Summer School 2018

The course will be taking place at "Polo Nobili” in the Granduca Building,

located in A. Volta Street n°4/6, Pisa.

Lessons: ES1 classroom, Practice: PN2 lab.

EXPERIMENTAL COURSE: GENE EXPRESSION ANALYSIS IN SITU

Lecturer: Alessandra Salvetti

Lesson: Overview of RNAse-free treatments, probe synthesis and in situ hybridization techniques (tissue fixation, hybridization, immunolocalization of probes, double labelling).

Practice: Students will focus their practical experience on RNA probes synthesis and whole mount in situ hybridization using animal model systems. Students will fix animals and will hybridize them using different cell/tissue specific RNA probes. In particular, students will use probes specific for stem cells, nervous system, eyes and gut.

EXPERIMENTAL COURSE: THE STUDY OF GENE FUNCTION BY TRANSIENT GENE SILENCING TECHNIQUES

Lecturer: Leonardo Rossi

Lesson: Overview of loss of function techniques for transient gene silencing in invertebrates, lower vertebrates and mammalian cell cultures.

Practice: Student will focus their practical experience on RNAi techniques using animal model systems. Students will synthesize double-strand (ds) RNA and will use it to feed animals and obtain gene silencing. Gene silencing will be verified by real time RT-PCR analysis of RNA extracted from dsRNA feeded animals and untreated controls. Students will also be provided with animals already silenced for the expression of specific genes “in blind”. With these animals, students will carry on some assays aimed to discover the function of the silenced genes. In particular, students will analyze: (i) the expression of tissue specific markers; (ii) regenerative capabilities and formation of morphological aberration.

EXPERIMENTAL COURSE: BASIC PROCEDURES IN TRANSMISSION ELECTRON MICROSCOPY

Lecturer: Alessandra Falleni

Lesson: The potential value of transmission electron microscopy (TEM) in life sciences. Short history of electron microscopy in Italy. Overview on basic techniques in conventional TEM, cytochemical and immunolabelling techniques.

Practice: Students will focus their pratical experience in basic transmission electron microscopy techniques using biological samples. Specimens will be fixed, dehydrated and embedded in Epoxy resin. Hardened resin blocks will be cut by students with a ultramicrotome equipped with a diamond knife. Ultrathin sections will be collected on formvar/carbon coated copper grids, double stained with uranyl acetate and lead citrate and finally observed with a Jeol 100 Sx transmission electron microscope. Students will discuss the results of their own samples together with teacher. The discussion of electron micrographs of different biological samples is also planned.

EXPERIMENTAL COURSE : GENOTOXICITY BIOMARKERS

Lecturer: Giada Frenzilli

Lesson: Overview on principles and potential applications of Comet assay and Cytokinesis-block micronucleus Cytome assay for the evaluation of DNA primary and chromosomal damage, respectively.

Practice: Students will focus their practical experience in setting up and analyzing biological samples. Cells embedded in agarose will be layered on conventional microscope slides. Cell and Nuclear membranes will be lysed and slides will undergo alkali unwinding and electrophoresis step. After neutralization slides will be stained and scored under fluorescence microscope. An image analyzer will allow the detection of DNA damage evaluated by different parameters. A parallel smear of fish nucleated erythrocytes will be carried out, fixed and stained to be evaluated by Cytome assay. Students will be also provided with other microscope slides obtained by human pulmonary epithelial cells (A549) and murine macrophages (RAW264.7) exposed to nanomaterials.