Unit 1. INTRODUCTION TO CELL BIOLOGY.pdf

VIROIDS Example: cadang-cadang Not virus • Viroids, are naked, circular, singlestranded RNA molecules that do not encode protein • Viroids only infect plants; some cause economically important diseases of crop plants, while others appear to be benign. Viroids: • • Smallest known pathogen (1/10 o...

VIROIDS Example: cadang-cadang Not virus • Viroids, are naked, circular, singlestranded RNA molecules that do not encode protein • Viroids only infect plants; some cause economically important diseases of crop plants, while others appear to be benign. Viroids: • • Smallest known pathogen (1/10 of virus size). • The 30 known viroids have been classified in two families. RNA dont catalitik propriety ALL enz. = prot • • • • small genome size (to avoid error catastrophe caused by error-prone replication), G-C STRONGER A-T high G+C content (for greater thermodynamic stability), circular genomes (to avoid the need for mechanisms to prevent loss of information at the ends of linear genomes), no protein content, presence of a ribozyme. Logarithmic scale Technology used Atom distribution Limit of human eye: 100 μm X ray difraction 1m 1 mm Eye and simple lenses 10 µm 1 µm Optical microscope (1-200 μm) 100 nm 10 nm Electron microscope Tissues Cell components Cells Viruses Bacteria 1 nm Limit Resolution of 0,2 µm (200 nm) Optical microscope REQUIREMENTS: First, a bright light must be focused onto the specimen by lenses in the condenser. Second, the specimen must be carefully prepared to allow light to pass through it. Third, an appropriate set of lenses (objective and eyepiece) must be arranged to focus an image of the specimen in the eye. TYPES: 1) Bright field 2) Phase-contrast MAGNIFICATION cells up to 1000 times. 2) Fluorescence Bright-field conventional optical microscope TYPE OF SAMPLE: A) LIVING ANIMAL CELL (fibroblast) in culture viewed A stained section of onion in mitosis B) FIXED AND STAINED sections of tissues. Most tissues are neither small enough nor transparent enough to examine directly in the microscope. Tissues therefore must be chemically fixed and cut into very thin slices, or sections, that can be mounted on a glass microscope slide and stained to reveal different components of the cells. Optical microscope of fluorescence GREEN: 488-546nm Emission filter Excitting filter sample Exciting at a specific wavelenght and emission at a different wavelenght REQUIREMENTS: The illuminating light is passed through two sets of filters. The first (exciting) filters the light before it reaches the specimen, passing only those wavelengths that excite the particular fluorescent dye. The second (emission) blocks out this light and passes only those wavelengths emitted when the dye fluorescens. OUTPUT: Dyed objects show up in bright color on a dark background. Optical microscope of fluorescence TYPE OF SAMPLE: 1) FIXED AND STAINED sections of tissues of cells using Fluorescent dyes Fluorescent dyes : 1) absorb light at one wavelength and emit it at another, longer wavelength. 2) bind specifically to particular molecules in cells and can reveal their location. An example is the stain for DNA shown here (blue ). Other dyes can be coupled to antibody molecules, which then serve as highly specific and versatile staining reagents that bind selectively to particular large molecules, FLUORESCENCE MICROSCOPY allowing us to see their distribution in the cell. shows the location of DNA and multiple This approach is named proteins within the same cell. IMMUNOFLUORESCENCE Fluorescent tagging and staining techniques use different fluorescent 2) LIVING CELLS molecules to reveal different structures In electron microscopy, images are formed from electrons that pass through a specimen or are scattered from a metal-coated specimen LIMITATIONS: Not living specimens could be observed Transmission electron microscope ULTRASTRUCTURE Limit Resolution of 0,1 nm Scanning electron microscope TRIDIMENSIONAL Limit Resolution of 10 nm TRANSMISSION ELECTRON MICROSCOPE GO THROUGH (TEM) ↓ Is in principle similar to a light microscope, but it uses a beam of electrons instead of a beam of light, and magnetic coils to focus the beam instead of glass lenses. The sample, which is placed in a vacuum, must be very thin. Sample must be fixed, and the contrast is usually introduced by staining the specimen with electron-dense heavy metals that locally absorb or scatter electrons, removing them from the beam as it passes through the specimen. MAGNIFICATION: a million-fold and can resolve details as small as about 1 nm. LOOK ORGANITE INSIDE CELL(MITOCHONDRI.. SCANNING ELECTRON MICROSCOPE (SEM) The specimen must be coated with a very thin film of a heavy metal. 1) The sample is bombarded by a beam of electrons. 2) The quantity of electrons scattered or emitted by each successive point on the surface of the specimen is measured by the detector. 3) And an image is built up on a video screen. The microscope creates striking images of three-dimensional objects with great depth of focus and can resolve details between 3 nm and 20 nm. LOOK SHAPE , SURFACE, CONTOUR: EXT PART IN 3D DIFFERENCES BETWEEN SEM and TEM • SEM is based on scattered electrons while TEM is based on transmitted electrons. • SEM focuses on the sample’s surface and its composition whereas TEM provides the details about ultrastructure (internal composition). Therefore TEM can show many characteristics of the sample, such as morphology, crystallization, stress or even magnetic domains. On the other hand, SEM shows only the morphology of samples. • The sample in TEM has to be cut thinner whereas there is no such need with SEM sample. • TEM has much higher resolution than SEM. • SEM also provides a 3-dimensional image while TEM provides a 2dimensional picture. CELL CULTURES: It is the growth and proliferation of cellular models under controlled conditions. HISTORY OF CELL CULTURE 1907- Frog embryo nerve fiber outgrowth by Harrison. 1943- Development of mouse lymphocyte cell line by Earle, et al. • Cell culture is performed on 1952- Development of HeLa cell line by Gey, et al. 1955- Development of defined cell culture media by Eagle. artificial media prepared by mixing purified components or complex 1961- Demonstration of the finite lifespan of normal human organic solutions, cells by Hayflick and Moorhead. 1964- Discovery of pluripotency of embryonic stem cells by Kleinsmith and Pierce. 1976- Totipotency of embryonic stem cells described by Illmensee and Mintz. 2007- Use of viral vectors to reprogram adult cells to embryonic state (induced pluripotent stem cells) by Yu et al. 2008 and beyond- Era of induced pluripotent stem cells – promises and challenges. • Using equipment to maintain the appropriate physico-chemical conditions and on containers that isolate them from A culture medium consists of four elements 1. The nature of the substrate or growing phase. 1 .- Adherent: The majority of cells maintained in culture derived from tissue disintegration or tumors formed by adherent cells and maintain this characteristic: they need to adhere to the substrate to be maintained. (derived from organs: muscle, liver, nerve cells ...) 2 .- Suspension: The suspension cultures often coincide with those of cells that "in vivo" are circulating, overall blood cells, immune cells. Lab equipment will depend on this characteristic 2. The physico-chemical and physiological conditions of the medium 3. The nature and composition of the gas phase 4. Incubation conditions, specially humidity and temperature Cell Culture • Gaseous phase. The most significant components of the gas phase are oxygen and carbon dioxide. • Physical properties. Environmental characteristics are: pH, osmolarity, temperature, viscosity and surface tension. • Physiological conditions. They refer to the composition of the medium. The main difficulty in the establishment of cell lines is to obtain adequate nutritious media to be able to replace the "natural" environment such as embryonic extracts, protein hydrolysates or sera. Some of the main media used are: Basal Medium Eagle (BME), Eagle's Minimum Essential Medium (MEM), RPMI 1640 and MEM modified by Dulbeco Medium (DMEM). BIOCHEMICAL COMPOSITION OF CULTURE MEDIUM EXAMPLE: DMEM - Bicarbonate buffer, -à Maintenance of pH Salts, pyruvate, glucose, aas, vitamins…-à Nutrients (Primary cultures have more requirements) - Fetal Bovine Serum (5-15%)à Growth factors-à Proliferation - Antibiotics/Antimicotics (10%) à sterility - Phenol red, to observe medium acidification--à Control of the general status of the culture