00062 - Applied Biochemistry

Learning outcomes

At the end of the course the student:

- knows and understands the principles and applications of biochemical investigation methodologies, both in the research laboratory and in the field of clinical biochemistry, for the purpose of separation, identification, characterization and analysis of biomolecules , of the mechanisms of the metabolic activities of biological phenomena, also in relation to the action of drugs and diagnostic devices for biological analyses.

- will be able to design or evaluate experimental protocols used in biochemical research.

The 2 credits of exercises combined with the 2 credits of laboratory will allow the student to acquire theoretical / practical skills of some methodologies applied in biochemical and clinical biochemical experimentation with particular attention to the logic of setting up experiments and data collection.

Course contents

Applied Biochemistry-1 (3 credits) - Prof. Cecilia Prata

Introduction. Principles of biochemical research. Biochemical experimentation. Introduction to Microscopy.

Cell cultures

Biological samples

pH-buffer solutions: choice of buffers for biochemistry. Amino acids and isoelectric point.

Homogenization: choice of methods and media.

Centrifugal techniques: principles of sedimentation, RCF, sedimentation coefficient and sedimentation times. Centrifuges and rotors. Differential centrifugation: separation and analysis of subcellular fractions; differential flotation of lipoproteins. Density gradient centrifugation, zonal and isopycnic. Applications: separation of cells, subcellular organelles, proteins, nucleic acids. Evaluation of yield and enrichment, "markers" subcellular

Fractional precipitation of proteins: fractional precipitation with ammonium sulfate and isoelectric precipitation with organic solvents and polymers; heat precipitation.

Dialysis and ultrafiltration: principles and applications.

Extraction (outline)

Chromatographic techniques: general principles, resolution and theoretical plates. Column chromatography.

Adsorption chromatography. Notes on TLC: two-dimensional chromatography. Hydrophobic chromatography of proteins (HIC).

Ion-exchange chromatography of proteins. Automatic amino acid analyzer.

Exclusion chromatography (gel filtration).

Affinity chromatography. Immuno affinity. Chromatography with dyes and lectins. Chromatography with metals; purification of histidine-tagged proteins and of recombinant proteins.

Applied Biochemistry-2 (2 credits_ Exercitations in class) – Prof. Romana Fato

HPLC.

Purification of enzymes: specific activity and yield. Immobilized enzymes.

Determination of the protein sequence (Edman sequencing and mass spectrometry).

Electrophoretic techniques: general principles, factors that influence the electrophoretic mobility. Electrophoresis free phase. Zone electrophoresis on cellulose acetate, serum proteins. Gel Electrophoresis: PAGE, SDS-PAGE, Agarose. Methods of detection and quantitative assessments. Blotting - Western blotting in the study of proteins: biochemical and diagnostic applications.

Isoelectric focusing (IEF). Two-dimensional electrophoresis.

Spectroscopic techniques

UV-VIS in Biochemistry. Report of Lambert-Beer. Colorimetric assays.

Enzymatic techniques. Elements of enzyme kinetics: initial speed, effect of the concentration of enzyme and substrate, pH and temperature. Reversible and irreversible inhibition. Competitive, non-competitive and a-competitive inhibitors. Chemical modification of proteins: inhibitors as drugs. Measurement of enzyme activity: continuous methods, discontinuous, direct, indirect and coupled.

Evaluation of enzymes and metabolites plasma / serum diagnostic purposes. Types of clinical laboratory biochemical analysis. The blood sample. Enzymes present in biological materials, their dosage and clinical significance of the evaluation of the activity. General characteristics, biochemical role and methods for the determination of the main enzymes of clinical interest: LDH, CPK, AST and ALT, alkaline phosphatase, cholinesterase. Enzymatic profiles: heart, liver and pancreatic. Metabolites of clinical interest: glucose (blood sugar, glucose tolerance test, glycated hemoglobin) cholesterol and lipid disorders. Plasma proteins: nature, function, and meaning of the quantitative determination of diagnostic importance. Plasma lipoproteins: significance of quantitative determination and diagnostic significance.

Spectrofluorimetry (general principles and applications) and Chemiluminescence; flow-cytometry and "cell sorting". Elements of luminometry.

Electrochemical techniques. Oxygen electrode; studies on mitochondrial respiration. Biosensors.

Molecular biology techniques. Recombinant DNA technology: general principles. Polymerase Chain Reaction (PCR): principles and diagnostic applications, forensic and paleobiological. VNTR sequences: paternity test. Identification of specific sequences of DNA and RNA: Southern hybridization (Southern blotting) and applications in the diagnosis of genetic diseases. Northern blotting and applications in the study of gene expression. Evaluation of gene expression: the technique of DNA microarray. Notes on proteomics.

Of each technique are discussed instrumental aspects and applications with related examples.

Applied biochemistry-3 (2 credits_Laboratory activities)- Dr.Manuela Sollazzo

P

Students will be divided into two groups:

Group A/Group B

During the lab module, the student will apply and learn different techniques for the study of biochemical and molecular effects induced by a compound inhibiting the respiratory chain, using a tumor cell line as a model.

The laboratory will be structured in different exercises, interconnected to each other, associated with some hours of theoretical lectures.

CELL CULTURES

During this experience, students will acquire the basic techniques for the culture of immortalized tumor cell lines as in vitro models. An ovarian cancer cell line will be used for the study of biochemical and molecular effects upon a treatment with a respiratory complex I inhibitor.

CELL VIABILITY ASSAY

The aim of this experience is to determine the effect on the proliferative capacity of tumor cells upon treatment with a respiratory complex I inhibitor. To determine viable cells, the sulforodamine B (SRB) assay will be performed.

PROTEIN EXTRACTION AND PROTEIN CONTENT EVALUATION USING BRADFORD METHOD

The aim of this laboratory is the extraction of proteins starting from a cellular pellet and the determination of the protein mixture concentration using the Bradford colorimetric method. The result obtained will be used to determine the volume of proteins for the next experience.

PROTEIN SEPARATION USING GEL-ELECTROPHORESIS (SDS-PAGE) AND DETERMINATION OF SIGNALING PATHWAYS ACTIVATION

The aim of this experience is to investigate the activation of AMPK pathway, the main sensor of energy stress, following inhibition of the respiratory chain. The technique used is based on protein separation using SDS-page and on the determination of the phosphorylation status of the AMPK protein by western blotting.

ENZYMATIC DETERMINATION OF CELLS GLUCOSE CONSUMPTION UPON INHIBITION OF RESPIRATORY COMPLEX I

During this experience, students will be able to calculate the ability of the ovarian cancer cells to use glucose upon inhibition of respiratory Complex I, using an enzymatic method for detection of glucose in cell culture medium.

 

Readings/Bibliography

K.Wilson and J.Walker - Biochemistry and Molecular Biology: Principles and Techniques - Cortina Editore, 2006

K.Wilson e J.Walker - Biochimica e Biologia Molecolare: Principi e tecniche – NUOVA EDIZIONE (VIII) 2019

M.Maccarrone - Metodologie biochimiche e biomolecolari. Strumenti e tecniche per il laboratorio del nuovo millennio. Zanichelli 2019

 

Teaching methods

The course includes 5 frontal credits (module 2 with discussion of experimental protocols in class) and 2 laboratory credits.

During the lectures will be discussed the methods connected with the trial in both biochemical and instrumental aspects of the applications. For each technique will be presented and discussed some practical examples.

In the 2 laboratory credits problems such as: determination of the protein content of a solution, determination of enzymatic kinetic parameters, cell viability measurements will be addressed from a practical point of view.

 

*** As concerns the teaching methods of this course unit, all students (including all the international incoming exchange students, i.e. ERASMUS) must attend Module 1, 2 online [https://www.unibo.it/it/servizi-e-opportunita/salute-e-assistenza/salute-e-sicurezza/sicurezza-e-salute-nei-luoghi-di-studio-e-tirocinio], while Module 3 on health and safety is to be attended in class. Information about Module 3 attendance schedule is available on the website of your degree programme ("studiare"--"formazione obbligatoria su sicurezza e salute")***

Assessment methods

Modules 1 and 2: Final exam consists of MCQs and Open-ended questions to assess:

- Knowledge of the basic principles of the main biochemical methodologies used in the separation, identification, characterization and analysis of biomolecules;

- Abilities to design or evaluate experimental protocols used in biochemical research.

 

Laboratory Module (modules 3 and 4) : the final exam will consist in a test with 10 Quizzes/short answer questions

The The overall rating is a weighted average of the two tests.

Teaching tools

Video, PC, Overhead Projector

Office hours

See the website of Cecilia Prata

See the website of Romana Fato

See the website of Manuela Sollazzo

See the website of Manuela Sollazzo