B021423 -

Lectures are held in Italian. It is however advised that students have medium/good knowledge of the English language, also because several slides are in English. Furthermore, in some lectures clips/videos in English will be shown

Molecular Biology technologies employed in Cancer Research. technologies will be rather presented according to general themes (gene, oncogens, animal models) or sometimes grouped as technologies themselves (PCR, cytogenetics, NGS etc.)

Textbooks/Didactic Material:
Watson, JD; Caudy AA; Myers RM, Witkowski JA DNA RICOMBINANTE (from Recombinant DNA, 3rd Edition (WH Freeman and Company) Trad. Zanichelli
ULTERIORI LETTURE:
Rovigatti (2016). "The long Journey of Cancer Modeling: Ubi Sumus ? Quo Vadimus ?" Journal of Cancer Science 1(1): 001.
Rovigatti, U. (2015). "Cancer modelling in the NGS era - Part I: Emerging technology and initial modelling." Critical Reviews in Oncology/Hematology 96(2): 274-307.

Students should acquire by the end of classes a landscape/overview -sometimes in greater details- of different technologies employed in cancer research

It is suggested -but not requested- that students have already passed the Molecular Biology I exam.

Frontal lessons of 2-2.5 hours each. 1-2 in-class exercises will be performed during some lectures (see program)

not applicable

written examination with open questions in the due dates according to our Academic calendar

Lect. 1 VIRUSES . Focus Formation Assay (FFA) developed by R. Dulbecco for oncogenic DNA viruses and H. Rubin for Retroviruses, further optimized by H. Temin. DNA extraction technologies and DNA sedimentation/banding (illustrated by the work on SV-40 integration by J. SAmbrook and R. Dulbecco). Clonality of SV-40-transformed/tumorigenic cells as shown by restriction enzyme mapping (also Adeno/Polyoma virus). Fundamental experiments in H. Temin and D. Baltimore laboratories have characterized Retroviruses replicative cycle.

Lect. 2 THE GENE. General Landscape on Molecular Biology from Mendel to Correns- von Tshermack and De Vries. First genetic maps in the fly-room (Morgan / Sturtevant / Bridges). Barbara McClintock definition of recombination by crossing-over. The fine-mapping of S. Benzer provides the first definition of gene at the nucleotide level. Technology developed by J. Beckwith allowed to firstly isolate and visualize a gene (LacZ of LAC Operon) by hybridization/EM. Studies of M. Wigler and R. Axel: calculation of frequency of restriction enzyme sites. Isolation and transfection of HSV-TK fragments using the selection imposed by H A T.

Lect. 3 ONCOGENE’s. Chemical Carcinogenesis: technologies employed to decipher it (Yamagiwa, Itchikawa, Kenneway etc.). Bruce Ames and its test + similar tests based on auxotrophs/prototrophs mutants (Beadle and Tatum). Transfection technologies as initially developed by G. Van der Eb for Adenoviruses etc. R. Weinberg applies transfection to retroviruses genes and oncogenes. The “oncogene hunt” starts a real race (“Racing to the beginning of the road” by R. Weinberg): M. Barbacid, J. Cooper, R. Weinberg and M. Wigler. Initial identification and purification of the first human oncogene with Alu probes. Similar techniques identify the gene for Von Recklinghaus Disease (NF-1/GAP).

Lect. 4 MMUTATIONS. Landscape from Mendel and De Vries to Morgan and Hermann Mueller. Techniques/experiments used by Mueller in Deosophila. Thechniques used in order to induce and study mutations in N. Crassa / A. Nidulans (Beadle & Tatum). Techniques used by S. Luria and M. Delbrueck to molecularly define mutation. John Cairns and the technology utilized in order to define adaptive mutations (Foster, Rosenberg, Roth and Tlsty). NGS can provide today the most direct approach to detect mutations (Vogelstein / Stratton / MArdis).

Lect. 5 GENE EXPRESSION. The seminal work of J. Monod, F. Jacob and A. Lwoff defined the concept of gene as unity of gene expression and regulation (techniques of genetics, biochemistry, microbiology, virology, the biostat etc.). Southern and Northern blotting – classroom exercise and testing from Watson’s MBOG. Microarrays: B. Vogelstein and I. David pioneering work on GEP by dot-blotting. Artificial DNA synthesis with phosphoramidites pioneered by Hunkapiller’s, Leroy Hood, M. Caruthers and others. Pat Brown and UniCalif optimized the first Micro-Array systems. Further developments and state of the art: S. Fodor and Affimetrix matrices (lithography). Sthephen Friend and Laura Van ‘t Veer studies on mammary carcinoma/lynphonode negative women.

Lect. 6 PCR AND CYTOGENETIC TECHNOLOGIES FOR CANCER RESEARCH. Kary Mullis, eccentric Nobel, and its creation of PCR technology. Roche further quantitative developments and applications for sequencing. Beacon primers and associated technologies: RT and Q-PCR. Digital PCR and its applications to cancer studies. Great pioneers have set the stage for our understandings of cytogenetic aberrations in cancer: Hungerford, Nowell, Rowley, JJ Yunis, D. Burkitt. High resolution banding developed by JJ Yunis: see Rovigatti and Yunis: Science 1986, also as an example of in situ hybridization (ISH). T(9;22) in Chronic Myelogenous Leukemia (CML) Ph’ positives; t(8;14) etc. in Burkitt’s Lymphoma (BL); t(14;18) in Follicular Lymphoma, etc.

Lect. 7 SEQUENCING IN CANCER RESEARCH. Methods of Maxam and Gilbert based on DNA degradation steps. Methods developed by Fred Sanger and J. Soulston based upon dideoxynucleotides incorporation. Pyrosequencing: phosphatases quantum e production allows detection; S454 machines. Sequencing methods based on so-called Next Generation Sequencing (NGS) methods. Very fast sequencers: the goal of $ 1000 genome sequencing, DNA sequence of Jim Watson see also my publication: “Cancer Modeling in the NGS Era”, CROH 2015. Synthesis and Massive Parallel Sequencing by Illumina. Examples of Sequencing by Ligation with SOLiD (Sequencing by Oligonucleotide Ligation and Detection) developed by ABI (Applied Biosystems).

Lect. 8 MODEL ORGANISMs Some of the most utilized and useful models are well described in the last chapter of the last Edition of MBOG by Jim Watson & colleagues. Bacteriophages. E. Coli. Barbara McClintock and trasposons. Baker’s and other yeasts. The small warm Caenorhabditis Elegans has been essential for studying apoptosis (S. Brenner, J. Soulston, R. Horvitz) and other pathways. Drosophila: organism of Molecular Biology first steps and among the first genomes to be sequenced. The Balancer chromosome; introduction to recombinase FLT and FRT signals. Transforming elements P and hybrid dysgenesis. Mus musculus and Rattus rattus . Embryonal manipulations and transgenic animals. Mario Capecchi and knocked-out mice. Imprinting. Zebra fish as interesting model for differentiation and more. New great interests in models based on species with extreme longevities (Mathusa): Naked Mole Rat (NMR)and Blind Mole Rat (BMR) display longevities 20X up to 32X and question our current modeling of carcinogenesis.