Aula Te

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Aula Teórica Nº 2

• Organelos Celulares

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Microscopia

• Ampliação, Contraste, Resolução
• Microscopia óptica (200 nm)
• Campo claro
• Fluorescente
• Avançada (M.Confocal, contraste fase, etc)
• Microscopia electrónica (1 nm)
• Transmissão (TEM)
• Scanning (SEM)

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O Microscópio óptico
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O microscópio de campo claro

• Problem Most cells are colorless transparent
• To visualize structures ? stain with dyes
• Must preserve (fix), embed, section
• New problem ? these actions
• Alter cell structure/molecules
• Only give snapshot of dead cells

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Specimen preparation for brightfield microscopy
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Fluorescent microscopy

• Permits localization of specific cellular
  molecules
• Fluorescent dyes glow against dark background
• Dye may be indirectly or directly associated with
  
• the cellular molecule
• Multiple fluorescent dyes may be used
  simultaneously
• Cells may be fixed or living

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O Microscópio de Fluorescência
Figure 5-6
Figure 5-5
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Microscopia óptica de objectos 3D

• Confocal Scanning or Deconvolution Microscopy
• Generates 3D images of living cells
• Removes out-of-focus images ? optical sectioning
• Can look inside thick specimens (eggs, embryos,
  tissues)

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Advanced light microscopy

• Permits observation of transparent living cells
• Light phase shifts induced by specimen are used
  to
• generate contrast
• Phase contrast (refracted and unrefracted light)
• Differential interference contrast (two light
  beams)

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Transmission electron microscopy (TEM)

• Operates in vacuum
• Specimen usually fixed, embedded, sectioned, and
  stained with an electron-dense material
• Special techniques
• Metal shadowing visualize surface structures,
  cell components
• Cryoelectron visualize unfixed, unstained
  samples
• Freeze fracture, freeze etch visualize membrane
  interior
• Freeze etch visualize cell interior

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The transmission electron microscope
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Imunomarcação em TEM
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Scanning electron microscopy

• Can visualize surfaces of tissues, cells,
  isolated cell parts
• Specimen is fixed and coated with thin layer of
  heavy metal
• Images secondary electrons, resolution 10 nm

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SEM
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Criofractura
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Purification of specific cells by flow cytometry
Requires fluorescent tag for desired cell
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Purification of cell parts

• Understanding the roles of each each cell
  component depends on methods to break open (lyse)
  cells and separate cell components for analysis
• Cell lysis is accomplished by various techniques
  
• blender, sonication, tissue homogenizer,
  hypotonic solution
• Separation of cell components generally involves
  centrifugation

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Cell fractionation by differential centrifugation
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Organelle separation by equilibrium
density-gradient centrifugation
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Biomembranas

• Fundamental structure and function of all cell
  membranes depends on lipids (phospholipids,
  steroid derivatives)
• Specific function of each membrane depends on the
  membrane proteins that are present in that
  specific membrane
• Membrane lipids and proteins may be glycosylated

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Biomembranas

• Bicamada de fosfolípidos
• Fluidez
• Colesterol
• Aumenta a resistência
• Diminui a fluidez
• Flip-flop
• Assimetria
• Glicolípidos
• Proteínas
• Integrais
• periféricas
• Ancoradas covalentemente em lípidos

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Phospholipid structure
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Due to the amphipathic nature of phospholipids,
these molecules spontaneously assemble to form
closed bilayers
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Each closed compartment has two faces
The two faces of a membrane are asymmetric in
terms of lipid and protein composition
Figure 5-31
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Lipids and integral proteins demonstrate lateral
mobility in biomembranes
The Fluid Mosaic Model

• Mobility (diffusion) of a given membrane
• components depends on
• the size of the molecule
• its interactions with other molecules
• temperature
• lipid composition (tails, cholesterol)
• Mobility can be measured by FRAP

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Fluorescence recovery after photobleaching (FRAP)
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Concentração de Proteínas em domínios de membrana
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The freeze fracture, freeze etch method
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Functions of the plasma membrane

• Regulate transport of nutrients into the cell
• Regulate transport of waste out of the cell
• Maintain proper chemical conditions in the cell
• Provide a site for chemical reactions not likely
  to occur in an aqueous environment
• Detect signals in the extracellular environment
• Interact with other cells or the extracellular
  matrix
• (in multicellular organisms)

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Complexidade celular
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Animal cell structure
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Plant cell structure
Figure 5-43
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Organelles of the eukaryotic cell

• Lysosomes
• Peroxisomes
• Mitochondria
• Chloroplasts
• the Endoplasmic Reticulum (ER)
• the Golgi complex
• the Nucleus
• the Cytosol

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Lysosomes

• Responsible for degrading
• certain cell components
• material internalized from the extracellular
  environment
• Key Features
• single membrane
• pH of lumen ? 5
• acid hydrolases carry out degradation reactions

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Peroxisomes

• Responsible for degrading
• fatty acids
• toxic compounds
• Key Features
• single membrane
• contain oxidases and catalase

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Peroxisoma
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Mitochondria

• Site of ATP production via aerobic metabolism
• Key Features
• outer membrane
• intermembrane space
• inner membrane
• matrix

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Mitocondria
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Cloroplasto

• Site of photosynthesis in plants and green algae
• Key Features
• outer membrane
• intermembrane space
• inner membrane
• stroma
• thylakoid membrane
• thylakoid lumen

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Cloroplasto
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O Retículo endoplasmático (ER)

• Responsible for
• most lipid synthesis
• most membrane protein synthesis
• Ca ion storage
• detoxification
• Key Features
• network of interconnected closed membrane tubules
  and vesicles
• composed of smooth and rough regions

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Retículo Endoplasmático
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Ribossomas
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O complexo de Golgi

• Modifies and sorts most ER products
• Key Features
• series of flattened compartments vesicles
• composed of 3 regions
• cis (entry), medial, trans (exit)
• each region contains different set of modifying
  enzymes

Figure 5-49
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O complexo de Golgi
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Secretory proteins are synthesized in the ER and
pass through the Golgi on the way to the
extracellular environment
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O núcleo

• Separa
• DNA do citosol
• Transcrição da tradução
• Características essenciais
• Dupla membrana
• Lâmina nuclear
• Poros nucleares
• Nucléolo
• cromatina

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Núcleo

• Territórios cromossómicos bem definidos
• Cromatina altamente organizada
• Nucléolo com domínios definidos

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Poro Nuclear

• Estrutura supramolecular
• 2 aneis coaxiais
• Ligados em estrutura octogonal
• Grânulo central
• Filamentos ligam ao citoplasma
• 1 anel intranuclear
• Menor
• Ligado aos 2 maiores
• Forma um cesto

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The cytosol

• The portion of the cell enclosed by the plasma
  membrane but not part of any organelle
• Key Features
• the cytoskeleton
• polyribosomes
• metabolic enzymes

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citoesqueleto
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Microtubulos
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Parede Celular
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Vírus