96027 - Methods in Cell Biology

Learning outcomes

With this course, the student acquires knowledge on methods to study different aspects of cell and developmental biology. In particular, the student learns: - to propose up to date experimental set up by means of optical and electron microscopy; - how to choose a proper experimental model system and to apply the comparative approach; - how to carry out an immunohistological analysis, from biological sample preparation to its observation. Also, the student becomes autonomous in the interpretation, comparison, and communication of scientific data on these topics.

Course contents

Lessons

Methods to study cell and developmental biology

Molecular approaches 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. Light sheet fluorescence 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).

Electron microscopy. Transmission electron microscopy (TEM). Scanning electron microscopy (SEM).

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 elements by piRNAs (Drosophila, topo). Study of chromatin morphology with FLIM-FRET at the two-photon microscope. Metabolic characterization of male germ cells by assaying FLIM of coenzymes NAD(P)H and FAD at the two-photon microscope.

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. New model systems to study ageing.

Wet lab: 1 cfu

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.

Readings/Bibliography

Slides of the lessons and articles/reviews made available on Virtuale.

Teaching methods

Lessons and wet lab.

In consideration of the types of activities and teaching methods adopted, the attendance of this training activity requires all students to carry out e-learning of modules 1 and 2 [https://www.unibo.it/en/services-and-opportunities/health-and-assistance/health-and-safety/online-course-on-health-and-safety-in-study-and-internship-areas] and participation in module 3 of specific training on safety and health in study places. Information on dates and methods of attendance of module 3 can be consulted in the specific section of the degree program website.

Assessment methods

The final exam consists of an oral interview and aims to evaluate the achievement of the didactic objectives: knowledge of the methodologies for the study and understanding of various problems of cell biology.

Teaching tools

Slides of the lessons (with the use of video projector and PC).

Laboratory instrumentations (wet lab).

Office hours

See the website of Liliana Milani