Methods in endocrinology

Mind Map by , created over 6 years ago

Endocrinology Mind Map on Methods in endocrinology, created by maisie_oj on 04/12/2013.

Created by maisie_oj over 6 years ago
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Methods in endocrinology
1 Areas of study
1.1 Glands
1.2 Target tissues/cells
2 General methods
2.1 Surgical manipulation - e.g. removal/replacement of a gland
2.1.1 Physiological alterations In target tissue Loss of activity Chnages in blood composition?
2.1.2 e.g. castration or adrenalectomy reversed by hormone replacement therapy
2.2 Tissue extract preparation
2.3 Histological methods - ID target cells (immunohistochemistry, staining)
2.3.1 Cytological characteristics Hypertrophic - enlarged tissue (not number of cells) Organelles - number present, size etc. Dilated ER/golgi = increased activity Atrophy (wasting of tissue) Hyperplasia (increased number of cells) Disease status
2.3.2 Stains (of chemical components in cell) Glycoproteins Glycogen Lipid Can indicate function
2.4 Gene expression
2.5 Pharmacological - mimic/inhibit activity
2.6 Anima models
3 General considerations
3.1 Source
3.2 Structure and synthesis
3.3 Control of secretion and mechanism
3.4 Circulation and metabolism
3.5 Actions and roles
3.6 Pathophysiological roles
3.7 Comparative endocrinology
4 Studying the endocrine control of a physiological process
4.1 For hormones secreted by a ductless gland
4.1.1 Remove the gland observe effects
4.1.2 Is effect restored after gland extract?
4.1.3 Purified/synthetic hormone induec same effect?
4.1.4 Secretion - mechanism and control
4.1.5 ID receptors on target cells Many receptors found - some are referred to as orphans as their ligands are not known
4.2 Gross observation of tissue
4.2.1 Anatomical location
4.2.2 Size
4.2.3 Vascularisation / innervation
4.2.4 Pathophysiologies - e.g. goitre - indicating a thyroid problem
4.2.5 Immunocytochemistry
5 Techniques
5.1 Immunocytochemistry
5.1.1 Can involve Ab's Very specific For peptide/protein hormones Conjugated With fluorescent tags/enzymes that produce a coloured prduct
5.1.2 Method Tissue sample prepared Incubated in primary Ab for specfic cell protein Incubated with secondary Ab carrying a fluorescent tag Visualisation Dependent on method Immunofluorescence (with fluorescent Ab's) Immunoperoxidase (if enzyme-conjugated)
5.2 Fluorescent in situ hybridisation (FISH)
5.2.1 Detects gene amplification using a fluorescently labeleld gene probe Aids treatment selection in breast cancer (HER2 amplification? - herceptin appropriate?)
5.3 Radioisotopes
5.3.1 Considerations Half life of isotope Trace distribution throughout the body e.g. using radioisotopic iodine in the diagnosis (and treatment) of hyperthyroidism Autoradiography Uptake and incorporation of isotope into cells metabolic pathways - e.g. nucleotide synthesis / glucose metabolism Cellular site of action Steroids (gene expression) - intracellular peptide hormones - extracellular
5.4 Radioimmunoassay (RIA)
5.4.1 Sensitive and highly specific (due to use of Ab's)
5.4.2 In vitro assay used to determine levels of hormone in sample (e.g. blood)
5.4.3 Method Known amount of antigen is made radioactive and mixed with known amount of Ab = hot antigen Next a sample (from the patient) of unknown antigen concentration is added = cold antigen The unbound cold antigen competes for the Ab and displaces bound hot antigen the amount of displacement correlates to the amount of cold antigen in the sample The solution containing the unbound antigens (both hot and cold) is then separated and its radioactivity measured (geiger counter) The level of radiation is proportionate to the amount of displaced radioactive antigen This can be compared to a standard curve of solution radiation against known amounts of cold antigen (done previously)
5.4.4 Can be affected by episodic/diurnal release
5.5 Tissue extracts
5.5.1 Crude extraxcts (preparation from tissue - not purified) Replace excised tissue Can be purified - i.e. hormonal supplements Can be used as treatment (i.e. insulin) Tissue extract can be analysed using subcellular fractionisation Using differential centrifugation Subcellular fractions separated based on density/size Then tested for receptor/hormone of interest (determines cellular location)
5.6 Detecting protein or mRNA of interest
5.6.1 Nothern blot Detects mRNA Simliar to western blot Radiolabelled/fluorescent RNA primer used intead of Ab
5.6.2 Western blot Detects proteins Extract all proteins (lysis) give negative charge or denature Run on SDS page Produce Ab Transfer SDS page to nitrocellulose Incubate with primary antibody Wash off excess Ab and incubate with secondary Ab (+ fluorescent tag) Detect fluorescence
5.7 PCR
5.7.1 Amplification of short mRNA sequence (testing transcription level) Convert mRNA from cell into cDNA using a reverse polymerase
5.8 Bioassays
5.8.1 Use of a biological response to detect presence of a hormone in serum/tissue extract
5.8.2 The level of response to a hormone can be measured and a standard produced for known concetrations of hormone Patient serum containing an unknown concentration of the hormone can then be tested and compared against the standard curve
5.8.3 Structure-activity assays Looking at the structure of a compound and comparing this to its funtion Antagonist or agonist? Use known amounts of a hormone to produce a dose response curve (standard curve) - forms a sigmoid curve due to ligand receptor kinetics Repeat with test compound Is max response reached quicker/at all?
5.8.4 Site-directed mutagensis Induce genetic mutation To detect functional domains Can be tested for using a reporter assay Constructed gene with a hormone response element, basic promotor region and reporter gene (e.g. beta-galactosidase or luciferase) If mutation affects a functional domain of the hormone/hormone receptor (e.g. steroid hormone receptor) then there is no trasncription of the reporter Visual result: beta-galactosidase expressing cells grow blue on X-gal medium; and luciferase produces light

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