April 10, 2012

Participant: Soren B. Hayrabedyan, MD, PhD

Visited institution: School of Biological Sciences, Imaging Unit and Proteomics Unit, University of Essex, Wivenhoe Park, Colchester CO4 3SQ, Essex, UK

Host Principal Investigator: Prof. Nelson Fernández

Duration: 05.03.2012 - 03.04.2012

Purpose of the visit: High-resolution confocal microscopy and nano proteomics studies of Sertoli cells in activated innate immunity NOD signalling context.

The Program of Research visit included:

• Functional Ligand challenge studies with mouse Sertoli cells followed by high-resolution confocal microscopy imaging for subsequent molecular level 3D object segmentation and co-localization

          - cultivation and maintenance of mouse Sertoli cell line;

          - NUNC cell chambers cultivated and ligand challenged (ie-DAP, LPS) Sertoli cells immunostaining with antibodies against NOD receptors, inflammasome and autophagy pathway members;

          - accessory HLA immunophenotyping of Sertoli cell line;

          - Nikon Eclipse Ti confocal digital imaging - single plain and z-stack acquisition of multiple combinations of ligand stimulation and antibody co-staining;

          - the subsequent analysis of the digital images was scheduled for and is currently undergoing in the partner lab in IBIR, BAS, Bulgaria (Soren Hayrabedyan)  (an example image is provided on this page);

• Nano proteomics comparison of differential changes in TLR4 vs NOD1 signalling pathways stimulated Sertoli cells membrane proteomes. Implications on blood-testis barrier protein dynamics (using topological and protein enrichment bioinformatics analysis)

          - cultivation and mono stimulation of Sertoli cells;

          - Thermo Scientific LTQ Orbitrap Nanospray system analysis of protein lysates of differentially challenged or intact Sertoli cells;

          - the primary analysis of the raw data, including the computationally heavy probabilistic peptide signatures mapping over different protein databases as well as ms/ms spectra and relative units expression level analyses were performed in Essex Proteomics Unit courtesy of Dr. Metodi Metodiev, and the subsequent protein enrichment and topological analyses are currently undergoing in the partner lab in IBIR, BAS, Bulgaria (Soren Hayrabedyan);

• Attendance of lecture on multiple omics integration analysis - by Thomson Reuters company
• Designing a collaborative plan for future publications on the current data acquired and the subsequent analysis performed with the Partner labs - Prof. Nelson Fernández and Dr. Metodi Metodiev and Gergana Metodieva

In Conclusion:

Dr Soren Hayrabeydan would like to express his deep gratitude and appreciation of the visit at the Essex University, and especially to his host - Prof. Nelson Prof. Nelson Fernández  who has supported our project on Sertoli cell innate signaling biology not only logistically and resourcefully, but most of all - cordially!  Prof. Fernández is the founder of the bioimaging facility at Colchester and as such had profound record of research in the field of co-localization molecular level bioimaging, and has inspired this research approach with a very insightful speech on bioimaging in a Reproductive Immunology Symposia many years ago. Current newly purchased state of the art equipment allowed for even higher resolution of co-localization studies and the application of recently published in Nature Methods approach of 3D object segmentation and co-localization. A collaborative research paper utilizing this approach and substantial amount of experimental findings of the research group at IBIR, BAS is currently in active preparation.