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The Na+,K+-ATPase was discovered more than 50 years ago, but even today the pumpcycle and its partial reactions are still not completely understood. In this thesis, Voltage Clamp Fluorometry was used to monitor the conformational changes that are associated with several electrogenic partial reactions of the Na+,K+-ATPase. The conformational dynamics of the ion pump were analyzed at different concentrations of internal Na+ or of external K+ and the influences on the conformational equilibrium were determined. To probe the effect of the internal Na+ concentration on the Na+ branch of the ion pump, oocytes were first depleted of internal Na+ and then loaded with Na+ using the epithelial sodium channel which can be blocked by amiloride. The conformational dynamics of the K+ branch were studied using different external K+ concentrations in the presence and in the absence of external Na+ to yield additional information on the apparent affinity of K+. The results of our Voltage Clamp Fluorometry experiments demonstrate that lowering the intracellular concentration of Na+ has a comparable effect on the conformational equilibrium as increasing the amount of K+ in the external solution. Both of these changes shift the equilibrium towards the E1/E1(P) conformation. Furthermore, it can be shown that the ratio between external Na+ and K+ ions is also a determinant for the position of the conformational equilibrium: in the absence of external Na+, the K+ dependent shift of the equilibrium towards E1 was observed at a much lower K+ concentration than in the presence of Na+. In addition, indications were found that both external K+ and internal Na+ bind within an ion well. Finally, the crucial role of negatively charged glutamate residues in the 2nd extracellular loop for the control of ion-access to the binding sites could be verified.
The present study consists of two parts: The first part is made up of questions concerning the cognitive underpinnings of auditory verbal hallucinations in schizophrenia. As this thesis framed schizophrenia as a multivariate problem, neural correlates to auditory verbal and visual hallucinations were investigated in the second part. The main finding is that vividness of mental imagery was increased in all putative high-risk groups as well as the patients themselves, compared with low-schizotypy controls. Therefore, it seems that vivid imagery is a trait rather than a state marker, and may be related to the genetic liability to develop schizophrenia. However, no evidence was found for a linear relationship between vividness of mental imagery and predisposition to hallucinate. Self-reported imagery vividness and predisposition to hallucinate did not depend on psychomotor speed or intelligence. In addition, individual psychopathology ratings did not correlate significantly with the mental imagery scores. Furthermore, the analysis of the control orientation and the degree of dysfunctional psychopathological status across the schizophrenia spectrum, showed an independence of control orientation and dysfunctional status from each other, as well as from other markers of schizophrenia or schizophrenic-like individuals. As a conclusion, external control orientation seems to be a symptom or a trait marker of schizophrenia. The results lead to the assumption that, beside schizophrenic individuals, first-degree relatives and schizotypy controls have some impairments and visible signs without suffering from the illness directly. This would lead to the further assumption that the illness schizophrenia is not only genetic but also dependent on environmental factors. In the second part of the study, we investigated anatomical and functional brain abnormalities in the schizophrenia patients compared with first-degree relatives and healthy controls. Here, the results followed the continuum of healthy controls, first-degree relatives and schizophrenic patients in the functional and anatomical data sets, and in the language lateralization. The decrease of lateralisation correlated with the severity of symptoms in the patient group. The investigation of visual hallucinations showed activity in higher visual areas during the experience of visual hallucinations in a schizophrenia patient and in a blindfolded subject. The activity in higher visual areas followed the boundaries of category-selective areas in both subjects. In contrast to the memory-related areas found in the schizophrenic patient experiencing visual hallucinations, we did not observe memory-related areas during visual hallucinations induced by blindfolding. This suggests that the neural mechanisms underlying hallucinations in schizophrenia are at least partly distinct from those operational in cortical deafferentation. It is proposed that individual differences in psychopathology, as well as neuropsychological and psychosocial functioning may provide further means to understand the complex and highly dynamic aspects of hallucinations specifically and schizophrenia in general. The enlargement of the subject sample to high-schizotypy controls and first-degree relatives of patients allowed new insights into the mental imagery debate and the dysfunctional connectivity pattern known to be responsible for psychotic symptoms. Further topics of research are discussed.
Structuring participles
(2008)
In this paper we discuss three types of adjectival participles in Greek, ending in -tos and –menos, and provide a further argument for the view that finer distinctions are necessary in the domain of participles (Kratzer 2001, Embick 2004). We further compare Greek stative participles to their German (and English) counterparts. We propose that a number of semantic as well as syntactic differences shown by these derive from differences in their respective morpho-syntactic composition.
In this work the nuclear structure of exotic nuclei and superheavy nuclei is studied in a relativistic framework. In the relativistic mean-field (RMF) approximation, the nucleons interact with each other through the exchange of various effective mesons (scalar, vector, isovector-vector). Ground state properties of exotic nuclei and superheavy nuclei are studied in the RMF theory with the three different parameter sets (ChiM, NL3, NL-Z2). Axial deformation of nuclei within two drip lines are performed with the parameter set (ChiM). The position of drip lines are investigated with three different parameter sets (ChiM, NL3, NL-Z2) and compared with the experimental drip line nuclei. In addition, the structure of hypernuclei are studied and for a certain isotope, hyperon halo nucleus is predicted.
RNA interference (RNAi) is triggered by recognition of double-stranded RNA (dsRNA), and elicits the silencing of gene(s) complementary to the dsRNA sequence. RNAi is thought to have emerged as a way of safeguarding the genome against mobile genetic elements and viral infection, thus maintaining genomic integrity. dsRNA is first processed into small interfering RNAs (siRNA) by the enzyme Dicer. siRNAs are ~21 to 25 -nt long, and contain a signature 5’ phosphate group and a two nucleotide long 3’ overhang (Bernstein et al., 2001). The siRNA is then loaded into the RNA-induced si-lencing complex (RISC), of which Argonaute is the primary catalytic component (Liu et al., 2004). Energetic asymmetry of the siRNA ends allows for its directional loading into RISC (Khvorova et al., 2003; Schwarz et al., 2003). Argonaute cleaves the passen-ger strand of the siRNA, leaving the guide strand of the siRNA bound to RISC (Gregory et al., 2005; Matranga et al., 2005; Rand et al., 2005). This single-stranded guide strand siRNA bound to Argonaute is able to recognize target mRNA in a sequence-specific manner, and cleaves the mRNA. Argonaute 2 in complex with single-stranded siRNA is sufficient for mRNA recognition and cleavage, thus forming a minimal RISC (Rivas et al., 2005). miRNAs, endogenously expressed small RNA genes which typically contain mismatches and non-Watson-Crick base pairing, are processed by this general pathway, although typically modulate gene expression by translational repression as opposed to cleavage of their target mRNA. The number of Argonaute genes is highly variable between species, ranging from one in S. pombe to twenty-seven in C. elegans. Earlier crystal structures of Argonaute apoen-zymes show the architecture of Argonaute to be a multidomain protein composed of N terminal, PAZ, MID, and PIWI domains (Song et al., 2004; Yuan et al., 2005). These multi-domain proteins are present in both prokaryotic and eukaryotic organisms. The role of Argonaute proteins in prokaryotes is still unknown, but based similarity to eu-karyotic Argonautes, they may also be involved in nucleic acid-directed regulatory pathways. These proteins have served as excellent models for learning about the struc-ture and function of this family of proteins. RNAi has found a widespread application for the simple yet effective knockdown of genes of interest. The catalytic cycle of RISC requires the binding of a number of different nucleotide structures to Argonaute, and we expect Argonaute to undergo a number of conforma-tional changes during the cycle of mRNA recognition by RISC (Filipowicz, 2005; Tom-ari and Zamore, 2005). Nevertheless, it remains unclear how the multi-domain ar-rangement of Argonaute recognizes and distinguishes between single-stranded and dou-ble-stranded oligonucleotides, which correspond to the Dicer-processed siRNA product, guide strand siRNA, and the guide strand / mRNA duplex. The Argonaute protein from Aquifex aeolicus was cloned, expressed, crystallized and solved by molecular replacement. Relative to earlier Argonaute structures, a 24° reorientation of the PAZ domain in this structure opens a basic cleft between the N-terminal and PAZ domains, exposing the guide strand binding pocket of PAZ. A 5.5-ns molecular dynamics simulation of Argonaute showed a strong tendency of the PAZ and N-terminal domains to be mobile. Binding of single-stranded DNA to Argonaute was monitored by total internal reflection fluorescence spectroscopy (TIRFS). The experi-ments showed biphasic kinetics indicative of large conformational changes, and re-vealed a hotspot of binding energy corresponding to the first 9 nucleotides, the so-called “seed region” most crucial for sequence-specific target recognition. As RNAi may have evolved as a way of safeguarding the genome viral infection, it is not surprising that viruses have evolved different strategies to suppress the host RNAi response in the form of viral suppressor protein. (Hock and Meister, 2008; Lecellier and Voinnet, 2004; Rashid et al., 2007; Song et al., 2004; Vastenhouw and Plasterk, 2004). These viral suppressors are widespread, having been identified in a number of different viral families. Not surprisingly, they generally share little sequence homology with one another, although they appear to exist as oligomers built upon a ~ 100-200 amino acid protomer. Tomato aspermy virus, a member of the Cucumoviruses, encodes for protein 2B (TAV 2B, 95 a.a., ~11.3 kDa) that acts as an RNAi suppressor. Intriguingly, a similar genomic arrangement is seen in RNAi suppressors in the Nodaviruses, a family of viruses that can infect both plants and animals, such as Flock house virus b2 (FHV b2). The 2B and b2 proteins are both derived from a frameshifted ORF within the RNA polymerase gene (Chao et al., 2005). In spite of this genomic similarity, the 2B and b2 proteins share little sequence identity, and it is not well understood how the Cucumovirus 2B proteins suppress RNAi. To address how TAV 2B suppresses RNAi, the oligonucleotide-binding properties of TAV 2B were studied. TAV 2B shows a preference for double-stranded RNA oligonucleotides corresponding to siRNAs and miRNAs, and also binds to single-stranded RNA oligonucleotides. A stretch of positively charged residues between amino acids 20-30 are critical for RNA binding. Binding to RNA oligomerizes and induces a conformational change in TAV 2B into a primarily helical structure. These studies sug-gest that suppression of RNAi by TAV 2B may occur by targeting different stages of the RNAi pathway. TAV 2B falls under the category of more general RNAi suppres-sors, with potentially multiple targets for suppression.
Structural analysis of the enzyme N-formylmethanofuran:tetrahydromethanopterin formyltransferase
(2008)
Archaea represent a third domain of life and some archaea exhibit a high degree of tolerance to extreme environmental conditions. Several members are methanogens and present in many anaerobic environments. Most methanogens are able to maintain growth simply on H2 and CO2 via the enzymatically catalyzed reaction 4H2 + CO2 > CH4 + 2 H2O. The archaeon Methanopyrus kandleri grows optimally at temperatures of 84°C to 110°C, pH values of 5.5 to 7.0 and NaCl concentrations 0.2% to 4%. The enzyme N-formylmethanofuran tetrahydromethanopterin formyltransferase (MkFTR) catalyzes the transfer of a formyl group from the cofactor N-formylmethanofuran (FMF) to the cofactor tetrahydromethanopterin (H4MPT), the second step of the above reaction. X-ray crystallographic analysis yielded insights into the structure and function of MkFTR, (1) the MkFTR monomer exhibits a pseudo-two fold structure suggestive of an evolutionary gene duplication. (2) The structure is a D2 homo-tetramer with prominent cleft-like surface features. Analysis of the interface contacts showed that the tetramer is best described as a dimer of dimers. The clefts were associated with the monomer:monomer interface and were weakly occupied by extra electron density which might be attributed to the H4MPT analog folate. (3) This suggested that the clefts are active sites and their association with oligomer interfaces suggested a basis for the dependence of activity on oligomerization. (4) The thermal stability of MkFTR most likely arises from the greater number of H- and ionic-bonds within the monomer and between monomers with respect to mesophilic protein structures. (5) The structure showed a large number of surface exposed negatively charged, glutamate and aspartate residues. These residues explain the salt dependent oligomerization, as only at high enough salt concentration is the electrostatic charge compensated by cation binding and neutralized allowing oligomerization. (6) These residues also improve the solubility of MkFTR at high salt concentration by increased charge repulsion. (7) Comparison of MkFTR structures from low and hight salt conditions showed that surface glutamate residues bind slightly more water molecules at high salt conditions further contributing to MkFTR solubility at high salt concentration.
We present the status of runs performed in the twisted mass formalism with Nf =2+1+1 flavours of dynamical fermions: a degenerate light doublet and a mass split heavy doublet. The procedure for tuning to maximal twist will be described as well as the current status of the runs using both thin and stout links. Preliminary results for a few observables obtained on ensembles at maximal twist will be given. Finally, a reweighting procedure to tune to maximal twist will be described.
It has been recognized that molecular classifications will form the basis for neuropathological diagnostic work in the future. Consequently, in order to reach a diagnosis of Alzheimer's disease (AD), the presence of hyperphosphorylated tau (HP-tau) and beta-amyloid protein in brain tissue must be unequivocal. In addition, the stepwise progression of pathology needs to be assessed. This paper deals exclusively with the regional assessment of AD-related HP-tau pathology. The objective was to provide straightforward instructions to aid in the assessment of AD-related immunohistochemically (IHC) detected HP-tau pathology and to test the concordance of assessments made by 25 independent evaluators. The assessment of progression in 7-µm-thick sections was based on assessment of IHC labeled HP-tau immunoreactive neuropil threads (NTs). Our results indicate that good agreement can be reached when the lesions are substantial, i.e., the lesions have reached isocortical structures (stage V–VI absolute agreement 91%), whereas when only mild subtle lesions were present the agreement was poorer (I–II absolute agreement 50%). Thus, in a research setting when the extent of lesions is mild, it is strongly recommended that the assessment of lesions should be carried out by at least two independent observers.
Spiders were collected at the massif 'Panský diel' near the city of Banská Bystrica (Central Slovakia). We recorded 252 spider species for the territory and one new species for Slovakia. Although the summit reaches an altitude of 1.100 m a.s.l., more or less thermophilous species apparently prevail here, especially at lower moderate sites. On the other hand, only several typical oreophilous species were documented. Many recorded species are scarce or even very rare. This indicates the very high value of this territory from both a genetic and an environmental perspective.
Spider fauna of semi-dry grasslands on a military training base in Northwest Germany (Münster)
(2008)
The spider fauna of semi-dry grasslands on the military training area of Dorbaum near Münster (North Rhine-Westphalia) was investigated. From 2002 to 2003 a total of 11,194 mature spiders from 141 species and 20 families was caught by pitfall trapping and hand sampling. Among them are 18 species listed in the Red Data Book of North Rhine-Westphalia, four species are rare or previously rarely recorded. Most of the spiders are habitat generalists that extend their occurrence into all types of habitats, while the number of species which are stenotopic to sand habitats is noticeably low (n = 13). The spider data were analysed with Principal Component Analysis (PCA). It is possible to distinguish spider communities of neighbouring forested habitats from species groups of open habitats, but there is no uniform spider community which is characteristic for semi-dry grassland.
We suggest a new method to compute the spectrum and wave functions of excited states. We construct a stochastic basis of Bargmann link states, drawn from a physical probability density distribution and compute transition amplitudes between stochastic basis states. From such transition matrix we extract wave functions and the energy spectrum. We apply this method toU(1)2+1 lattice gauge theory. As a test we compute the energy spectrum, wave functions and thermodynamical functions of the electric Hamiltonian and compare it with analytical results. We find excellent agreement. We observe scaling of energies and wave functions in the variable of time. We also present first results on a small lattice for the full Hamiltonian including the magnetic term.
The main objective of the study presented in this paper was to develop an evaluation scheme which is suitable for spatially explicit groundwater vulnerability assessment according to the Water Framework Directive (WFD). Study area was the Hase river catchment, an area of about 3 000 km2 in north-west Germany which is dominated by livestock farming, in particular pig and poultry production. For the Hase river catchment, the first inventory of the WFD led to the conclusion that 98% of the catchment area is "unclear/unlikely" to reach a good groundwater status due to diffuse nitrogen emissions from agriculture. The groundwater vulnerability assessment was embedded in the PartizipA project ("Participative modelling, Actor and Ecosystem Analysis in Regions with Intensive Agriculture", www.partizipa.net), within which a so-called actors´ platform was established in the study area. The objective of the participatory process was to investigate the effects of the WFD on agriculture as well as to discuss groundwater protection measures which are suitable for an integration in the programme of measures. The study was conducted according to the vulnerability assessment concept of the Intergovernmental Panel on Climate Change, considering sensitivity, exposure and adaptive capacity. Sensitivity was computed using the DRASTIC index of natural groundwater pollution potential. Exposure (for a reference scenario) was computed using the STOFFBILANZ nutrient model. Several regional studies were analysed to evaluate the adaptive capacity. From these studies it was concluded that the adaptive capacity in the Hase river catchment is very low due to the economic importance of the agricultural sector which will be significantly affected by groundwater protection measures. As a consequence, the adaptive capacity was not considered any more in the vulnerability assessment. A groundwater vulnerability evaluation scheme is presented which enjoys the advantage that both exposure and sensitivity can be operationalized in a spatially resolved manner (500×500 m grid) by the two models mentioned above. The evaluation scheme was applied in the Hase river catchment. 21% of the catchment was classified as highly vulnerable, another 73% as medium vulnerable. Only 6% of the Hase river catchment has low vulnerability. Grid cells of the high vulnerability class are considered as priority areas for groundwater protection measures in the programme of measures of the WFD. Measures will be particularly effective in the north-eastern part of the catchment where groundwater vulnerability is mainly due to high nitrogen emissions.
Classificatory changes are made for some taxa of New World Melolonthinae based on the examination of specimens (including type specimens) and a reevaluation of some of the characters used to justify previous classification decisions. Blepharotoma angustata (Blanchard) (new combination) is transferred from the genus Aplodema Blanchard. The Neotropical members of the genus Heteronyx Guérin-Méneville are transferred to the genus Blepharotoma resulting in the following new combinations: Blepharotoma boliviana (Moser), Blepharotoma corumbana (Moser), Blepharotoma cuyabana (Moser), Blepharotoma heynei (Moser), and Blepharotoma schencklingi (Moser). The genus Blepharotoma is transferred from the tribe Liparetrini to the tribe Sericoidini. The genus Aplodema and the junior synonym Haplodema Harold are transferred from the tribe Liparetrini to the tribe Sericoidini and synonymized with the genus Sericoides Guérin-Méneville. Sericoides magellanica (Blanchard) (new combination) is transferred from the genus Aplodema and placed as a senior synonym of Apterodema acuticollis Fairmaire (new synonymy). Ampliodactylus (new genus) is described for two southern South American species: Ampliodactylus marmoratus (Curtis) (new combination) and Ampliodactylus vestitus (Philippi) (new combination). The genus Chremastodus Solier is placed in synonymy with the genus Macrodactylus Dejean (Macrodactylini) and Chremastodus pubescens Solier is placed as a junior synonym of Macrodactylus chilensis Solier (new synonymy). Macrodactylus crassipes Philippi and Macrodactylus nigrinus Philippi are placed as junior synonyms of Macrodactylus farinosus Philippi (new synonymies). The genus Astaenosiagum Martínez is placed in synonymy with the genus Pristerophora Harold (Macrodactylini). Pristerophora longipes (Philippi) (new combination) is transferred from the genus Astaenosiagum and Schizochelus ursulus Philippi is placed as a junior synonym of this species (new synonymy). Pristerophora paulseni (new species) is described. Pristerophora picipennis (Solier) is placed as a senior synonym of Schizochelus breviventris Philippi (new synonymy) and Schizochelus serratus Philippi (new synonymy). Pusiodactylus (new genus) is described for two southern South American species: Pusiodactylus mondacai (new species) and Pusiodactylus flavipennis (Philippi) (new combination). The genus Paulosawaya Martínez and D'Andretta is placed in synonymy with the genus Clavipalpus Laporte (Macrodactylini) resulting in the new combination Clavipalpus ornatissima (Martínez and D'Andretta). The following replacement names are proposed for three junior secondary homonyms the genus Plectris LePeletier and Serville: Plectris evansi (new name) for Plectris cinerascens Moser (junior secondary homonym of Plectris cinerascens (Blanchard)), Plectris katovichi (new name) for Plectris bonariensis Frey (junior secondary homonym of Plectris bonariensis (Bruch)), and Plectris tacoma (new name) for Plectris comata (Blanchard) (junior secondary homonym of Plectris comata (Blanchard)).
Souls Forgotten
(2008)
' ''One day, Mama Ngonsu told her son: ''''Normally, a child grew up and stayed around to help his parents. The world has changed, and things are no longer as they used to be. Things must not be normal all the time, otherwise life would not be life.'''' When Emmanuel Kwanga gets a University scholarship, he travels from the lake and hills of Abehema to the Great City. Everyone in the village has invested in him their hopes for the good life. When the life they've imagined is cut short by the University guillotine, Emmanuel Kwanga must struggle to make sense of what the good life means - for himself and for Abehema - in a world where things are no longer as they used to be. This novel is about coming of age and coming to terms in Mimboland. It is also about the fragility of life and the strength of the human spirit. The filth and screaming splendor of the city and the perplexed tranquility of the village are juxtaposed, as the tension and conviviality between tradition and modernity are lived and explored. Roads and drivers, dreams and public transport link different geographies. Faltering along or speeding away, these spaces of risk, frustration and solidarity are filled with popular songs as vehicles for understanding events and relationships. With every crossing of the Pont de Maturit? the story flows, and its mysteries surge. In this novel, the worlds of the living and the dead intermingle, as do the natural and the supernatural, the visible and the invisible.'''
A series of production and perception experiments investigating the prosody and well-formedness of special sentences, called Wide Focus Partial Fronting (WFPF), which consist of only one prosodic phrase and a unique initial accented argument, are reported on here. The results help us to decide between different models of German prosody. The absence of pitch height difference on the accent of the sentence speaks in favor of a relative model of prosody, in which accents are scaled relative to each other, and against models in which pitch accents are scaled in an absolute way. The results also speak for a model in which syntax, but not information structure, influences the prosodic phrasing. Finally, perception experiments show that the prosodic structure of sentences with a marked word order needs to be presented for grammaticality judgments. Presentation of written material only is not enough, and falsifies the results.
Cytotoxic T lymphocytes eliminate infected cells upon surface display of antigenic peptides on major histocompatibility complex I molecules. To promote immune evasion, UL49.5 of several varicelloviruses interferes with the pathway of major histocompatibility complex I antigen processing. However, the inhibition mechanism has not been elucidated yet. Within the macromolecular peptide-loading complex we identified the transporter associated with antigen processing (TAP1 and TAP2) as the prime target of UL49.5. Moreover, we determined the active oligomeric state and crucial elements of the viral factor. Remarkably, the last two residues of the cytosolic tail of UL49.5 are essential for endoplasmic reticulum (ER)-associated proteasomal degradation of TAP. However, this process strictly requires additional signaling of an upstream regulatory element in the ER lumenal domain of UL49.5. Within this new immune evasion mechanism, we show for the first time that additive elements of a small viral factor and their signaling across the ER membrane are essential for targeted degradation of a multi-subunit membrane complex.
Methodology and Objects: Methodologically, from a diachronic linguistics perspective regarding the concept of the shin, spirits in folk belief in China and neighbouring cultures, we compare texts that comprise meanings a) historically in the local language and b) compared to the meanings of equivalent terms in languages of other cultures. Comparing sources of this belief, we examine if and how the shin belief can serve as an example of communication across cultural borders including practical forms of worshipping. Argumentation: We argue that the concept of the shin is across cultural and national borders a result from folk culture transcending political or cultural borders transmitted via migration of ethnic groups. Although similar, mind concepts of different cultures and groups never melted; evidence for this independence gives the Islamic distinctive separation between shin and jinn in this area in the Chinese Quran and other spiritual Chinese writings. On the other hand, the practice of worshipping is similar. Conclusions: A spiritual concept like shin varies in practice in different areas. Central Asia as the melting pot of Chinese and Middle East culture shows the cultural practice of Shamanism with shin belief, complex mind concepts like in Daoism, and religions incorporating shin belief (Islam). Observed changes in the particular local languages show the continuity of the local set of meanings. Multilingual and multicultural areas such as Central Asia rather integrate new words to increase their thesaurus with new meanings than to change the set of previous existing meanings in the languages. Arabic as a language of conquerors in Central Asia is a typical example for such a language that serves as a tool to set up new meanings.
Self-inactivating gammaretroviral vectors for the gene therapy of chronic granulomatous disease
(2008)
Chronic granulomatous disease (CGD) is a rare inherited primary immunodeficiency characterized by defective intracellular oxidative killing of ingested invading microbes by PMN and monocytes. It is caused by mutations in one of the four genes coding for the essential subunits of the NADPH oxidase (gp91phox, p47phox, p67phox and p22phox). Approximately 75% of the CGD cases are due to mutations in the gp91phox gene. If regular care and conventional therapy fail, the recommended therapy is allogeneic bone marrow transplantation (BMT), but only if a matched donor is available. A therapeutic option for patients lacking suitable donors is the genetic modification of autologous hematopoietic stem cells. The gene therapy offers an interesting alternative to BMT since it implies a less invasive treatment and represents a possibly unique curative option for patients with no suitable donor. Gammaretroviral vectors were already used in some gene therapy trials for CGD and other immunodeficiencies showing relevant clinical benefit. However, these trials uncovered an unexpected mutagenic side effect. If the retrovial integration ocurrs near to, or into proto-oncogenes this might lead to clonal dominance or even malignant transformation (Hacein-Bey-Abina et al., 2003a; Ott et al., 2006). Therefore, there was a need to further improve the safety of these vectors and to this end the self-inactivating gammaretroviral vectors were engineered. Non essential sequences for virus infectivity and integration, which might influence the surrounding gene expression, were deleted in these vectors. In the first set of experiments, a series of SIN gamma retroviral vectors was cloned driving the expression of the wild-type gp91phox cDNA under the control of a viral constitutive SFFV promoter. However initial studies with these vectors failed because the titers of the virus produced by transient transfection protocols were extremely low (<5x105 TU/ml). Therefore, a codon optimization of the gp91phox cDNA was considered as an alternative. The codon optimized synthetic gp91phox gene was used to construct a SIN gammaretroviral vector, again under the control of the SFFV promoter (Schambach et al., 2006c). With this vector an increase in titer was observed compared to the native gp91phox sequence, which was due to the improved transcription in 293T transfected cells. The enhancement of the synthetic gp91phox transcription led to a higher internal transcript production and protein expression. An enhanced superoxide production in transduced myelomonocytic X-CGD PLB-985 populations was also detected. All these data indicate that the synthetic gp91phox might represent an excellent alternative to those former constructs expressing the native gp91phox transgene. Since it was postulated that the SFFV promoter could still cause transactivation of neighboring genes due to its strength (Modlich et al., 2006), three different non-viral promoters were tested, one constitutive (the EFs promoter) and two myeloid-specific promoters (the c-fes and MRP8 promoter). The three SIN gammaretroviral vectors were able to generate high titers after transient transfection of 293T packaging cells, to efficiently transduce the X-CGD PLB-985 cell line and to reconstitute the NADPH oxidase activity to a high degree. In mouse transplantation experiments, the EFs promoter showed a high variable transgene expression in the different lineages analyzed, and the c-fes promoter showed also a ubiquitinous expression. In contrast, the MRP8 promoter showed a high myeloid specificity since gp91phox expression in mSca-1+ cells and lymphoid B cells from transplanted mice was extremly low and even absent. However, the lowest levels of transgene expression were observed in the myeloid populations both in bone marrow and peripheral blood with this vector. When the oxidase reconstitution ability of these promoters was tested, the numbers of superoxide producing cells obtained were similar than those observed in the clinical X-CGD trial conducted by the groups of Dr. M. Grez and Prof. R. A. Seger (over 35% in one patient and ~15% in the second), which led to the eradication of therapy refractory infections (Ott et al., 2006). Between the three constructs, the MRP8 promoter was less effective in restoring the NADPH oxidase activity than the EFs and c-fes promoters. The c-fes promoter reached the highest levels of DHR reactive cells in the highest number of mice. Overall, these data showed that between all constructs tested, the c-fes containing construct in combination with the codon optimized gp91phox sequence showed the best performance within the SIN gammaretroviral backbone. It generated the highest titers in combination with a better NADPH oxidase reconstituting ability. One main goal in the development of SIN gammaretroviral vectors is reducing the genotoxic effect due to random vector integration. An improved gene transfer and expression, and a constant performance are also highly desirable. The present study shows that the c-fes SIN vector in combination with the synthetic gp91phox may be considered as an effective gene therapy strategy for the restoration of the NADPH oxidase activity in CGD. It allows the use of a cellular promoter generating adequate physiological levels of the therapeutic protein and reduces the number of vector copies required for a therapeutic effect.
Science in Wonderland
(2008)
Lewis Carroll's Alice, who first explores Wonderland (1865) and later on the country behind the Looking-Glass (1872), belongs to the most well-known characters in world literature. [...] The scientific reception of Carroll's stories – concerning physics as well as the humanities – has taken place on different levels. On the one hand, […] various Carrollian ideas and episodes obviously correspond to topics, subjects and models that are treated in the contexts of scientific discourses. Therefore, they can be quoted or alluded to in order to represent theories and questions […] – as […] physical models of the world […]or theoretical models of language and communication. […] On a more abstract level of observation, Carroll's stories have been used in order to explain and to discuss the pre-conditions, the procedures, and the limits . of scientific modeling as such. Above all, they make it possible to narrate on the problem of defining and observing an 'object' of research. […] According to Deleuze, the paradox structures of the world that Alice experiences give an idea of all meaning being groundless and all logic being subverted by the illogical. Finally, besides all affinities of Alice's adventures to scientific attempts to explain the world, the absolutely incomprehensible is present in Carroll's books as well. Especially the self proves to be something profoundly incomprehensible […].
Agricultural intensification is a major threat to biological diversity worldwide. Land management activities enhancing landscape diversity are therefore regarded as a key strategy to halt species loss in cultural landscapes. Diverse and abundant communities of predatory arthropods, e.g. spiders (Araneae), have a high potential to suppress pest populations (Symondson et al. 2002) and could therefore contribute to allow reductions of pesticide use. Crop fields alone are usually not able to sustain diverse and individual-rich populations of predatory arthropods, because agricultural management results in disturbances and habitat deteriorations (harvest, soil cultivation, pesticide application) that kill or drive away large parts of the populations. Therefore semi-natural and perennial habitats in agricultural landscapes are considered to be of great importance for beneficial arthropods. On the one hand they offer refuge habitats in times when arable fields are hostile, e.g. fields with bare grounds during winter (Schmidt & Tscharntke 2005). On the other hand, viable populations of predatory arthropods in semi-natural habitats can serve as sources for (re-) colonisation of arable fields (Schmidt & Tscharntke 2005). Because of these exchanges between crop and non-crop areas it is important to include the surrounding landscape when investigating field-scale processes. We investigated the relations between spider assemblages in arable fields and the surrounding landscape in 29 fields of winter oilseed rape (OSR) in an agricultural landscape in eastern Austria. The objectives of this study were to estimate (1) how much spider assemblages in oilseed rape fields are influenced by the surrounding landscape, (2) the relative influence of landscape variables compared to field-scale variables and (3) at which spatial scales landscape variables are effective.
Hypoxic pulmonary vasoconstriction (HPV) redistributes pulmonary blood flow from areas of low oxygen partial pressure to areas of normal or relativity high oxygen availability, thus optimising the matching of perfusion to ventilation and preventing arterial hypoxemia. Generalised alveolar hypoxia results in a sustained increase in pulmonary artery pressure which in turn leads to structural changes in the walls of the pulmonary vasculature (pulmonary vascular remodelling). Recent findings have indicated a role for cytochrome P450 (CYP) epoxygenase-derived epoxyeicosatrienoic acids (EETs) in hypoxia-induced pulmonary vasoconstriction. Given that the intracellular concentration of EETs is determined by the soluble epoxide hydrolase (sEH), which metabolises EETs to their less active dihydroxyeicosatrienoic acids (DHETs), we assessed the influence of the sEH and EETs on pulmonary artery pressure, acute and chronic HPV, and pulmonary vascular remodelling in the mouse lung. In isolated lungs from wild-type mice, acute HPV was significantly increased by sEH inhibition, an effect abolished by pre-treatment with CYP epoxygenase inhibitors and the EET antagonist 14,15-EEZE. The acute hypoxia-induced vasoconstriction and EET production were greater in lungs from sEH-/- mice than from wild-type mice and sEH inhibition had no further effect on HPV in lungs from the former animals, while MSPPOH (CYP epoxygenase inhibitor) and 14,15-EEZE decreased the response. Exogenous application of 11,12-EET increased pulmonary artery pressure in a concentration-dependent manner and enhanced acute HPV in wild-type lungs, while 14,15-EET and 11,12-DHET were without significant effect on pulmonary artery pressure. 5-HT2A receptor antagonism or Rho kinase inhibition shifted the EET concentration-response curve to the right and abrogated the EET- and sEH inhibition-induced potentiation of acute hypoxic vasoconstriction. In lungs from wild-type and sEH-/- mice, hypoxic preconditioning (hypoxic ventilation for 10 minutes) enhanced the 5-HT response. 1-Adamantyl-3-cyclohexylurea (ACU), a sEH inhibitor, further amplified the hypoxia-induced 5-HT-hypersensitivity in wild-type mice. However, after hypoxic preconditioning, the sEH-/- lungs displayed a striking leftward shift in the 5-HT response. 11,12-EET can activate TRPC6 channels in endothelial cells by eliciting its translocation to the plasma membrane, more specifically to membrane domains enriched with the caveolae marker caveolin-1. This effect was also observed in rat pulmonary artery smooth muscle cells overexpressing the channel. Exposure of the latter cells to acute hypoxia also stimulated the intracellular translocation of TRPC6 to caveolae, an effect that was sensitive to the EET antagonist. The EET-induced translocation of TRPC6 channels was prevented by a 5-HT2A receptor antagonist but not by a Rho kinase inhibitor. Moreover, while acute hypoxia and 11,12-EET increased pulmonary pressure in lungs from TRPC6+/- mice, lungs from TRPC6-/- mice did not respond to either stimuli. These results indicate that the sEH and CYP-derived EETs are involved in acute HPV and that EET-induced pulmonary contraction under normoxic and hypoxic conditions involves a TRPC6 channel, a 5-HT2A receptor-dependent pathway and Rho kinase activation. In the second part of the study the role of the sEH in the development of pulmonary hypertension and vascular remodelling induced in mice by exposure to hypoxia (10% O2) for 21 days was analysed. In wild-type mice, chronic hypoxia decreased the pulmonary expression/activity of the sEH, induced right heart hypertrophy and erythropoiesis, and increased the number of partially and fully muscularised pulmonary resistance arteries (by 3-fold). Moreover, in HEK 293 cells, hypoxia (1% O2 up to 24 h) decreased sEH promoter activity by 50%. In isolated lungs, pre-exposure to chronic hypoxia significantly increased baseline perfusion pressures and potentiated the acute HPV. While an sEH inhibitor, ACU, potentiated acute HPV in lungs from mice maintained in normoxic conditions, it had no effect on HPV in lungs from mice exposed to hypoxia. The EET antagonist, 14,15-EEZE, abolished the sEH inhibitor-dependent increase in acute HPV in normoxic lungs and decreased HPV in chronic hypoxic lungs. Hypoxia-induced right heart hypertrophy and erythropoiesis were more pronounced in sEH-/- than in wild-type mice. Under normoxic and hypoxic conditions the muscularisation of resistance pulmonary arteries was greater in lungs from sEH-/- mice than in lungs from wild-type mice. sEH-/- mice also displayed an enhanced acute HPV, compared to that observed in wild-type mice and chronic exposure to hypoxia did not further potentiate acute HPV. However, in the presence of 14,15-EEZE responses returned to levels observed in normoxic lungs from wild-type animals. Furthermore, immunohistochemistry demonstrated an extensive expression of the sEH in the medial wall of pulmonary arteries from human donor lungs. Whereas sEH expression was not detectable in samples from pulmonary hypertension patients, indicating that the sEH is involved in hypoxia-induced pulmonary vascular remodelling and hypoxic pulmonary vasoconstriction. Taken together, the results presented in this thesis indicate that the expression/activity of the sEH is an important determinant of the magnitude of acute and chronic hypoxia-induced pulmonary vasoconstriction and pulmonary vascular remodelling by inactivating vasoconstrictor CYP-derived EETs. As sEH inhibitors are currently being developed for the treatment of human systemic hypertension, it should be noted that these compounds may even promote the development of pulmonary hypertension.
The growth of blood vessels is crucial for organ growth in the embryo and repair of wounded tissues in the adult. An imbalance in this process contributes to numerous malignant, inflammatory, ischemic, infectious and immune disorders (Ferrara et al., 2003). Postnatal neovascularization occurs through the recruitment of progenitor cells and angiogenesis. Integrins are heterodimeric cell surface molecules and are the main receptors for extracellular matrix proteins. Regulation of integrin activation is crucial during embryonic development and during adult life. Dysregulation of integrin activity leads to severe diseases. In this study, we have demonstrated that Rap1, a small GTPase regulating integrin activity, and its GEF Epac1 are expressed in both EPC and endothelial cells. Moreover, the pharmacological activator of Epac activates the small GTPase Rap1 in progenitor cells. In parallel the angiogenic growth factors VEGF and bFGF activate Rap1 in endothelial cells. In addition, the regulation of Rap1 activity in EPC and in endothelial cells plays an important role in the regulation of migration and adhesion to matrix proteins, by regulating the activity of different integrins, a mechanism known as integrin inside‐out signaling. Furthermore, regulation of Rap1 activity affects probably indirectly through outside‐in signaling of integrins the activity of several and crucial proteins such PKB/Akt and focal adhesion kinase in endothelial cells. In line with these results, we have demonstrated that Rap1 activity affect angiogenesis, homing of EPC to ischemic tissues and thereby postnatal neovascularization. The understanding how Rap1 regulates integrin activity in endothelial cells is still not completely clear, for example we have demonstrated that the known effectors of Rap1 mediating the increase of integrin activity in T and B cells, such as RAPL and RIAM are, respectively, either not increasing integrin activity or not expressed in endothelial cells. We aim to find the effector of Rap1 promoting integrin activity in endothelial cells and how RAPL regulates integrin functions and angiogenesis. Moreover data from us and others using genetic models and generation of Rap1a or Rap1b deficient mice or deficient for Rap1a and Rap1b led to embryonic lethality suggesting that Rap1 is a key node protein during embryonic development. The development of conditionnal Rap1a/b endothelial/pericytes restricted deficient mice will help us to decipher more precisely the role of Rap1 during vascular development and angiogenesis.
Prion diseases or transmissible spongiform encephalopathies (TSEs) are rare neurological disorders that may be of genetic or infectious origin, but most frequently occur sporadically in humans. Their outcome is invariably fatal. The infectious agent has been defined as prion (from proteinaceous infectious only) in 1992 by Stanley B. Prusiner and represent mainly, if not solely, an abnormal, protease-resistant isoform (PrPSc) of a cellular protein, the prion protein or PrPC. According to the “protein only” hypothesis, the prion is devoid of informational nucleic acids and consists of an “infectious” protein that is capable of converting the normal host protein PrPC into a likeness of itself. TSEs can be distinguished from other neurodegenerative diseases because of their infectivity and transmission capability. The only organ system in which severe histopathological damage can be demonstrated as a consequence of infection with prions is the nervous system. The communal lesions are neuronal loss, spongiosis and astrogliosis, accompanied by an intra- and extracellular accumulation of PrPSc, occasionally in form of amyloid plaques. Even if a strong activation of microglia and astrocytes occurs, no immunological response is usually detectable as consequence of prion infection. Despite the considerable attention for its involvement in TSEs, the physiological role of the cellular, nonpathogenic isoform of PrPC, has not yet been determined. In the last years, several putative cellular functions have been attributed to PrPC: its localization in “lipid rafts” is consistent with a possible role in cell adhesion, transmembrane signalling or as a recognition molecule. Furthermore, PrPC has been implicated in protection against oxidative stress, copper metabolism, apoptosis, cell proliferation and in the regeneration of blood precursors stem cells in the adult. It has also been shown that PrPC interacts with the neuronal cell adhesion molecule NCAM, promoting neurite outgrowth. However, both the PrPC-mediated effects and the role of PrPC-dependent pathways on neuronal differentiation are still not elucidated. First objective of this Ph.D thesis was the establishment of a novel in vitro cellular model for the study of the role of PrPC in neuronal differentiation and neurite outgrowth. Furthermore, an additional goal of this project was the indentification of the PrPC domains responsible for the induction of neuronal differentiation. A novel PrPC-depleted cell line (PrP0/0 ML) was derived from murine primary PrP-knockout neuronal cells by SV40 large T antigen-mediated immortalization. A temperature sensitive form of this oncogenic protein was used, allowing a temperature-mediated regulation of its expression. This cell line was then characterised for its growth potential, for the expression of specific cellular markers and for its ability to differentiate. It was found that, under culture conditions promoting the expression of the temperature-sensitive SV40 large T antigen, the cells expressed nestin, a specific marker of neuronal precursor cells. Therefore, the PrP0/0 ML cell line was identified as a potential neuronal stem cell line. In fact, under nonpermissive culture conditions when the expression of the temperature-sensitive SV40 large T antigen is downregulated, the PrP0/0 ML cells differentiated into neurons. Noteworthy, maintenance of the cells in conditions that promote cell differentiation induced a progressive reduction in the expression levels of nestin, an event that strongly correlated with the appearance of the specific neuronal markers MAP-2b and NeuN. In order to investigate the role of PrPC in the process of neuronal differentiation, the PrP0/0 ML cells were then reconstituted for the expression of either the full-length PrP or a N-terminal truncated PrPC form (PrPdel32-134). The differentiation potential of both reconstituted cell lines under nonpermissive culture conditions was then compared with that of the parenteral PrP0/0 ML cells. This in vitro study clearly highlights that PrPC expression in the PrP0/0 ML cell line accelerates neuronal differentiation and that the N-terminal domain of the prion protein is not necessary for this PrP-mediated function. Prion diseases like BSE, vCJK, Kuru and the majority of iatrogenic cases of CJK are caused by a peripheral infection. Infectious prions accumulate in the central and peripheral nervous system as well as in extracerebral tissues, such as the secondary lymphoid organs and muscles. The prion pathogenesis is a dynamic process which can be defined temporary and spatially in different phases: i) infection and peripheral replication, ii) neuroinvasion, transport of prions from the periphery to the central nervous system (CNS), and iii) neurodegeneration. In the last years, progresses in the elucidation of the peripheral prion pathogenesis were achieved. The identification of the cell types involved in the lymphoreticular prion replication phase and the recognition of the role of the peripheral nervous system in the process of prion spread from the periphery to the CNS have elucidated some of the cellular mechanisms that are involved in prion uptake, replication and propagation. However, relatively little information is available about the mechanism(s) underlying intercellular prion transfer and tissue-to tissue prion spread. Microvesicles (MVs) are submicron vesicles (0,03-1 microm.) with a single membrane and are shed from most eukaryotic cells undergoing activation or apoptosis. The segregation of specific proteins is followed by blebbing of the membrane surface, leading to the formation of MVs and their release in the extracellular environment. MVs can be also secreted upon fusion of multivesicular endosomes with the plasma membrane (exosomes). The secretion of MVs is the result of a complex cellular process involving changes in the metabolism of lipids and proteins. The functional role of MVs is still largely unknown. However, there is evidence showing that they are important modulators of cell-to-cell communication, participate in a variety of intracellular adhesion processes and are able to induce cellular response(s). The release of PrPC and infectious PrPSc by prion infected epithelial, neuroglial and neuronal cells in association with exosomes has recently been highlighted. Furthermore, it has been shown that exosomes can propagate prion infectivity both in vitro and in vivo, suggesting that PrPSc-bearing exosomes may provide a mechanism for intercellular transmission of infectious prions in addition to cell-to-cell contact. Second objective of this Ph.D thesis was to determine the possible role of plasma membrane-derived microvesicles in the propagation and transmission of prions. The release of MVs was first studied in different murine neuronal cell lines. Here it is shown for the first time that neurons also shed plasma membrane derived MVs, in addition to exosomes. Immunoelectron microscopy and immunoblot analyses clearly demonstrated the presence of PrPC on the membrane of MVs released from PrPC-expressing cells. Characterization of lipid rafts components in MVs highlighted the presence of the ganglioside GM2, the tyrosine kinase p59Fyn, flotillin-2 and the neuronal protein GAP-43. In order to investigate whether MVs are involved in the intercellular transmission of prions, MVs were first isolated from two prion infected murine neuronal cell lines, namely the Neuro-2a PK1 and the N2a58 cells, and then used for in vitro and in vivo infection assays. Immunoblot analyses after proteinase K treatment demonstrated the association of PrPSc with the secreted MVs. The PrPSc-bearing MVs were then used to perform infection experiments on noninfected cells. By the use of cell blot assay, a method that allows the detection of PrPSc-amplification and -accumulation in cultured cells, the kinetic of prion infection in the de novo infected cells was followed. Noteworthy, it was found that PrPSc-bearing MVs were capable to transmit prions in vitro and to stably infect the recipient cells. In order to investigate the role of MVs in the transmission of infectivity in vivo, PrPSc-bearing MVs as well as MVs isolated from noninfected cells (as negative control) were injected intracerebrally in PrPC-overexpressing indicator mice (tga20). The development of clinical disease was followed in a time-dependent manner. Clinical symptoms could be observed only in the group of indicator mice inoculated with the PrPSc-bearing MVs, which then succumbed to desease. These findings clearly demonstrated that MVs are biological carriers of both PrPSc and prion infectivity. MVs could therefore participate in vivo in the processes of intercellular prion transmission and propagation.
Cytochrome P450 epoxygenases of the 2C family (CYP2C) are highly expressed in the endothelium and metabolize arachidonic acid to different regioisomers of epoxyeicosatrienoic acids (EET). They have a number of roles in the regulation of vascular tone and homeostasis by activating different signal transduction pathways and have recently been reported to be involved in proliferation and angiogenesis. However, the exact mechanisms by which epoxygenases regulate angiogenesis are still unclear. Therefore, the initial aim of the present study was to characterize the relevance of major signalling molecules that are involved in angiogenesis and to investigate possible signalling pathways involved. Initially the effect of CYP2C9 overexpression on expression levels of EphB4, a tyrosine kinase that plays a role in a number of developmental processes, was investigated. EphB4 protein expression was increased in CYP2C9 overexpressing cells without any effects on expression levels of its ligand ephrinB2. To clarify whether EphB4 is a critical determinant of CYP2C9-induced angiogenesis, endothelial cell sprouting was assessed using a collagen gel-based in vitro angiogenesis assay. Following transfection with EphB4 antisense or scrambled oligonucleotides, capillary-like structures were clearly present after 24 hours in cells overexpressing CYP2C9, while EphB4 downregulation abolished CYP2C9-induced sprouting. In addition stimulation of human umbilical vein endothelial cells with VEGF resulted in an increase in CYP2C expression and a subsequent increase of 11,12-EET production; an effect that was abolished by the CYP epoxygenases inhibitor MSPPOH as well as when cells were infected with a dominant negative mutant of AMPK. In vivo 11,12-EET treatment increased EphB4 expression in mesenteric arteries as well as in Matrigel plugs; an effect that was abolished when plugs were impregnated at the same time with small interfering RNA (siRNA) for EphB4. Furthermore, impregnation of Matrigel plugs with VEGF resulted in endothelial cell and smooth muscle cell recruitment into a Matrigel plug and this effect was mediated by CYP2C9-derived EETs as it was prevented by 14,15-EEZE. When infiltration of EET impregnated plugs with endothelial cells and pericytes/smooth muscle cells in vivo was compared to the effects seen in VEGF treated plugs, it was apparent that only EET treatment resulted in the formation of tube like structures that were covered by smooth muscle cells. Therefore, the final aim of the study was to further define the consequences of EET signalling in vivo as well as to characterize its physiological relevance. This hypothesis could be assessed by isolectin injection through the tail-vein where isolectin was taken up only by the EET-impregnated plug. Moreover ultrasound measurements revealed accumulation of contrast agent in EET impregnated plugs compared to control plugs. Taken together our findings emphasize that CYP2C plays a crucial role in the vessel formation process by modulating the effects mediated by two important control elements of the angiogenic response, namely VEGF and EphB4. CYP2C-derived EETs not only participate as second messengers in the angiogenic response, but have the potential to influence much more than angiogenesis by enhancing smooth muscle cell/pericyte recruitment to endothelial cell tubes to promote vascular maturation.
Risk transfer with CDOs
(2008)
Modern bank management comprises both classical lending business and transfer of asset risk to capital markets through securitization. Sound knowledge of the risks involved in securitization transactions is a prerequisite for solid risk management. This paper aims to resolve a part of the opaqueness surrounding credit-risk allocation to tranches that represent claims of different seniority on a reference portfolio. In particular, this paper analyzes the allocation of credit risk to different tranches of a CDO transaction when the underlying asset returns are driven by a common macro factor and an idiosyncratic component. Junior and senior tranches are found to be nearly orthogonal, motivating a search for the whereabout of systematic risk in CDO transactions. We propose a metric for capturing the allocation of systematic risk to tranches. First, in contrast to a widely-held claim, we show that (extreme) tail risk in standard CDO transactions is held by all tranches. While junior tranches take on all types of systematic risk, senior tranches take on almost no non-tail risk. This is in stark contrast to an untranched bond portfolio of the same rating quality, which on average suffers substantial losses for all realizations of the macro factor. Second, given tranching, a shock to the risk of the underlying asset portfolio (e.g. a rise in asset correlation or in mean portfolio loss) has the strongest impact, in relative terms, on the exposure of senior tranche CDO-investors. Our findings can be used to explain major stylized facts observed in credit markets.
Risk transfer with CDOs
(2008)
Modern bank management comprises both classical lending business and transfer of asset risk to capital markets through securitization. Sound knowledge of the risks involved in securitization transactions is a prerequisite for solid risk management. This paper aims to resolve a part of the opaqueness surrounding credit-risk allocation to tranches that represent claims of different seniority on a reference portfolio. In particular, this paper analyzes the allocation of credit risk to different tranches of a CDO transaction when the underlying asset returns are driven by a common macro factor and an idiosyncratic component. Junior and senior tranches are found to be nearly orthogonal, motivating a search for the where about of systematic risk in CDO transactions. We propose a metric for capturing the allocation of systematic risk to tranches. First, in contrast to a widely-held claim, we show that (extreme) tail risk in standard CDO transactions is held by all tranches. While junior tranches take on all types of systematic risk, senior tranches take on almost no non-tail risk. This is in stark contrast to an untranched bond portfolio of the same rating quality, which on average suffers substantial losses for all realizations of the macro factor. Second, given tranching, a shock to the risk of the underlying asset portfolio (e.g. a rise in asset correlation or in mean portfolio loss) has the strongest impact, in relative terms, on the exposure of senior tranche CDO-investors. Our findings can be used to explain major stylized facts observed in credit markets.
Introduction: Healthcare workers (HCWs) are exposed to bloodborne pathogens (e.g., contaminated devices). In the healthcare environment, needlestick injuries (NSI) represent a major risk factor in the transmission of hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus (HIV). Medical students are at risk of occupational exposure to bloodborne viruses following needlestick injuries during medical education. Reporting of needlestick injuries is an important step for initiating early prophylaxis or treatment. In the case of a bloodborne infection, pursuant to insure law could result in a claim. The objective of the present study was to describe occupational blood exposure of medical students through needlestick injuries.
Methods: Sixth-year medical students were invited to complete an anonymous questionnaire.
Results: In our study, 58.8% (n=183/311) of medical students recalled at least one needlestick injury during their studies. Overall, 284 needlestick injuries were reported. Only 38.3% of medical students reported all NSI to the appropriate hospital personnel. The main reason (54.0%) for not reporting NSI was being ashamed of having an NSI.
Conclusions: Occupational exposure to blood is a common problem among medical students. Efforts are required to ensure greater awareness among medical students about the risk of bloodborne pathogens. Proper training in procedures and how to act in case of injury should be offered to reduce the number of needlestick injuries.
Ribavirin in combination with peginterferon alfa shows strong clinical efficacy against chronic hepatitis C, and is now established as the standard of care. However, the precise role of ribavirin is still being defined, suggesting that optimal ribavirin dose should be maintained over the whole treatment period. Ribavirin dosage varies by bodyweight for genotype 1 disease (1000 mg/day in patients ⩽75 kg and 1200 mg/day in patients >75 kg), whereas 800 mg/day is sufficient to ensure optimal response in all genotype 2/3 patients. Similarly, genotype 1 patients benefit from 48 weeks of therapy, while 24 weeks is sufficient for genotype 2/3 disease.
Recent data suggest treatment success is dependent on cumulative ribavirin exposure, as patients who receive <60% of the planned dose have lower response rates, regardless of whether reductions are from temporary interruptions or premature cessation of therapy. All patients should be monitored for hemolytic anemia, as early diagnosis allows management through small dose reductions and stepwise return to the target dose, maximizing cumulative exposure. Despite these recent advances in our knowledge, many questions remain, such as whether the role of ribavirin will change or even be eliminated as new therapies are developed.
Rhetoric and global justice
(2008)
Activists have long recognized the importance of rhetoric and emotional appeal in building support for the global justice movement. However, many political theorists worry that the use of rhetoric may obstruct clear presentation of the issues at stake, and may result in our policies being determined by where the sympathies of the best rhetoricians lie. In this article I examine the ways in which contemporary theorists try to accommodate the need for rhetoric and emotional appeal, and I argue that their attempts are unsatisfactory because they view rhetoric as a tool or skill that can be used to manipulate people to support any position. Yet if we question the sharp separation between rhetoric and reason, then the aims of building support for a cause, identifying the causes we ought to support, and treating others with respect need no longer conflict. Re-examining the radical liberal theories of J.S. Mill, L.T. Hobhouse and J.A. Hobson, will help us to see how this separation might be questioned and rhetoric recovered as a respectful and respectable form of argument.
The genus Efferia Coquillett from the Bahamas, Cayman Islands, Cuba, and Jamaica is reviewed. The fauna now totals 16 species with 6 new species described (Ef. bellardii n. sp., Ef. bromleyi n. sp., Ef. hinei n. sp., Ef. insula n. sp., Ef. pina n. sp., and Ef. vinalensis n. sp.). Cuba has the greatest diversity with 10 species, Jamaica 3, the Bahamas 2, and the Cayman Islands 1. Efferia stylata (Fabricius) is removed from the species list of these West Indian islands. The wings of Ef. caymanensis Scarbrough and Ef. bromleyi, spermathecae of Ef. bromleyi, Ef. cubensis (Bromley), Ef. insula, Ef. nigritarsis (Hine), and terminalia of all species are illustrated. Keys for the identification of the species are provided. Specimens of two additional species from Cuba are in too poor a condition to be described but their terminalia are illustrated and the species are included in the key to the males.
The Brazilian scarab beetle genus Microchilus Blanchard (Rutelinae: Geniatini) is reviewed. Based on examination of type specimens, Microchilus beckeri Martínez is transferred to the genus Leucothyreus becoming L. beckeri (Martínez), new combination; Leucothyreus bucki Machatschke is a new junior synonym of L. beckeri (Martínez).Microchilus is limited to two species, M. beckeri (Martínez) and a new species,Microchilus rodmani Jameson, here described. Characters that circumscribe the genus, biology, and species distribution are discussed. A key to the species is provided.
Background: A number of the deeper divergences in the placental mammal tree are still inconclusively resolved despite extensive phylogenomic analyses. A recent analysis of 200 kbp of protein coding sequences yielded only limited support for the relationships among Laurasiatheria (cow, dog, bat and shrew), probably because the divergences occurred only within a few million years from each other. It is generally expected that increasing the amount of data and improving the taxon sampling enhance the resolution of narrow divergences. Therefore these and other difficult splits were examined by phylogenomic analysis of the hitherto largest sequence alignment. The increasingly complete genome data of placental mammals also allowed developing a novel and stringent data search method. Results: The rigorous data handling, recursive BLAST, successfully removed the sequences from gene families, including those from well-known families hemoglobin, olfactory, myosin and HOX genes, thus avoiding alignment of possibly paralogous sequences. The current phylogenomic analysis of 3,012 genes (2,844,615 nucleotides) from a total of 22 species yielded statistically significant support for most relationships. While some major clades were confirmed using genomic sequence data, the placement of the treeshrew, bat and the relationship between Boreoeutheria, Xenarthra and Afrotheria remained problematic to resolve despite the size of the alignment. Phylogenomic analysis of divergence times dated the basal placental mammal splits at 95–100 million years ago. Many of the following divergences occurred only a few (2–4) million years later. Relationships with narrow divergence time intervals received unexpectedly limited support even from the phylogenomic analyses. Conclusion: The narrow temporal window within which some placental divergences took place suggests that inconsistencies and limited resolution of the mammalian tree may have their natural explanation in speciation processes such as lineage sorting, introgression from species hybridization or hybrid speciation. These processes obscure phylogenetic analysis, making some parts of the tree difficult to resolve even with genome data.
Prolonged treatment of leukemic cells with chemotherapeutic agents frequently results in development of drug resistance. Moreover, selection of drug-resistant cell populations may be associated with changes in malignant properties such as proliferation rate, invasiveness, and immunogenicity. In the present study, the sensitivity of cytarabine (1-β-d-arabinofuranosylcytosine, araC)-resistant and parental human leukemic cell lines (T-lymphoid H9 and acute T-lymphoblastic leukemia Molt-4) to natural killer (NK) cell-mediated killing was investigated. The results obtained demonstrate that araC-resistant H9 and Molt-4 (H9rARAC100 and Molt-4rARAC100) cell lines are more sensitive to NK cell-mediated lysis than their respective parental cell lines. This increased sensitivity was associated with a higher surface expression of ligands for the NK cell-activating receptor NKG2D, notably UL16 binding protein-2 (ULBP-2) and ULBP-3 in H9rARAC100 and Molt-4rARAC100 cell lines. Blocking ULBP-2 and ULBP-3 or NKG2D with monoclonal antibody completely abrogated NK cell lysis. Constitutive phosphorylated extracellular signal-regulated kinase (ERK) but not pAKT was higher in araC-resistant cells than in parental cell lines. Inhibition of ERK using ERK inhibitor PD98059 decreased both ULBP-2/ULBP-3 expression and NK cell cytotoxicity. Furthermore, overexpression of constitutively active ERK in H9 parental cells resulted in increased ULBP-2/ULBP-3 expression and enhanced NK cell lysis. These results demonstrate that increased sensitivity of araC-resistant leukemic cells to NK cell lysis is caused by higher NKG2D ligand expression, resulting from more active ERK signaling pathway.
Requirements for the interaction of mouse Polkappa with ubiquitin and its biological significance
(2008)
Polkappa protein is a eukaryotic member of the DinB/Polkappa branch of the Y-family DNA polymerases, which are involved in the tolerance of DNA damage by replicative bypass. Despite universal conservation through evolution, the precise role(s) of Polkappa in this process has remained unknown. Here we report that mouse Polkappa can physically interact with ubiquitin by yeast two-hybrid screening, glutathione S-transferase pulldown, and immunoprecipitation methods. The association of Polkappa with ubiquitin requires the ubiquitin-binding motifs located at the C terminus of Polkappa. In addition, Polkappa binds with monoubiquitinated proliferating cell nuclear antigen (PCNA) more robustly than with non-ubiquitinated PCNA. The ubiquitin-binding motifs mediate the enhanced association between monoubiquitinated PCNA and Polkappa. The ubiquitin-binding motifs are also required for Polkappa to form nuclear foci after UV radiation. However, the ubiquitin-binding motifs do not affect Polkappa half-life. Finally, we have examined levels of Polkappa expression following the exposure of mouse cells to benzo[a]pyrene-dihydrodiol epoxide or UVB radiation.
Background One of the central issues in ecology is the question what allows sympatric occurrence of closely related species in the same general area? The non-biting midges Chironomus riparius and C. piger, interbreeding in the laboratory, have been shown to coexist frequently despite of their close relatedness, similar ecology and high morphological similarity. Methodology/Principal Findings In order to investigate factors shaping niche partitioning of these cryptic sister species, we explored the actual degree of reproductive isolation in the field. Congruent results from nuclear microsatellite and mitochondrial haplotype analyses indicated complete absence of interspecific gene-flow. Autocorrelation analysis showed a non-random spatial distribution of the two species. Though not dispersal limited at the scale of the study area, the sister species occurred less often than expected at the same site, indicating past or present competition. Correlation and multiple regression analyses suggested the repartition of the available habitat along water chemistry gradients (nitrite, conductivity, CaCO3), ultimately governed by differences in summer precipitation regime. Conclusions We show that these morphologically cryptic sister species partition their niches due to a certain degree of ecological distinctness and total reproductive isolation in the field. The coexistence of these species provides a suitable model system for the investigation of factors shaping the distribution of closely related, cryptic species.
In 2007, a project named Adriatic Montenegro 2007 was initiated and realised under the protection of IDF. Stimulated by the suggestion of Vincent Kalkman from the European Invertebrate Survey, The Netherlands, distribution data for the Odonata of Adriatic Montenegro were collected and used to create distribution maps. These data and maps should then be used in the evaluation of conservation measures for individual odonate species as well as for the IUCN Red List of the Mediterranean countries (organized by the IUCN Centre for Mediterranean Cooperation) and the Project on an atlas of European dragonflies (a co-operation of all European countries organised by the European Invertebrate Survey, The Netherlands).
It has been suggested that the existence of a non-Gaussian fixed point in general relativity might cure the ultraviolet problems of this theory. Such a fixed point is connected to an effective running of the gravitational coupling. We calculate the effect of the running gravitational coupling on the black hole production cross section in models with large extra dimensions.
Religious conversion has become a dangerous social and individual problem. In Latin America, a traditional Catholic area, Protestant sects are successfully con-verting more and more Catholics into their own communities. Therefore the Pope demands a strict control of these activities. In India e.g., the Catholic hierarchy is critizising the Indian governments which have forbidden conversion on non-spiritual reasons. Hindu organizations have started even very successfully to re-convert Indian Christians particularly of Dalit and tribal background. Buddhists are very successful in indirect and even direct conversion of many Westerners. Wah-habit missionaries spread their Neo-Islam in the Muslim societies and get more and more even non-Muslim converts. We should add the forcible and sometimes ex-tremely cruel conversions the atheistic states had executed since the last century. ...
Background Medical students come into contact with infectious diseases early on their career. Immunity against vaccine-preventable diseases is therefore vital for both medical students and the patients with whom they come into contact. Methods The purpose of this study was to compare the medical history and serological status of selected vaccine-preventable diseases of medical students in Germany. Results The overall correlation between medical history statements and serological findings among the 150 students studied was 86.7 %, 66.7 %, 78 % and 93.3 % for measles, mumps, rubella and varicella, conditional on sufficient immunity being achieved after one vaccination. Conclusions Although 81.2 % of the students medical history data correlated with serological findings, significant gaps in immunity were found. Our findings indicate that medical history alone is not a reliable screening tool for immunity against the vaccine-preventable diseases studied.
Ataxin-2 is a novel protein, within which the unstable expansion of a polyglutamine domain can cause Spinocerebellar Ataxia type 2 (SCA2), a neurodegenerative disease which belongs to the group of polyglutamine disorders. SCA2 is characterised by a progressive loss of neurons that first affects the cerebellum and brain stem and then may extend to other areas of the brain, like substantia nigra, motoneurons and thalamus. Several lines of research have attempted to determine therole of ataxin-2 in its normal and mutant version. Different animal models and cell culture approaches to study ataxin-2 function implicated ataxin-2 in RNA processing, embryonic development, apoptosis and cytoskeleton. However, the function of ataxin-2 still remains unclear. In this thesis, a protein interaction approach was chosen as an alternative to gain insights into the cellular function of ataxin-2. Full-length ataxin-2 was used as bait in a yeast two-hybrid screen of human adult brain cDNA. Among five candidate interactor proteins identified, two were the endophilins A1 and A3, proteins involved in vesicle endocytosis. Co-immunoprecipitation studies confirmed the association of these proteins in an endogenous complex of mouse brain. In vitro binding experiments narrowed the binding interfaces down to two proline-rich domains on ataxin-2, which interacted with the SH3 domain of endophilins A1/A3. Ataxin-2 and endophilins A1/A3 colocalised at the endoplasmic reticulum as determined by immunofluorescence microscopy of transfected cell lines, and by centrifugation fractionation studies of mouse brain. Importantly, the pattern observed in transfected cells was conserved in untransfected rat hippocampal neurons. In mouse brain, associations of ataxin-2 with endocytic proteins such as the adaptor CIN85, the ubiquitin ligase c-Cbl and also GRB2, in the last case by means of a SH3 domain array chip, were also demonstrated. GST pull-down assays showed ataxin-2 to interact directly with the SH3 domains A and C of CIN85, the C-terminal SH3 domain of GRB2, and the SH3 domain of Src, a kinase activated after receptor stimulation. Functional studies demonstrated that ataxin-2 affects endocytic trafficking of the epidermal growth factor receptor (EGFR) by reducing the EGFR internalisation after EGF stimulation. Taken together, these data implicate ataxin-2 to play a role in endocytic receptor cycling.