Comparing properties of natural biogenic with biomass burning particles in Amazonia.

The Large Scale Biosphere Atmosphere Experiment in Amazonia (LBA) is a long-term (20 years) research effort aimed at the understanding of the functioning of the Amazonian ecosystem. The strong biosphere-atmosphere interaction is a key component of the ecosystem functioning. Two aerosol components are the most visible: The natural biogenic emissions of particles and VOCs, and the biomass burning emissions. Two aerosol and trace gases monitoring stations were operated for 4 years in Manaus and Porto Velho, two very distinct sites, with different land use change. Manaus is a very clean and pristine site and Porto Velho is representative of heavy land use change in Amazonia. Aerosol composition, optical properties, size distribution, vertical profiling and optical depth were measured from 2008 to 2012. Aerosol radiative forcing was calculated over large areas. It was observed that the natural biogenic aerosol has significant absorption properties. Organic aerosol dominates the aerosol mass with 80 to 95%. Light scattering and light absorption shows an increase by factor of 10 from Manaus to Porto Velho. Very few new particle formation events were observed. Strong links between aerosols and VOC emissions were observed. Aerosol radiative forcing in Rondonia shows a high -15 watts/m² during the dry season of 2010, showing the large impacts of aerosol loading in the Amazonian ecosystem. The increase in diffuse radiation changes the forest carbon uptake by 20 to 35%, a large increase in this important ecosystem.

In vivo assessment of specific cytotoxic T lymphocyte killing

The direct killing of target cells by cytotoxic T lymphocytes (CTLs) plays a fundamental role in protective immunity to viral, bacterial, protozoan and fungi infections, as well as to tumor cells. In vivo cytotoxic assays take into account the interaction of target and effector cells in the context of the proper microenvironment making the analysis biologically more relevant than in vitro cytotoxic assays. Thus, the development, improvement and validation of in vivo methods are necessary in view of the importance of the results they may provide. We describe and discuss in this manuscript a method to evaluate in vivo specific cytotoxic T lymphocyte killing. We used as model system mice immunized with human recombinant replication-deficient adenovirus 5 (HAd5) containing different transgenes as the trigger of a CTL-mediated immune response. To these mice, we adoptively transferred syngeneic cells labeled with different vital fluorescent dyes. Donor cells were pulsed (target) or not (control non-target) with distinct CD8 T-cell epitopes, mixed in a 1:1 ratio and injected i.v. into immunized or non-immunized recipient mice. After 18-24h, spleen cells are collected and analysed by flow cytometry. A deviation from the 1:1 ratio of control and target cell populations indicates antigen specific lysis of target cells

Oligonucleotide Adsorption Affects Phase Transition but Not Interdigitation of diC14-Amidine Bilayers

Oligonucleotides have been extensively used in basic research of gene expression and function, vaccine design, and allergy and cancer therapy. Several oligonucleotide-based formulations have reached the clinical trial phase and one is already on the market. All these applications, however, are dependent on suitable carriers that protect oligonucleotides against degradation and improve their capture by target cells. The cationic lipid diC14-amidine efficiently delivers nucleic acids to mammalian cells. It was recently shown that diC14-amidine bilayers present an interdigitated phase which strongly correlates with a potent fusogenic activity at low temperatures. Interdigitated phases correspond to very ordered gel phases where the two bilayer leaflets are merged; they usually result from perturbations at the interfacial region such as modifications of the polar headgroup area or dehydration of the bilayer. Interdigitation has been described for asymmetric lipids or mixed-chain lipids of different chain lengths and for lipids with large effective headgroup sizes. It has also been described for symmetric lipids under pressure modifications or in the presence of alcohol, glycerol, acetonitrile, polymyxin B, or ions like thiocyanate. Surprisingly, the role of polyelectrolytes on membrane interdigitation has been only poorly investigated. In the present work, we use dynamic light scattering (DLS), differential scanning calorimetry (DSC), and electron spin resonance (ESR) to explore the effect of a small single-stranded oligonucleotide (ODN) polyelectrolyte on the structure and colloid stability of interdigitated diC14-amidine membranes.

Isolation and functional characterization of Hrp65-binding proteins in Chironomus tentans

It is well-established that the organization of nuclear components influences gene expression processes, yet little is known about the mechanisms that contribute to the spatial co-ordination of nuclear activities. The salivary gland cells of Chironomus tentans provide a suitable model system for studying gene expression in situ, as they allow for direct visualization of the synthesis, processing and export of a specific protein-coding transcript, the Balbiani ring (BR) pre-mRNA, in a nuclear environment in which chromatin and non-chromatin structures can easily be distinguished. The RNAbinding protein Hrp65 has been identified in this model system as a protein associated with non-chromatin nucleoplasmic fibers, referred to as connecting fibers (CFs). The CFs associate with BR RNP particles in the nucleoplasm, suggesting that Hrp65 is involved in mRNA biogenesis at the post-transcriptional level. However, the function of Hrp65 is not known, nor is the function or the composition of CFs. In the work described in this thesis, we have identified by yeast two-hybrid screening and characterized different proteins that bind to Hrp65. These proteins include a novel hnRNP protein in C. tentans named Hrp59, various isoforms of Hrp65, the splicing- and mRNA export factor HEL/UAP56, and a RING-domain protein of unknown function. Immuno-electron microscopy experiments showed that Hrp59 and HEL are present in CFs, and in larger structures in the nucleoplasm of C. tentans salivary gland cells. Hrp59 is a C. tentans homologue of human hnRNP M, and it associates cotranscriptionally with a subset of pre-mRNAs, including its own transcript, in a manner that does not depend quantitatively on the amount of synthesized RNA. Hrp59 accompanies the BR pre-mRNA from the gene to the nuclear envelope, and is released from the BR mRNA at the nuclear pore complex. We have identified the preferred RNA targets of Hrp59 in Drosophila cells, and we have shown that Hrp59 binds preferentially to exonic splicing enhancer sequences. Hrp65 self-associates through an evolutionarily conserved domain that can also mediate heterodimerization of Hrp65 homologues. Different isoforms of Hrp65 interact with each other in all possible combinations, and Hrp65 can oligomerize into complexes of at least six molecules. The interaction between different Hrp65 isoforms is crucial for their intracellular localization, and we have discovered a mechanism by which Hrp65-2 is imported into the nucleus through binding to Hrp65-1. Hrp65 binds to HEL/UAP56 in C. tentans cells. We have analyzed the distribution of the two proteins on polytene chromosomes and in the nucleoplasm of salivary gland cells, and our results suggest that Hrp65 and HEL become associated during posttranscriptional gene expression events. HEL binds to the BR pre-mRNP cotranscriptionally, and incorporation of HEL into the pre-mRNP does not depend on the location of introns along the BR pre-mRNA. HEL accompanies the BR mRNP to the nuclear pore and is released from the BR mRNP during translocation into the cytoplasm.