HOW TO FIND THE PROJECT. Different ways Reference from your Mentor National Societies ESMO pathway Try to find your way
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2 HOW TO FIND THE PROJECT Different ways Reference from your Mentor National Societies ESMO pathway Try to find your way
3 Educational Fellowships Translational Research Unit Visit Clinical Unit Visits Palliative Care Fellowship Research Fellowships Translational Research Fellowships Clinical Research Fellowships Georges Mathé Translational Research
4 APPLY Completed online application form Digital photograph Detailed description of research project proposal including timeline Brief statement of project goals and how the acquired techniques will be used in the home institute/country Name of host institute and acceptance letter from the person responsible for the project at the host facility Letter of recommendation from the candidate s department head
5 CLUES Personal Profile - Well structured CV MOTIVATION LETTER!!! The training program The research project
6 Translational Research Program Molecular characterisation of the stromal microenvironment in pancreatic adenocarcinoma and its role in tumour progression and prognosis Caveolin and SHH Interaction in pancreatic cancer
7 Caveolae and Caveolins Caveolin-1 From B Alberts Molecular Biology of the cell CAVEOLAE
8 Central role of Hedgehog signaling in biology DEVELOPMENT HOMEOSTASIS Organizing centers Patterning Proliferation Angiogenesis Survival Sonic (Shh) Indian (Ihh) Desert (Dhh) Repair/regeneration Angiogenesis Proliferation Survival CANCER Angiogenesis Proliferation Survival Stroma
9 Hh HEDGEHOG SIGNALING On SMOOTHENED PATCHED CDO/BOC Sufu PKA Gli GSK3 P CK-1 G Gli P Gli-R GBS Target genes: Gli1, Ptc1
10 Breast Cancer Fibrosis caused by lack of caveolin and associated with poor prognosis CAF without caveolin Induce cancer cells to EMT Feed cancer cells through nutrients secretion
11 Pancreatic Cancer Pancreatic ductal adenocarcinoma (PDA) Among the most lethal human cancers Highly heterogeneous Insensitive to many chemotherapeutic drugs
12 PDA? Br J Cancer Nov 4;87(10): Impact of caveolin-1 expression on prognosis of pancreatic ductal adenocarcinoma tumour diameter (P=0.0079) histopathologic grade (P=0.0272) poor prognosis (P=0.0008). + CAV-1 expression was shown to be an independent negative predictor for survival (P=0.0358)
13 Hedgehog is an early and late mediator of pancreatic cancer tumorigenesis a. Immunohistochemical identification of SHH. a, Normal human pancreas. No specific staining for SHH protein was identified in acini, islets (I) or ducts (D) (X125 magnification). No SHH expression is detected in normal ductal epithelium (x500 magnification). Thayer SP, Nature. 2003
14 Science 2009 June (5993) Inhibition of Hedgehog Signaling Enhances Delivery of Chemotherapy in a mouse model of Pancreatic Cancer
15 Experimental Plan Do PdA cell lines with different caveolin expression have different Shh signaling? BXPC3 AsPc1
16 Do PdA cell lines with different caveolin expression have different Shh signaling? 300 CAV GLI PTCHD Basal Conditions BXPC3 ASPC-1
17 3 1 SSC Counts 128 R1 Silencing CAV FSC Region Count % Hist % All Total R (G1: R1) Kostas_04_OCT_2011_ R3 Counts FL R FSC Region Count % Hist % All Bounds Mode Count Total (0.00, ) (255.00,1.00) 164 R (16.00, ) (255.00,128.64) 164 R (23.00,9.65) (255.00,1.04) 101
18 0 Do PdA cell lines with different caveolin expression have different Shh signaling? CAV CAV BxPc3 Basal Conditions GLI PTCH GLI PTCH CAV GLI PTCH MOCK ctrl 0 MOCK ctrl MOCK transf sicav ctrl MOCK transf sicav ctrl sicav transf sicav transf 50
19 Control Shh 100 sicav+ Shh sicav % area % area hours hours
20 summary PdA cells expressing Caveolin have increased Shh signalling and are more sensitive to Shh stimulation Extracellular Shh induces only cav positive cells to proliferate/migrate faster, whereas it has an opposite effect on caveolin negative cells
21 % Growth Rate Tumor Size (mm 3 ) TUMORS' CROWTH RATE BxPC3 Bx97 GEMCITABINE 0, BxMOCK BxPC3 Bx97 TUMOR SIZE (MM 3 ) % Growth Rate DAYS POST CELLS' INOCULATION -100 Concentration (μμ) 1400 Tumors' Growth Rate BxPC BxMOCK Doxorubicin BxPC3MOCK Bx97 Bx97 BxPC Days Post Inoculation (DPI) Concentration (μμ) -100
22 Fibroblast Isolation 8 months
23 Different tumors Different cell lines Chemosensitivity In vivo Analysis Coculture
24 Lessons I have learned Many Lab Techniques Primary tissue culture techniques quantitative Real Time PCR to Extract whole cell protein-western Blot Cloning of expression vectors to induce transient overexpression of target molecules (i.e. Gli2 and PTCH1) Plasmid Transfection Infecting primary cell lines with lentivirus expressing short hairpin RNA for stable silencing of target genes ( i.e. caveolin 1) Fibroblast Isolation Shorting cells Confocal Imaging-Immunofluorescence.
25 Lessons I have learned Understand MATERIALS and METHODS Lab work matured my clinical thinking Lab world is different from Clinical world Exciting -Enthusiasm Disappointment Be patient There is no time in the lab Have to Ask for help and collaborate Have to repeat Have to be consistent More questions than answers A good translational paper needs a lot of effort (years?)
26 Not only. Participate in Phase I and Gastrointestinal clinics
27 Networking
28 Suggestions BE MOTIVATED SEARCH-STUDY APPLY AND RE-APPLY BE PATIENT AND PERSISTENT BE OPTIMISTIC WORK HARD FORM PARTICIPATE in NETWORKS IMPLEMENT what you have learned
29 Enjoy the adventure K. P. Kavafis ITHAKA When you set out on your journey to Ithaca, pray that the road is long, full of adventure, full of knowledge.
30 Acknowledgments Lab Members Francesco DelGaldo Justin Gillespie Vasiliki Liakouli Filomena Esteves Giuseppina Abignano Gemma Migneco University Hospital of Larissa Christos N. Papandreou Kostas Dimas The Leeds Teaching Hospitals Alan A. Anthoney Alan Melcher Alison Cairns, Chris Menon Andy Smith Shh Lab collaboration Natalia A. Riobo Karolinska Institute Caroline Verbeke
31 Last but not least BURNOUT PROJECT IMAGE OF THE MONTH
32
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