@phdthesis{Metzger2008,
  author    = {Metzger, Alexandra},
  title     = {Molekulargenetische Charakterisierung tumorigener Chromosomenaberrationen in extranodalen Marginalzonenlymphomen vom MALT-Typ der Lunge},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-28676},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2008},
  abstract  = {MALT-Lymphome der Lunge geh{\"o}ren als extranodale Marginalzonen-B-Zell-Lymphomen zu den malignen Non-Hodgkin-Lymphomen. Zahlreiche Untersuchungen haben gezeigt, dass rekurrenten genetischen Translokationen eine wichtige Rolle bei der Tumorgenese zukommt. Eine zentrale Rolle nimmt die Aktivierung des Transkriptionsfaktors NFkappaB ein, die Folge solcher rekurrenter Translokationen (z.B. t(11;18)(q21;q21), t(14;18)(q32;q21), t(1;14)(p22;q32)) und numerischer Chromosomenaberrationen (Trisomie 3, Trisomie 18) ist. In der vorliegenden Arbeit haben wir 60 F{\"a}lle extranodaler Marginalzonen-B-Zell-Lymphome der Lunge bez{\"u}glich der genannten genetischen Translokationen untersucht. Es ist dies die gr{\"o}ßte bisher in der Literatur beschriebene Serie von MALT-Lymphomen in dieser Lokalisation. Die Untersuchung der t(11;18) ergab in der vorliegenden Arbeit eine geringere H{\"a}ufigkeit als in der Literatur beschrieben, wobei zu ber{\"u}cksichtigen ist, dass die bisher vorgestellten Studien deutlich geringere Fallzahlen aufwiesen. Bez{\"u}glich der Translokationen t(14;18), t(1;14), t(3;14) und der Trisomie 3 waren in der vorliegenden Studie {\"a}hnliche H{\"a}ufigkeiten zu finden, wie sie in der Literatur beschrieben sind. Als m{\"o}glichen alternativen Aktivierungsweg des Zellzyklus zeigte sich in dieser Studie neben den genannten Translokationen sowohl eine Trisomie 3 als auch eine Amplifikation der Genkopienzahl von MALT1. Im Vergleich der genetischen und immunhistochemischen Ergebnisse bez{\"u}glich der FOXP1- und der BCL10-Expression zeigte sich f{\"u}r FOXP1 eine hohe Korrelation zwischen immunhistologischer Expression und genetischem Nachweis einer Genaktivierung, w{\"a}hrend f{\"u}r BCL10 eine starke Diskrepanz bez{\"u}glich Sensitivit{\"a}t und Spezifit{\"a}t gefunden wurde, so dass die immunhistologische Analyse nur einen Hinweis auf das Vorliegen einer genetischen Translokation zu geben vermag, aber nicht als Surrogatmarker zu verwenden ist.},
  subject      = {Chromosomenaberration},
  language  = {de}
}
@phdthesis{Kronhardt2012,
  author    = {Kronhardt, Angelika},
  title     = {Channel Formation, Binding and Translocation Properties of Anthrax, CDT and Related Toxins of the AB7 type},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-71559},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2012},
  abstract  = {The ability to produce toxins is spread among a huge variety of bacterial strains. A very prominent class of bacterial protein toxins is the family of binary AB toxins sharing a common mode of intoxication. A pore forming component B binds and translocates an enzymatic component A into the cytosol of target cells exhibiting a fatal mode of action. These components are supposed to be not toxic themselves but both required for cell toxicity. Anthrax toxin produced by the Gram-positive bacteria Bacillus anthracis is the best studied binary toxin especially since its use as a biological weapon in the context of the attacks of 9/11 in 2001. In contrast to other binary toxins, Anthrax toxin possesses two different enzymatic components, edema factor (EF), a calcium- and calmodulin-dependent adenylat-cyclase and lethal factor (LF), a zinc-dependent metalloprotease. Protective antigen (PA) is the pore-forming component responsible for binding and translocation. Clostridium botulinum possesses in addition to the well known botulinum toxin (Botox) a variety of other toxins, such as the binary C2 toxin. C2 toxin is composed of the binding and translocation moiety C2II and the enzymatic moiety C2I acting as an actin-ADP-ribosyltransferase. In this study, the mode of translocation and the binding kinetics to the enzymatic component were studied in a biophysical experimental setup. In chapter 2, the binding of the N-terminal fractions EFN and LFN to the PA channel are analyzed in artificial bilayer membranes revealing lower binding affinity compared to full-length EF and LF. Other biophysical properties like voltage-dependency and ionic-strength dependency are not influenced. The results suggest that additional forces are involved in the binding process, than those concerning the N-terminus exclusively, as it was supposed previously. As the treatment of an Anthrax infection with antibiotics is often medicated very late due to the lack of early symptoms, tools to prevent intoxication are required. 4-aminoquinolones like chloroquine are known to block the PA channel, thereby inhibiting intoxication but they also lead to severe side-effects. In chapter 3 new promising agents are described that bind to PA in artificial bilayer systems, elucidating common motives and features which are necessary for binding to PA in general. The possible interaction of Anthrax and C2 toxin is investigated by measuring the binding of one enzymatic component to the respective other toxin's pore (chapter 4). Interestingly, in vitro experiments using the black lipid bilayer assay show that PA is able to bind to C2I resulting in half saturation constants in the nanomolar range. Furthermore, in vivo this combination of toxin components exhibits cell toxicity in human cell lines. This is first-time evidence that a heterologous toxin combination is functional in in vitro and in vivo systems. In contrast, C2II is able to bind to EF as well as to LF in vitro, whereas in in vivo studies almost no toxic effect is detected. In the case of PA, an N-terminal His6-tag attached to the enzymatic subunit increased the binding affinity (chapter 5). A His6-tag attached to not related proteins also led to high binding affinities, providing the possibility to establish PA as a general cargo protein. In chapter 6 a set of different molecules and proteins is summarized, which are either related or not related to binary toxins, PA is able to bind. In first line, the presence of positive charges is found to be responsible for binding to PA which is in accordance to the fact that PA is highly cation selective. Furthermore, we present evidence that different cationic electrolytes serve as a binding partner to the PA channel. In the last decade another toxin has aroused public attention as it was found to be responsible for a rising number of nosocomial infections: Clostridium difficile CDT toxin. The mode of action of the enzymatic subunit CDTa is similar to C2I of C2 toxin, acting as an ADP-ribosylating toxin. The channel forming and binding properties of CDT toxin are studied in artificial bilayer membranes (chapter 7). We found that two different types of channels are formed by the B component CDTb. The first channel is similar to that of iota toxin's Ib of Clostridium perfringens with comparable single channel conductance, selectivity and binding properties to the enzymatic subunit CDTa. The formation of this type of channel is cholesterol-dependent, whereas in the absence of cholesterol another kind of channel is observed. This channel has a single channel conductance which is rather high compared to all other binary toxin channels known so far, it is anion selective and does not show any binding affinity to the enzymatic component CDTa. The results reveal completely new insights in channel formation properties and the flexibility of a pore-forming component. Additionally, these findings suggest further possibilities of toxicity of the pore forming component itself which is not known for any other binary toxin yet. Therefore, the pathogenic role of this feature has to be studied in detail.},
  subject      = {Bacillus anthracis},
  language  = {en}
}
@phdthesis{Dunkel2008,
  author    = {Dunkel, Marcel},
  title     = {Untersuchungen zur Translokation und Funktion von Tandem-Poren Kaliumkan{\"a}len der TPK-Familie aus Arabidopsis thaliana},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-34743},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2008},
  abstract  = {• Die Modellpflanze der Pflanzenphysiologen, Arabidopsis thaliana, besitzt mindestens 15 verschiedene kaliumselektive Kan{\"a}le, von denen 5 der Strukturklasse der Tandemporen-Kaliumkan{\"a}le angeh{\"o}ren und daher TPK-Kan{\"a}le genannt werden. • Tandemporenkan{\"a}le findet man nur bei eukaryontischen Organismen. Die pflanzlichen Tandemporen Kaliumkan{\"a}le haben einen gemeinsamen phylogenetischen Ursprung und unterscheiden sich von den Tierischen und denen der Pilze und Einzeller. Die pflanzlichen TPK-Kan{\"a}le lassen sich wiederum in die TPK1-Unterfamilie und die TPK2-Unterfamilie unterteilen. Die weitere Evolution der TPK2-Unterfamilie von A. thaliana, TPK2, TPK3, TPK4 und TPK5, l{\"a}sst sich eindeutig auf bestimmte Duplikationsereignisse im Genom von A. thaliana und dessen Ahnen zur{\"u}ckf{\"u}hren. Auch der Ein-Poren Kaliumkanal KCO3 geht sehr wahrscheinlich auf die Duplikation des TPK2 und einer anschließenden Deletion und nicht auf einen der prokaryontischen Ein-Poren-Kaliumkanal-Prototypen zur{\"u}ck. • Vier der A. thaliana TPK-Kan{\"a}le (TPK1, 2, 3 und 5) lokalisieren in der Vakuolenmembran, w{\"a}hrend einer, TPK4, zum großen Teil im ER, aber auch in der Plasmamembran zu finden ist. Die Translokation des TPK1 folgt dem sekretorischen Pfad vom ER, durch den Golgi und m{\"o}glichen intermedi{\"a}ren Kompartimenten hin zur Membran der lytischen Vakuole. Von entscheidender Bedeutung ist dabei der zytoplasmatische Carboxy-Terminus (CT) des TPK1. Deletionsmutanten des TPK1 CT zeigen, dass die Translokation mindestens zwei Sortierungsschritten, am Ausgang des ER und des Golgi, unterliegt. Fehlt der CT komplett bleibt der Kanal im ER. Die Sortierungssignale des TPK1 CT konnten auf die EF-Hand Dom{\"a}ne I eingegrenzt werden. Anschließende Punktmutationen in diesem Bereich konnten zeigen, dass TPK1 in der eigentlich f{\"u}r die Ca2+ Bindung zust{\"a}ndigen Dom{\"a}ne ein di-azidisches ER-Export Motiv bestehend aus Asparagins{\"a}ure, Leucin und Glutamins{\"a}ure enth{\"a}lt. Andere Arbeiten legen nahe, dass der Mechanismus des ER-exports von TPK1 auf der Interaktion mit COPII Vesikelh{\"u}llproteinen beruht; TPK1 also in Vesikel sortiert wird, die sich am ER abschn{\"u}ren und mit dem cis-Golgi fusionieren. Der Vergleich mit anderen pflanzlichen TPK Kan{\"a}len l{\"a}sst vermuten, dass TPK1 Orthologe, nicht aber die A. thaliana Homologen ein di-azidisches ER-Exportmotiv besitzen. Die Translokation des TPK3 erwies sich dementsprechend als unabh{\"a}ngig von dessen CT. Weitere Experimente schließen außerdem eine Beteiligung der 14-3-3 Bindung an der Translokation aus. • TPK4 ist der einzige TPK der heterolog in Xenopus Oozyten funktionell exprimiert werden kann. Wie Mutationen an einem essentiellen Aspartat (Asp86, Asp200) in der Pore zeigten, sind beide tandem repetierten Porendom{\"a}nen einer Kanaluntereinheiten an der Porenbildung beteiligt. Somit formt sich TPK4 {\"a}hnlich wie die tierischen TPK-Kan{\"a}le voraussichtlich aus zwei Untereinheiten. Ein Austausch der zweiten Porendom{\"a}ne von TPK4 konnte zeigen, dass TPK2, TPK3 und TPK5, mit ihrer zweiten Porendom{\"a}ne und TPK4 mit seiner ersten Porendom{\"a}ne den TPK4 zu einem funktionellen Kaliumkanal komplementieren k{\"o}nnen. Da keine der TPK4 Eigenschaften, außer geringf{\"u}gig die relative Permeabilit{\"a}t f{\"u}r Rb+, ver{\"a}ndert wurde, kann man absehen, dass die homologen TPK2, TPK3 und TPK5 als instantan aktivierte, spannungsunabh{\"a}ngige Kaliumkan{\"a}le der Vakuolenmembran fungieren. Dazu kommt wahrscheinlich {\"a}hnlich wie bei TPK1 ein 14-3-3 und Ca2+ abh{\"a}ngiges {\"O}ffnen und Schließen. • Weiterf{\"u}hrende elektrophysiologische Untersuchungen am TPK4 zeigten eine Beteiligung einer transmembranen Asparagins{\"a}ure (Asp110) an der Kaliumpermeation und der schwachen Einw{\"a}rtsgleichrichtung. Der Aspartatrest ist in die wassergef{\"u}llte Aussparung der zytoplasmatischen Porenh{\"a}lfte orientiert. Damit kann er {\"u}ber ionische Wechselwirkungen sowohl Kalium in der Pore konzentrieren als auch potentielle Kanalblocker wie Mg2+ oder Polyamine binden. Die Konservierung des Aspartats unter anderem bei TPK2, TPK3 und TPK5 deutet daraufhin, dass auch die vakuol{\"a}ren TPK-Kan{\"a}le eine Einw{\"a}rtsgleichrichtung vermitteln, die auf einem spannungsabh{\"a}ngigen Block von zytoplasmatischer Seite basiert. • Im Gegensatz zum zytoplasmatischen Block ist das Schließen des TPK4 durch zytoplasmatische Ans{\"a}uerung spannungsunabh{\"a}ngig und ist daher von einer Protonierungsreaktion abh{\"a}ngig. {\"U}ber zahlreiche Deletionen und Chim{\"a}ren des TPK4 wurde der Bereich, in dem sich pH-Sensor und pH-Tor befinden, auf den Bereich zwischen transmembranen und zytoplasmatischen Dom{\"a}nen eingegrenzt. Dar{\"u}ber hinaus fungieren Histidine nicht als pH-Sensor.},
  subject      = {Vakuole},
  language  = {de}
}
@phdthesis{Beitzinger2011,
  author    = {Beitzinger, Christoph},
  title     = {Binding-, Blocking- and Translocation-Processes Concerning Anthrax-Toxin and Related Bacterial Protein-Toxins of the AB7-Family},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-70052},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2011},
  abstract  = {Bacterial protein toxins belong to the most potent toxins which are known. They exist in many different forms and are part of our every day live. Some of them are spread by the bacteria during infections and therefore play a crucial role in pathogenicity of these strains. Others are secreted as a defense mechanism and could be uptaken with spoiled food. Concerning toxicity, some of the binary toxins of the AB7-type belong to the most potent and dangerous toxins in the world. Even very small amounts of these proteins are able to cause severe symptoms during an infection with pathogen species of the genus Clostridium or Bacillus. Apart from the thread the toxins constitute, they exhibit a unique way of intoxication. Members of the AB7-toxin family consist of a pore-forming subunit B, that acts as a molecular syringe to translocate the enzymatic moieties A into the cytosol of target cells. This complex mechanism does not only kill cells with high efficiency and therefore should be studied for treatment, but also displays a possibility to address certain cells with a specific protein cargo if used as a molecular delivery tool. Concerning both issues, binding and translocation of the channel are the crucial steps to either block or modify the system in the desired way. To gain deeper insight into the transport of binary toxins the structure of the B subunit is of great importance, but being a membrane protein, no crystal could be obtained up to now for either protective antigen (PA) of Anthrax toxin or any other AB7-type binding domain. Therefore, the method of choice in this work is an electro-physical approach using the so-called black-lipid-bilayer system for determination of biophysical constants. Additionally, diverse cell based assays serve as a proving method for the data gained during in vitro measurements. Further information was gathered with specially designed mutants of the protein channel. The first part of this thesis focuses on the translocation process and its possible use as a molecular tool to deliver protein cargo into special cell types. The task was addressed by measuring the binding of different effector proteins related and unrelated to the AB7 toxin family. These proteins were tested in titration experiments for the blockage of the ion current through a membrane saturated with toxin channels. Especially the influence of positively charged His-tags has been determined in detail for PA and C2II. As described in chapter 2, a His-tag transferred the ability of being transported by PA, but not by C2II, to different proteins like EDIN (from S. aureus) in vitro and in cell-based experiments. This process was found to change the well-known voltage-dependency of PA to a huge extend and therefore is related to membrane potentials which play a crucial role in many processes in living cells. Chapter 3 sums up findings, which depict that binding partners of PA share certain common motives. These could be detected in a broad range of substrates, ranging from simple ions in an electrolyte over small molecules to complex protein effectors. The gathered information could be further used to design blocker-substrates for treatment of Anthrax infections or tags, which render PA possible as a molecular syringe for cargo proteins. The deeper insight to homologies and differences of binary toxin components is the core of chapter 4, in which the cross-reactivity of Anthrax and C2-toxin was analyzed. The presented results lead to a better understanding of different motives involved in binding and translocation to and via the B components PA and C2II, as well as the enzymatically active A moieties edema factor (EF), lethal factor (LF) and C2I. In the second part of the thesis, the blockage of intoxication is the center of interest. Therefore, chapter 5 focuses on the analysis of specially designed blocker-substrate molecules for PA. These molecules form a plug in the pore, abolishing translocation of the enzymatic units. Especially, if multi-resistant strains of Anthrax (said to be already produced in Russia as a biological weapon) are taken into consideration, these substrates could stop intoxication and buy time, to deal with the infection. Chapter 6 describes the blockage of PA-channels by anti-His antibody from the trans-side of the porin, an effect which was not described for any other antibody before. Interestingly, even mutation of the estimated target amino acid Histidine 310 to Glycine could not interfere with this ionic strength dependent binding.},
  subject      = {Bacillus anthracis},
  language  = {en}
}