- Docente: Liliana Milani
- Credits: 6
- SSD: BIO/06
- Language: Italian
- Moduli: Liliana Milani (Modulo 1) Liliana Milani (Modulo 2)
- Teaching Mode: Traditional lectures (Modulo 1) Traditional lectures (Modulo 2)
- Campus: Bologna
- Corso: Second cycle degree programme (LM) in Molecular and cellular biology (cod. 8021)
Learning outcomes
With this course, the student will acquire knowledge on the methods to the study different aspects of cell and developmental biology. In particular, the student: will be able to propose up to date experimental set up to address specific issues in cell and developmental biology; will become autonomous in the interpretation, comparison, and communication of scientific researches on the topic.
Course contents
Lessons
Methods to study cell and developmental biology
Molecular approaches (transcriptomics/proteomics) and the use of microscopy will be described, as well as their application to classical animal model systems and to non-model organisms that have specific features making them of basic importance to investigate basic biological mechanisms.
Tools & techniques
Fluorochromes. Fluorescence microscopy. Laser scanning confocal microscopy. Two-photon microscopy.
Bioimages. Signal sampling, signal quantification, digital imaging and compression techniques. Acquisition, transformation, reduction, analysis, and interpretation of bioimages. Sampling Theorem. Transformations on bioimages (punctual, local, global). Sources of noise. Software for the analysis of bioimages (ImageJ).
FRAP (Fluorescence Recovery After Photobleaching). FRET (Fluorescence Resonance Energy Transfer). FLIM (Fluorescence Lifetime Imaging). Example of application 1: metabolic characterization of male germ cells by assaying FLIM of coenzymes NAD(P)H and FAD at the two-photon microscope. Example of application 2: study of chromatin morphology with FLIM-FRET at the two-photon microscope.
Electron microscopy. Transmission electron microscopy (TEM). Scanning electron microscopy (SEM).
Techniques for high-throughput sequencing. Pyrosequencing. Reversible-dye Terminator Sequencing-by-Synthesis. Semiconductor Sequencing. Single-Molecule Real-Time (SMRT) Sequencing. How to choose the platform. Sequencing strategies (direct, shotgun, single-end, paired-end, mate-pair sequencing, multiplexing). Workflow: sequencing output, quality scores, assembly, annotation. RNA-sequencing. Applications.
Model system choice
The problem with overgeneralization. The choice of the right model system given the biological problem to investigate. Surrogate and exemplary models.
Specification, maintenance and differentiation of the germline
Animal germline specification (preformation and epigenesis). Examples of preformation (Drosophila melanogaster, Caenorhabditis elegans). Examples of epigenesis (axolotl, mouse). Evolutionary aspects. Germline quiescence. Transcriptional regulation and chromatin remodelling (Drosophila, C. elegans, mouse). Silencing of transposable by piRNAs (Drosophila, topo).
Ageing as the last phase of development
The ageing process and its causes. The role of telomeres in ageing. Telomere shortening. Telomeres and associated proteins: the shelterin complex. Telomerase and its regulation. Methods to measure telomere length (Q-FISH). Methods to measure telomerase activity (using FRET). Senescence and ageing. The role of mitochondria in ageing. Free radical theory of ageing. Mitochondrial DNA replication in the theory of ageing. Stem cell hypothesis of ageing.
Wet lab: 1 cfu, 15 hours
Preparation of invertebrate gonadal tissues for confocal laser scanning microscopic analysis of germ cell morphology at different stages of differentiation (protocol of immunohistochemistry: agar embedding, sectioning, permeabilization, blocking of non-specific binding sites, incubation with primary and secondary antibody, nuclear staining, mounting on slides, observation at the confocal microscope.
Observation of biological samples at the electron microscopes (TEM e SEM).
Readings/Bibliography
Articles and reviews available on Insegnamenti Online (IOL), https://iol.unibo.it/.
Teaching methods
Lessons and wet lab.
Assessment methods
An oral exam at the end of the course to verify the acquisition of knowledge and expected skills.
Teaching tools
Video projector, PC (lessons).
Laboratory instrumentations (wet lab).
Office hours
See the website of Liliana Milani