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No description available.

This study deals with the effects of botulinum toxin A treatment on pathological muscular hyperactivity in a group of 53 children. The thesis objective was to evaluat eeffect of botulinum toxin on muscle tone, range of movement and motor performance of the patient. The theoretical part summarizes the knowledge about this theme, characterizes the term spasticity and an impact of botulinum toxin on abnormal muscular hyperactivity. The practical part evaluates the effect of botulinum toxin in two groups of patients. The first group consists of 14 children, who were tested before and at least one month after botulinum toxin application. Clinical assessment involved range of movement measurement, the evaluation of spasticity with Modified Ashworth scale, Global Assesment of Spasticity Scale (GSA), assessment of motor performance with the use of GMFM test and Functional Independence Meassure for children WeeFIM and summarized physical therapeutic methods used in treatment of patients before and after the application. The second group consists of 39 patients who administered the GSA survey forms within a period of one month. The results confirmed positive effect of botulinum toxin treatment on reduction of spasticity, increasing of range of movement and improvement of motor performance of children. Powered by...

Bacteria are under constant threat by their viral parasites, the bacteriophages (phages) and have evolved a range of anti-phage systems to defend themselves. One of these systems is termed abortive infection (Abi) where, upon phage infection, an Abi system may be activated which initiate a bacteriostatic or bactericidal response. While the infected bacteria do not obviously benefit from the activation of these systems, the cessation of bacterial growth or premature cellular suicide prevents the release of phage progeny. Thus Abi can be viewed as an altruistic process as only the remaining clonal bacterial population benefits. The Type III toxin-antitoxin systems have previously been shown to be involved in Abi, however the mechanisms through which these systems are activated are still poorly understood. A common approach to reveal the phage product involved in triggering these systems is to first determine the mutations that a previously sensitive phage evolves to escape after exposure to an Abi system. Analysis of viral "escape" mutants has been used in this study to try to elucidate the activation mechanism(s) of two Type III systems (ToxIN$_P$$_a$ and TenpIN$_P$$_l$) of several environmental phages. Several new phage products were identified in escape mutants as candidate factors involved in circumventing Abi - and possible roles in phage metabolism predicted. Furthermore, the genomes of several phages that could not evolve escapes, or were insensitive to Abi, are sequenced and these data exposed interesting curiosities regarding Abi (as well as the discovery of several novel and rare phages). Previously, no coliphage was identified that was capable of escape of the ToxIN$_P$$_a$ or TenpIN$_P$$_l$ systems. However, this study defined and characterised the first ToxIN$_P$$_a$ and TenpIN$_P$$_l$ coliphage escapes as well as a new method for isolating host-dependent coliphage escapes. Finally, multiple phages that infect the insect pathogen $\textit{Photorhabdus luminescens}$ TT01 (the bacterial strain from which the TenpIN$_P$$_l$ system originated) were isolated, genomically sequenced and characterised in terms of host range. The results revealed a large superfamily of flagellum-dependent phages that exhibit remarkable host promiscuity, possibly defining the most promiscuous phages thus far identified.

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. / Bakterielle Protein-Toxine gehören zu den wirksamsten bekannten Toxinen. In vielfältigen Variationen findet man sie in allen Bereichen des Lebens. Einige werden von den Bakterien während einer Infektion freigesetzt und übernehmen einen wichtigen Part in der Pathogenität. Andere werden zu Verteidigungszwecken sekretiert und können in verdorbenen Lebensmitteln gefunden werden. Was die Wirkung binärer Toxine der AB7-Gruppe angeht, so gehören diese zu den potentesten und gefährlichsten Giften weltweit. Selbst kleine Mengen dieser Proteine können schwerste Symptome während einer Infektion mit Bakterien der Gattung Clostridium oder Bacillus verursachen. Abgesehen von der Bedrohung die durch die Toxine ausgeht, zeichnen sie sich durch einen einzigartigen Intoxikationsmechanismus aus. AB7-Toxine sind aus einer porenformenden Domäne B, die als eine Art molekulare Injektionskanüle fungiert, und enzymatisch aktiven Proteinen A zusammengesetzt. Der komplexe Wirkmechanismus ermöglicht es nicht nur Zellen in höchst effektiver Weise abzutöten und sollte deswegen zu Behandlungszwecken untersucht werden, sondern könnte auch als molekulares Werkzeug umfunktioniert werden, um spezielle Zellen mit gewünschten Proteinen zu beladen. Für beide Zwecke (Blockierung und gezielter Transport) ist die Bindung an, und der Transport durch die porenformende Domäne von größter Bedeutung. Die Struktur der B-Domäne ist wichtig um tiefere Einsicht in den Transportprozess der binären Toxine zu ermöglichen. Leider ist es bisher nicht gelungen die Kristallstruktur des Membranproteins protective antigen (PA) von Anthrax oder irgendeiner anderen Bindedomäne eines AB7-Toxins zu lösen. Deshalb wurde in dieser Arbeit ein elektrophysiologischer Ansatz zur Bestimmung der biophysikalischen Konstanten des Prozesses gewählt, die Black-lipid-Bilayer Methode. Zusätzliche Versuche an Zellen und mit Mutanten der Proteine dienen zur Absicherung der in vitro Ergebnisse. Im ersten Teil der Arbeit wird der Translokationsmechanismus, und die mögliche Nutzung dessen als molekulares Werkzeug näher behandelt. Dies erfolgte durch Bindungsstudien an PA und C2II mit Effektoren (sowohl aus der AB7-Familie, als auch nicht näher verwandt). In Kapitel 2 wird beschrieben, dass ein His-Tag es EDIN (von S. aureus) und anderen Effektoren ermöglicht, dass ein Transport durch PA-Poren in vitro und in vivo stattfindet (nicht aber durch C2II). Ebenfalls konnte eine starke Abweichung in der bekannten Spannungsabhängigkeit von PA festgestellt werden, die den Prozess eindeutig mit den Membranpotentialen in Verbindung bringt, die häufig eine wichtige Rolle im Metabolismus spielen. Kapitel 3 fasst zusammen, dass Bindungspartner von PA bestimmte Motive beinhalten, die von Substraten wie Ionen in Elektrolyten, über kleine Moleküle, bis hin zu komplexen Proteinen reichen. Diese Erkenntnisse könnten genutzt werden um Blockersubstanzen zur Behandlung von Anthrax, oder Tags zur Aufnahme durch Anthrax zu designen. Neueste Befunde zu Homologien und Unterschieden zwischen den Komponenten der binären Toxine sind der Kern von Kapitel 4, in dem die Kreuzreaktivität von Anthrax und C2-Toxin analysiert wurde. Die enthaltenen Daten ermöglichen einen tieferen Einblick in die verschiedenen Stufen der Bindung und Translokation des edema factor (EF), des lethal factor (LF) und von C2I an und durch PA und C2II. Im zweiten Teil rückt die Blockierung der Intoxikation in den Fokus. Die Analyse speziell designter Blockersubstanzen für PA wird in Kapitel 5 behandelt. Diese formen einen Porenverschluss, der weiteren Transport von Toxinkomponenten verhindert. Eine besondere Bedeutung könnten diese Substanzen im Zusammenhang mit Multiresitenz bei Anthrax Stämmen (vermutlich in Russland als biologische Waffe hergestellt) zur Verhinderung von Symptomen und der Verlängerung der Zeit spielen, die man hat um neue Antibiotika zu erzeugen. Kapitel 6 beschreibt zum ersten Mal die Blockierung von PA-Poren mittels eines Anti-His Antikörpers von der trans-Seite aus. Interessanterweise trat diese Ionenstärke abhängige Blockierung, auch bei einer Histidin zu Glycin Mutation an der Stelle 310 (vermutete Bindeposition) auf.

A large number of metabolic waste products accumulate in the blood of patients with renal failure. Since these solutes have deleterious effects on the biological functions, they are called uremic toxins and have been classified in three groups: 1) small water soluble solutes (MW < 500 Da), 2) small solutes with known protein binding (MW < 500 Da), and 3) middle molecules (500 Da < MW < 60 kDa). Protein bound uremic toxins are poorly removed by conventional hemodialysis treatments because of their high protein binding and high distribution volume. The prototypical protein bound uremic toxins indoxyl sulfate (IS) and p-cresyl sulfate (pCS) are associated with the progression of chronic kidney disease, cardiovascular outcomes, and mortality of patients on maintenance hemodialysis. Furthermore, these two compounds are bound to albumin, the main plasma protein, via electrostatic and/or Van-der-Waals forces. The aim of the present thesis was to develop a dialysis strategy, based on the reversible modification of the ionic strength in the blood stream by increasing the sodium chloride (NaCl) concentration, in order to enhance the removal of protein bound substances, such as IS and pCS, with the ultimate goal to improve clinical patient outcomes. Enhancing the NaCl concentration ([NaCl]) in both human normal and uremic plasma was efficient to reduce the protein bound fraction of both IS and pCS by reducing their binding affinity to albumin. Increasing the ionic strength was feasible during modified pre-dilution hemodiafiltration (HDF) by increasing the [NaCl] in the substitution fluid. The NaCl excess was adequately removed within the hemodialyzer. This method was effective to increase the removal rate of both protein bound uremic toxins. Its ex vivo hemocompatibility, however, was limited by the osmotic shock induced by the high [NaCl] in the substituate. Therefore, modified pre-dilution HDF was further iterated by introducing a second serial cartridge, named the serial dialyzers (SDial) setup. This setting was validated for feasibility, hemocompatibility, and toxin removal efficiency. A better hemocompatibility at similar efficacy was obtained with the SDial setup compared with the modified pre-dilution HDF. Both methods were finally tested in an animal sheep model of dialysis to verify biocompatibility. Low hemolysis and no activation of both the complement and the coagulation systems were observed when increasing the [NaCl] in blood up to 0.45 and 0.60 M with the modified pre-dilution HDF and the SDial setup, respectively. In conclusion, the two dialysis methods developed to transitory enhance the ionic strength in blood demonstrated adequate biocompatibility and improved the removal of protein bound uremic toxins by decreasing their protein bound fraction. The concepts require follow-on clinical trials to assess their in vivo efficacy and their impact on long-term clinical outcomes. / Eine große Zahl von Stoffwechselprodukten akkumuliert im Blut urämischer Patienten mit Nierenversagen. Da diese Moleküle schädliche Wirkungen auf die biologischen Funktionen haben, werden sie als Urämietoxine bezeichnet. Man teilt sie in drei Gruppen ein: 1) kleine wasserlösliche Substanzen (MG < 500 Da), 2) kleine, proteingebundene Substanzen (MG < 500 Da), 3) Mittelmoleküle (500 Da < MG < 60 kDa). Proteingebundene Urämietoxine werden wegen ihrer starken Proteinbindung und ihres Verteilungsvolumen durch klassische Hämodialyseverfahrens nur schlecht entfernt. Die prototypischen proteingebundenen Urämietoxine Indoxylsulfat (IS) und p-Cresylsulfat (pCS) sind bei chronischen niereninsuffizienten Patienten mit dem Fortschreiten der Niereninsuffizienz, Herz-Kreislauf-Erkrankungen und der Mortalität verbunden. Außerdem sind diese beiden Toxine an Albumin, dem wichtigsten Plasmaprotein, durch elektrostatische und/oder Van-der-Waals-Kräfte gebunden. Das Ziel der vorliegenden Arbeit war es, ein Dialyseverfahren basierend auf einer reversiblen Modifikation der Ionenstärke im Blut durch Erhöhung der Natriumchlorid (NaCl)-Konzentration zu entwickeln, um die Entfernung von proteingebundenen Molekülen wie IS und pCS zu erhöhen und dadurch eine Verbesserung des klinischen Verlauf der Patienten zu erreichen. Die Erhöhung der NaCl-Konzentration ([NaCl]) sowohl in normalem als auch in urämischem menschlichem Plasma war geeignet, um den proteingebundenen Anteil von IS und pCS durch Schwächung ihrer Bindungsaffinität zu Albumin zu verringern. Die Erhöhung der Ionenstärke während einer modifizierten Prädilutions-Hämodiafiltration (HDF) konnte durch eine Erhöhung der [NaCl] in der Substitutionslösung umgesetzt werden; dabei wurde der NaCl-Überschuss innerhalb des Dialysators vollständig entfernt. Dieses Verfahren war effektiv, um die Entfernungsrate beider proteingebundenen Urämietoxine zu steigern; seine Ex-vivo-Hämokompatibilität war allerdings aufgrund des osmotischen Schocks infolge der hohen [NaCl] im Substituat begrenzt. Deshalb wurde eine Iteration der modifizierten Prädilutions-HDF durch Einbau eines zweiten, seriellen Dialysators vorgenommen, bezeichnet als serielles Dialysator System (SDial). Diese letzte Methode wurde dann bezüglich der Durchführbarkeit, der Hämokompatibilität und Toxinentfernung validiert. Durch das SDial-System konnte, verglichen mit der modifizierten Prädilutions-HDF, eine bessere Hämokompatibilität bei ähnlicher Wirksamkeit erzielt werden. Beide Methoden, modifizierte Prädilutions-HDF und SDial System, wurden abschließend in ein Tierdialysemodell mit Schafen transferiert, wobei eine zufriedenstellende Biokompatibilität demonstriert werden konnte. Beide, zur vorübergehenden Erhöhung der Ionenstärke im Blut entwickelten Dialyseverfahren zeigten bei zufriedenstellender Biokompatibilität eine verbesserte Entfernung proteingebundener Urämietoxine durch Reduktion ihrer proteingebundenen Fraktion. In einem nächsten Schritt sind klinische Studien erforderlich, die diese Konzepte bezüglich ihrer In-vivo-Wirksamkeit und ihrer langfristigen Wirkung auf den Krankheitsverlauf untersuchen.

Streptococcus agalactiae CAMP factor is a protein exotoxin that contains 226 amino acid residues and forms oligomeric pores on susceptible cell membranes and liposomes. In this study, fragments of CAMP factor were created and recombinantly expressed to identify functional domains that are involved in membrane binding, oligomerization, and membrane insertion. Altogether, six truncated forms of CAMP factor were created and assayed. CAMP1-113, CAMP1-170, CAMP57-226, and CAMP171-226 showed different levels of hemolytic activity. CAMP1-56 and CAMP114-226 did not show hemolytic activity or oligomerization ability, but showed binding ability. CAMP114-226 inhibited the hemolytic activity of wild-type CAMP factor, most likely through ‘one-sided’ oligomerization. From the comparison of these fragments, it emerges that the region between residues 57 and 113 plays a crucial role in oligomerization and membrane insertion. The high binding efficiency of CAMP114-226 suggests this region has great responsibility on membrane binding. The hemolytically inactive fragments showed higher binding efficiency than some of the active fragments. For the hemolytic fragments, higher binding efficiency gave stronger hemolysis. These findings support that CAMP factor has different functional regions for pore-formation.

Streptococcus agalactiae CAMP factor is a protein exotoxin that contains 226 amino acid residues and forms oligomeric pores on susceptible cell membranes and liposomes. In this study, fragments of CAMP factor were created and recombinantly expressed to identify functional domains that are involved in membrane binding, oligomerization, and membrane insertion. Altogether, six truncated forms of CAMP factor were created and assayed. CAMP1-113, CAMP1-170, CAMP57-226, and CAMP171-226 showed different levels of hemolytic activity. CAMP1-56 and CAMP114-226 did not show hemolytic activity or oligomerization ability, but showed binding ability. CAMP114-226 inhibited the hemolytic activity of wild-type CAMP factor, most likely through ‘one-sided’ oligomerization. From the comparison of these fragments, it emerges that the region between residues 57 and 113 plays a crucial role in oligomerization and membrane insertion. The high binding efficiency of CAMP114-226 suggests this region has great responsibility on membrane binding. The hemolytically inactive fragments showed higher binding efficiency than some of the active fragments. For the hemolytic fragments, higher binding efficiency gave stronger hemolysis. These findings support that CAMP factor has different functional regions for pore-formation.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

Type II toxin-antitoxin (TA) systems are composed of a stable toxin and its cognate unstable antitoxin that impedes the toxin through direct interaction. The human oral pathogen Streptococcus mutans uses a quorum-sensing peptide (CSP) as a stress-inducible pheromone to synchronize gene expression in response to specific stressors. The objectives of this study were to investigate the role of S. mutans MazEF TA in cell survival and characterize the functionality of CSP-inducible chromosomal type II TAs. Our results suggest that MazEF represents a stress-response element. Interestingly, S. mutans negatively regulates its MazEF system under high-cell-density environment that is characteristic of oral biofilms. S. mutans also encodes a novel chromosomal type II TA involved in biofilm formation and development of dormant persister cells. The results from this study suggest a complex interplay between quorum-sensing (signal), type II TA activation (response), and persister formation (phenotype) as a reaction to environmental perturbations.