CELLION – kick off meeteing Wojciech Dabros Jagiellonian University Medical College, Deptment of Pathomorphology Krakow

Our goal is to investigate the reaction of a cell to irradiation by a single ion (or a known number of ions), depending on the location of the ion trajectory within the cell, the type and status of the cells, the type and energy of the ion, etc. The study shall include the following phenomena: cell viability, type of damage, induction of apoptosis, repair processes (especially in the case of DNA damage), inter-cellular signaling processes, especially when associated with E-cadherin mutations (including the processes that induce the “bystander effect”), the character of genetic changes in damaged cells and the transference of genetic changes into daughter cells. The investigations shall be carried out based on the premise that their results shall be helpful in determining the risk of neoplastic diseases developing in effect of low radiation doses (radon, cosmic radiation, diagnostic management), as well as in the selection of optimal doses in radiotherapy and ion radiotherapy of neoplastic lesions.

Our Department can be involved in the following activities: Studies on viability of irradiated cells determinations of genetic changes triggered by such damages. Studies on repair processes in radiation damage Studies on the effect of signal-transference between irradiated and non-irradiated cells (including the “bystander effect”)

The investigations shall be carried out in the following laboratories: Immunohistochemistry Lab Immunohistochemical reactions are employed to supplement the histopathological diagnosis. Such reactions allow for a detailed diagnostic management that is necessary for appropriate clinical management in treating neoplastic lesions. The EnVision method is routinely used in immunohistochemical tests. Numerous (over 100) primary antibodies supplied by various companies (DAKO, Novocastra, SantaCruz, LabVision, BDBiosciences) are employed Histology Lab routine & special staining

Molecular Pathology Lab The list of methods/tests employed: Routine molecular biology Techniques (amplification and detection of DNA/RNA) DNA and RNA isolation from fresh, frozen tissue and PETs (paraffin embedded tissue) PCR (polymerase chain reaction) RT-PCR (reverse transcriptase PCR) Semi-nested PCR AP-PCR (fingerprinting) RFLP (restriction fragment length polymorphism) PAGE (DNA and proteins) – Vertical electrophoresis/acrylamide gels SSCP (single strand conformation polymorphism) Agarose EP (Horizontal electrophoresis) Protein truncation test Western blotting Direct sequencing Genotyping ISH – in situ hybridization FISH – fluorescent in situ hybridization CISH – Chromogenic in situ hybridization Tests: DNA fingerprinting STR analysis (Short tandem repeats) BRCA1 mutations analysis CDH1 mutations analysis APC mutations analysis MSI/LOH Analysis EBV, CMV (Ventana Medical Systems discovery)

Electron Microscopy Lab Cytoplasmic swelling: Expected ultrastructural changes Nuclear changes: Swollen nucleus or condensed nucleus chromatin margination clumping or cupping of chromatin disrupted nuclear envelope – perinuclear cisternae Giant cells with pleomorphic nuclei (irregular nuclear profile) or with multiple nuclei (Karyolysis, Pyknosis, Kariorhexis – need stronger dose of radiation) Cytoplasmic swelling: Mitochondrial cloudy swelling - Cristolysis swelling of ER (SER and RER) lysosomal changes cytoskeletal changes disrupted cell membrane – (one of the most sensitive structure) Apoptosis

The project will be implemented by four laboratories, ten research workers, ten technicians and one postgraduate fellow. The composition of the team that is to implement the project may change, depending on the tasks. The research is coordinated by Professor J. Stachura. The Faculty has long-term experience in the methodology of studies on morphological changes in cell cultures and suspensions, as well as in investigations on the effect of ionizing radiation. The first studies in this field were carried out by Professor Stachura 40 years ago.

Following irradiation with single ions (or a known number of ions), the cellular material shall be subjected to detailed observations and studies in our labs: Structural changes in DNA and RNA shall be investigated in the Molecular Pathology Lab, Changes in the structure of proteins may be pinpointed in the Immunohistochemistry Lab & Histology Lab while Cellular and Subcellular changes shall be documented by the Histochemistry Lab and Electron Microscopy Lab.

Fellows from Western and Eastern Europe shall participate in the studies. One postgraduate fellow seeking the title of Doctor of Medical Sciences shall be employed for 3 years, and additionally each year we shall offer a 3-month training to one individual. The Department and its Faculty are ready to commence project implementation.