PLASMA POSTER COMPETITION

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2. Mitochondrial DNA? Western Blots?

3. Bladders?  Prostates?  Urine? 

4. Non-invasive assessment of bladder function Jennifer Caffarel

5. 1/3 of men will have bothersome urinary symptoms at some point in their life

6. … then you might have “Bladder Outlet Obstruction”

7. The prostate gland surrounds the urethra With increasing age, it increases in size and can obstruct the urethra…

8. … and can cause a slow or “weak” urine flow. Urine flow can be quantified by a technique called “uroflowmetry”.

9. Uroflowmetry is performed in clinics, using expensive electronic flowmeters Attending a flow clinic can be a long and tedious experience…

10. … and having to fill and empty your bladder several times in a short period of time is rather unnatural!

11. The data obtained from flow clinics is variable, due to a number of factors: • Time of day • Volume of urine in the bladder • Emotional state of patient

12. The aim is to improve the diagnosis made using uroflowmetry. Electronic Flowmeter Basic Flowmeter vs. For example, we tested a very basic flowmeter, to obtain more representative results.

13. This is how the basic flowmeter is used: • The patient takes the device home • Makes multiple measurements over several days, in his own time

14. This home flowmeter provided more representative results than in-clinic flows. • And 2 patients who could not urinate in the clinic were able to use this device.

15. Thank you for your time

16. PLASMA POSTER COMPETITION All Staff and Students are invited to use the ballot papers below to vote for your favourite plasma poster

17. PLASMA POSTER COMPETITION All Staff and Students are invited to use the ballot papers below to vote for your favourite plasma poster

18. ? Questions (and some answers) on research into stomach cancer… ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? Claire Worrall

19. Who gets stomach cancer? So biological markers of early disease would be very useful. It is difficult to predict. It depends on: • If you have H. pylori infection • What you eat • If you have stomach ulcers • How much alcohol you drink • If you smoke • Your Body Mass Index • What country you live in • Your genetic background • Your gender

20. What 100 % % of cases expressing TFIZ1 0 % Normal stomach Stomach cancer 0.005 % % of normal TFIZ1 expression AGS NUGC3 MKN74 KATO III SNU 1 SNU 5 SNU 16 0 % changes occur in stomach cancer? Expression levels of many proteins are altered… We are studying one protein whose expression is lost in the majority of stomach cancers: TFIZ1 is expressed in mucin-secreting cells and secreted into the mucous layer in the stomach. It is virtually undetectable by RT-PCR in our panel of 7 gastric cancer cell lines.

21. Where can we study stomach cancer? These behaviours in patient cancer cells help determine tumour growth and the ability to metastasize. There is limited supply of patient samples. However, we can grow stomach cancer cells in the lab to perform tests on. These cells grow and divide, allowing us to measure the effects of different conditions on: • How fast they grow and divide • How easily they die • How quickly they move

22. When TFIZ1 TFF1 Involved in ulcer repair 30% null mice get stomach cancer Binds to H. pylori TFF1 TFF1 do we study a protein in stomach cancer? When we have evidence linking it to stomach cancer… TFIZ1 is bound in the stomach to TFF1, which has many links to cancer: High expression in oestrogen responsive breast cancer

23. How are we studying this protein? By introducing controllable TFIZ1 expression into stomach cancer cells: 1. Transfect in regulator plasmid neoR tTA TFIZ1 Off TFIZ1 On The cells now express TFIZ1 pTet-Off TRE neoR tTA TFIZ1 2. Transfect in TFIZ1 plasmid Adding doxycycline turns off TFIZ1 expression TRE TFIZ1 pTRE-Tight TFIZ1 Stomach cancer Cell line

24. Why do we study a protein in gastric cancer? If we know • WHY the expression changes • WHAT the protein does • In the normal stomach • In stomach cancer • WHEN the expression changes It can Help us decide whether it might be a good early indicator of cancer. Help us decide whether it might be a good therapeutic target.

25. Acknowledgements: • Felicity May • Herbie Newell • Northern Institute for Cancer Research Claire Worrall claire.worrall@ncl.ac.uk

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27. RADIOTHERAPY INDUCED BREAST CANCER Farida Sufi INTRODUCTION: Chemotherapy and radiotherapy- induced second cancers are the leading cause of death in patients cured of Hodgkin Lymphoma (cancer of the lymphatic system). Young women treated for Hodgkin Lymphoma with radiotherapy at the chest are at a high risk of developing breast cancer Farida.begum@ncl.ac.uk ‘MRI scan of the breast reveals two discrete areas of abnormality, which proved to be cancer.’ AIM: To develop an in vitro model system of radiogenic breat cancer, so that the molecular genetic events associated with transformation and the development of a radio-resistant phenotype can be investigated. • PROCEDURE: • This project will involve exposing transformed (MCF-7) and non-transformed (MCF-10) breast cell lines to increasing doses of ionising radiation. • Post treated cell lines will be compared to untreated parental cells across the whole genome to identify regions characterised by loss of heterozygosity, copy number alterations and uniparental disomy. • Candidate locations of interest will be further investigated in treated cell lines and in breast tissue from patients with breast cancer following radiotherapy for Hodgkin Lymphoma. http://www.breastcancer.org/pictures/diagnosis/mammogram/mri_2.jsp FUNDED BY: Northern Institute for Cancer Research

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29. Forkhead Proteins & Cancer Frances Purtill iCAMB

30. Forkhead Proteins & Cancer Unregulated cell proliferation is a characteristic of all cancers Understanding the mechanisms behind cell proliferation will help in the treatment and prevention of cancer

31. Forkhead Proteins & Cancer Cell proliferation is controlled by a series of events known as the Cell Cycle The cell cycle consists of 4 phases: 2 M Phase (Mitosis) Replicated DNA is equally distributed into 2 cells M 3 & 4 G1 and G2 are ‘gap’ phases which prepare the cell for its next step G2 G1 S 1 S Phase DNA is replicated Genetic material is copied and 2 identical cells are formed

32. Forkhead Proteins & Cancer TARGET PROTEIN MADE Forkhead Transcription Factors (FKH-TF) arecritical for the regulation of the cell cycle Forkhead proteins have been associated with various human cancers They bind upstream of genes involved in mitosis, and activate their expression FKH-TF GENE SWITCHED ON TARGET GENE

33. Forkhead Proteins & Cancer Forkhead proteins are vital for mitosis in all eukaryotes, from yeast to man Therefore it is a valuable model to study forkhead proteins, and improve our knowledge of the cell cycle in humans Yeast is easy to manipulate, genetically and biochemically In the future, this knowledge could potentially be used to improve existing cancer therapies

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35. The Development of Small Molecule Inhibitors of the MDM2-p53 InteractionJunfeng Liu

36. p53 is a Tumour Suppressor % mice with tumour p53-/- 74% at 6 months p53+/- 2% at 9 months p53+/+ 1% at 18 months Donehower et al. Nature, 1992 Also suggest to read Donehower et al. Nature genetics, 1993

37. MDM2 controls p53 through an auto- regulatory negative-feedback loop p53 MDM2 p53’s proteasomal degradation p53 MDM2 mdm2 mRNA mRNA of other targets • Three mechanism to repress p53 by activation of MDM2-expression: • MDM2 binds p53 at transactivation domain and blocks its ability to activate transcription • MDM2 acts as an E3 ubiquitin ligase that promotes p53’s proteasomal degradation • MDM2 is involved in the nuclear export of p53

38. Breaking the negative-feedback loop with antagonists p53 p53 MDM2 p53’s proteasomal degradation MDM2 mdm2 mRNA mRNA of other targets • Therapeutic potential Rescue of p53 Function by Disrupting the p53–MDM2 Interaction. • Blocking MDM2 Expression • Inhibiting MDM2 Ubiquitin Ligase Activity • Disruption of the p53–MDM2 binding

39. Possible Methods • Disruption of the p53–MDM2 Binding Interaction • Antibody microinjection • Peptide analogues corresponding to the MDM2 binding domain of p53 • Small molecular weight compounds

40. The p53-MDM2 interaction: Unique binding site Unique & Good for Small Molecule Drug Design and Development

41. Structure based drug design MDM2 Binding Domain (Binding area high lighted) PyMOL Software were applied for the followed cartoons. MDM2 protein and p53 peptide structures come from Protein Data Bank.

42. Structure based drug design 8mer p53 peptide Binding with MDM2

43. Structure based drug design Three key residues of p53 in the hydrophobic pocket of MDM2

44. Structure based drug design Three key residues of p53 were picked out for drug design

45. Structure based drug design Drug design based on the three key residues of p53

46. Structure based drug design Designed small molecule scaffold

47. Structure based drug design Manual docking of the compound with MDM2 binding domain

48. Biotinylated IP3 Peptide Streptavidin M M M M M Incubation with MDM2 Y Y Y Addition of: MDM2 primary antibody, Y Y Y Y M M M HRP conjugated secondary antibody Y Inhibitor Screen Using ELISA

49. Cellular activity Confirmation via Western Blotting Assay Effect of MDM2 inhibitors on the cellular levels of p53, MDM2 and p21 Nutlin-3 NU8XXX DMSO 0.5 1 10 1 10 20 mM MDM2 p53 p21 Actin

50. Thank you for your attention!