Post-doctoral Fellowship in Parasitology / Virology

Location
Brazil
Posted
Aug 15, 2019
Closes
Sep 14, 2019
Ref
116190
Organization Type
University and College
Hours
Full Time
The Pasteur-USP Scientific Platform has one post-doctoral position available under Thematic Project Integrated strategy for the study of infectious agents that cause emerging and / or neglected diseases transmitted by vectors of global impact.

The opportunity is related to the subproject number four of the Thematic Project entitled Impact of Trypanosoma vivax infections on blood brain barrier crossing / disruption in murine systems. The selected candidate will receive a 24 month Post-Doctoral fellowship granted by FAPESP, the São Paulo Research Foundation.

Subproject four aims at deciphering the T. vivax brain impairment process and the functional / lethal consequences of the infection process, especially the main histopathological lesions of the brain, thus forming a base of studies focused on neuroinflammation and the breakdown of the blood-brain barrier. The project will serve as the basis for interfacing with other subprojects of a thematic study involving infections with Zika virus infectious clones, modified by reporter genes for the analysis of the infectious process in vitro and in vivo using 2D- and 3D- imaging.

Subproject 4 Summary

To better study T. vivax infections in well-established experimental models, our group had revisited the state-of-the-art of available in vitro, in vivo and ex vivo imaging tools, generating new and more powerful essential and specific tools for imaging trypanosomatids (Goyard et al., 2013; Rouault et al., 2016; Dias de Melo et al., 2017). These efforts allow us today to quantify in real time the particularities, abundance and magnitude of trypanosomal infections, to reveal original data related to the development of the pathology, thus opening new research and innovation opportunities. Our preliminary results described the development of a reference model for T. vivax, especially due to the high virulence of the mouse and the parasite's difficulty to grow in axenic cultures. However, to study animal trypanosomosis, Nagana, and to characterize some of the major actors in T. vivaximmunopathology, our laboratory has developed further the complex T. vivax axenic cultures of an African strain and today masters the process of parasitic differentiation into in vitro infectious forms (D'Archivio et al., 2011). Reproducible infections were obtained in mice of several strains, which presented parasitological, histological and pathological parameters very similar to those observed in the field, characteristic of cattle trypanosomosis - severe acute anemia, thrombocytopenia and a significant reduction of B lymphocytes after infection (Chamond et al. (2010; Blom-Potar et al., 2010). The in vitro cultivated T. vivaxstages allowed the laboratory to construct the first specific expression vectors, which led us to develop reverse genetics for this parasite (D'Archivio et al., 2011). Thus, establishment of axenic cultures was useful for establishing and optimizing parasite transfections and selecting stable mutants that continue metacyclogenesis while maintaining virulence for immunocompetent mice. The region containing the T. vivaxribosomal promoter was inserted into the plasmid to improve expression of the luciferase reporter gene transgene and to allow integration of the vector into the genome. With these tools, the group analyzed the evolution of the infectious process and the distribution of parasites in mouse tissues. The results corroborated the use of realtime biophotonic analysis to study and monitor the infectious process in vivo. Through these experiments, we could suggest that the blood-brain barrier (BBB) is crossed by trypanosomes in the later stages of infection (D'Archivio et al. 2013). T. vivax, in fact, penetrate the brain parenchyma shortly before the death of the animals.

Main methodologies

Infections in vivo. Trypanosoma vivax parasites (IR1392) will be used for mouse infections as an experimental model. Outline and follow-up of infections in vivo and the choice of appropriate animal models will be performed in the laboratory. The participation of CD5B cells in the resistance and susceptibility of the chosen mouse strains, as well as the analysis of the in vivo infectious process (cytokines, markers of cell differentiation) will be performed by standard techniques involving FCP (Cytometric Bead Array , Becton Dickinson) for Th1 / Th2 / Th17 cytokines, monoclonal antibodies directed against adhesion molecules, cell surface markers, etc., and flow cytometry (AccuriTm C6 Cytometer System, Becton Dickinson).
Flow cytometry. Specific antibodies for proteins of interest (leukocytes, glial cells, mediators, cytokines, chemokines, adhesion molecules such as S100beta, NSE, GFAP, albumin) will be used. Flow cytometry will be evaluated using a FACS Accuri (Becton Dickinson), while events will be acquired and analyzed using the FlowJo program (Tree Star, Inc). When necessary, specific neuroinflammatory markers will be quantified by radioimmunoassay.

Quantitative PCR. Cells will be subjected to RNA extraction using Trizol Reagent (Life Technologies) following the manufacturer's instructions. The cDNA obtained will be subjected to quantitative PCR (qPCR) using SYBR green Supermix (Bio-Rad - CA, USA).
Immunofluorescence assays will be performed to label the various neural cell types for synaptogenesis verification, with antibodies that specifically recognize presynaptic and postsynaptic buttons. After marking the synapses, cells will be analyzed by fluorescence microscope and quantifications will be made using IMARIS software (Bitplane, MA, USA). Multiple-factor secretion analysis in cell culture supernatants with reference to panel 27-plex: IL-1β, IL-1ra, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8 , IL-9, IL-10, IL12 (p70), IL-13, IL-15, IL-17, eotaxin, basic FGF, GCSF, GM-CSF, IFN-γ, IP-10, MCP-1 (MCAF), MIP-1α, MIP-1β, PDGF-BB, RANTES, TNFα, VEGF.

Imaging studies in vivo. Mice will be anesthetized and analyzed on the Newton 7.0 (Vilber) imaging camera. When animals are infected with luminescent parasites, the animals will be previously injected i.p. with D-luciferin (122799, PerkinElmer, Walthan, MA, USA) - luciferase substrate. Immunofluorescence or luminescence 2D images will be acquired and regions of interest (ROI) of the organs of interest will be determined using the Living Image program (Perkin Elmer).

RT-PCR studies. RT-PCR will be used when necessary to quantify inflammatory response mediators transcripts or parasitic tissue load. Reference oligonucleotides for the transcripts to be analyzed will be chosen from our experience.

Assessment of the blood-brain barrier permeability and / or neuronal lesions resulting from the infection will be made, for example, by the measurement of only brain or serum proteins in the cerebrospinal fluid, serum and plasma of mice. Annexin 1, produced by brain microvascular endothelial cells, as well as markers of apoptosis, hypoxia and fluorescent bleeding processes, or markers of inflammation such as cytokines and specific markers of neuroinflammation will be quantified. The privileged techniques will be: immunohistochemistry to detect microglia (anti-Iba-1) and astrocytes (GFAP) and flow cytometry for inflammatory cytokines, chemokines and adhesion molecules (CBA r luminex). Cytokines involved in blood-brain barrier rupture will be particularly studied (IL-1-beta, IL-6 and TNFalpha).

Histopathology studies will be performed in parallel for analysis of neuroinflammation by the presence also of T, B lymphocytes, macrophages with appropriate cell differentiation antibodies.

Dosage of circulating and brain proteins. S100beta, NSE, GFAP, and albumin proteins will be analyzed in cerebrospinal fluid and serum, and their presence compared to plasma levels. Monoclonal antibodies directly labeled and specifically directed against these brain markers will be used in immunoassay (Elisa, Westernblot) and immunohistochemical (anti-Iba-1 and GFAP) tests.

Research project activities:
  1. Adapt on the new platform the Trypanosoma vivax cultures previously developed (PLoS Negl Trop Dis. 2011 Dec; 5 (12): e1461), clone, sequence and express parasite, host and / or reporter genes;
  2. Maintain cell cultures and perform transfection experiments;
  3. Inoculate animals with parasites expressing reporter genes and study the inflammatory (immune) process and its consequences on the impairment of brain and blood brain barriers using noninvasive imaging techniques in real time and immunological approaches;
  4. Interact with colleagues from associated subprojects for generation of infectious ZIKV clones;
  5. Participate in infections and analyses of the in vitro and in vivo experimental infectious processes with viral clones under NB2 and NB3 confinement conditions.

The candidate will practice writing manuscripts for submission.

Requirements:

The candidate for PD must have previous experience in parasitology and / or virology, techniques in immunology, microscopy, molecular and cellular biology that allow the exercise: 1) cloning, sequencing and gene expression, 2) prokaryotic and eukaryotic cell cultures and transfection experiments and 3)animal inoculation. Priority will be given to candidates who have proficiency in English and French.

Rationale for the Plan in terms of the objectives of the FAPESP PD Scholarship Program

Contribute to the knowledge, development and technological adaptation aimed at combating the understanding of neuroinvasivity and neurodegeneration caused by pathogen infections; establish and increase the training of researchers involved in the process, as well as the development of specific skills; highly qualified human resources training for cutting-edge research in Brazil; put Brazil in evidence as a promoter of quality research and relevance in the world scientific scenario.

The post-doctoral opportunity is open to Brazilians and foreigners. The candidate will receive a FAPESP Post-Doctoral Fellowship in the amount of R $ 7,373.10 per month and a Technical Reserve equivalent to 15% of the annual scholarship amount to meet unforeseen expenses directly related to the research activity.

More information about the fellowship is at: fapesp.br/oportunidades/3049 .

Eligible destination country/ies for fellows: Brazil

Eligibility of fellows: country/ies of residence: All

Eligibility of fellows: nationality/ies: All

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