Contributions of demography and dispersal parameters to the spatial spread of a stage-structured insect invasion

Stage-structured models that integrate demography and dispersal can be used to identify points in the life cycle with large effects on rates of population spatial spread, information that is vital in the development of containment strategies for invasive species. Current challenges in the application of these tools include: (1) accounting for large uncertainty in model parameters, which may violate assumptions of ‘‘local’’ perturbation metrics such as sensitivities and elasticities, and (2) forecasting not only asymptotic rates of spatial spread, as is usually done, but also transient spatial dynamics in the early stages of invasion. We developed an invasion model for the Diaprepes root weevil (DRW; Diaprepes abbreviatus [Coleoptera: Curculionidae]), a generalist herbivore that has invaded citrus-growing regions of the United States. We synthesized data on DRW demography and dispersal and generated predictions for asymptotic and transient peak invasion speeds, accounting for parameter uncertainty. We quantified the contributions of each parameter toward invasion speed using a ‘‘global’’ perturbation analysis, and we contrasted parameter contributions during the transient and asymptotic phases. We found that the asymptotic invasion speed was 0.02–0.028 km/week, although the transient peak invasion speed (0.03– 0.045 km/week) was significantly greater. Both asymptotic and transient invasions speeds were most responsive to weevil dispersal distances. However, demographic parameters that had large effects on asymptotic speed (e.g., survival of early-instar larvae) had little effect on transient speed. Comparison of the global analysis with lower-level elasticities indicated that local perturbation analysis would have generated unreliable predictions for the responsiveness of invasion speed to underlying parameters. Observed range expansion in southern Florida (1992–2006) was significantly lower than the invasion speed predicted by the model. Possible causes of this mismatch include overestimation of dispersal distances, demographic rates, and spatiotemporal variation in parameter values. This study demonstrates that, when parameter uncertainty is large, as is often the case, global perturbation analyses are needed to identify which points in the life cycle should be targets of management. Our results also suggest that effective strategies for reducing spread during the asymptotic phase may have little effect during the transient phase. Includes Appendix.

O profissional da informação na elaboração de políticas públicas: uma análise do Plano Nacional do Livro e da Leitura (PNLL)

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Demography and evolution of regional inequality

This paper addresses the importance of life cycle aspects in explaining the evolution of regional income inequality. The analysis of household microdata organized in age cohorts shows that Brazilian regional income inequality has different dynamics across generations, with income convergence being observed only for the older generations. The larger income share of younger generations produces a low speed of convergence in the country. When retirement payments, pensions, and other government transfers are excluded from income, convergence is not observed even for the older generations.

Leishmania Metacyclogenesis Is Promoted in the Absence of Purines

Leishmania parasites, the causative agent of leishmaniasis, are transmitted through the bite of an infected sand fly. Leishmania parasites present two basic forms known as promastigote and amastigote which, respectively, parasitizes the vector and the mammalian hosts. Infection of the vertebrate host is dependent on the development, in the vector, of metacyclic promastigotes, however, little is known about the factors that trigger metacyclogenesis in Leishmania parasites. It has been generally stated that "stressful conditions" will lead to development of metacyclic forms, and with the exception of a few studies no detailed analysis of the molecular nature of the stress factor has been performed. Here we show that presence/absence of nucleosides, especially adenosine, controls metacyclogenesis both in vitro and in vivo. We found that addition of an adenosine-receptor antagonist to in vitro cultures of Leishmania amazonensis significantly increases metacyclogenesis, an effect that can be reversed by the presence of specific purine nucleosides or nucleobases. Furthermore, our results show that proliferation and metacyclogenesis are independently regulated and that addition of adenosine to culture medium is sufficient to recover proliferative characteristics for purified metacyclic promastigotes. More importantly, we show that metacyclogenesis was inhibited in sand flies infected with Leishmania infantum chagasi that were fed a mixture of sucrose and adenosine. Our results fill a gap in the life cycle of Leishmania parasites by demonstrating how metacyclogenesis, a key point in the propagation of the parasite to the mammalian host, can be controlled by the presence of specific purines.

Trypanosoma cruzi Gene Expression in Response to Gamma Radiation

Trypanosoma cruzi is an organism highly resistant to ionizing radiation. Following a dose of 500 Gy of gamma radiation, the fragmented genomic DNA is gradually reconstructed and the pattern of chromosomal bands is restored in less than 48 hours. Cell growth arrests after irradiation but, while DNA is completely fragmented, RNA maintains its integrity. In this work we compared the transcriptional profiles of irradiated and non-irradiated epimastigotes at different time points after irradiation using microarray. In total, 273 genes were differentially expressed; from these, 160 were up-regulated and 113 down-regulated. We found that genes with predicted functions are the most prevalent in the down-regulated gene category. Translation and protein metabolic processes, as well as generation of precursor of metabolites and energy pathways were affected. In contrast, the up-regulated category was mainly composed of obsolete sequences (which included some genes of the kinetoplast DNA), genes coding for hypothetical proteins, and Retrotransposon Hot Spot genes. Finally, the tyrosyl-DNA phosphodiesterase 1, a gene involved in double-strand DNA break repair process, was up-regulated. Our study demonstrated the peculiar response to ionizing radiation, raising questions about how this organism changes its gene expression to manage such a harmful stress.

A SURVEY OF NEOSPORA CANINUM AND TOXOPLASMA GONDII INFECTION IN URBAN RODENTS FROM BRAZIL

Toxoplasma gondii is a protozoan parasite that infects humans and other warm-blooded animals; it uses feral and domestic cats as the definitive hosts. Neospora caninum is a protozoan parasite of animals whose life cycle is very similar to T. gondii but uses canids as definitive hosts. Small rodents play an important role in the life cycle of T. gondii, and a few findings indicated that they may be natural intermediate hosts for N. caninum. The present study was aimed at identifying infections by T. gondii and N. caninum in urban rodents. Infections by T. gondii were quantified using isolation of the parasite by bioassay in mice; molecular methods were also used for both parasites. Overall, 217 rodents were captured. Brain and heart tissues of all rodents were bioassayed in mice for the detection of T. gondii infection. Brain and heart tissues of 121 rodents had the DNA extracted for molecular analysis. Toxoplasma gondii was isolated by bioassay from a single rodent. From the 121 rodents tested for the presence of T. gondii DNA, 2 animals were positive. In contrast, DNA of N. caninum was not detected in any of the samples. In conclusion, the surveys of N. caninum and T. gondii infection in Rattus rattus, Rattus norvegicus, and Mus musculus captured in urban areas of Sao Paulo reveal a striking low frequency of occurrence of these infections.

Radiation of the Tnt1 retrotransposon superfamily in three Solanaceae genera

Abstract Background Tnt1 was the first active plant retrotransposon identified in tobacco after nitrate reductase gene disruption. The Tnt1 superfamily comprises elements from Nicotiana (Tnt1 and Tto1) and Lycopersicon (Retrolyc1 and Tlc1) species. The study presented here was conducted to characterise Tnt1-related sequences in 20 wild species of Solanum and five cultivars of Solanum tuberosum. Results Tnt1-related sequences were amplified from total genomic DNA using a PCR-based approach. Purified fragments were cloned and sequenced, and clustering analysis revealed three groups that differ in their U3 region. Using a network approach with a total of 453 non-redundant sequences isolated from Solanum (197), Nicotiana (140) and Lycopersicon (116) species, it is demonstrated that the Tnt1 superfamily can be treated as a population to resolve previous phylogenetic multifurcations. The resulting RNAseH network revealed that sequences group according to the Solanaceae genus, supporting a strong association with the host genome, whereas tracing the U3 region sequence association characterises the modular evolutionary pattern within the Tnt1 superfamily. Within each genus, and irrespective of species, nearly 20% of Tnt1 sequences analysed are identical, indicative of being part of an active copy. The network approach enabled the identification of putative "master" sequences and provided evidence that within a genus these master sequences are associated with distinct U3 regions. Conclusion The results presented here support the hypothesis that the Tnt1 superfamily was present early in the evolution of Solanaceae. The evidence also suggests that the RNAseH region of Tnt1 became fixed at the host genus level whereas, within each genus, propagation was ensured by the diversification of the U3 region. Different selection pressures seemed to have acted on the U3 and RNAseH modules of ancestral Tnt1 elements, probably due to the distinct functions of these regions in the retrotransposon life cycle, resulting in both co evolution and adaptation of the element population with its host.

A quantitative view of the transcriptome of Schistosoma mansoni adult-worms using SAGE

Abstract Background Five species of the genus Schistosoma, a parasitic trematode flatworm, are causative agents of Schistosomiasis, a disease that is endemic in a large number of developing countries, affecting millions of patients around the world. By using SAGE (Serial Analysis of Gene Expression) we describe here the first large-scale quantitative analysis of the Schistosoma mansoni transcriptome, one of the most epidemiologically relevant species of this genus. Results After extracting mRNA from pooled male and female adult-worms, a SAGE library was constructed and sequenced, generating 68,238 tags that covered more than 6,000 genes expressed in this developmental stage. An analysis of the ordered tag-list shows the genes of F10 eggshell protein, pol-polyprotein, HSP86, 14-3-3 and a transcript yet to be identified to be the five top most abundant genes in pooled adult worms. Whereas only 8% of the 100 most abundant tags found in adult worms of S. mansoni could not be assigned to transcripts of this parasite, 46.9% of the total ditags could not be mapped, demonstrating that the 3 sequence of most of the rarest transcripts are still to be identified. Mapping of our SAGE tags to S. mansoni genes suggested the occurrence of alternative-polyadenylation in at least 13 gene transcripts. Most of these events seem to shorten the 3 UTR of the mRNAs, which may have consequences over their stability and regulation. Conclusion SAGE revealed the frequency of expression of the majority of the S. mansoni genes. Transcriptome data suggests that alternative polyadenylation is likely to be used in the control of mRNA stability in this organism. When transcriptome was compared with the proteomic data available, we observed a correlation of about 50%, suggesting that both transcriptional and post-transcriptional regulation are important for determining protein abundance in S. mansoni. The generation of SAGE tags from other life-cycle stages should contribute to reveal the dynamics of gene expression in this important parasite.

Parapoxvirus: basi genomiche della patogenesi

Parapoxvirus (PPV) are member of a genus in the family poxviridae which currently encompasses four species: the prototype orf virus (OV), bovine papular stomatitis virus (BPSV), pseudocowpox virus (PCPV) and parapoxvirus of New Zealand red deer (PVNZ). PPVs cause widespread, but localized diseases of small and large ruminants and they can also be transmitted to man. Knowledge of the molecular biology of PPV is still limited as compared to orthopoxviruses, especially vaccinia virus (VACV). The PPV genome displays a high G+C content and relatively small size for poxvirus. Coventional electron microscopy displays PPV virions with ovoid shape and slightly smaller in size than the brickshaped orthopoxviruses. The most striking feature, which readily enables identification of PPV, is a tubule-like structure that surrounds the particle in a spiral fashion. PPV genome organization and content is very similar to that of other poxviruses, the central region contain 88 genes which are present in all poxviruse, in contrast the terminal regions are variable and contain a set of genes unique to the genus PPV. Genes in the near-terminal regions of the genome are frequently not essential for growth in cultured cells encoding factors with important roles in virushost interactions including modulating host immune responses and determining host range. Recently it was suggested that the open reading frames (ORFs) 109 and 110 of the OV genome have a major role in determining species specificity during natural infection in sheep and goats. This hypothesis is based on the analysis of a few number of sequences of different sheep and goats viral isolates. PPV replicate into the cytoplasm of infected cells and produce three structurally different infectious particles: the intracellular mature virions (IMV), intracellular enveloped virions (IEV) and the extracellular enveloped virions (EEV). The vaccinia A33R and A34R hotologue proteins encoded by the ORFS 109 and 110 are expressed in the envelope of the IEV and EEV. The F1L immunodominant protein of orf virus is the major component of the surface tubule structure of the IMV and can post-translationaly insert into membranes via Cterminal, hydrofobic anchor sequence like its orthologue VACV H3L protein. Moreover the F1L protein binds to glycosaminoglycans on the cell surface and has an important role in IMV adsorption to mammalian cells. In this study we investigated the morphogenesis of the PPV through the construction of a mutant virus deleted of the F1L protein. A study of the deleted virus life cycle was conducted in different type of cells and its morphology was observed with electron microscopy. It was demonstared that F1L protein have important role in morphogenesis and infectivity. Moreover it is essential to determine the spiral fashion of the tubule like structure of the virion surface. Some pathogenetic aspects of the PPV infection were studied, in particular the protein implicated in the host range were analysed in detail. An experimental infection with OV and PCPV was conducted in goats and sheep. After infection, the severity of the lesions were comparable in both the animal species. The OV did not result in severe disease neither in sheep nor in goats, suggesting that host factors, rather than virus strain characteristics, may play an important role in the pathogenesis of the Parapoxvirus infections. The PCPV failed to produce any lesion in both sheep and goats, ruling out the possibility of any recombination between PCPV and OV during natural infection in these animal species. The phylogenetic analysis of the ORFs 109 and 110 from several goats and sheep viral isolates showed a clustering based on the antigenic content of the protein that was independent from species and geographic origin.

Asset management of wastewater networks

Asset Management (AM) is a set of procedures operable at the strategic-tacticaloperational level, for the management of the physical asset’s performance, associated risks and costs within its whole life-cycle. AM combines the engineering, managerial and informatics points of view. In addition to internal drivers, AM is driven by the demands of customers (social pull) and regulators (environmental mandates and economic considerations). AM can follow either a top-down or a bottom-up approach. Considering rehabilitation planning at the bottom-up level, the main issue would be to rehabilitate the right pipe at the right time with the right technique. Finding the right pipe may be possible and practicable, but determining the timeliness of the rehabilitation and the choice of the techniques adopted to rehabilitate is a bit abstruse. It is a truism that rehabilitating an asset too early is unwise, just as doing it late may have entailed extra expenses en route, in addition to the cost of the exercise of rehabilitation per se. One is confronted with a typical ‘Hamlet-isque dilemma’ – ‘to repair or not to repair’; or put in another way, ‘to replace or not to replace’. The decision in this case is governed by three factors, not necessarily interrelated – quality of customer service, costs and budget in the life cycle of the asset in question. The goal of replacement planning is to find the juncture in the asset’s life cycle where the cost of replacement is balanced by the rising maintenance costs and the declining level of service. System maintenance aims at improving performance and maintaining the asset in good working condition for as long as possible. Effective planning is used to target maintenance activities to meet these goals and minimize costly exigencies. The main objective of this dissertation is to develop a process-model for asset replacement planning. The aim of the model is to determine the optimal pipe replacement year by comparing, temporally, the annual operating and maintenance costs of the existing asset and the annuity of the investment in a new equivalent pipe, at the best market price. It is proposed that risk cost provide an appropriate framework to decide the balance between investment for replacing or operational expenditures for maintaining an asset. The model describes a practical approach to estimate when an asset should be replaced. A comprehensive list of criteria to be considered is outlined, the main criteria being a visà- vis between maintenance and replacement expenditures. The costs to maintain the assets should be described by a cost function related to the asset type, the risks to the safety of people and property owing to declining condition of asset, and the predicted frequency of failures. The cost functions reflect the condition of the existing asset at the time the decision to maintain or replace is taken: age, level of deterioration, risk of failure. The process model is applied in the wastewater network of Oslo, the capital city of Norway, and uses available real-world information to forecast life-cycle costs of maintenance and rehabilitation strategies and support infrastructure management decisions. The case study provides an insight into the various definitions of ‘asset lifetime’ – service life, economic life and physical life. The results recommend that one common value for lifetime should not be applied to the all the pipelines in the stock for investment planning in the long-term period; rather it would be wiser to define different values for different cohorts of pipelines to reduce the uncertainties associated with generalisations for simplification. It is envisaged that more criteria the municipality is able to include, to estimate maintenance costs for the existing assets, the more precise will the estimation of the expected service life be. The ability to include social costs enables to compute the asset life, not only based on its physical characterisation, but also on the sensitivity of network areas to social impact of failures. The type of economic analysis is very sensitive to model parameters that are difficult to determine accurately. The main value of this approach is the effort to demonstrate that it is possible to include, in decision-making, factors as the cost of the risk associated with a decline in level of performance, the level of this deterioration and the asset’s depreciation rate, without looking at age as the sole criterion for making decisions regarding replacements.

An evolutive approach for smile recognition in video sequences

Facial expression recognition is one of the most challenging research areas in the image recognition ¯eld and has been actively studied since the 70's. For instance, smile recognition has been studied due to the fact that it is considered an important facial expression in human communication, it is therefore likely useful for human–machine interaction. Moreover, if a smile can be detected and also its intensity estimated, it will raise the possibility of new applications in the future

Specification and Verification of Declarative Open Interaction Models - A Logic-based framework

The advent of distributed and heterogeneous systems has laid the foundation for the birth of new architectural paradigms, in which many separated and autonomous entities collaborate and interact to the aim of achieving complex strategic goals, impossible to be accomplished on their own. A non exhaustive list of systems targeted by such paradigms includes Business Process Management, Clinical Guidelines and Careflow Protocols, Service-Oriented and Multi-Agent Systems. It is largely recognized that engineering these systems requires novel modeling techniques. In particular, many authors are claiming that an open, declarative perspective is needed to complement the closed, procedural nature of the state of the art specification languages. For example, the ConDec language has been recently proposed to target the declarative and open specification of Business Processes, overcoming the over-specification and over-constraining issues of classical procedural approaches. On the one hand, the success of such novel modeling languages strongly depends on their usability by non-IT savvy: they must provide an appealing, intuitive graphical front-end. On the other hand, they must be prone to verification, in order to guarantee the trustworthiness and reliability of the developed model, as well as to ensure that the actual executions of the system effectively comply with it. In this dissertation, we claim that Computational Logic is a suitable framework for dealing with the specification, verification, execution, monitoring and analysis of these systems. We propose to adopt an extended version of the ConDec language for specifying interaction models with a declarative, open flavor. We show how all the (extended) ConDec constructs can be automatically translated to the CLIMB Computational Logic-based language, and illustrate how its corresponding reasoning techniques can be successfully exploited to provide support and verification capabilities along the whole life cycle of the targeted systems.

Analysis of the influence of epitope flanking regions on MHC class I restricted antigen presentation

Peptides presented by MHC class I molecules for CTL recognition are derived mainly from cytosolic proteins. For antigen presentation on the cell surface, epitopes require correct processing by cytosolic and ER proteases, efficient TAP transport and MHC class I binding affinity. The efficiency of epitope generation depends not only on the epitope itself, but also on its flanking regions. In this project, the influence of the C-terminal region of the model epitope SIINFEKL (S8L) from chicken ovalbumin (aa 257-264) on antigen processing has been investigated. S8L is a well characterized epitope presented on the murine MHC class I molecule, H-2Kb. The Flp-In 293Kb cell line was transfected with different constructs each enabling the expression of the S8L sequence with different defined C-terminal flanking regions. The constructs differed at the two first C-terminal positions after the S8L epitope, so called P1’ and P2’. At these sites, all 20 amino acids were exchanged consecutively and tested for their influence on H-2Kb/S8L presentation on the cell surface of the Flp-In 293Kb cells. The detection of this complex was performed by immunostaining and flow cytometry. The prevailing assumption is that proteasomal cleavages are exclusively responsible for the generation of the final C-termini of CTL epitopes. Nevertheless, recent publications showed that TPPII (tripeptidyl peptidase II) is required for the generation of the correct C-terminus of the HLA-A3-restricted HIV epitope Nef(73-82). With this background, the dependence of the S8L generation on proteasomal cleavage of the designed constructs was characterized using proteasomal inhibitors. The results obtained indicate that it is crucial for proteasomal cleavage, which amino acid is flanking the C-terminus of an epitope. Furthermore, partially proteasome independent S8L generation from specific S8L-precursor peptides was observed. Hence, the possibility of other existing endo- or carboxy-peptidases in the cytosol that could be involved in the correct trimming of the C-terminus of antigenic peptides for MHC class I presentation was investigated, performing specific knockdowns and using inhibitors against the target peptidases. In parallel, a purification strategy to identify the novel peptidase was established. The purified peaks showing an endopeptidase activity were further analyzed by mass spectrometry and some potential peptidases (like e.g. Lon) were identified, which have to be further characterized.

Charakterisierung redundanter Kerntransportsignale des minoren Kapsidproteins L2 humaner Papillomviren

Das minore Kapsidprotein L2 humaner Papillomviren wird im Laufe des HPV-Lebenszyklus zweimal in den Zellkern importiert und akkumuliert dort an den Nukleären Domänen 10 (ND10). Der erste Kernimport erfolgt in der frühen Phase der Infektion zusammen mit der Virus-DNA. Für den Zusammenbau von Virionen wird neu synthetisiertes L2-Protein ein weiteres Mal in den Kern transportiert. Im Rahmen dieser Arbeit konnte die Domäne von L2 identifiziert werden, die für den Kernimport von HPV16 L2 absolut notwendig ist. Dabei gelang es diesen Bereich auf 25 Aminosäuren einzuengen. Sowohl während der frühen Phase der Infektion als auch während der Morphogenese scheint die zentrale, basische Aminosäureregion 291-315 (mNLS) hauptverantwortlich für die Interaktion mit Kernimportrezeptoren zu sein. Möglicherweise leisten dabei flankierende Sequenzen einen Beitrag zur Stabilisierung der notwendigen Konformation. Des Weiteren gelang die Identifizierung der Aminosäuren, die für die Funktionalität des mNLS essentiell sind. Hierbei handelt es sich um ein zentrales Arginin-Motiv, bestehend aus vier dicht beieinander liegenden Argininen, dessen Mutation den Kernimport von L2 während Infektion und Morphogenese verhindert. Untersuchungen mit HPV16 und HPV18 L2-Proteinen verdeutlichten, dass es möglicherweise ein universelles Motiv zu sein scheint und in verschiedenen HPV-Typen konserviert ist. Flankiert wird dieses Arginin-Motiv von konservierten Serinen und Threoninen. Wie die Analyse von Punktmutationen zeigte, sind diese Aminosäuren für den Kernimport von L2 ohne Bedeutung. Interessanterweise verhinderte aber die Mutation TS295/6A die Kolokalisation von L2 mit ND10 im Zellkern. L2wt rekrutiert den transkriptionellen Regulator Daxx. Auch diese Funktion ging bei der Mutante TS295/6A verloren. Diese Ergebnisse zeigen, dass nicht nur die ND10-Lokalisationsdomäne (AS 390-420) in L2 sondern auch weitere Aminosäuren oder Domänen für die Assoziation mit ND10 und die Rekrutierung von Daxx verantwortlich sein könnten. Auf der Suche nach zellulären Faktoren, die eine Rolle im mNLS-vermittelten Kernimport spielen, wurde zunächst die Bedeutung von Hsc70 untersucht. Während der Morphogenese maskiert Hsc70 den C-Terminus von L2 und verhindert damit unerwünschte Interaktionen mit Mikrotubuli im Zytoplasma. Es existieren aber weitere noch unbekannte Hsc70-Bindedomänen in L2, die möglicherweise den Kernimport ebenfalls beeinflussen können. Wie die Untersuchungen deutlich machten, ist der zentrale, basische Bereich von L2 aber nicht mit Hsc70 assoziiert und der mNLS-vermittelte Kernimport findet unabhängig von Hsc70 statt. In einem siRNA-Screen wurde anschließend die Rolle von Karyopherinen während der Infektion untersucht. Sowohl Kapß2-siRNA als auch Kapß3-siRNA waren in der Lage unabhängig voneinander die Infektion von HPV16-Pseudovirionen zu reduzieren. Für beide Karyopherine konnte in der Vergangenheit in vitro die Interaktion mit HPV16 L2 nachgewiesen werden. Das L2-Protein ist das einzige virale Protein, das während der Infektion die Virus-DNA in den Kern begleitet (Day et al., 2004). Demzufolge ist es auch das einzige virale Protein, das mit Importinen während der Infektion interagiert. Möglicherweise sind also beide Karyopherine in der Lage sein L2 während der Infektion in den Kern zu importieren. Abschließend wurden Präzipitationsversuche durchgeführt, die zur Identifizierung möglicher Bindungspartner des mNLS führen sollten. In diesen Versuchen konnte eine erhöhte Bindungsaffiniät zu den beiden Importinen Kapß1 und Kapß2 festgestellt werden. Möglicherweise ist das L2-Protein mit seiner mNLS in der Lage mehrere Importrezeptoren zu binden und für den Kernimport zu nutzen. Eines dieser Importine ist Kapß2. Dieser Importrezeptor scheint sowohl bei der Infektion als auch während der Morphogenese den Kernimport von L2 durch die Bindung an das mNLS zu vermitteln.

Charakterisierung zellulärer Interaktionspartner des großen Hüllproteins des Hepatitis-B-Virus

Im Mittelpunkt dieser Arbeit stand das große L-Hüllprotein (L) des Hepatitis B - Virus. L bildet eine ungewöhnliche duale Topologie in der ER-Membran aus, welche auch im reifen Viruspartikel erhalten bleibt. In einem partiellen, posttranslationalen Reifungsprozess wird die sogenannte PräS-Region von der zytosolischen Seite der Membran aus in das ER-Lumen transloziert. Aufgrund seiner dualen Topologie und der damit verbundenen Multifunktionalität übernimmt L eine Schlüsselfunktion im viralen Lebenszyklus. Ein Schwerpunkt dieser Arbeit lag deshalb darin, neue zelluläre Interaktionspartner des L-Hüllproteins zu identifizieren. Ihre Analyse sollte helfen, das Zusammenspiel des Virus mit der Wirtszelle besser zu verstehen. Hierfür wurde das Split - Ubiquitin Hefe - Zwei - Hybrid System eingesetzt, das die Interaktionsanalyse von Membranproteinen und Membran-assoziierten Proteinen ermöglicht. Zwei der neu identifizierten Interaktionspartner, der v-SNARE Bet1 und Sec24A, die Cargo-bindende Untereinheit des CoPII-vermittelten vesikulären Transports, wurden weitergehend im humanen Zellkultursystem untersucht. Sowohl für Bet1 als auch für Sec24A konnte die Interaktion mit dem L-Hüllprotein bestätigt und der Bindungsbereich eingegrenzt werden. Die Depletion des endogenen Bet1 reduzierte die Freisetzung L-haltiger, nicht aber S-haltiger subviraler Partikel (SVP) deutlich. Im Gegensatz zu Bet1 interagierte Sec24A auch mit dem mittleren M- und kleinen S-Hüllprotein von HBV. Die Inhibition des CoPII-vermittelten vesikulären Transportweges durch kombinierte Depletion der vier Sec24 Isoformen blockierte die Freisetzung sowohl L- als auch S-haltiger SVP. Dies bedeutet, dass die HBV - Hüllproteine das ER CoPII-vermittelt verlassen, wobei sie aktiv Kontakt zur Cargo-bindenden Untereinheit Sec24A aufnehmen. Der effiziente Export der Hüllproteine aus dem ER ist für die Virusmorphogenese und somit für den HBV - Lebenszyklus essentiell. rnEin weiterer Schwerpunkt dieser Arbeit basierte auf der Interaktion des L-Hüllproteins mit dem ER-luminalen Chaperon BiP. In der vorliegenden Arbeit wurde überprüft, ob BiP, ähnlich wie das zytosolische Chaperon Hsc70, an der Ausbildung der dualen Topologie des L-Hüllproteins beteiligt ist. Hierfür wurde BiP durch die ektopische Expression seiner Ko-Chaperone BAP und ERdj4 in seiner Substrat-bindenen Kapazität manipuliert. ERdj4, ein Mitglied der Hsp40 - Proteinfamilie, stimuliert die ATPase-Aktivität von BiP, was die Substratbindung stabilisiert. Der Nukleotid - Austauschfaktor BAP hingegen vermittelt die Auflösung des BiP - Substrat - Komplexes. Die Auswirkung der veränderten in vivo-Aktivität von BiP auf die posttranslationale PräS-Translokation wurde mit Proteaseschutz - Versuchen untersucht. Die ektopische Expression des positiven als auch des negativen Regulators von BiP resultierte in einer drastischen Reduktion der posttranslationalen PräS-Translokation. Ein vergleichbarer Effekt wurde nach Manipulation des BiP ATPase - Zyklus durch Depletion der zellulären ATP - Konzentration beobachtet. Dies spricht dafür, dass das ER-luminale Chaperon BiP, zusammen mit Hsc70, eine zentrale Rolle in der Ausbildung der dualen Topologie des L-Hüllproteins spielt. rnZwei weitere Proteine, Sec62 und Sec63, die sich für die posttranslationale Translokation in der Hefe als essentiell erwiesen haben, wurden in die Analyse der dualen Topologie des L-Hüllproteins einbezogen. Interessanterweise konnte eine rein luminale Ausrichtung der PräS-Region nach kombinierter Depletion des endogenen Sec62 und Sec63 beobachtet werden. Dies deutet an, dass sowohl Sec62 als auch Sec63 an der Ausbildung der dualen Topologie des L-Hüllproteins beteiligt sind. In Analogie zur Posttranslokation der Hefe könnte Sec62 als Translokon-assoziierter Rezeptor für Substrate der Posttranslokation, und damit der PräS-Region, dienen. Sec63 könnte mit seiner J-Domäne BiP zum Translokon rekrutieren und daraufhin dessen Substrat-bindende Aktivität stimulieren. BiP würde dann, einer molekularen Ratsche gleich, die PräS-Region durch wiederholtes Binden und Freisetzen aktiv in das ER-Lumen hereinziehen, bis eine stabile duale Topologie des L-Hüllproteins ausgebildet ist. Die Bedeutung von Sec62 und Sec63 für den HBV - Lebenszyklus wird dadurch untermauert, dass sowohl die ektopische Expression als auch die Depletion des endogenen Sec63 die Freisetzung L-haltiger SVP deutlich reduziert. rn