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The aim of this retrospective observational study is to describe and discuss various complications that can arise after insertion of alloplastic materials in the field of urogynecology that require further surgical interventions in order to manage them or to at least improve the quality of life in those women. We were able to collect data on 77 patients who fulfilled the criteria. Medical history, data of clinical findings, and outcomes were collected and analyzed. The most common complication seen as an indication for resecting slings or meshes was de novo overactive bladder syndrome (40%). Other indications seen were lower urinary tract obstruction or obstructive voiding symptoms (21%), chronic pain (21%), and de novo dyspareunia (13%). 36% of the patients had recurrent symptoms (failure) after insertion of alloplastic materials in the form of urinary incontinence or prolapse, 32% presented with vaginal erosions, 2 women had severe signs of infection with abscess formation, another 3 women had urogenital fistulae. Other rare complications after mesh or sling insertion are perforations of the urinary bladder or urethra. Proper case selection is the key factor. The use of meshes and slings seems justified only in patients with known connective tissue weakness and recurrences after native tissue repair. Otherwise, patients will be exposed to unnecessary risk without any expectable improvement to their quality of life. Most of the complications are mainly caused by wrong and inadequate surgical techniques, wrong indications, or missed diagnosis of the underlying problem. In addition, lack of long-term follow-up is usually the cause behind the negligence towards many complications. Therefore, only experienced physicians should be allowed to perform such procedures, and long-term postoperative follow-up is strongly recommended. As slings and meshes are used for procedures of choice as means to improve quality of life, and not for life threatening situations, there is a need for intensive informed consent. All possible alternatives have to be discussed, as do the pros and cons of selected procedures, even the rare complications. Mesh or sling resection is considered to be an effective solution for the management of such complications. It has shown a high success rate in comparison to conservative treatment, and the majority of patients were satisfied and experienced a big improvement in their quality of life. The most common complication after resection is the recurrence of primary symptoms, either urinary incontinence or prolapse. Major or serious intra- or postoperative complications are very rare. All complications were classified and given a code according to the classification system of the international urogynecological association and the international continence society (IUGA/ICS) on 2011. The applicability and practicability of this code were evaluated, looking for ways to possibly improve it or to identify missing parameters. Many patients had more than one code, a problem that entirely torpedoed the idea of “simple” classification. Some complications are not covered individually in the classification, such as failure and recurrence or overactive bladder syndrome. These complications should be included. Many cases began with the same code, despite having different complications. Further sub-classifications should be considered to enable the reader to easily recognize the complication at hand. Patients who came with complications more than one year after mesh or sling insertion were categorized as (T4), regardless of whether the complication arose after 1 year of after 10. Therefore, sub-classifications in the (T4) category are recommended. The “site” category was not applicable in many cases. Furthermore, it is necessary that the severity of a complication is discernible, and should be mentioned in the code. We did not find any correlation between the code given and patient satisfaction. After re-modification and completion, the IUGA/ICS code could be more practical for clinical use, which would allow for the comparison of complications and make the assessment of adverse effects easier for research purposes.
Dilated cardiomyopathy (DCM) is a myocardial disorder characterised by ventricular dilation with reduced left ventricular ejection fraction (LVEF). Immunoadsorption (IA) followed by immunoglobulin (IgG) substitution (IA/IgG) has been shown to be a promising therapeutic intervention to recover myocardial functions in DCM patients. The beneficial effects of IA/IgG therapy are associated with increased LVEF, decreased left ventricular inner diameter at diastole (LVIDd) and reduced myocardial inflammation. Despite knowing the cardiac benefits of IA/IgG, the precise molecular mechanism induced by therapy is still elusive. Additionally, only ≈60 % DCM patients treated with IA/IgG demonstrated improved heart function. Moreover, the reasons for this differential outcome among DCM patients after treatment have not been clearly understood. In this study, efforts were made to uncover the therapy induced proteomic changes in the heart of responders (relative change in LVEF ≤ 20%, LVEF < 5% absolute value) and non-responders using a global proteomic approach. Apart from it, proteomic profiling of endomyocardial biopsies and plasma was performed to find protein biomarker candidates which might be useful to distinguish responder and non-responder DCM patients before immunoadsorption therapy and support a selective and individualized treatment. To reveal therapy induced myocardial proteomic changes, endomyocardial biopsies of DCM patients before and after therapy were compared. LVEF increased (32 ± 8 to 45±7, p<0.002) and LVIDd decreased (66 ± 6 to 60±6, p<0.040) after therapy in responders, whereas non-responders did not show any significant changes in these clinical parameters. To address the changes in the myocardial proteome induced by therapy, a label-free proteomic approach was applied. The most prominent proteomic differences between both subgroups were observed in cytoskeletal, fibrosis, and extracellular matrix proteins. Therapy linked benefit in responders seems to be highly associated with the lower abundance of fibrotic and extracellular matrix proteins which seems to reflect a lower activity of transforming growth factor-β signaling. To elucidate proteomic differences between responders and non-responders at baseline, endomyocardial biopsies and plasma proteome profiling were performed. Responder and non-responder DCM patients did not show any significant differences in the clinical parameters (LVEF, LVIDd, age, inflammation, etc.) before IA/IgG therapy except for disease duration that was in tendency higher among non-responders. Proteomics profiling of endomyocardial biopsies revealed 54 differentially abundant proteins between responders and non-responders. Among those proteins, Protein S100-A8 and kininogen-1 was found higher whereas perilipin-4 was found lower abundant in responders. Plasma profiling of these subgroups revealed five proteins (S100-A8, S100-A9, C-Reactive protein, lipopolysaccharide-binding protein, and cysteine-rich secretory protein) displaying strong discriminative power between responders and non-responders. Higher abundance of Protein S100-A8 was observed in myocardium as well as in plasma among responders. Protein S100-A8 might be a potential candidate to distinguish responders and non-responders at baseline, and its potential utility at clinical levels must be evaluated. The last objective of the thesis was to establish a workflow for the relative quantitation of phosphopeptides for samples generally obtained in small amounts like myocardial biopsies. To address this question, optimization was performed with HL-1 cardiomyocytes using a PolyMAC phosphopeptide enrichment kit and the effect of TGF-β1 on the phosphoproteome was evaluated as a proof-of-principle study. Using only 200µg protein of each sample up to 2000 phosphopeptides with an efficiency of >90 percent could be covered. In total, upon TGF-β1 incubation alterations of 214, 92, and 53 phosphopeptides were observed after 1, 6 and 24 hours, respectively. Differentially altered phosphopeptides belonged to many signaling pathways including the ubiquitin-proteasome pathway, cytoskeletal regulation by Rho GTPase, calcium signaling, and TGF-β signaling. Thus, in this study a workflow for relative quantitation of phosphopeptides was established that may be later applied to precious biopsy samples. Along with this, TGF- β1 induced phosphoproteome was analysed in HL-1 cardiomyocytes.
The thesis deals with ions stored in an electrostatic ion beam trap. In the first part of the thesis the so-called self-synchronization effect is discussed. It is demonstrated that the time a bunch of injected ions is conserved by the self-synchronization effect depends on the number of injected ions. In the second part of the thesis the cooling of small anionic cobalt and copper clusters is addressed. Measurements on anionic copper clusters consisting of four to seven atoms are presented and the decay of hot clusters is observed in order to draw conclusions on the internal temperature and the cooling process itself. Afterwards measurements on Co4- are discussed and a measurement scheme based on laser induced delayed electron emission is presented enabling to monitor the internal energy distribution of the clusters over storage time in a temperature-controlled environment. The cooling of initially hot clusters as well as the heating of initially cold clusters were observed.
Achieving commercial production of electricity by magnetic confinement fusion requires improvements in energy and particle confinement. In order to better understand and optimise confinement, numerical simulations of plasma phenomena are useful. One particularly challenging regime is that in which long wavelength MHD phenomena interact with kinetic phenomena. In such a regime, global electromagnetic gyrokinetic simulations are necessary. In this regime, computational requirements have been excessive for Eulerian methods, while Particle-in-Cell (PIC) methods have been particularly badly affected by the "cancellation problem", a numerical problem resulting from the structure of the electromagnetic gyrokinetic equations. A number of researchers have been working on mitigating this problem with some significant successes. Another alternative to mitigating the problem is to move to a hybrid system of fluid and gyrokinetic equations. At the expense of reducing the physical content of the numerical model, particularly electron kinetic physics, it is possible in this way to perform global electromagnetic PIC simulations retaining ion gyrokinetic effects but eliminating the cancellation problem. The focus of this work has been the implementation of two such hybrid models into the gyrokinetic code EUTERPE. The two models treat electrons and the entire bulk plasma respectively as a fluid. Both models are additionally capable of considering the self-consistent interaction of an energetic ion species, described gyrokinetically, with the perturbed fields. These two models have been successfully benchmarked in linear growth rate and frequency against other codes for a Toroidal Alfvén Eigenmode (TAE) case. The m=1 internal kink mode, which is particularly challenging in terms of the fully gyrokinetic cancellation problem, has also been successfully benchmarked using the hybrid models with the MHD eigenvalue code CKA. Non-linear simulations in this TAE case have been performed confirming the analytical prediction of a quadratic relationship between the linear growth rate of the TAE and the saturated amplitude of the TAE for a range of moderate values of the linear growth rate. At higher linear growth rate, a slower scaling of saturated amplitude with linear growth rate is observed. This analysis has been extended to include the non-linear wave-wave coupling between multiple TAE modes. It has been shown that wave-wave coupling results in a significant reduction in the saturated amplitude. It has been demonstrated that both plasma elongation and ion kinetic effects can exert a stabilising influence on the internal kink mode. A population of energetic particles can also exert a stabilising influence at low normalised pressure. At high normalised fast particle pressure the stabilised kink mode has been shown to give way to the m=1 EPM, which has been simulated both linearly and non-linearly (the "fishbone" mode). The first self-consistent simulations of global modes in the magnetic geometry of the optimised stellarator Wendelstein 7-X have been performed both linearly and non-linearly. Limitations have been encountered in performing simulations in 3D geometry. A hypothesis for the cause of these problems is outlined and ideas for mitigation are briefly described. In addition to the hybrid model simulations, some of the first utilisations of a new scheme for mitigating the cancellation problem in the fully gyrokinetic regime have been carried out in the framework of this thesis. This scheme, which was developed separately, is concisely described in this work. The new scheme has been benchmarked with existing gyrokinetic and hybrid results. The linear Wendelstein 7-X simulations and linear and single mode non-linear TAE simulations have been repeated with the new model. It is shown that bulk plasma kinetics can suppress the growth rate of global modes in Wendelstein 7-X. The results of fully gyrokinetic TAE simulations, the first to have been performed to our knowledge, are shown to be in close agreement with those results obtained using hybrid models. In the TAE case, the hybrid models are an order of magnitude less computationally demanding than the new gyrokinetic scheme, which is in turn at least an order of magnitude less computationally demanding than the previous gyrokinetic scheme.
Myxomycetes are fungus-like protists of the supergroup Amoebozoa found to be abundant in all terrestrial ecosystems. Mainly based on its macroscopically visible fruit bodies, our knowledge on ecology and diversity of myxomycetes is better than for most other protistean groups, but there is still a lacking knowledge about global diversity patterns since tropical regions, especially the old world tropics, are still understudied. In this thesis a combination of classical ecological analyses and modern molecular methods were used to expand the current knowledge on myxomycete diversity and biogeography in the Paleotropics. A number of surveys in the Philippine archipelago are conducted to provide and to add information about the distribution of myxomycetes in the Southeast Asian region. A combination of field collecting and ca. 2500 moist chamber cultures from four unexplored areas in the Philippines, namely, the Bicol Peninsula (746 records, 57 taxa), Puerto Galera (926 records, 42 taxa), Quezon National Park (205 records, 35 taxa), and Negros Province (193 records, 28 taxa), now brings the number of species recorded for Philippines to 150; with one record, Stemonaria fuscoides, noted as new for the Asian Paleotropics. Collecting localities that have more diverse plant communities showed as well higher species diversity of myxomycetes. In congruence with studies from the Neotropical forests, it seems also that anthropogenic disturbances and the type of forest structure affect the occurrence of myxomycetes for the Philippines. Another survey carried out in another paleotropical region, the highlands of Ethiopia, revealed a total of 151 records, with all 39 species found as new for the country. Three records of Diderma cf. miniatum with a strong bright red peridium and one record of Didymium cf. flexuosum with a conspicuous broad reticulation in the spore ornamentation were described and barcoded, since both may represent morphospecies new to science. A number of rarely recorded species, like Didymium saturnus, Metatrichia floripara, Perichaena areolata, and Physarina echinospora showed that resembling to its unique flora, the east African mountain ranges harbor a diverse and distinctive myxomycete assemblage. One incentive of this study was to compile a solid large dataset for the Paleotropical region that is comparable to data obtained from comprehensive studies performed in the Neotropical areas a decade ago. A total of eight surveys (with four comprehensive regional surveys, two from lowland and two from highland, for each region, the Neo- and the Paleotropics) were used, to compare the myxomycete assemblages of both regions. Each survey comes from a region with fairly homogenous vegetation, and includes specimens from both field and moist chamber cultures component. A statistical analysis of species accumulation curves revealed that only between 70 and 95% of all species to be expected have been found. Even for >1000 specimens per survey these figures seem hardly to increase with increasing collection effort, since a high proportion of species is always represented by a single or a few records only. Both ordination and cluster analysis suggests that geographical separation explains differences in species composition of the myxomycete assemblages much better than elevational differences. 5 The molecular component of this thesis is a phylogeographic study of the widely distributed tropical myxomycete Hemitrichia serpula. It is a morphologically distinct species with golden-yellow fructifications forming a reticulum. However, subtle variation in spore ornamentation points to cryptic speciation within this myxomycete. Using two independent molecular markers, 135 partial sequences of the small subunit (SSU) rRNA (a nuclear but extrachromosomal gene) and 30 partial sequences of the elongation factor 1 alpha gene (EF1A) (a nuclear gene), a study of 135 Hemitrichia serpula specimens collected worldwide revealed the existence of four clades that are likely to represent reproductively isolated biospecies, since each clade shows a unique combination of SSU and EF1A genotypes. A Mantel test with the partial SSU sequences indicated geographical differentiation, giving a correlation coefficient of 0.467 between the pairwise computed geographic and genetic distances, compared with the 95% confidence interval from 999 permutations (-0.013 to 0.021). Biogeographical analysis of the 40 SSU ribotypes showed clear intraspecific variation and geographic differentiation demonstrating a limited gene flow among the world population. We argue that the distribution of cryptic species in the different clade can be explained by ongoing, but still incomplete speciation. An event-based ancestral area reconstruction using the software S-DIVA employed in RASP showed that the probable origin of the ribotypes was a global dispersal event in the Neotropics. Additional species distribution models that were implemented for the three most prominent clades show different putative ranges. As such H. serpula supports the moderate endemicity hypothesis for protists. In summary, myxomycete assemblages in the Paleotropics (1) displayed a higher diversity than for Neotropical forests, (2) harbor unique taxa that differentiates those assemblages in spite of the expected similar macroecological all over the Tropics, (3) are affected by geographical barriers that likely causes speciation both at a morphospecies and biospecies level, and (4) follow the ubiquitous model in the sense that gene flow mediated by long-distance dispersal of spores is high enough that a species can fill out its entire putative range, but (5) the gene flow is not high enough to prevent variation in regional gene pools, which may lead to speciation and is better explained by the moderate endemicity model. Our data are still too limited to draw a comprehensive picture of the diversity of tropical myxomycetes, but the baseline information compiled with the aid of both classical ecology and molecular approaches from this study are first major steps towards this goal.
The antigen in heparin-induced thrombocytopenia (HIT) is expressed on platelet factor 4 (PF4) when PF4 complexes with polyanions. In recent years, biophysical tools (e.g. circular dichroism spectroscopy, atomic force microscopy, isothermal titration calorimetry, x-ray crystallography, electron microscopy) have gained an important role to complement immunological and functional assays for better understanding the interaction of heparin with PF4. This allowed identification of those features that make PF4 immunogenic (e.g. a certain conformational change induced by the polyanion, a threshold energy of the complexes, the existence of multimeric complexes, a certain number of bonds formed by PF4 with the polyanion) and to characterize the morphology and thermal stability of complexes formed by the protein with polyanions. These findings and methods can now be applied to test new drugs for their potential to induce the HIT-like adverse drug effect by preclinical in vitro testing. The methods and techniques applied to characterize the antigen in HIT may also be helpful to better understand the mechanisms underlying other antibody-mediated disorders in thrombosis and hemostasis (e.g. acquired hemophilia, thrombotic thrombocytopenic purpura). Furthermore, understanding the mechanisms making the endogenous protein PF4 immunogenic may help to understand the mechanisms underlying other autoimmune disorders.
Hantaviruses are enveloped viruses with a single-stranded RNA genome of negative polarity. The genome consists of three segments: small (S), medium (M) and large (L). As zoonotic pathogen, hantaviruses are worldwide responsible for 150,000 to 200,000 human disease cases per year. Two forms of human disease are currently distinguished: In the Americas the hantavirus cardiopulmonary syndrome (HCPS) and in Europe and Asia the hemorrhagic fever with renal syndrome (HFRS). Since the introduction of the German Protection against Infection Act in 2001 until now a total of 10,082 disease cases have been reported. As a result, hantavirus infections currently rank as the fifth frequent notifiable disease in Germany. More than 80% of these infections were caused by the hantavirus species Puumala virus (PUUV), transmitted by the bank vole Myodes glareolus. Besides temporal oscillations, an unequal geographical distribution of human PUUV cases was noticed in Germany and in other countries of Central Europe. This is reflected in the presence of endemic and non-endemic regions as well as of so-called outbreak years. Therefore, the overall objective of this study was to find out possible reasons for the inhomogeneous distribution of PUUV in Central Europe, in particular in Poland, Germany and certain districts of Baden-Wuerttemberg. The basic working hypothesis was that PUUV spread in Central Europe after the last glaciation with different evolutionary lineages of the bank vole and that the current emergence of PUUV in bank vole populations is determined by local geographical and ecological factors. Very little was known about the presence of PUUV in Poland. Earlier studies were based exclusively on serological detection of PUUV, but a molecular detection with subsequent phylogenetic investigation was missing so far. Therefore, 45 bank voles from the northeastern part of Poland were investigated by serological and molecular assays. In three animals from a forest region close to the city of Miko³ajki PUUV-reactive antibodies and/or PUUV RNA were detected. Phylogenetic analysis indicated the presence of a Latvian (LAT) PUUV strain. Viral RNA was detected in one bank vole of the Eastern evolutionary lineage and two animals of the Carpathian lineage. Thereby it could be demonstrated for the first time that the distribution of the LAT PUUV lineage ranges from Latvia south-west to the northeastern part of Poland. An inhomogeneous spatial distribution of human disease cases has been observed even for Baden-Wuerttemberg, a long time known endemic federal state of Germany. Therefore 660 bank voles were trapped during the outbreak and non-outbreak years 2012 and 2013 in four districts with high incidences (H) and in four districts with low incidences or lacking PUUV cases (L). During the outbreak year 2012 PUUV-positive bank voles were detected by serological and molecular investigations in seven of eight districts. In contrast, in the following year only in one district PUUV infected bank voles were detected. Furthermore, it was demonstrated that after a beech mast, i.e., a massive fructification of beech trees, in H districts with a higher percentage of beech forest coverage a higher number of human cases was notified, but not in L districts with a lower percentage of beech forest coverage. For the future development of early warning modules it is therefore necessary to have a long-term bank vole monitoring established that incorporates beech mast data and information on beech forest coverage. High endemic regions for PUUV are mainly located in the southern and western parts of Germany, whereas in the eastern and northern parts only low numbers or even no human cases are recorded. To find out possible reasons for this inhomogeneous distribution, 1,774 bank voles from different regions of Germany were investigated for PUUV infections and in parallel for the corresponding bank vole evolutionary lineage (Western, Eastern, Carpathian). The PUUV investigations indicated positive voles in the known endemic regions with an easternmost and northernmost occurrence in western Saxony-Anhalt, western Thuringia and in Osnabrück. In the northern and eastern part of Germany none of the 1,210 investigated bank voles showed a PUUV infection. In the southern and western parts of Germany only the Western bank vole lineage was identified, whereas the Eastern lineage was exclusively found in the eastern and northern part and the Carpathian lineage in the South-East and North-East of Germany. PUUV infections were found almost exclusively in bank voles of the Western lineage. Individuals of the other two vole lineages were found to be PUUV infected only in regions with sympatric occurrence of the Western lineage. The previously described contact zone of the different bank vole phylogroups ranges from Poland to the entire northern part of Germany. In conclusion, the results of this investigation indicate two potential major reasons for the inhomogeneous distribution of PUUV in Germany: First, PUUV of the CE lineage seems to be associated with the Western bank vole lineage. The current geographical distribution of virus and host might be explained by a post-glacial northern expansion of the bank vole starting at the western refuge. Second, the missing detection of PUUV in bank voles of the Western lineage in areas close to high endemic regions might be explained by the extinction of the virus due to a limited winter survival of infected animals during long and harsh winters. The virus stability outside the host or ecological barriers, such as isolated forest areas or broad rivers, might also influence the distribution of PUUV in bank vole populations.
The presented study was dedicated to outstanding issues in regard to the safety and efficacy of the LAV “CP7_E2alf”, during the final licensing process and towards its putative implementation in outbreak scenarios as emergency vaccine. (I) For application of a genetically engineered virus under field conditions, knowledge about its genetic stability is mandatory. Therefore, the genetic stability of “CP7_E2alf” needed to be assessed in vivo and in vitro. Mutation rates were compared to the parental pestivirus strains (BVDV-1 “CP7” and CSFV “Alfort/187”), and BVDV or CSFV field-strains. There was no indication that “CP7_E2alf” could be more prone to mutational events than its parental viruses or representative field-strains. Moreover, no recombination events were observed in in vitro experiments. In conclusion, the data obtained in this study confirm a strong genetic stability of “CP7_E2alf” as an important safety component. (II) Since vaccination of breeding animals is often discussed, this study was conducted to assess the safety of “CP7_E2alf” vaccination of breeding male pigs. The study with “CP7_E2alf” vaccinated boar demonstrated that the new CSFV marker vaccine is suitable for application in reproductive boar. Neither in organs of the uro-genital tract related to sperm production nor in urine or feces, vaccine virus genome was detectable. Dissemination of “CP7_E2alf” through semen, and shedding with urine and feces, is therefore highly unlikely. (III) In order to investigate the influence of pre-existing pestivirus antibodies of the efficacy of “CP7_E2alf”, a vaccination-challenge-trial was conducted with “CP7_E2alf” (Suvaxyn® CSF Marker) and the “gold-standard” of live-modified CSFV vaccines, the C-strain (RIEMSER® Schweinepestvakzine). Pre-existing antibodies against BVDV-1 were provoked through intramuscular inoculation of a recent field isolate from Germany. Seven days after the vaccination, all animals were challenged with highly virulent CSFV strain “Koslov”. It was demonstrated that pre-existing anti- BVDV-1 antibodies do not impact the efficacy of both live attenuated vaccines against CSFV. Both C-strain “Riems” and marker vaccine “CP7_E2alf” were able to confer full protection against the highly virulent challenge. However, slight interference was seen with serological DIVA diagnostics accompanying “CP7_E2alf”. Amended sample preparation and combination of test systems was able to resolve most cases of false positive reactions. However, in such a coinfection scenario, optimization and embedding in a well-defined surveillance strategy is clearly needed for marker vaccination scenarios. (IV) To supplement the data about the kinetic of maternally derived antibodies in piglets from sows vaccinated during outbreaks, a single “emergency-type” vaccination of two pregnant sows was done. Focus was laid on the kinetics of maternally derived antibodies (MDA) in the screening assays of their offspring with screening assays that would be used in case of CSFV outbreaks, i.e. CSFV E2 and Erns antibody ELISA. Upon vaccination with “CP7_E2alf” 21 days before farrowing, MDAs were measurable in all piglets born to vaccinated sows. The E2- ELISA reactivities showed an almost linear decrease over ten weeks after which all piglets were tested negative in the ELISA. Future studies should investigate, if MDA are able to protect offspring of vaccinated sows or whether the piglets should also be vaccinated.
Abstract
Nanoscale multilayer thin films of W and PC (Polycarbonate) show, due to the great difference of the components’ characteristics, fascinating properties for a variety of possible applications and provide an interesting research field, but are hard to fabricate with low layer thicknesses. Because of the great acoustic mismatch between the two materials, such nanoscale structures are promising candidates for new phononic materials, where phonon propagation is strongly reduced. In this article we show for the first time that W/PC-multilayers can indeed be grown with high quality by pulsed laser deposition. We analyzed the polymer properties depending on the laser fluence used for deposition, which enabled us to find best experimental conditions for the fabrication of high-acoustic-mismatch W/PC multilayers. The multilayers were analyzed by fs pump-probe spectroscopy showing that phonon dynamics on the ps time-scale can strongly be tailored by structural design. While already periodic multilayers exhibit strong phonon localization, especially aperiodic structures present outstandingly low phonon propagation properties making such 1D-layered W/PC nano-structures interesting for new phononic applications.
Background: Microvascular Decompression represents an effective treatment for hemifacial spasm. The use of lateral spread responses (LSRs) monitoring remains a useful intraoperative tool to ensure adequate decompression of the facial nerve. Objective: To assess the value of LSRs intraoperative monitoring as a prognostic indicator for the outcome of microvascular decompression in hemifacial spasm. Methods: Our study included 100 patients prospectively. The patients were classified into 4 groups whether LSRs were totally, partially, not relieved or not detected from the start. According to clinical outcome, the patients were classified into 4 groups depending on the clinical course after surgery and the residual symptoms if any. Then, correlations were made between LSRs events and treatment outcome to detect its reliability as a prognostic indicator. Results: LSRs were relieved totally in 56% of the patients, partially relieved in 14%, not relieved in 10% and were not detected in 20% of the patients from the start. HFS was relieved directly after operation in 62% with clinical improvement of 90-100%. 31% described 50-90% improvement over the next 3 months after surgery. Almost all of these 31% (28 out of 31 patients) reported further clinical improvement of 90-100% within one year after surgery. 3% suffered from a relapse after a HFS-free period and 4% reported minimal or no improvement describing 0-50% of the preoperative state. The percentage of the satisfied patients with the clinical outcome who reported after one year a clinical improvement of 90-100% was 90%. Statistical analysis did not find a significant correlation between the relief of LSRs and clinical outcome. Conclusion: LSRs may only represent an intraoperative tool to guide for an adequate decompression but failed to represent a reliable prognostic indicator for treatment outcome.
Myxomycetes (Amoebozoa, plasmodial slime molds) are one of the last larger groups of organisms where the biodiversity is not yet investigated by molecular methods, except for a very few cultivable model species. Based on the first phylogenies for the group produced in 2012 and 2013, this thesis work explores the genetic diversity of wild populations of myxomycetes, addressing two questions: 1. Does diversity and phylogenetic trees found with barcode markers fit the current morphological species concept, and do barcode markers reveal a lower or higher diversity than found by morphological characters? In the first case, morphological characters seen as decisive for species differentiation would be plastic (shaped by the environment), in the second case we must assume the existence of cryptic species. 2. Can genetic markers be used to see if natural populations of myxomycetes reproduce mainly sexual or asexual? Sexuality is proven to occur in the Amoebozoa, but asexual reproduction should be advantageous for habitat colonization. Experiments with cultivable species have shown that both reproductive modes occur in the myxomycetes. Two species complexes were chosen for an in-depth investigation. The first species is the common wood-inhabiting myxomycete Trichia varia (Pers. ex J.F. Gmel.) Pers., one of the first myxomycetes to be described and always seen as a variable, yet single, species. The second example involves a snowbank species so far known as Lamproderma atrosporum Meyl., which was recently transferred to a genus on its own, Meriderma Mar. Mey. & Poulain, and a morphological species concept, including several taxa, was proposed. Trichia varia belongs to the bright-spored myxomycetes. Partial sequences of three independent markers (nuclear small-subunit ribosomal RNA gene, SSU, extrachromosomal; protein elongation factor 1 alpha gene, EF1A, chromosomal; cytochrome oxidase subunit 1 gene, COI, mitochondrial) from 198 specimens resulted in a three-gene phylogeny containing three groups, within each group combinations of the single-marker genotypes occurred exclusively. Complete SSU sequences were generated for 66 specimens, which revealed six positions that can carry group I introns and putatively functional or degenerated homing endonuclease genes in two groups. All observations (genotypic combinations of the three markers, signs of recombination, intron patterns) fit well into a pattern of three cryptic biological species that reproduce predominantly sexual but are reproductively isolated. The pattern of group I introns and inserted homing endonuclease genes mounts evidence that the Goddard-Burt intron life cycle model applies to naturally occurring myxomycete populations. A total of 89 specimens of the dark-spored myxomycete genus Meriderma from five European mountain ranges were sequenced for partial genes of SSU and EF1A. The latter gene includes an extremely variable spliceosomal intron. Three clades, the two morphologically recognizable taxa M. fuscatum, M. aggregatum, and the morphologically complicated complex species M. atrosporum agg., were recovered. The EF1A-based phylogeny of the 81 specimens of M. atrosporum agg. resulted in seven subclades, with the two EF1A-haplotypes of a sequence sharing always one subclade for each of the 50 heterozygous specimens, a pattern consistent with the existence of several independent but sexually reproducing biospecies. Identical EF1A genotypes occurred more often within a regional population than in between. A simulation assuming panmixis within a biospecies but not in between, and isolation between mountain ranges suggested that similar numbers of shared genotypes can be created by chance through sexual reproduction alone. Numbers of haplotypes shared between mountain ranges correlate with geographical distance, suggesting occasional long-distance dispersal by spores. An enlarged data set containing 227 partial SSU sequences of Meriderma spp. identified 53 ribotypes, with a ribotype accumulation curve indicating 68.4±14.5 ribotypes to expect according to the Chao2 estimator. The topology of the SSU phylogeny generally confirms results from the partial SSU and EF1A data set of 89 specimens, where several putative biospecies could be recognized. A novel method for automated analyses of SEM images allows to derive quantitative descriptors for spore ornamentation, which were subjected to multivariate analyses. Spore ornamentation provided traits with the highest explanatory power in a multivariate statistics, whereas spore size and stalk length were much less significant. For some but not all putative biospecies a unique combination of morphological characters was found, which is in accordance with the hypothesis of instant sympatric 8 speciation via mutations creating incompatible strains splitting from existing biospecies. The morphologically recognizable taxa of the genus are described and a key for the genus Meriderma is given. To compare morphological and molecular diversity in lignicolous myxomycetes, all specimens found in a study covering the late-autumn aspect were sequenced, using partial SSU gene as a barcode marker. A total of 161 logs in the old-growth forest Eldena, northeastern Germany, was surveyed, resulting in 530 collections representing 27 taxa from 14 genera. Bright-spores species were far more abundant than dark-spored taxa. A phylogeny based on partial SSU sequences for bright-spored myxomycetes revealed morphospecies to be largely consistent with phylogenetic groups. Most but not all morphospecies may contain multiple ribotypes that cannot be differentiated by light microscopy. This first study backing up a traditional morphology-based survey by a full molecular component demonstrates that partial SSU sequences can function as reliable barcode markers for myxomycetes, but reveals as well a significant, yet not infinite, amount of hidden diversity. The main conclusions of this work, set up in the frame of a project funded by the German Research Council (DFG), are the following: 1. Sexual reproduction seems to be an important, if not the dominating mode (apart from clonal myxamoebal populations built up by binary fission) of reproduction in naturally occurring populations of myxomycetes. 2. From the two investigated species complexes we can expect many, if not most, morphopecies to be composed of reproductively isolated, sexually reproducing, biospecies. 3. Partial SSU sequences, as most widely used in this study, seem to represent suitable barcode markers for the group and can be used to distinguish the (usually cryptic) biospecies, although they alone do not allow any conclusions about reproductive isolation and speciation processes. 4. We have to expect a significant amount of hidden diversity in myxomycetes, which will increase the number of taxa from ca. 1000 recognized morphologically by a factor between two and ten.
Enzymatic evolution and the corresponding relationship to substrate scope and catalytic promiscuity were targeted in this thesis. As enzyme examples, pig liver esterase (PLE), oleate hydratases and linoleate isomerases, as well as epoxide hydrolases (EH) and haloalkane dehalogenases (HLD) were used. The substrate scope and the enantiopreference of PLE was analyzed by molecular modeling and substrate docking, since different enantiomeric excesses were detected for the conversion of malonate diethyl esters, depending on the PLE isoenzyme. Additionally, fatty acid converting enzymes with high identity were found and analyzed to comprehend the switch of both activities. Furthermore, the evolutionary connection between EH and HLD was investigated by interconversion studies to implement an HLD acitivity in an EH. By directed evolution and rational design, both possibilities of protein engineering were realized. Finally, a new methodology for targeted, continuous in vivo evolution was established by a temperature-dependent mutagenesis frequency.
Background: Pancreatic cancer is the fourth leading cause of cancer-related mortality in both genders. More than 80% of patients suffer from significant weight loss at diagnosis and over time develop severe cachexia. Early nutritional support is therefore essential. Summary: This review evaluates the different nutritional therapies, such as enteral nutrition, parenteral nutrition and special nutritional supplements, on nutritional status, quality of life and survival. Key Message: Due to the high prevalence of malnutrition and the rapid development of anorexia-cachexia-syndrome, early nutritional intervention is crucial and supported by clinical data. Practical Implications: Enteral nutrition should be preferred over parenteral nutrition. Omega-3 fatty acids and <smlcap>L</smlcap>-carnitine are promising substances for the prevention of severe cachexia, but further randomized controlled trials are needed to establish generally accepted guidelines on nutrition in pancreatic cancer.
Background: Cardiovascular disease (CVD) remains the major cause of mortality and morbidity worldwide and produces large productivity loss. The majority of CVD mortality could be prevented with changes in modifiable risk factors including tobacco use, physical inactivity, unhealthy diet and harmful use of alcohol. Successful behavioral prevention of CVD requires the identification of relevant target behaviors and reach of populations at risk. Presenteeism i.e. attending work while ill is discussed as a work-related risk factor for CVD. However, little is known about the interplay of presenteeism with established health risk behaviors. The first aim of this dissertation was to examine the association of presenteeism with health behaviors (study 1). The second aim was to examine factors that can enhance the public health impact of CVD prevention efforts. Therefore, the effect of recruitment strategy used on reach (study 2) and of communication channel used on intervention usage (study 3) was examined. Methods: Study 1 comprised data from 710 Australian employees aged 18 years and older who completed an online-survey. Linear regression analysis was used to examine the association of health behaviors (physical activity, work and non-work-related sitting time, sleep duration and sleep quality) with presenteeism. For study 2 individuals aged 40-65 years were invited to a two-stage cardio-preventive program including an on-site health screening and a cardiovascular examination program (CEP) using face-to-face recruitment in general practices (n = 671) and job centers (n = 1,049), and mail invitations from a health insurance company (n = 894). Recruitment strategies were compared regarding three aspects of reach: (1) participation rate, (2) participants’ characteristics i.e. socio-demographics, self-reported health and CVD risk factors, and (3) predictors of program participation. Study 3 compromised 16,948 users (aged 18 years and older) of the feely available physical activity promotion program 10,000 Steps. Users were grouped based on which platform (website, app) they logged their physical activity: Web-only, App-only, or Web-and-app. Groups were compared on socio-demographics, engagement parameters and logged physical activity. Non-usage attrition i.e. discontinued program usage over the first three months was examined using Kaplan-Meier survival curves. A Cox regression model was used to determine predictors of non-usage attrition. Results: Analyses from study 1 revealed that presenteeism was associated with poor sleep quality and suboptimal sleep duration after controlling for socio-demographics, work and health-related variables. Engaging in three health risk behaviors was associated with higher presenteeism compared with engaging in none or one. Study 2 showed screening participation rates of 56.0%, 32.8%, 23.5% for general practices, job centers and the health insurance company, respectively. Participation rate for the CEP among eligible individuals was 80.3%, 65.5%, and 96.1%, respectively. Job center clients showed the lowest socio-economic status and the most adverse CVD risk pattern. Whereas being female predicted screening participation across all strategies, higher age predicted screening participation only within individuals recruited via the health insurance company. Within general practices and job centers CEP participants were less likely to be smokers than non-participants. Study 3 revealed that engagement with the program was highest for Web-and-app users. Cox regression showed that user group predicted non-usage attrition: Web-and-app users (hazard ratio = 0.86; P < .001) and App-only users (hazard ratio = 0.63; P < .001) showed a reduced attrition risk compared to Web-only users. Further, older age, being male, being non-Australian, higher program engagement and higher number of steps logged were associated with reduced non-usage attrition risk. Conclusion: The results of this dissertation have three implications for designing CVD behavioral interventions with a high public health impact. First, employees suffering from presenteeism may require interventions addressing health risk behaviors including suboptimal sleep behaviors. Second, implementing prevention efforts in job centers may be especially useful to reduce health inequalities induced by social gradient. Third, the population impact of web-based interventions may be increased when using mobile delivery channels.
Members of the species Bacillus pumilus get more and more in focus of the biotechnological industry as potential new production strains. Based on secretome analysis, Bacillus pumilus strain Jo2, possessing high secretion capability, was chosen for an omics based investigation. The physiology of Bacillus pumilus cells growing either in minimal or complex medium was analyzed by a combination of proteomic and metabolomic methods. Master gels of the cytosolic and the secreted proteome covering major parts of the main metabolic pathways were created by means of 2D gel electrophoresis. Quantification of 2D gels allowed displaying the most abundant proteins in these sub-proteomes. Application of the GeLC-MS/MS technique tripled the number of identified proteins and enabled detection of many intrinsic membrane proteins. In total, 1542 proteins were identified in growing B. pumilus cells, among them 1182 cytosolic proteins, 297 membrane and lipoproteins and 63 secreted proteins. This accounts for about 43 % of the 3616 proteins encoded in the B. pumilus Jo2 genome sequence. By using GC-MS, IP-LC/MS and H-NMR methods numerous metabolites were analyzed and assigned to the reconstructed metabolic pathways. Our data indicate that applying a combination of proteomic and metabolomic techniques a comprehensive view of the physiology of growing B. pumilus cells can be gained. In addition, selected production-relevant genome features such as the restriction modification system, NRPS clusters and the secretory system of B. pumilus Jo2 are discussed. In their natural habitat, the soil, B. pumilus cells are often exposed to growth limiting conditions due to the lack of sufficient amounts of nutrients. Such limitations can also occur during fermentation conditions and will negatively influence the efficiency of the process. Glucose is the main carbon and energy source of B. pumilus. Thus, a deficiency of glucose has an enormous impact on cell growth. A 1D LC-MS/MS approach was performed to quantify the proteins using an N14/N15 labeling and to analyze the changes in the protein equipment when B. pumilus cells stop their exponential growth and become stationary due to limitation of glucose. 1033 proteins in the cytosolic fraction of B. pumilus cells were quantified and 272 of them appeared to be upregulated when the cells experience glucose starvation. 2D-PAGE was used to analyze the exoproteome of those cells. Glucose starving B. pumilus cells seemed to focus on usage of proteins and peptides as alternative carbon and energy sources instead of other carbohydrates. Especially the exoproteome of glucose starving cells is dominated by proteases and peptidases. Furthermore, cells used fatty acids as carbon source indicated by upregulation of enzymes involved in β-oxidation and the methylcitrate pathway. Bacillus pumilus is characterized by a higher oxidative stress resistance than other comparable industrially relevant Bacilli such as B. subtilis or B. licheniformis. In this study the response of B. pumilus to oxidative stress was investigated during a treatment with high concentrations of hydrogen peroxide at the proteome, transcriptome and metabolome level. Genes/proteins belonging to regulons, which are known to have important functions in the oxidative stress response of other organisms, were found to be upregulated, such as the Fur, Spx, SOS or CtsR regulon. Strikingly, parts of the fundamental PerR regulon responding to peroxide stress in B. subtilis are not encoded in the B. pumilus genome. Thus, B. pumilus misses the catalase KatA, the DNA-protection protein MrgA or the alkyl hydroperoxide reductase AhpCF. Data of this study suggests that the catalase KatX2 takes over the function of the missing KatA in the oxidative stress response of B. pumilus. The genome-wide expression analysis revealed an induction of bacillithiol (Cys-GlcN-malate, BSH) relevant genes. An analysis of the intracellular metabolites detected high intracellular levels of this protective metabolite, which indicates the importance of bacillithiol in the peroxide stress resistance of B. pumilus. Using the physiological knowledge gained during our studies, we analyzed samples taken during an industrial fermentation process. Five samples were taken during the processes using a protease overexpressing B. pumilus strain and a non-overexpressing B. pumilus reference strain. 2D-PAGE was employed to analyze the samples. 448 proteins could be identified in the samples from the protease overexpressing stain as well as 453 proteins in the reference strain. The proteins were quantified relatively comparing the different growth phases of each strain as well as comparing the strains to each other. The physiological knowledge gained from the shake flask studies enabled us to interpret the findings. Both strains showed an induction of proteins involved in acquisition of alternative carbon sources and of proteins involved in degradation and usage of fatty acids, e.g. the methylcitrate pathway, when they stop exponential growth. This is comparable to the results gained from the analysis of B. pumilus cells under glucose limitation, indicating similar conditions during the processes. Especially in the late phases of the fermentation processes the cells were obviously exposed to severe stress conditions. Our results demonstrated that overexpressing cells showed a significantly stronger oxidative stress response at the end of the fermentation process compared to non-overexpressing cells, which indicated that not only the high cell densities but also the overproduction of the target protein might be responsible for these conditions.
High-Frequency Binaural Beats Increase Cognitive Flexibility: Evidence from Dual-Task Crosstalk
(2016)
Increasing evidence suggests that cognitive-control processes can be configured to optimize either persistence of information processing (by amplifying competition between decision-making alternatives and top-down biasing of this competition) or flexibility (by dampening competition and biasing). We investigated whether high-frequency binaural beats, an auditory illusion suspected to act as a cognitive enhancer, have an impact on cognitive-control configuration. We hypothesized that binaural beats in the gamma range bias the cognitive-control style toward flexibility, which in turn should increase the crosstalk between tasks in a dual-task paradigm. We replicated earlier findings that the reaction time in the first-performed task is sensitive to the compatibility between the responses in the first and the second task—an indication of crosstalk. As predicted, exposing participants to binaural beats in the gamma range increased this effect as compared to a control condition in which participants were exposed to a continuous tone of 340 Hz. These findings provide converging evidence that the cognitive-control style can be systematically biased by inducing particular internal states; that high-frequency binaural beats bias the control style toward more flexibility; and that different styles are implemented by changing the strength of local competition and top-down bias.
Because of the vital role of the liquid as interface in plasma medicine, this work is focused on the elucidation of the interaction of plasmas with biologically relevant liquids. The results of this thesis are an important step in the direction of the applications to real biological liquids such as blood and wound secretion ex vivo as well as in vivo. In this thesis the following questions are investigated and answered with the special focus on the free radicals as highly reactive and, therefore, hard to detect relevant group of chemical species: What is the impact of the atmospheric-pressure argon plasma jet on biologically relevant solutions? Which species are generated due to the plasma treatment of liquids? What is an appropriate detection procedure for the qualification and quantification of the short-lived species? Does the surrounding conditions influence the formation of liquid-phase reactive species and can this influence be used to tailor a desired liquid composition? What is the influence of the plasma surroundings? What is the influence of feed gas manipulation regarding the reactive species generation? Can these impacts be used for a selected reactive species composition generation? Does the treated liquid medium affect the plasma-generated reactive species output and in what way? Which are the underlying mechanisms and origins of the plasma-caused chemical changes in the solutions? Do reactive species exist, which origin is located in the gaseous phase? What is the impact of the plasma jet radiation?
Protamine is administered as protamine sulfate to reverse the anticoagulant effect of heparin following cardiopulmonary bypass surgery. Immunogenicity of protamine has been recognized for decades in several patient groups including vasectomized men, diabetic patients on protamine-containing insulin and patients undergoing cardiopulmonary bypass surgery. Anti-protamine/heparin antibodies are a newly described class of heparin-dependent antibodies found in about 30% of patients exposed to protamine and heparin during cardiac surgery. A subset of seropositive patients especially who tested positive for platelet-activating anti-protamine/heparin immunoglobulin G (IgG) antibodies before surgery have prolonged postoperative thrombocytopenia with an increased risk for arterial occlusions. Studies presented in this thesis shed light on potential approaches that may prevent antibody-mediated platelet activation by anti-protamine/heparin antibodies. Two approaches are presented in this thesis, partially desulfated heparin (ODSH) and low molecular weight protamine (LMWP). Our studies demonstrated the ability of ODSH to inhibit anti-protamine/heparin antibody-mediated platelet destruction in the NOD/SCID mouse model by: i) reduction of antibody binding to preformed protamine/heparin complexes, as shown by enzyme immunoassay, ii) interfering with the binding of protamine/heparin complexes to platelets as shown by flow cytometry and fluorescence microscopy, and iii) inhibition of antibody-mediated platelet activation. Interestingly, ODSH was also able to block ongoing platelet destruction by displacing pre-bound complexes from the platelet surface. In addition, our data suggest the use of synthesized LMWP as a substitute for protamine in heparin reversal. The in vitro investigations showed that synthesized LMWP efficiently neutralizes heparin using the activated partial thromboplastin time. Anti-protamine/heparin antibodies have low binding properties to LMWP/heparin complexes as indicated in enzyme immunoassay. The ability of platelet-activating anti-protamine/heparin antibodies to induce platelet activation in the functional assay was significantly reduced in the presence of LMWP/heparin compared to protamine/heparin complexes. Owing to findings obtained in our studies, both approaches might be a promising future option to reduce anti-protamine/heparin antibody-mediated adverse effects.
An interesting subclass of the SLs are Cers, the simplest SLs. Cers are assigned a special role within SLs because of their involvement in many cellular and biophysical processes.In literature Cers are describe to modulate many events in signaling including apoptosis. Besides its role as second messenger and therefore the involvement in many signal cascades, Cers are also known to be essential in physical modifications and structural alternations of membranes. Such regulatory functions on membrane formation are e.g. domain formation with other lipids (i.g. SM and Chol), phase separation with sterols (Chol), vesicular trafficking, fusion, membrane curvature fluidity and thickness and the induction of membrane leakiness. In contrast to phospholipids, Cers can move from one side of the membrane leaflet to the other, due to their strong hydrophobicity. This movement is called flip-flop or as transbilayer movement and is controversially discussed. Consequently, no exact value has been reported about the flip-flop property of Cers, which probably plays an important role during the transmission of an extra cellular signal through the membrane.In order to probe the biophysical properties of ceramides, a synthetic access to 1-thioceramides (1-SHCer) analogues with different N-acyl chain length has been developed in this study. With 1SHCer the flip-flop was investigated on pre-formed liposomes and the data indicated a very rapid flip-flop of Cers with a half time t1/2 <10s in raft- and non-raft like membrane models. Furthermore, the acyl chain length exhibited no measurable impact on the speed of the flip-flop. Utilizing the same probes the importance of hydrogen bond donor and acceptor properties of Cers upon interaction with sphingomyelin in the presence or absence of cholesterol (Chol) has been probed. Performed fluorescent quenching experiments (P.Slotte) proposed the following relative preference in interaction with pSM:pSM:DAGs > pSM:Cer > pSM:Chol > pSM: 1-pCerSH.Most strikingly, the importance of the 1-OH H-bond acceptor functionality to replace Chol around and above the melting temperature of pSM has been demonstrated. Recently, an unusual subclass of SLs, named 1-deoxysphingoids have come to the foreground, as biomarker for metabolic disorders. 1-doxSA is physiologically generated (10-40nM) due to substrate promiscuity of SPT and shown to be elevated in patients with metabolic disorders. In this study an organic synthetic access to fluorescent DSB derivatives was established, featuring a fluorescent moiety at the lipid tail, such as FITC 26. Comprehensive fluorescent studies of 26 revealed an unusual subcellular distribution. Exogenous 1-doxSA analogues, such as FB1 and 1-doxSA-FITC, enter via specific entry points. During the next few hours these lipids accumulate within the cytosol prior to N-acylation by CerS. Upon N-acylation, the newly formed 1-doxdhCer and its analogues insert into the ER membrane.The fluorescent probe and most likely FB1 analogues accumulate within the late endosomal and lysosomal system, probably via a direct connection with the ER. Analysis of the lipid metabolism of unlabeled 1-doxSA and FB1 revealed a strikingly similar behavior, pointing towards a common pharmacological effect. Complete consumption of TG within 24h in epithelia cells combined with GO analysis of 1-doxSA interacting lipids indicates significant modulation of fatty acid degradation, pointing towards regulation of the energy metabolism. This is in good agreement with the observed induction of autophagy. Together, this rapid and similar metabolic change of both 1-doxSA and FB1, points toward direct 1-doxSA head-group related lipid-protein interaction and less toward the influence of FB1 on CerS activity. This work suggests the biological significance of 1-doxSA as a primary nutrient sensor to maintain nutrient homeostasis and its role in the pathophysiology of metabolic diseases.
Alcohol dehydrogenases as biocatalysts for the production of enantiomerically pure chiral alcohols
(2016)
Summary Enantiomerically pure chiral alcohols are key compounds in the production of certain chemicals including pharmaceuticals. Chemical synthesis allows to obtain maximal yield of 50% for one enantiomer ( >50% yield is achievable with chiral catalysts used in chemical synthesis), whereas biosynthesis leads to nearly 100% yield. Hence, expensive and time consuming resolution of racemic mixture can be avoided. Alcohol dehydrogenases are the most popular enzymes used in the chiral alcohols synthesis due to high activity with appropriate aldehydes or ketones. ADHs require a cofactor which has to be regenerated after the conversion of aldehyde/ketone to the respective alcohol. Thereby, different regeneration methods were used in the practical work to compare and choose the better one. R. erythropolis and C. hydrogenoformans alcohol dehydrogenases were chosen based on the literature screening. Each gene was cloned into Xplor2 vector and pFPMT vector. Xplor2 vector was used for the transformation of A. adeninivorans and pFPMT vector was used for the transformation of H. polymorpha. Chemically synthesized alcohol dehydrogenase sequences from R. erythropolis (ReADH) and C. hydrogenoformans (ChADH) were cloned between TEF1 promoter and PHO5 terminator which are components of Xplor2 vector or between FMD promoter and MOX terminator which are genetic elements of pFPMT vector. Moreover, ChADH and ReADH sequences with His-tag encoding sequence at the 5’ or 3’ end were constructed and the most active form of the protein was selected for further studies. ReADH-6H was used for the synthesis of 1-(S)-phenylethanol and ethyl (R)-4-chloro-3-hydroxybutanoate whereas ChADH-6H was used for the production of ethyl (R)-mandelate. ReADH-6H synthesized in A. adeninivorans and H. polymorpha was fully biochemically characterized. The enzymes from the two yeast species showed some differences in their pH and temperature optima, thermostability and activity levels. A-ReADH (A. adeninivorans) and H-ReADH (H. polymorpha) were highly active with the same substrates which were: acetophenone, 4-hydroxy-3-butanone and ethyl 4-chloroacetoacetate for reduction reaction along with 1-phenylethanol and 1,6-hexanediol for oxidation reaction. Recombinant A-ReADH-6H and H-ReADH-6H were synthesized in A. adeninivorans and H. polymorpha, respectively. Both enzymes were used for the synthesis of 1-(S)-phenylethanol and ethyl (R)-4-chloro-3-hydroxybutanoate with the use of substrate-coupled cofactor regeneration system. The enantiopurity of the products was >99%. Moreover, A. adeninivorans whole cell catalyst was also used for the synthesis of both chiral alcohols. BmGDH (Bacillus megaterium glucose dehydrogenase) was co-expressed with ReADH-6H for NADH cofactor regeneration. Comparison between isolated enzymes and permeabilized whole cell catalysts indicate that cell biocatalysts are more suitable for the production of 1-(S)-phenylethanol with 92% of acetophenone being converted in 60 min. However, cells did not show any significant advantage over isolated enzymes in the synthesis of ethyl (R)-4-chloro-3-hydroxybutanoate although the velocity of the synthesis of ethyl (R)-4-chloro-3-hydroxybutanoate was slightly improved using whole-cell catalysts, giving an 80% substrate conversion in 120 min. Recombinant C. hydrogenoformans alcohol dehydrogenase was synthesized in A. adeninivorans and biochemically characterized. Enzyme showed high activity only with one substrate, ethyl benzoylformate. The A. adeninivorans and H. polymorpha cell catalysts synthesizing ChADH and BmGDH (Bacillus megaterium glucose dehydrogenase) were constructed and used in the synthesis of ethyl (R)-mandelate (reduction product of ethyl benzoylformate) with the enantiopurity of the reaction product being >98%. H. polymorpha catalysts were more effective in the synthesis than A. adeninivorans cells. The first were able to convert 93% of ethyl benzoylformate within 180 min and the latter were converting 94% of the substrate within 360 min. Re-use of non-immobilized cells and catalysts entrapped in Lentikat® was performed and the improvement of the stability of immobilized catalysts was reported. Space time yield of 3.07 mmol l-1 h-1 and 6.07 mmol l-1 h-1 was achieved with A. adeninivorans and H. polymorpha cell catalysts, respectively. Alcohol dehydrogenase 1 from A. adeninivorans was analyzed concerning the synthesis of enantiomerically pure chiral alcohols. The enzyme did not synthesize industrially attractive products. However, based on biochemical characterization enzyme plays a role in the synthesis of 1-butanol or ethanol and thereby it is of biotechnological interest.