and a molecular level in affected neurons.
The role of lipid transfer proteins (LTPs) during the fertilization process in Arabidopsis thaliana
(2021)
Double fertilization is a defining characteristic of flowering plants (angiosperms). As the sperm cells of higher plants are non-motile, they need to be transported to the female gametophyte via the growing pollen tube. The pollen-tube journey through the female tissues represents a highly complex process. To provide for successful reproduction it demands intricate communication between the cells of the two haploid gametophytes - the polar growing pollen tube (carrying the two non-motile sperm cells) and the ovule (hosting the egg cell/synergid cells). The polar growth of the pollen tube towards the female gamete is guided by different signaling molecules, including sugars, amino acids and peptides. Some of these belong to the family of lipid transfer proteins (LTPs), which are secreted cysteine-rich peptides. Depending on the plant species several lines of evidence have also suggested potential roles for LTPs during pollen germination or pollen-tube guidance. Although Arabidopsis thaliana has 49 annotated genes for LTPs, several of which are involved in plant immunity and cell-to-cell communication, the role of most members of this family during fertilization is unknown.
The aim of this project was therefore to systematically identify LTPs which play a role in the fertilization process in A. thaliana, particularly during pollen tube guidance. To identify candidate proteins, the expression profile of LTPs in reproductive tissue was investigated. This was accomplished by in-silico bioinformatic analysis using different expression databases. Following confirmion of these results by qRT-PCR analysis, seven Type-I nsLTPs (LTP1, LTP2, LTP3, LTP4, LTP5, LTP6 and LTP12) were found to be exclusively expressed in pistils. Except for LTP12, all other pistil expressed LTPs were transcriptionally induced upon pollination. Using reporter-based transcriptional and translational fusions the temporal and spatial expression patterns together with protein localizations for LTP2, 3, 4, 5, 6, and 12 were determined in planta. Stable transgenic plants carrying PromLTP::GUS constructs of the six different LTP candidates showed that most of LTPs were expressed in the stigma/stylar region and were induced upon pollination. With respect to protein localization on the cellular level, they split into two categories: LTP2, LTP5 and LTP6 were localized in the cell wall, while LTP3, LTP4 and LTP12 were specifically targeted to the plasma membrane.
For the functional characterization of the candidate LTPs, several T-DNA insertion mutant plant lines were investigated for phenotypes affecting the fertilization process. Pollen development and quality as well as their in-vitro germination rate did not differ between the different single ltp mutant lines and wildtype plants. Moreover, in-vivo cross pollination experiments revealed that tube growth and fertilization rate of the mutant plants were similar to wildtype plants. Altogether, no discernible phenotype was evident in other floral and vegetative parts between different single ltp mutant lines and wildtype plants. As there was no distinguishable phenotype observed for single ltp-ko plants, double knock out plants of the two highly homologous genes LTP2 (expressed in the female stigma, style and transmitting tract) and LTP5 (expressed in the stigma, style, pollen pollen-tube and transmitting tract) were generated using the EPCCRISPR-Cas9 genome editing technique. Two ltp2ltp5 mutant transgenic-lines (#P31-P2 and #P31-P3) with frameshift mutations in both the genes could be established. Further experiments showed, that the CRISPR/Cas9-mediated knock-out of LTP2/LTP5 resulted in significantly reduced fertilization success. Cell biological analyses revealed that the ltp2ltp5 double mutant was impaired in pollen tube guidance towards the ovules and that this phenotype correlated with aberrant callose depositions in the micropylar region during ovule development. Detailed analysis of in-vivo pollen-tube growth and reciprocal cross pollination assay suggested that, the severely compromised fertility was not caused by any defect in development of the pollen grains, but was due to the abnormal callose deposition in the embryo sac primarily concentrated at the synergid cell near the micropylar end. Aberrant callose deposition in ltp2ltp5 ovules pose a complete blockage for the growing pollen tube to change its polarity to enter the funiculus indicating funicular and micropylar defects in pollen tube guidance causing fertilization failure.
Our finding suggests that female gametophyte expressed LTP2 and LTP5 play a crucial role in mediating pollen tube guidance process and ultimately having an effect on the fertilization success. In line with the existence of a N-terminal signal peptide, secreted LTPs might represent a well-suited mobile signal carrier in the plant’s extracellular matrix. Previous reports suggested that, LTPs could act as chemoattractant peptide, imparting competence to the growing pollen tube, but the molecular mechanism is still obscure. The results obtained in this thesis further provide strong evidence, that LTP2/5 together regulate callose homeostasis and testable models are discussed. Future work is now required to elucidate the detailed molecular link between these LTPs and their potential interacting partners or receptors expressed in pollen and synergid cells, which should provide deeper insight into their functional role as regulatory molecules in the pollen tube guidance mechanism.
Kardiovaskuläre Erkrankungen sind unverändert die häufigste Ursache für Morbidität und Mortalität in den Industrienationen [1]. Die Risikoprädiktion und -prävention dieser Erkrankungen ist von großer Bedeutung, unter anderem deswegen weil primäre Ereignisse bei bis dato asymptomatischen Personen auftreten können [2]. Die zugrundeliegende Pathogenese, die Arteriosklerose, ist immer besser erforscht und zugleich sind Risikofaktoren identifiziert, die einen schädlichen Einfluss haben [3, 4]. Durch die Messung der Karotis-Intima-Media-Dicke (Carotid-Intima-Media-Thickness, CIMT) mittels B-Mode Ultraschall steht eine weit verbreitete, sichere und anerkannte Methode zur Verfügung, mit der bereits subklinische Formen der Arteriosklerose erfasst werden können [5]. Die CIMT ist als Surrogatparameter für eine generalisierte Arteriosklerose im gesamten Gefäßsystem etabliert und ihre Zunahme wird mit dem Vorliegen von kardiovaskulären Risikofaktoren assoziiert [6-8]. In der Risikoprädiktion mit Hilfe der CIMT bilden geschlechts-, alters- und regionalspezifische Normwerte die Basis [5]. Die aktuellen internationalen Leitlinien empfehlen in ihren neusten Fassungen, nicht mehr die CIMT zur kardiovaskulären Risikoprädiktion in der Allgemeinbevölkerung einzusetzen [1, 9]. Die Experten berufen sich auf Studien, in denen lediglich ein singuläres Messsegment betrachtet wurde [1, 9-11]. Das Ziel der vorliegenden Arbeit war es den Einfluss spezifischer kardiovaskulärer Risikofaktoren auf die verschiedenen Segmente der A. carotis zu erfassen und – davon ausgehend – den Stellenwert der vorhandenen Modelle zur Risikoprädiktion zu evaluieren. Des Weiteren wurden Normwerte aus einer repräsentativen Gruppe der Würzburger Allgemeinbevölkerung gebildet und die Reproduzierbarkeit der Ultraschalluntersuchung im Bereich der Halsschlagader überprüft.
Den Berechnungen liegen Daten der STAAB-Kohortenstudie (Häufigkeit und Einflussfaktoren auf frühe STAdien A und B der Herzinsuffizienz in der Bevölkerung) zugrunde, einer große Bevölkerungsstudie, die seit 2015 Daten der Würzburger Bevölkerung erhebt [12]. Es wurden Probanden zwischen mit einem Alter zwischen 30 und 79 Jahren eingeschlossen. Die CIMT wurde auf beiden Seiten des Halses auf der schallkopffernen Seite an drei vorab definierten Lokalisationen des Gefäßes, der A. carotis communis (ACC), dem Bulbus und der A. carotis interna (ACI), vermessen. Es wurden die fünf Risikofaktoren Diabetes mellitus, Dyslipidämie, Hypertonie, Rauchen und Übergewicht berücksichtigt. Mittels einer logistischen Regression wurde der spezifische Einfluss dieser Faktoren auf die individuelle, alters- und geschlechtsbasierte 75. Perzentile der CIMT in den einzelnen Lokalisationen betrachtet. Diese Grenzwerte stammten aus den eigens erstellten Normwerten für die Allgemeinbevölkerung. Es wurde eine „gesunde“ Subpopulation zur Erstellung dieser Normwerte gebildet, die keine der oben genannten Risikofaktoren sowie keine manifesten kardiovaskulären Erkrankungen aufwiesen.
Die Auswertung umfasste die Daten von insgesamt 2492 Probanden. Die segmentspezifische CIMT war am größten im Bereich Bulbus, gefolgt von der ACC und der ACI. Männer hatten höhere Wanddickenwerte und mehr Risikofaktoren als Frauen. Die Reproduzierbarkeit zwischen den einzelnen Untersuchern war insgesamt moderat bis stark. Im Vergleich zu anderen Studien zeigte sich jedoch insgesamt eine schwächere Übereinstimmung, so dass von einer potentiellen Verbesserung des Schulungsprotokolls für unerfahrene Personen ausgegangen wird. Die Ergebnisse der Reproduzierbarkeitsanalyse verdeutlichen den Bedarf eines standardisierten, international anerkannten Protokolls zur Schulung von Untersuchern der CIMT und eines exakten Messprotokolls [5, 13]. Die erhobenen Normwerte der „Gesunden“ zeigten eine Konsistenz mit verschiedenen, auf vergleichbare Weise erhobenen Werten und bildeten die Basis für die weiteren Untersuchungen. Die CIMT nahm mit dem Alter und – unabhängig davon – ebenfalls mit der Anzahl an Risikofaktoren zu. Die Faktoren Dyslipidämie, Rauchen und Hypertonie hatten einen statistisch signifikanten Einfluss für das Überschreiten des Grenzwertes der 75. Perzentile (OR (95 % KI) zwischen 1,28 (0,98 – 1,65), ACC, und 1,86 (1,53 – 2,27), Bulbus) [14]. Die Faktoren Diabetes mellitus und Übergewicht zeigten im verwendeten Modell keinen Effekt auf die CIMT. Insgesamt konnte, bis auf eine mögliche Interaktion zwischen dem Risikofaktor Rauchen und der ACI, kein segmentspezifischer Effekt beobachtet werden [14]. Daraus resultierend wurde die Hypothese aufgestellt, dass zur Erfassung des kardiovaskulären Risikos einer Person die Messung eines singulären Segments möglicherweise ausreicht [14]. Dies stärkt die neusten Empfehlungen der Leitlinien, die sich auf Studien berufen, welche eben nur ein Segment betrachteten. Die identifizierten Risikofaktoren spiegeln sich darüber hinaus in den gängigen Modellen zur Risikoprädiktion und -prävention wider. Demnach gerät der Einsatz der CIMT zur Bestimmung des individuellen Risikos von Personen der Allgemeinbevölkerung in den Hintergrund [15].
Peptide receptor radionuclide therapy (PRRT) is a molecular targeted radiation therapy involving the systemic administration of radiolabeled somatostatin receptor binding peptides designed to target with high affinity and specificity receptors overexpressed on tumors. Peptides are applied which either target as agonist (with internalization) or antagonist (little to no internalization). Recently, two novel antagonistic agents have been developed for clinical use: OPS202 and OPS201. 68Ga-labelled OPS202 is used for diagnostic purposes with positron emission tomography and 177Lu-labelled OPS201 is used for the therapy in patients with neuroendocrine tumors (NETs). Both agents are presently under clinical evaluation. Despite the very low internalization rate, the use of somatostatin receptor antagonists which target more binding sites on receptors are expected to result in higher specificity, more favorable pharmacokinetics and higher tumor retention and better visualization than the agonists. The main goal of this thesis was analyzing the biodistribution, biokinetics and internal dosimetry of the recently developed somatostatin receptor antagonists (OPS201 and OPS202) for therapeutic and diagnostic purposes in different species (mice, pigs and patients). In addition, an analysis of the influence of image quantification and the integration of time activity curves on kidney dosimetry in a pig model was carried out. Furthermore, extrapolation methods, which are used for predicting organ absorbed doses for humans based on preclinical animal models, were systematically compared for blood, liver, and kidneys of OPS201 injected species. Based on the OPS202 injected patients’ investigations, 68Ga-OPS202 shows promising biodistribution and imaging properties with tumor contrast which is optimal one hour after injection of the radiotracer. OPS202 is well tolerated and delivers absorbed doses to organs that are lower than those by 18F-FDG and similar to other 68Ga-labeled somatostatin receptor ligands. As a result of 68Ga OPS202 injection, the highest absorbed doses were observed in the urinary bladder (0.10 mGy/MBq) and kidneys (0.84 mGy/MBq). The calculated mean effective dose coefficient of 68Ga-OPS202 injected patients was 0.024 mSv/MBq (3.6 mSv for 150 MBq 68Ga-OPS202 injection) which is similar to other 68Ga-labeled compounds. Based on the OPS201 biokinetics and dosimetry investigations, after the injection of 177Lu-OPS201, a fast blood clearance of the compound is observed in the first phase (half-life: 1.83 h) for each species. 10 min after injection, less than 5% of the injected activity per milliliter of blood circulates in pigs and humans. The analysis of the mice, pig and preliminary patient data provides evidence that, patients enrolled in a phase 1 177Lu-OPS201 trial would not be at risk of overexposure. Based on our results, for 177Lu labelled studies, late time points after 72 h have a great impact on absorbed dose calculations. That is why follow-up times especially at late time points (more than 72 h) are required for the time-integrated activity coefficient (TIAC) calculations in order to represent the area under the curve appropriately and to analyze both biokinetics and dosimetry accurately. In addition, to find the most adequate extrapolation methods that minimize the interspecies differences of dosimetry data, several extrapolation methods from animal to human have been tested. For OPS201 time scaling or combination of relative mass and time scaling results in most similar TIAC values, if the organ mass ratios between the species are high. In time scaling, the scan/sampling time is scaled by using the ratio of the whole body masses of the respective species. In relative mass scaling, the TIACs are scaled based on the ratio of the whole body and organ mass of respective species. Other methods tested showed higher deviations. For the study on the influence of image quantification and the choice of the optimal scanning time points, a study in a pig model, which was performed in collaboration with Aalborg University and Octreopharm Sciences GmbH, was reanalyzed. As kidneys are organs-at-risk in PRRT with 177Lu labelled peptides, several quantification methods, based on 2D and 3D quantitative imaging were chosen. For this purpose, a 3D printed pig kidney phantom was prepared and measured with/without background activities representing the activities in the pig SPECT/CT scans. The phantom dosimetry data based on multiple SPECT/CT images and based on multiple planar images in combination with one SPECT/CT scan (MP1S Imaging) were compared to the pig dosimetry. The calculated TIACs of the phantom with background based on multiple SPECT/CT and MP1S imaging were quite similar to the multiple SPECT/CT based pig TIAC. In addition, in order to investigate the effect of late time points on dosimetry and absorbed dose values in 177Lu therapies, the difference, associated with eliminating the late two scan time points, on the TIACs was analyzed. When the TIACs (including all time points) of the pig based on multiple SPECT/CT and MP1S imaging were investigated, the use of MP1S imaging results in considerably lower TIAC values to the kidney (by a factor of 1.4). With eliminating late time points from the created time activity curve, the factor increases up to 2.4 times with a corresponding increase in TIAC uncertainties. As a consequence, further evaluation of 68Ga-OPS202 for PET/CT imaging and 177Lu-OPS201 for the treatments of NET patients is necessary. In particular, a head-to-head comparison of agonists and OPS peptides with respect to biokinetics, biodistribution and dosimetry would be helpful. In addition, the influence of the late scan time points on dosimetry needs further attention in particular for kidney dosimetry
In dieser Dissertation wird der MEK5/ERK5- Signalweg als möglicher Angriffspunkt in der zielgerichteten Melanomtherapie identifiziert. Die Adressierung von ERK5 bietet eine Alternative, um einer Resistenzentwicklung gegenüber Inhibitoren des MAPK- Signalwegs entgegenzuwirken. Das maligne Melanom ist ein hochaggressiver Tumor mit steigender Inzidenz. Zunehmende Sonnenstunden im Rahmen des Klimawandels mit erhöhter Belastung der Haut durch UV-Strahlung werden die Problematik des malignen Melanoms für den Menschen in den nächsten Jahren weiter zunehmen lassen.
Die Aktivierung des MEK5/ERK5- Signalwegs scheint eine Reaktion von Tumorzellen auf Therapiestress zu sein. Diese Aktivierung liefert den Melanomzellen einen Überlebensvorteil und verhindert ein langfristiges Therapieansprechen. ERK5 beeinflusst den Zellzyklus von Melanomzellen und ist somit möglicherweise von wichtiger Bedeutung in der Tumorgenese des malignen Melanoms.
Patienten mit NRAS- Mutation profitieren auffallend weniger von einer gezielten MEKi-Therapie als solche mit BRAF Mutation. Für ersteres Patientenkollektiv steht aktuell lediglich die Immuntherapie zur Verfügung, wodurch oft nur ein kurzes, progressionsfreies Intervall erreicht werden kann und die Patienten häufig unter schweren Nebenwirkungen leiden. Grund für die problematische Behandlung könnte das häufige Auftreten einer basalen ERK5- Aktivierung in NRAS- mutierten Melanomen sein. Diese Arbeit liefert eine positive Prognose über den Nutzen einer ERK5- Inhibition als Erweiterung des Therapieschemas. Diese These gilt auch für Melanompatienten mit einer BRAF- Mutation. Patienten, die an einem malignen Melanom erkrankt sind, weisen zu 80% eine Mutation in einem dieser beschriebenen Onkogene auf. Die Arbeit lässt darauf schließen, dass eine ERK5- Inhibition in der Therapie von beiden Gruppen erfolgreich sein könnte und somit das Leben nahezu aller Melanompatienten betrifft.
DD is a cardiac disturbance, which has gained increasing importance in recent years due to its important role in different cardiac disease and cardiomyopathies including ischemic cardiomyopathy, arterial hypertension and diabetic cardiomyopathy.
ECG-gated 18F-FDG PET is an imaging technique, that can distinguish between districts of myocardial viability and myocardial scars and further provides information of great interest on the efficacy of experimental approaches designed to improve the cardiac function and/or myocardial metabolism in experimental small animal models. However, ECG-gated 18F-FDG PET is a technique whose feasibility in the assessment of the LV diastolic function in small animals has not been a subject of study.
In this thesis, the ability of the ECG-gated 18F-FDG PET for the assessment of both the systolic and diastolic function in eight control rats and in seven ZDF rats, which are an experimental animal model mimicking T2DM conditions and diabetic related complications in humans including DCM, has been investigated The ECG-gated 18F-FDG PET imaging was performed under hyperinsulinemic-euglycemic clamping and the data were stored in list mode files and retrospectively reconstructed. The systolic and diastolic parameters were achieved from the time/volume and the time/filling curve calculated from the software HFV. Additionally, the influence of the number of gates per cardiac cycle on the LV volumes and function parameters has been studied.
Hyperinsulinemic-euglycemic clamp procedure and blood glucose measurement did confirm the development of a manifest diabetes in the ZDF rats at the timepoint of the experiments.
Regarding the systolic parameters, no significant difference could be detected between the ZDF and ZL rats. The values for the CO were similar in both groups, which demonstrates a similar LV systolic function in the ZDF and the ZL rats at the age of 13 weeks. Values for the systolic parameters are in good line with previous PET, MRI and cardiac catheterization-based studies in diabetic rats.
The main finding of this study was that by using in vivo ECG-gated 18F-FDG PET and the software HFV, reliable diastolic parameters could be calculated. Moreover, it was possible to detect the presence of a mild impaired diastolic filling in the ZDF rats in absence of any systolic alteration. This impaired diastolic function in an early stage of diabetes could also be detected by other investigators, who used echocardiography or cardiac catheterization. Therefore, this is the first study showing, that the assessment of the diastolic function in rats can be carried out by ECG-gated 18F-FDG PET imaging.
In conclusion, additionally to calculating LV volumes and LV EF, ECG-gated 18F-FDG PET can evaluate the diastolic function of healthy and diabetic rats and is able to detect a DD in ZDF rats.
G-protein-coupled receptors (GPCRs) regulate diverse physiological processes in the human body and represent prime targets in modern drug discovery. Engagement of different ligands to these membrane-embedded proteins evokes distinct receptor conformational rearrangements that facilitate subsequent receptor-mediated signalling and, ultimately, enable cellular adaptation to altered environmental conditions. Since the early 2000s, the technology of resonance energy transfer (RET) has been exploited to assess these conformational receptor dynamics in living cells and real time. However, to date, these conformational GPCR studies are restricted to single-cell microscopic setups, slowing down the discovery of novel GPCR-directed therapeutics. In this work, we present the development of a novel generalizable high-throughput compatible assay for the direct measurement of GPCR activation and deactivation. By screening a variety of energy partners for fluorescence (FRET) and bioluminescence resonance energy transfer (BRET), we identified a highly sensitive design for an α2A-adrenergic receptor conformational biosensor. This biosensor reports the receptor’s conformational change upon ligand binding in a 96-well plate reader format with the highest signal amplitude obtained so far. We demonstrate the capacity of this sensor prototype to faithfully quantify efficacy and potency of GPCR ligands in intact cells and real time. Furthermore, we confirm its universal applicability by cloning and validating five further equivalent GPCR biosensors. To prove the suitability of this new GPCR assay for screening purposes, we measured the well-accepted Z-factor as a parameter for the assay quality. All tested biosensors show excellent Z-factors indicating outstanding assay quality. Furthermore, we demonstrate that this assay provides excellent throughput and presents low rates of erroneous hit identification (false positives and false negatives). Following this phase of assay development, we utilized these biosensors to understand the mechanism and consequences of the postulated modulation of parathyroid hormone receptor 1 (PTHR1) through receptor activity-modifying protein 2 (RAMP2). We found that RAMP2 desensitizes PTHR1, but not the β2-adrenergic receptor (β2AR), for agonist-induced structural changes. This generalizable sensor design offers the first possibility to upscale conformational GPCR studies, which represents the most direct and unbiased approach to monitor receptor activation and deactivation. Therefore, this novel technology provides substantial advantages over currently established methods for GPCR ligand screening. We feel confident that this technology will aid the discovery of novel types of GPCR ligands, help to identify the endogenous ligands of so-called orphan GPCRs and deepen our understanding of the physiological regulation of GPCR function.
The family of trypanosomatid parasites, including the human pathogens Trypanosoma brucei and Leishmania, has evolved sophisticated strategies to survive in harmful host environments. While Leishmania generate a safe niche inside the host’s macrophages, Trypanosoma brucei lives extracellularly in the mammalian bloodstream, where it is constantly exposed to the attack of the immune system. Trypanosoma brucei ensures its survival by periodically changing its protective surface coat in a process known as antigenic variation. The surface coat is composed of one species of ‘variant surface glycoprotein’ (VSG). Even though the genome possesses a large repertoire of different VSG isoforms, only one is ever expressed at a time from one out of the 15 specialized subtelomeric ‘expression sites’ (ES). Switching the coat can be accomplished either by a recombination-based exchange of the actively-expressed VSG with a silent VSG, or by a transcriptional switch to a previously silent ES.
The conserved histone methyltransferase DOT1B methylates histone H3 on lysine 76 and is involved in ES regulation in T. brucei. DOT1B ensures accurate transcriptional silencing of the inactive ES VSGs and influences the kinetics of a transcriptional switch. The molecular machinery that enables DOT1B to execute these regulatory functions at the ES is still elusive, however. To learn more about DOT1B-mediated regulatory processes, I wanted to identify DOT1B-associated proteins.
Using two complementary approaches, specifically affinity purification and proximity-dependent biotin identification (BioID), I identified several novel DOT1B-interacting candidates. To validate these data, I carried out reciprocal co-immunoprecipitations with the most promising candidates. An interaction of DOT1B with the Ribonuclease H2 protein complex, which has never been described before in any other organism, was confirmed. Trypanosomal Ribonuclease H2 maintains genome integrity by resolving RNA-DNA hybrids, structures that if not properly processed might initiate antigenic variation. I then investigated DOT1B’s contribution to this novel route to antigenic variation. Remarkably, DOT1B depletion caused an increased RNA-DNA hybrid abundance, accumulation of DNA damage, and increased VSG switching. Deregulation of VSGs from throughout the silent repertoire was observed, indicating that recombination-based switching events occurred. Encouragingly, the pattern of deregulated VSGs was similar to that seen in Ribonuclease H2-depleted cells. Together these data support the hypothesis that both proteins act together in modulating RNA-DNA hybrids to contribute to the tightly-regulated process of antigenic variation.
The transmission of trypanosomatid parasites to mammalian hosts is facilitated by insect vectors. Parasites need to adapt to the extremely different environments encountered during transmission. To ensure their survival, they differentiate into various specialized forms adapted to each tissue microenvironment. Besides antigenic variation, DOT1B additionally affects the developmental differentiation from the mammalian-infective to the insect stage of Trypanosoma brucei. However, substantially less is known about the influence of chromatin-associated proteins such as DOT1B on survival and adaptation strategies of related Leishmania parasites. To elucidate whether DOT1B’s functions are conserved in Leishmania, phenotypes after gene deletion were analyzed. As in Trypanosoma brucei, generation of a gene deletion mutant demonstrated that DOT1B is not essential for the cell viability in vitro. DOT1B deletion was accompanied with a loss of histone H3 lysine 73 trimethylation (the lysine homologous to trypanosomal H3K76), indicating that Leishmania DOT1B is also solely responsible for catalyzing this post-translational modification. As in T. brucei, dimethylation could only be observed during mitosis/cytokinesis, while trimethylation was detectable throughout the cell cycle in wild-type cells. In contrast to the trypanosome DOT1B, LmxDOT1B was not essential for differentiation in vitro. However, preliminary data indicate that the enzyme is required for effective macrophage infection.
In conclusion, this study demonstrated that the identification of protein networks and the characterization of protein functions of orthologous proteins from related parasites are effective tools to improve our understanding of the parasite survival strategies. Such insights are a necessary step on the road to developing better treatments for the devastating diseases they cause.
Bacterial small non-coding RNAs (sRNAs) play fundamental roles in controlling and finetuning gene expression in a wide variety of cellular processes, including stress responses, environmental signaling and virulence in pathogens. Despite the identification of hundreds of sRNA candidates in diverse bacteria by genomics approaches, the mechanisms and regulatory capabilities of these posttranscriptional regulators have most intensively been studied in Gram-negative Gammaproteobacteria such as Escherichia coli and Salmonella. So far, almost nothing is known about sRNA-mediated regulation (riboregulation) in Epsilonproteobacteria, including the major human pathogen Helicobacter pylori. H. pylori was even thought to be deficient for riboregulation as none of the sRNAs known from enterobacteria are conserved in Helicobacter and since it lacks the major RNA chaperone Hfq, which is crucial for sRNA function as well as stability in many bacteria. Nonetheless, more than 60 cis- and trans-acting sRNA candidates were recently identified in H. pylori by a global RNA sequencing approach, indicating that this pathogen, in principle, has the capability to use riboregulation for its gene expression control. However, the functions and underlying mechanisms of H. pylori sRNAs remained unclear.
This thesis focused on the first functional characterization and target gene identification of a trans-acting sRNA, RepG (Regulator of polymeric G-repeats), in H. pylori. Using in-vitro and in-vivo approaches, RepG was shown to directly base-pair with its C/Urich terminator loop to a variable homopolymeric G-repeat in the 5’ untranslated region (UTR) of the tlpB mRNA, thereby regulating expression of the chemotaxis receptor TlpB. While the RepG sRNA is highly conserved, the length of the G-repeat in the tlpB mRNA leader varies among different H. pylori isolates, resulting in a strain-specific tlpB regulation. The modification of the number of guanines within the G-stretch in H. pylori strain 26695 demonstrated that the length of the homopolymeric G-repeat determines the outcome of posttranscriptional control (repression or activation) of tlpB by RepG. This lengthdependent targeting of a simple sequence repeat by a trans-acting sRNA represents a new twist in sRNA-mediated regulation and a novel mechanism of gene expression control, since it uniquely links phase variation by simple sequence repeats to posttranscriptional regulation.
In almost all sequenced H. pylori strains, tlpB is encoded in a two gene operon upstream of HP0102, a gene of previously unknown function. This study provided evidence that HP0102 encodes a glycosyltransferase involved in LPS O-chain and Lewis x antigen production. Accordingly, this glycosyltransferase was shown to be essential for mice colonization by H. pylori. The coordinated posttranscriptional regulation of the tlpB-HP0102 operon by antisense base-pairing of RepG to the phase-variable G-repeat in the 5’ UTR of the tlpB mRNA allows for a gradual, rather than ON/OFF, control of HP0102 expression, thereby affecting LPS biosynthesis in H. pylori. This fine-tuning of O-chain and Lewis x antigen expression modulates H. pylori antibiotics sensitivity and thus, might be advantageous for Helicobacter colonization and persistence.
Whole transcriptome analysis based on microarray and RNA sequencing was used to identify additional RepG target mRNAs and uncover the physiological role of this riboregulator in H. pylori. Altogether, repG deletion affected expression of more than 40 target gene candidates involved various cellular processes, including membrane transport and adhesion, LPS modification, amino acid metabolism, oxidative and nitrosative stress, and nucleic acid modification. The presence of homopolymeric G-repeats/G-rich sequences in almost all target mRNA candidates indicated that RepG hijacks a conserved motif to
recognize and regulate multiple target mRNAs in H. pylori.
Overall, this study demonstrates that H. pylori employs riboregulation in stress response and virulence control. In addition, this thesis has successfully established Helicobacter as a new model organism for investigating general concepts of gene expression control by Hfq-independent sRNAs and sRNAs in bacterial pathogens.