CELLS AND TISSUES IN HEALTH AND DISEASE Flashcards


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1

Where would you find simple squamous epithelium?

BLOOD VESSELS

HEART

ALVEOLI

(diffusion and filtration)

2

Where would you find simple cuboidal?

KIDNEY TUBULES

GLANDS

TERMINAL BRONCHIOLS

(some cilia, diffusion/secretion/absorption, particle movement)

3

Where would you find simple columnar?

GLANDS

DUCTS

BRONCHIOLES

STOMACH

INTESTINES

(move substances, secretion/absorption/protection)

4

Where would you find stratified squamous?

SKIN - KERATINISED AND NON

MOUTH

LARYNX

ESOPHAGUS

ANUS (lol)

(cuboidal in basal ---> flat at surface, protection, barrier, reduce H2O loss)

5

Where would you find stratified cuboidal?

SWEAT DUCTS

SALIVARY DUCTS

OVARIAN FOLLICULAR CELLS

(secretion/absorption/protection)

6

Where would you find stratified columnar?

MAMMARY DUCT

LARYNX

MALE URETHRA

(protection/secretion)

7

Where would you find pseudostratified columnar?

NASAL CAVITY

PHARYNX

TRACHEA

BRONCHI

(appear stratified due to nuclei being at different levels, ciliated, goblet cells, mucus mmm)

8

Where would you find transitional epithelium?

BLADDER

URETERS

SUPERIOR URETHRA

KIDNEY PELVIS

(stretch and recoil, non-stretched = cuboidal/columnar, stretched = flat/squamous)

9

Here is a nice helpful diagram

card image
10

Function:

OSTEO

CHONDRO

BLASTS

CYTES

CLASTS

OSTEO - Bone

CHONDRO - Cartilage

BLASTS - Create

CYTES - Maintain

CLASTS - Break

11

Major components of ground substance?

Proteins and polysaccharides - increase viscosity.

12

Function of cell adhesion proteins and proteoglycans in ECM (interstitial fluid)?

cell adhesion proteins – connective tissue glue.

Proteoglycans - macromolecule with protein core, GAGS attach.

Increase viscosity and compressability of joints, cushion cells.

ECM also contains insoluble collagen fibers, which provide strength and resilience.

(ECM can also be used to scaffold patient’s own cells eliminates adverse immune responses in artificial tissue and organ regeneration)

13

4 functions of connective tissue?

Separation: sheaths

Cushioning/insulation: adipose tissue

Storage: adipose tissue

Transportation: blood

14

What 6 cells are housed in connective tissue?

Adipose cells (adipocyte)

Mast cells (mastocyte) IMMUNE

Macrophages (phagocyte)

White blood cells (leukocytes)

Lymphocytes

Undifferentiated mesenchymal stem cells

15

Describe collagen fibres

3x chains of amino acids wind around each other forming rope-like collagen molecule (requires vitamin C).

In the ECM, triple helix modified into tropocollagen molecules which assemble into fibrils which are bundled into thick collagen fibres.

Type I most abundant, (tendons, ligaments etc),

Type II found in cartilage.

Reticular fibres are mainly Type III.

16

Describe elastic fibres

Form branching networks in ECM.

Found in areas where greater elasticity needed e.g. lungs, blood vessel walls.

17

Describe reticular fibres

Short, fine fibres (0.5 - 2 mm diameters)

Type III collagen fibres.

Branch extensively forming networks, fill spaces between tissues and organs (e.g. basement membrane of epithelial tissue and around capillaries).

They allow more “give” than collagen fibres.

Abundant in Liver, spleen and lymph nodes.

18

What are the 2 types of connective tissue proper?

LOOSE - fewer fibres more ground substance

DENSE - more fibres, less ground substance

19

Types of loose connective tissue proper?

card image

AREOLAR - hold organs in place and attaches epithelial tissue to other underlying tissues. SKIN

ADIPOSE - packed with many fat cells. MAMMARY GLAND

RETICULAR - network of reticular fibers. SPLEEN, LYMPH NODES.

20

Types of dense connective tissue?

card image

Regular collagenous - LIGAMENTS, TENDONS

Regular elastic - VOCAL CORDS

Irregular collagenous - SKIN

Irregular elastic - AORTA

21

What is scruvy?

Vitamin C deficiency

Results in unstable collagen molecules and therefore defective fibers.

22

What is Marfan's syndrome?

Defective elastic fibers

Destruction of elastic tissue due to increased elastase activity

Caused by air pollution and tobacco

23

What is systemic lupus erythematous?

Non-organ specific autoimmune disease

Inflammation of tissue, usually skin and joints

symptoms vary, 1:5000

24

What is fibrosis?

Scar tissue

Excess fibrous tissue in organ or tissue

Confluent fibrosis obliterates architecture of underlying organ or tissue

(Pulmonary fibrosis = scarring of the lungs)

25

Name some supporting connective tissue and fluid connective tissue

SUPPORTING - cartilage and bone (~vascular)

FLUID - blood and haemopoietic (marrow)

26

Components of cartilage

Chondroblasts and ECM.

Embedded chondroblasts = chondrocytes.

Chondrocytes sit in matrix spaces called lacunae.

27

Hyaline cartilage

Small, evenly dispersed collagen fibers

Matrix appears transparent

Covered externally by perichondrium (except: articular ends of bone and directly under skin).

FUNCTION -

Allows growth of long bones.

Provides rigidity with some flexibility.

Forms smooth flexible articulating surfaces.

TRACHEA/BRONCHI/RIBS/NOSE, GROWING LONG BONES, ARTICULATING SURFACES OF BONES, EMBRYONIC SKELETON.

28

Fibrocartilage

More numerous collagen fibres arranged in thick bundles.

Lacks a perichondrium.

FUNCTION -

Some flexibility.

Withstands heavy pressure & tensile forces.

INTER VERTEBRAL DISCS, PUBIC SYMPHYSIS, ARTICULAR DISCS OF TMJ, KNEE MENISCI

29

Elastic cartilage

Similar to hyaline.

Matrix also contains elastic fibers.

Has a perichondrium.

FUNCTION -

Provides rigidity with lots of flexibility.

Elastic fibers return to their original shape.

Extremely resilient.

Provides support and helps define and maintain the shape.

EXTERNAL EAR, AUDITORY TUBULES, EPIGLOTTIS

30

Components of bone?

Cells & mineralized matrix.

Organic portion (35%) of collagen protein fibers and proteoglycans.

Inorganic portion (65%) of calcium and phosphate salts.

31

Classification of bone - organisation of matrix collagen fibers.

card image

Woven bone Immature

  • Collagen fibers random.
  • Formed in foetal development/fracture repair.
  • Bone remodeled.

Lamellar bone Mature

  • Organised lamellae.
  • Parallel/ perpendicular fibers.
  • Osteocytes in lacunae between lamellae
32

Classification of bone - amount of bone matrix relative to amount of space within bone.

card image

SPONGY – aka TRABECULAR or CANCELLOUS

  • Porous, interconnecting trabeculae.
  • Trabeculae - several lamellae.
  • Osteocytes - between lamellae.
  • Osteocytes interconnected via canaliculi.
  • Trabecular surface covered with single layer of cells.

COMPACT - aka CORTICAL

  • Dense and vascular
  • Blood vessels & nerves parallel to long axis in central canals.
  • Concentric lamellae surround central canal.
  • Osteocytes in lacunae between concentric lamellae.
  • Basic unit is osteon (Haversian system): central canal, canal vessels, concentric lamellae and osteocytes.

Outer surface – circumferential lamellae.

Between osteons - interstitial lamellae.

  • Blood vessels from periosteum or medullary cavity enter through perforating canals - perpendicular to long axis (Volkmann canals).
33

Location of spongy and compact bone?

SPONGY:

Function – acts as scaffolding to provide strength and support without weight.

Location – Interior of skull bones, vertebrae, sternum & pelvis plus ends of the long bones.

COMPACT:

Function – provides great strength and support, forms a solid outer shell to prevent punctures &/ breaks.

Location – Outer portions of all bones and shafts of long bones.

34

LONG, SHORT, FLAT and IRREGULAR bone types

LONG - shaft with heads at both end, mainly compact.

SHORT - generally cube shaped, mainly spongy.

FLAT - thin, usually curved, thin layer of compact surrounding spongy.

IRREGULAR - don't fit other category.

35

Bone turnover and reabsorption?

Osteoblasts (secrete new bone) & osteoclasts (break bone down).

Osteoclasts move to resorb the surface of the bone, followed by deposition of bone by osteoblasts.

The space between osteons is occupied by interstitial lamellae, which are the remnants of osteons that were partially resorbed during the process of bone remodelling.

Bone remodelling (or bone metabolism) = lifelong process mature bone is removed via bone resorption and new bone is formed via ossification.

Control reshaping / replacement of bone following injuries e.g. fractures but also micro-damage from normal activity and functional demands of mechanical loading.

In the first year of life, almost 100% of the skeleton is replaced.

In adults, remodelling proceeds ~10% per year.

An imbalance in bone resorption vs bone formation = many metabolic bone diseases. e.g. Rickets.)

36

Too little or too much growth hormone?

TOO LITTLE - mutations of specific genes, damage to the pituitary gland, nutrition, stress.

Dwarfism = adult height of <4ft 10 inches.

TOO MUCH - pituitary tumour, usually diagnosed in adulthood, thickening of mandible and digits = Acromealgy.

Gigantism = height 7ft-9ft or top 1% of population.

37

What is bone marrow? Where are the 2 types found?

Soft spongy material in central cavity of larger bones and small spaces of spongy bone.

Produces all 3 types of blood cells:
- Red.
- White.
- Platelets.

Plus Lymphocytes – support the immune system.
Red – skull, vertebrae, ribs, sternum, heads of long bones.
Yellow – no longer produces blood.

As the child ages, hematopoietic (red) bone marrow is replaced by fatty, adipose marrow or yellow bone marrow.

By adulthood, hematopoietic bone marrow is largely confined to the skull, vertebrae, ribs, clavicles and sternum, pelvis, and proximal femora. Almost all of the bone marrow is yellow bone marrow by the time a person reaches old age.

38

What is leukemia?

high numbers of immature / abnormal WBC’s

Symptoms – anaemia, reduced clotting, excess bruising, tiredness, increased infections.

39

What is lymphoma?

abnormal lymphocytes.

40

Fibroconnective tissue arrangement around bundles of axonal projections?

Entire nerve bundles are surrounded by epineurium.

Branching and dividing bundles into fasicles = perineurium.

Each individual axon is surrounded by endoneurium.

41

What are the three FUNCTIONAL layers of the CEREBRAL cortex?

(SURFACE)

Supragranular

Internal granular

Infragranular

42

What are the layers of the CEREBELLAR cortex?

(SURFACE)

Molecular

Granular

Pyramidal

43

What is the function of intercalated discs?

Support synchronized contraction of tissue.

CARDIAC MUSCLE

44

How do skeletal muscle fibres form during development?

Fusion of undifferentiated myoblasts.

45

Name for the fibrous connective tissue surrounding the cartilage?

Perichondrium.

46

Name given to clusters of chondrocytes formed from a single chondroblast?

Isogenous group.

47

What is myeloma?

abnormal plasma cells.

48

How would you treat disease of the bone marrow? (lymphoma, myeloma, leukemia).

Radiotherapy.
Chemotherapy.
Bone marrow transplant.

49

5 functions of skin?

Protection

Thermoregulation

Water regulation

Sensation

Storage/synthesis

50

List the layers of thick skin (hands and feet), which one is not present in thin skin?

card image

Stratum corneum

Stratum lucidum (NOT PRESENT IN THIN SKIN)

Stratum granulosum

Stratum spinosum

Stratum basale

DERMIS

51

What do these skin cells do?

Keratinocytes

Melanocytes

Merkel cells

Langerhans cells

Keratinocytes - produce keratin (The protein that protects epithelial cells from damage or stress) CORNEUM

Melanocytes - produce melanin (The pigment that gives human skin, hair, and eyes their color) BASALE

Merkel cells - nerves, essential for light touch sensation BASALE

Langerhans cells - Dendritic cells (Antigen-presenting immune cells, protects against infections) ALL LAYERS BUT CORNEUM MOST PROMINENT IN SPINOSUM.

52

Describe the dermis

Strong, flexible connective tissue rich in collagen and elastin.

Contains cells, blood vessels, nerve endings, hair follicles, muscle and lymphatics.

53

What 3 factors determine skin colour?

Blood circulation (haemoglobin)

Thickness of stratum corneum

Pigments (melanin and carotene) - genetics and exposure to sunlight affect melanin.

54

Which protein is missing in Dychenne's muscular dystrophy and reduced or abnormal in Becker's muscular dystrophy?

Dystrophen - Stabilizes and protects muscle fibres in skeletal and cardiac muscle.

55

Which non-melanoma skin cancer starts with small nodules with pearly borders often on face and scalp and may become ulcerous?

Basal cell carcinoma.

56

Albinism: what determines light of darkly pigmented individuals?

MELANOCYTES' LEVEL OF ACTIVITY.

People with Oculocutaneous Albinism typically have a very low level of melanin production.

1 in 20,000 people worldwide are born with oculocutaneous albinism.

57

Name 4 accessory structures of the skin

Hair and hair follicles

Nails

Sebaceous glands

Sweat glands

Derived for epidermis but extend into dermis.

58

What are sebaceous glands?

Produce sebum by holocrine secretion.

Lubricates the skin keeping it soft/flexible, prevents drying out and makes it water tight.

Located in dermis, THIN SKIN.

59

2 types of sweat glands?

Eccrine - areas abundant in hair follicles, such as your armpits and groin, and they empty into the hair follicle just before it opens onto the skin surface.

Apocrine - secrete fluid onto the surface of your skin, where it cools your body as it evaporates.

60

Functional classification of glands?

TYPE OF SECRETION

Apocrine glands - a portion of the plasma membrane (apical region) containing the substance for excretion buds off the cell.

Holocrine glands - the entire cell disintegrates to excrete its substance.

Merocrine glands or (eccrine glands) - cells excrete their substances by exocytosis.

61

What 3 effects does aging have on skin?

Increased fragility

Loss of elasticity

Transparent quality

62

What effects does the sun have on skin?

Loss of elasticity

Roughness

Irregular pigmentation

Deep wrinkles

Due to changes in organisation and functionality of collagen and elastin in the dermis.

63

What does sunburn do to skin?

DNA damage in skin cells

-Triggers repair response

-Increases melanin production

-Inflammatory response

-Cell death

64

What are the functions of acini sebaceous glands?

Secrete oily wax/sebum.

Waterproof and lubricates skin.

65

3 types of skin cancer?

Basal cell carcinoma

Squamous cell carcinoma

Malignant melanoma

66

Treatment for non-melanoma skin cancer?

Non-melanoma

Surgical excision (+ skin graft)

Cryotherapy – Liquid Nitrogen.

Mohs micrographic surgery – high risk of the cancer spreading or returning and / or in an area where it would be important to remove as little skin as possible e.g. nose or eyes.

Topical Chemotherapy / immunotherapy / photodynamic therapy – via creams.

67

Treatment for malignant melanoma skin cancer?

Surgical excision (skin + lymph nodes).

Radiotherapy.

Chemotherapy.

68

What is eczema and how is it treated?

Atopic Dermatitis.

Cause = unknown (Genetic + environmental).

Treatment = steroid creams + immunosuppressants.

69

What is acne and how is it treated?

Comedones (blackheads and whiteheads) and pus-filled spots (pustules).

Sebaceous glands secrete an excess of oil in response to blood testosterone levels.

Dead keratinocytes are not shed properly and clog up the follicles.

Treatments:

Topical i.e. those that are applied directly to the skin.

Oral antibiotics or contraceptive pills.

Isotretinoin – instigates cell death in sebaceous gland cells.

70

What is a gland?

Epithelial cells that produce secretions (glandular epithelium)

A gland may be a single cell or a group of cells which secrete their products into a duct or surface or into the blood.

e.g. hormones, sweat, saliva, digestive enzymes

71

Endocrine gland

Produce & release secretions into surrounding interstitial fluid, which then enter the bloodstream.

Secretions – Hormones (e.g. Insulin / Somatostatin / Thyroid Stimulating Hormone)

Endocrine Cells:

  • Part of epithelial surface – e.g. lining of digestive tract.
  • Separate Organs – e.g. Thyroid / Pituitary Gland.

Widespread & Varied, regulation of bodily functions & maintenance of homeostasis.

72

Patterns of hormone secretion

card image

Chronic hormone regulation - Maintenance of relatively constant concentration of hormone. Thyroid hormone.

Acute hormone regulation - Epinephrine in response to stress.

Episodic (Cyclic) hormone regulation - Female reproductive hormones.

73

Regulation of hormonal secretion - HUMORAL, NEURAL, HORMONAL

HUMORAL – blood-borne molecules (e.g. high glucose levels stimulate insulin secretion)

NEURAL – neurons stimulate production (e.g. sympathetic nervous system)

HORMONAL – one hormone stimulates secretion of another (e.g. pituitary gland hormones stimulate testis to make testosterone)

74

Diseases of endocrine glands

GRAVES DISEASE (hyperthyroidism), CUSHING'S SYNDROME (hypercortisolism), PITUITARY HORMONES.

  • dysregulated hormone release (a productive pituitary adenoma).
  • inappropriate response to signalling (hypothyroidism).
  • lack of a gland.
  • structural enlargement in a critical site such as the thyroid.
75

Exocrine glands

Secretions released through ducts that open onto epithelial surface.

Classified by structure or method of secretion.

76

Classifying exocrine glands

STRUCTURE - Unicellular

- Multi-cellular, secretory sheet or pocket of glands set back from epithelial surface.

METHOD OF SECRETION - Merocrine (exocytosis), apocrine (hair follicles), holocrine (rupture of the plasma membrane)

77

Types of exocrine secretion

Serous - watery, contains enzymes.

Mucous - secrete mucins which hydrate

78

The pancreas secretions

ENDOCRINE - Islets of Langerhans - Alpha = Glucagon, Beta = Insulin.

EXOCRINE - Acinar cells - juicy juicy.

79

Pancreatitis

Pancreatic enzymes activated.

80% caused by alcoholism or gallstones.

Abdominal pain.

80

Pancreatic cancer

INVOLVES BOTH EXO & ENDOCRINE TISSUE.

PANCREATIC ADENOCARCINOMA – AFFECTS EXOCRINE PART = MOST COMMON.

DIFFICULT TO TREAT.

RISK FACTORS =

  • SMOKING
  • OBESITY
  • DIABETES
  • RARE GENETIC CONDITIONS

I DON'T KNOW WHY THIS ONES ALL CAPITALS

81

Diabetes mellitus (pancreas)

TYPE I

  • Juvenile diabetes or insulin-dependent diabetes
  • An autoimmune condition
  • Body attacks and destroys insulin-producing cells (β cells).
  • No insulin is produced
  • This causes glucose to quickly rise in the blood.

TYPE II

  • Interplay of genetic & environmental factors.
  • Body doesn’t make enough insulin / insulin made doesn’t work properly.
  • Elevated glucose levels in blood.
  • Up to 58% of cases can be delayed / prevented through a healthy lifestyle.
82

Characteristics of epithelium

Structurally & functionally distinct cell surfaces = polarity

free apical surface - not attached to other cells, often lines lumen of ducts & cavities.

lateral surface - attached to other epithelial cells.

basal surface - attached to basement membrane which attaches epithelia to underlying tissues.

83

Apical specializations

card image

Microvilli – cytoplasmic protrusions, ‘brush border’ Often found on epithelium lining internal passages. Increases surface area.

Stereocilia – similar to microvilli but longer. Non-motile unlike cilia. Limited distribution, epididymis/vas def & sensory hair cells inner ear

Cilia – motile hair-like protrusions. Several hundred per cell. Beat in coordination to move substances over them. Found in respiratory epithelium & fallopian tubes. Smoking reduces cilia movement = impaired movement of mucous = reduced protection against bacteria.

84

Basolateral specializations

Epithelial cells extremely cohesive & closely apposed

Specialisations at basolateral surface between cells or with underlying basement membrane = junctions

  • Maintain polarised state.
  • join cells together.
  • exchange information & metabolites.
85

Junctions types - occluding/tight, anchoring, communicating/gap

card image

Occluding/Tight junctions

  • Seals cells together, prevents leaking.
  • Zona occludens –apical part of lateral domain, almost fusing 2 cells.

Anchoring junctions

  • Mechanically attaches cells to neighbours.
  • Abundant in tissue subject to severe stress e.g. skin / cardiac muscle.
  • Zona adherens or Desmosomes – strong junctions joining cells at lateral domain.
  • Hemidesmosome – strong in basal domain.

Hemidesmosomes (HD) are very small stud- or rivet-like structures on the inner basal surface of keratinocytes in the epidermis of skin. They are similar in form to desmosomes when visualized by electron microscopy. While desmosomes link two cells together,hemidesmosomes attach one cell to the extracellular matrix.

Communicating/Gap junctions

  • passage of chemical/electrical signals between cells
86

What are the 4 types of tissue

Connective Tissues

  • packaging or supporting fabric

Nervous Tissue

  • nerve and glial cells

Muscle

  • generates force so produces movement

Epithelia

  • sheets of cells covering body surfaces
87

What are stem cells?

Capable of dividing and renewing themselves for long periods

Unspecialised

Can give rise to specialised cell types

Common to all multicellular organisms.

They help to maintain cell numbers and replace dead / injured cells.

88

What is asymmetrical division?

When a stem cell divides the daughter cell can either remain a stem cell or become a progenitor cell and commit to differentiation = ASYMMETRICAL OF FATE

89

What are the 6 stages of embryo development?

card image

ZYGOTE

2 CELL

4 CELL

MULTI CELL

MORULA

BLASTOCYST

90

What is the potency of: MORULA, BLASTOCYST, ADULT MESENCHYMAL, ADULT SKIN?

(POTENCY = potential to differentiate into different cell types)

MORULA - Totipotent - any tissue and placenta

BLASTOCYST - Pluripotent - any tissue

ADULT MESENCHYMAL - Multipotent - cells from 1 family (e.g. connective tissue)

ADULT SKIN - Unipotent - 1 cell type (e.g. keratinocytes)

91

What are adult stem cells?

Exist in many tissues – low numbers

~ 1 stem cell / 100,000 cells

Look like surrounding cells – hard to identify

Tissue maintenance & repair

92

Pros and cons of adult stem cells?

Pros

- High capacity of self-renewal into adulthood.

- Once committed to differentiate can undergo rapid divisions.

- One stem cell can produce lots of differentiated (specialised cells) of the same type / group.

Cons

- Unidirectional & irreversible differentiation.

- Cells divide rarely & slowly (self renewal).

- Isolating for culture is difficult and proliferating up to large populations is challenging.

93

Example of unipotent adult stem cells?

SKIN

Stratum Basale contains undifferentiated cells – epithelial stem cells.

These generate either more stem cells or Keratinocytes.

94

Example of multipotent adult stem cells?

INTESTINAL EPITHELIAL

OR

HEMATOPOIETIC CELLS - BLOOD COMPONENTS

95

Embryonic stem cells - PLURIPOTENT

Derived from embryos - ~5/6 days post fertilisation – blastocyst stage

Can differentiate into >200 cell types

First isolated in 1998.

Most are surplus from IVF clinics - full donor consent.

NOT derived from eggs fertilised in a woman’s body.

Proliferate in cell culture for 6 months without differentiating = embryonic stem cell line.

Some basic protocols for directed differentiation of ESC’s into specific cell types now established.

96

Pros and cons of embryonic stem cells?

Pros

- Easy to isolate and grow in culture.

- Can in theory create any cell type in the body.

Cons

- If allowed to clump together to form embryoid bodies they spontaneously differentiate.

- Current knowledge of differentiation not good enough to consistently produce pure populations of specific cells.

- Ethical considerations.

97

What is therapeutic cloning?

Cells from patient + egg donor

Nucleus removed from egg and replaced with nucleus from patient, egg is stimulated to divide.

Resulting embryo carries patients genetic material.

---> embryonic stem cells

98

What are induced pluripotent stem cells?

Adult stem cells genetically ‘reprogrammed’ back to an embryonic cell-like state.

Produced in 2006

Reprogrammed via viral introduction of 4 stem cell factors - important for maintaining the defining properties of ESC’s

  • Oct3/4
  • Sox2
  • c-Myc
  • klf4
99

Pros and cons of induced pluripotent stem cells?

Pros

  • Can generate ESC’s without the need for embryos.
  • Capable of producing cells from all 3 germ layers.
  • Used for drug development and modelling disease.

Cons

  • Not known if IPSC’s and ESC’s will differ clinically.
  • In animals the virus used to introduce stem cell factors has caused cancers.
100

Uses of embryonic stem cells?

  • Demand for transplants far outstrips donations.
  • Adult stem cells in particular are less likely to initiate immune rejection after transplant
    Embryonic – unknown reaction.
  • Drug testing – instead of testing on humans &/animals.
  • Genetic defects – e.g. create Cystic Fibrosis specific stem cells.
  • Veterinary medicine.
101

What must stem cells reproducibly be able to do?

  • Generate sufficient quantities of specific cells for making tissue.
  • Survive in the recipient after transplant.
  • Integrate into tissue after transplant.
  • Function appropriately for the duration of the recipients life.
  • Avoid harming the recipient in any way.
102

What was the first therapeutic use of stem cells?

Bone marrow transplant

Leukemia patients irradiated - destroys cancerous cells and healthy hemopoietic tissue.

--> Transfusion of healthy non cancerous hemopoietic stem cells.

Harvested from donor red bone marrow, can generate RBC, WBC and PLATELETS.

103

Parkinson's disease and potential stem cell use?

Cells in the midbrain which normally make Dopamine die

Dopamine = vital for movement control

Symptoms = tremors + uncontrolled movements

Could dead cells be replaced with fresh?

104

Studies of Parkinson's disease and stem cells

Researchers killed dopamine-producing neurons on one side of rats' brains.
Converted human embryonic stem cells into neurons that produced dopamine.
These were injected into the rats' brains, and provided evidence of damage reversal.

Similar method tried in a limited number of patients – year 2000
Brain tissue taken from multiple aborted foetuses.
Clinical trials abandoned after mixed results, but ~1/3 patients had foetal brain cells that functioned for >10 years.

105

What still needs to be controlled/discovered in using stem cells?

WE DO NOT….

  • Have any definitive markers for stem cells.
  • Understand what molecularly defines stem cell state.

NEED TO DISCOVER….

  • Factors that normally regulate stem cell proliferation and renewal
  • Does ‘transdifferentiation’ truly exist? E.g. can brain cells differentiate into blood cells?
  • Are adult SC’s just leftover ESC’s or do they arise in some other way?
  • Why do SC’s remain in an undifferentiated state when all those around them have differentiated? Stem-cell ‘niche’?
  • What are factors that stimulate SC’s to relocate to sites of injury and how can this be used in wound healing?
106

In what 6 situations is research on human embryos allowed?

To promote advances in the treatment of infertility

To increase knowledge about the causes of congenital (from birth) disease

To increase knowledge about the causes of miscarriages.

To develop methods for detecting the presence of gene or chromosome abnormalities.

To increase knowledge about the development of embryos.

To increase knowledge about and develop treatments for serious disease.

107

3 Laws for research on human embryos? (UK)

Licensed research can only take place on embryos that have developed from eggs fertilised outside the body (in vitro).

Licensed research can only take place on embryos up to 14 days. Stem cells usually isolated at 5-6 days.

Human reproductive cloning is illegal in the UK (Human Reproductive Cloning Act (2001)) nobody is allowed to use cell nuclear replacement, or any other technique, to create a child.

108

World laws on human embryo research?

The EU's 25 member states take different views reflecting diversity of ethical, philosophical and religious beliefs throughout Europe.

Belgium has a similar legal position to the UK.

Germany and Italy prohibit the procurement of human embryonic stem cells from human embryos.

Austria, Bulgaria, Cyprus, Ireland, Lithuania, Luxembourg, Malta, Poland, Romania and Slovakia have no specific legislation.

109

What is an advantage of bone remodeling? What does mechanical stress do to bones?

Remodelling gives each bone the ability to adapt to new stresses

Mechanical stress applied to bone increases osteoblast activity

Removal of mechanical stress decreases osteoblast activity

110

6 types of bone fracture?

card image

GREENSTICK - bone breaks incompletely, only one side of the shaft breaks, Children (more flexible bones).

COMMINUTED - bone fragments into 3 or more pieces. Older (more brittle bones).

EPIPHYSEAL - separates from the diaphysis (shaft) along the epiphyseal plate. (Where cartilage cells are dying and calcification is occurring).

SPIRAL - ragged break occurs when excessive twisting force is applied to bone. (Sports).

COMPRESSION - bone is crushed. (Porous bones - osteoporotic).

DEPRESSED - broken bone portion is pushed inwards. (Skull).

111

4 steps of fracture repair?

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1) Haematoma (blood clot) formation:

Bone breaks, blood vessels/tissue torn causing bleeding, cells deprived of nutrition die, tissue inflames.

2) Fibrocartilaginous callus formation (internal & external):

Soft granulation tissue forms, capillaries, phagocytes, fibroblasts & osteoblasts invade area.

3)Bony callus formation:

New woven bone converts callus into a hard callus.

4) Bone remodelling

Bony callus remodelled, woven bone replaced by lamellar bone.

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OSTEOARTHRITIS?

Joint inflammation as a result of cartilage degeneration.

Affects both men & women (8 million people in UK).

Commonly affects hands, feet, spine and large weight-bearing joints.

Symptoms and signs

  • Joint pain, tenderness, stiffness, burning in associate muscles & tendons.
  • Crepitus (popping sounds).
  • Heberden’s nodes and changes in x-ray appearance.
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Primary Osteoarthritis

  • not resulting from injury or disease (aging of joint, proteoglycan reduction, loss of water, collagen fibres degraded).
  • repetitive use of worn joint can cause joint pain, swelling & spurs.
  • in advance OA total loss of cartilage (pain & ltd joint mobility).
114

Secondary Osteoarthritis

  • caused by another disease or condition.
  • obesity, trauma or surgery, congenital abnormalities, hormone disturbances.
115

RHEUMATOID ARTHRITIS?

Autoimmune disease - chronic inflammation of joints

Small joints (hands, wrists and feet commonly affected)

Can affect multiple organs of the body and blood vessels

Progressive illness with potential to cause functional disability

Causes unknown (infectious agents suspected)

Symptoms and signs

  • Fatigue, fever, joint pain, tenderness, stiffness, inflammation of synovium.
  • Multiple joint usually inflamed in a symmetrical pattern.
  • Rheumatoid nodules.
  • Subluxation of metacarpophalangeal and proximal phalangeal joints.
  • Blood changes.
116

OSTEOPOROSIS?

Disease characterised by low bone mass and loss of bone tissue

May lead to weak and fragile bones

Increased risk of fractures

Bone mass decreases after the age of 35

Caused by imbalance between bone formation and resorption

Risks: genetic, environmental, some medications - Post menopausal – oestrogen levels decrease - In males a decrease of testosterone.

Symptoms and signs

  • No symptoms until bone fracture occurs.
  • Bone much thinner and lighter than normal bones.
  • Dual energy x-ray absorptiometry scan (DEXA).
  • Fractures of the spine/stress fractures/hip fractures.
117

Functions of the liver?

Protein, carbohydrate and fat metabolism

Plasma protein and enzyme synthesis

Production of bile

Detoxification

Storage of proteins, glycogen, vitamins and metals

Immune functions

118

What are liver hepatocytes?

Lie in plates and cords
Exchange material with blood at sinusoidal surface

Three types of surface:
Sinusoidal, intercellular & canalicular

119

5 cells of the liver?

  • Hepatocytes
  • Endothelial cells
  • Kupffer cells (macrophages)
  • Perisinusoidal cells (Ito, fat storing)
  • Liver-associated lymphocytes
120

What is cirrhosis?

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Diffuse process with Fibrosis & Nodule formation

End-stage liver disease

Result of chronic inflammation over many years

Persistence of the injury-causing agent

(Fibrous) scarring & hepatocyte regeneration

Irreversible

121

Pathogenesis of cirrhosis?

Hepatocyte injury ----> Progressive liver cell loss ---->

Chronic inflammation (Fibrosis) or Hepatocyte regeneration (Hyperplastic nodules)

----> Architectural abnormality ----> Cirrhosis or Ischaemia (which leads back to progressive liver cell loss)

122

Causes of cirrhosis: Acquired and inherited?

Freq. in West

Alcohol or alcohol-like 60-70%

Hepatitis incl. viral 10% or more

Biliary disease 5-10%

Unknown 10-15%

Haemochromatosis 5%

123

Steatohepatitis?

(fatty change plus inflammation) due to alcohol or alcohol-like disease

124

Complications of cirrhosis?

Portal hypertension

Portal-systemic shunts and varices

Ascites (accumulation of fluid in the peritoneal cavity)

Splenomegaly (spleen enlargement)

Liver failure

Hepatocellular (liver) cancer

125

Effects of liver failure?

Impaired production of secretory proteins

  • Albumin
  • Transport proteins
  • Coagulation and fibrinolysis proteins e.g. Factors II, V, VII-XIII
  • Complement
  • Protease inhibitors

Jaundice ( build-up of bilirubin in the blood)

Coagulation disorders (liquid/blood, changing to a solid or semi-solid state)

Altered intermediary and xenobiotic metabolism

Immune, circulatory and endocrine disturbances

126

Hepatocellular carcinoma?

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85% of malignant primary liver tumours

Geographical variation in incidence

Different age of onset in different areas

80% occur in males

80% arise in cirrhotic livers

127

Function of muscles?

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  • Movement
  • Posture and joint stability
  • Protein supply
  • Regulate organ volume
  • Propel fluids/food
  • Heat generation
128

Cardiac muscle?

Forms most of the heart wall

Contraction and relaxation involuntary

Has built in pacemaker for autorhythmicity

Speed and strength of contractions can be controlled by hormones and neurotransmitters

129

Smooth muscle?

Is non-striated

Contains actin and myosin

Action is involuntary

Some smooth muscle also has autorhythmicity (e.g. GI tract)

Regulated by ANS and endocrine system

130

Skeletal muscle?

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Striated muscle is surrounded by connective tissue – fascia

Forms compartments, separating individual muscles or muscle groups

Skeletal muscle has several layers of connective tissue

131

Skeletal muscle structure?

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muscle is surrounded by connective tissue – fascia.

Individual muscle cells are called fibres.

Fibres have many nuclei.

Develop by fusion of myoblasts.

Nuclei are in the periphery of the cell below the sarcolemma.

Myofibrils - 1-2μm in diameter. Composed of functional units called sarcomeres.

132

3 diseases of muscle?

Myopathy – Abnormal condition or disease of muscle tissue.

Neuromuscular disorders – A condition affecting any part of the motor unit (motor neurone, NMJ or muscle fibre).

Dystrophy – Muscle destroying disease, progressive degeneration of muscle fibres.

133

Muscular dystrophies?

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DECHENNE'S AND BECKER'S

  • Mutation in dystrophin gene
  • X-linked recessive
  • Necrotic fibres
  • Endomysial fibrosis
  • Muscle replaced by adipose and connective tissue

Progressive decrease in muscle function
Also affects cardiac muscle
Prognosis better in Becker’s

134

Metabolic Myopathies?

Defects in any stage of muscle ATP metabolism can lead to a myopathy.

Onset normally childhood/teenage years.

Symptoms include – Fatigue – Muscle weakness – Cramps and pains.

135

Inflammatory Myopathies?

Chronic muscle inflammation and weakness

Often idiopathic

Autoimmune

Progressive muscle weakness: Proximal ---> Distal

136

Cardiomyopathies?

Chronic disease of heart muscle

Deterioration in the functioning of the heart muscle

Can lead to heart failure and death

Symptoms include chest pain and arrhythmia

Cause often unknown but can be associated with other conditions

Little to no regeneration

137

What does the cell body of a neuron contain?

Central nucleus

Usual organelles

Nissl bodies – Clusters of free ribosomes and rER

Cytoskeleton – Neurofibrils: Microtubules & neurofilaments

No centrioles

138

Describe dendrites

Highly branched like trees.

Location of synapses.

Cytoplasm contains Nissl bodies, mitochondria and other organelles.

139

Describe the axon of a neuron

Long, thin, cylindrical projection.

Only one per neurone.

Specialised to conduct action potentials.

Contains mitochondria and cytoskeletal proteins.

140

What are neuroglia?

Smaller than neurones but more numerous.

Do not propagate action potentials*.

Able to divide and multiply.

In disease multiply to fill spaces previously occupied by neurones.

141

Name 6 neuroglial cells

CNS

  • Astrocytes
  • Microglia
  • Ependymal cells
  • Oligodendrocytes

PNS

  • Satellite cells
  • Schwann cells
142

What are astrocytes and what is their role?

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STAR SHAPED, CNS

  • Maintain chemical environment.
  • Blood-brain barrier.
  • Forms scar tissue after brain injury.
  • Provide nutrients to neurones.
  • Take up excess neurotransmitters.
143

What are microglia and what is their role?

SMALL, PHAGOCYTES, CNS

  • Protects the CNS by phagocytosis of invading microbes.
  • Clear away debris of dead cells.
144

What are ependymal cells and what is their role?

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EPITHELIAL, CNS

  • Epithelial cells which line the ventricles of the brain and central canal of the spinal cord.
  • Form cerebrospinal fluid (CSF) and assist in its circulation.
  • Cilia encourage movement of CSF.
  • Monitor composition of CSF.
  • Selectively permeable – allows exchange of fluid and substances across lining.
  • Barrier function.
145

What are oligodendrocytes and what is their role?

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CNS, Named because of their many (oligo) dendritelike processes

  • Provide structural support (scaffolding).
  • Forms myelin sheath for some CNS cells.
  • Foot process wraps around cell to be myelinated.
  • Not limited to one axon segment or axon.
146

What are schwann cells and what is their role?

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PNS

  • Only associated with one axonal segment.
  • Wrap “spiral” around axons of motor nerves.
  • Collectively called the myelin sheath.
  • Makes up white matter.
  • All other neural tissue is grey matter.
  • Acts like insulation on a wire.
  • Spaces between are called Nodes of Ranvier.
  • Intact sheath critical to proper nerve impulses.
  • Involved in repair and regeneration.
  • Damaged in some conditions
147

What are satellite cells and what is their role?

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PNS

  • Flattened cells arranged around the cell bodies of neurones.
  • Regulate chemical environment.
  • Involved in repair.
148

Nervous system tumours

(Disorders of the Nervous System)

In brain malignant and benign tumours can be equally serious

Most are gliomas.

Symptoms depend on location, size and growth rate of tumour.

149

What is demylination disorder?

(Disorders of the Nervous System)

Loss or destruction of myelin sheaths.

Can lead to paralysis e.g. Multiple Sclerosis.

Progressive destruction of myelin sheaths in CNS.

Autoimmune.

Can affect vision, speech, balance and motor co-ordination.

150

What is Guillain Barre Syndrome?

(Disorders of the Nervous System)

Immune response where macrophages strip myelin from axons in PNS.

Most patients recover completely or partially.

151

What happens in mild and sever head injury - trauma

(Disorders of the Nervous System)

Mild -> concussion – No visible bruising – May lead to headache, confusion, amnesia or temporary loss of consciousness.

Severe -> coma – State of unrousable unresponsiveness – GCS.

Diffuse Axonal Injury

  • Grey matter and white matter have different compositions.
  • Axons damaged when they move relative to one another.
  • Often caused by rapid acceleration/deceleration.
152

What is a stroke?

(Disorders of the Nervous System)

Third most common cause of death in developed world.

Abrupt onset of symptoms.

Disturbance in blood supply to brain.

Can be caused by – Haemorrhage – Emboli – Atherosclerosis.

Transient ischaemic attack (TIA) – Lasts up to 24 hours.]

Risk factors include – High BP – High blood cholesterol – Heart disease.

Treatments include – Clot dissolving drugs – Cold therapy.

153

What is dementia?

(Disorders of the Nervous System)

Chronic disorder of behaviour and higher intellectual function. E.g. Alzheimer’s Disease.

Confusion and memory loss.

Mood changes.

Lost ability to walk/talk/eat etc.

Plaques and tangles.

Brain atrophy.

154

What are the most noticeable differences between Prokaryotic and Eukaryotic cells ?

Eukaryotes are bigger and contain organelles (Eukaryotes range 10-100μm, with most 10- 40µm diameter). BUT adult RBCs are enucleated -mean diameter of 7.2µm.

Prokaryotic = 0.1-5μm.

155

What is epithelia?

  • Tightly cohesive sheets of cells.
  • Cover body surfaces.
  • Line hollow organs, body cavities and ducts.
  • Form glandular structures – secretory cells.
156

What is connective tissue?

  • Diverse and widely distributed group.
  • Loose/soft (e.g. blood, adipose tissue).
  • Hard (e.g. cartilage & bone).
  • Consist of cells and ECM.
  • Form the packaging or supporting fabric of body.
157

What is muscle?

Three types – skeletal, smooth and cardiac.

Capable of generating force – contraction.

Produce movement.

158

What is nervous tissue?

Nerve and glial cells.

Initiates and transmits action potentials.

Co-ordination.

159

What are the three lenses of a light microscope?

Condenser lens to focus light onto specimen.

Objective lens to collect light scattered by specimen.

Eye piece.

160

Light microscope limit of resolution?

Limit of human eye is about 200µm (0.2mm)

With light microscope, 2 structures in a specimen can be separated into 2 distinct images if distance of >0.2µm between them.

161

Two ways to prepare tissue for microscopy?

Tissue needs to be firm or hard – could prepare frozen sections, (faster),

or chemically fix material and embed in hard embedding agent. (routine, and easily stored).

FIXATION

DEHYDRATION

EMBEDDING

SECTIONING

162

What is fixation? (Microscopy)

For human tissue, use immersion fixation.

Small blocks of tissue improves penetration of fixative.

Routinely use formaldehyde or glutaraldehyde Often use second fixative - osmic acid, to preserve lipids.

163

What is dehydration? (Microscopy)

Dehydration: most subsequent stages require components immiscible with water so it is necessary to remove water content with series of alcohols or acetate.

For paraffin embedding, usually use 60% - 70% - 90% - 100% alcohol.

164

What is embedding? (Microscopy)

Embedding: Prevents specimen collapsing - medium has a fluid phase but has potential to harden allows cutting with glass or metal knives on microtome.

Choice of media, but usually resin or wax.

165

What is sectioning? (Microscopy)

Sectioning: Use paraffin wax or frozen sections for sections approx 7µm thick.

Glass slides used for LM.

With diamond knives and plastic resin embedded material can get 1µm sections -suitable for transmission electron microscopy.

Metal grids used for EM.

166

Silver stain?

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Certain biological components reduce silver nitrate to form black deposits of metallic silver at the site of chemical reduction. By modifying the conditions of the silver nitrate solution used, these methods can be used to demonstrate a wide range of structures, including reticular fibers.

Image = black reticular fibers.

167

Most common tissue stains?

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H and E (Haematoxylin and Eosin)

haematoxylin: basic dye with +ve charge and therefore binds to -ve charged species i.e. phosphate groups in nucleic acids; and proteins with large numbers of carboxyl or sulphate groups.

(Cell nuclei stain purple/black).

eosin: acid dye binds to +ve charged groups - amino groups in proteins

(e.g. collagen fibres, general cytoplasm pink).

168

Van Gieson method?

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Stains collagen pinkish-red and muscle yellow it is commonly used in combination with a stain for elastic fibers.

(Pulmonary veins (V) are thin-walled vessels that run in the fibrocollagenous septa with pulmonary lymphatics (L). Small venules resemble pulmonary arterioles, but the larger veins contain collagen and elastic fibers, as well as smooth muscle, outside the basement membrane)

169

Periodic acid-Schiff (PAS)?

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Many applications, particularly in the demonstration of various carbohydrates, either alone or combined with other molecules such as proteins which are stained magenta. It can therefore be used to indicate the exact position of basement membranes. The mucous cells of the stomach are strongly PAS positive.

170

Alcian blue dye method?

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Used mainly to demonstrate acidic mucins secreted by some epithelial cells, and can be combined with the PAS reaction to distinguish between acidic and neutral epithelial mucins.

It can also be used to demonstrate the extracellular glycosaminoglycan matrix.

171

Fluorescence and Immunofluorescence microscopy?

Fluorchromes usually tagged to secondary antibodies raised against species in which a primary antibody to a specific protein have been produced and therefore localise in a section.

Double or triple labelling is possible with this technique.

Fluorescence: Some molecules absorb light at one wavelength and re-emit it at a different wavelength – can view (e.g. blood cells autofluoresce).

Immunofluorescence: Can also couple fluorescent dyes (fluorchromes) as marker molecules to bind to specific molecules in cells and tissues and view by fluorescence microscope with filters at the respective wavelengths.

172

Confocal microscopy?

Offers several advantages over conventional optical microscopy, including controllable depth of field, the elimination of image degrading out -of-focus information, and the ability to collect serial optical sections from thick specimens.

The key to the confocal approach is the use of spatial filtering to eliminate out -of-focus light or flare in specimens that are thicker than the plane of focus.

Confocal microscopy, most frequently confocal laser scanning microscopy (CLSM), is an optical imaging technique for increasing optical resolution and contrast of a micrograph by means of adding a spatial pinhole placed at the confocal plane of the lens to eliminate out-of-focus light

173

Specimen preparation for EM?

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Sectioning (slice) and fractioning (frozen).

Electron beam used – not light Relies on density and not colour.

174

Scanning, Electron Microscopy?

Narrow bands of electrons used to build up image of surface of specimen (which has been coated with a thin film of metal to scatter electrons and collected to produce image).

i.e reveals surface rather than passing through specimen as in Transmission EM.

It splits along a line of weakness e.g. the centre of the membrane bilayer. Thus it splits the bilayer in two, but goes over or under membrane proteins. This gives two complementary faces.

A scanning electron microscope (SEM) is a type of electron microscope that produces images of a sample by scanning the surface with a focused beam of electrons. The electrons interact with atoms in the sample, producing various signals that contain information about the sample's surface topography and composition

175

Confocal Scanning Microscopy?

Uses narrow laser beam to scan specimens – computer builds up full image by collecting separate planes and merges them together.

Offers several advantages over conventional optical microscopy, including controllable depth of field, the elimination of image degrading out -of-focus information, and the ability to collect serial optical sections from thick specimens.

The key to the confocal approach is the use of spatial filtering to eliminate out -of-focus light or flare in specimens that are thicker than the plane of focus.

176

Growth medium for single celled organisms?

Carbon source such as glucose.

Nitrogen source such as NH4+ ions.

Salts: Na+, K+, Mg2+, Ca2+, Cl-, SO4-, PO4^3-

Trace elements.

177

Plant cell culture growth medium?

Unlike whole plants, isolated cells cannot make all their own nutrients.

Plant cells need:

  • carbon source e.g. sugar,
  • amino acids.
  • B vitamins and various elements.
  • plant hormones.

Originally the growth medium was coconut milk.

178

Bacteria growth medium vs Animal cell growth medium?

BACTERIA

  • Carbon source such as glucose.
  • Nitrogen source such as NH4 + ions.
  • Salts: Na+, K+ , Mg2+, Ca2+, Cl- , SO4 - , PO4 3-
  • Trace elements

ANIMAL CELLS

  • Balanced salt solution: Isotonic solution containing Na+, Ca2+, Mg2+, Cl- ions and a pH buffer.
  • An energy source such as glucose.
  • Essential amino acids and vitamins.
  • Serum; blood minus red and white blood cells & minus clotting proteins.
179

essential components of serum?

(Blood minus red and white blood cells & minus clotting proteins.).

Cell Spreading Factors.

Growth Factors.

Other serum components important for cell culture.

180

Cell culture?

Remove and dissociate cells

Add to culture vessel with G.M. -----> PRIMARY CULTURE

Cells divide

Cells fill dish: Confluent Culture

Remove cells from surface

Cell suspension

Add to new culture vessel

Sub-culture ----->SECONDARY CULTURE

Sub-culture

181

What are Established cell lines?

These are cultures of cells which have escaped the Hayflick limit and which will divide indefinitely.

They are potentially immortal.

They are the most widely used form of culture.

BHK21 cells

HeLa cells

182

What are Embryonic Stem Cells (ES cells)?

Embryonic stem cells are derived from cells in the early embryos of mammals (~4-day embryos).

Most of the earlier work on ES cells has been done with mouse embryos.

In some cases these cells when injected into the mouse will divide to give a population of the same cell type.

Work is now progressing using human ES cells from embryos produced by in vitro fertilisation.

The main aim of this work is to develop methods which allow cells which are lost in particular diseases (e.g. skeletal muscle cells in muscular dystrophy or nerve cells in Parkinson’s Disease.).

This research is very controversial and is banned in several countries

183

What did Avery–MacLeod–McCarty 1944 find?

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That DNA is the substance that causes bacterial transformation, in an era when it had been widely believed that it was proteins that served the function of carrying genetic information.

184

What did Hershey and Chase 1952 find?

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When bacteriophages, which are composed of DNA and protein, infect bacteria, their DNA enters the host bacterial cell, but most of their protein does not.

185

What is DNA Hybridisation?

Molecular biology technique that measures the degree of genetic similarity between pools of DNA sequences.

It is usually used to determine the genetic distance between two organisms.

DNA probe = single strand for particular sequence. (specificity depends on conditions e.g. temperature).

Label the probe - radioactive scattered along length or end (small so it doesn't interfere).

OR molecule attaches to probe - coloured or digoxigenin (good antibodies to identify and harmless.).

DNA can bind with label attached.

186

What is InSituHybridisation (ISH)?

Type of hybridization that uses a labeled complementary DNA, RNA or modified nucleic acids strand (i.e., probe) to localize a specific DNA or RNA sequence in a portion or section of tissue.

Used to reveal the location of specific nucleic acid sequences on chromosomes or in tissues, a crucial step for understanding the organization, regulation, and function of genes.

Fluorescent labels used.

Only 1 has label => sequence cut out/changed???

https://www.youtube.com/watch?v=w5l5SmKvS1o

187

Blotting technique?

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Southern blotting. A Southern blot is a method used for detection of a specific DNA sequence in DNA samples. Southern blotting combines transfer of electrophoresis-separated DNA fragments to a filter membrane and subsequent fragment detection by probe hybridization.

Single-stranded RNA molecules – Northern blotting.

Double-stranded DNA fragments – Southern blotting (must be denatured first).

For detection of a specific DNA sequence in DNA samples. Southern blotting combines transfer of electrophoresis-separated DNA fragments to a filter membrane and subsequent fragment detection by probe hybridization.

https://www.youtube.com/watch?v=DUw0ypjkSFk

https://www.youtube.com/watch?v=GPVf_AWMYZ4

188

What is PCR?

POLYMERASE CHAIN REACTION

To amplify a single copy or a few copies of a segment of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence.

Kary Mullis

C1 - Separate strands, primer (small specific probe), enzyme, nucleotide, replicates.

C2 - Same again, 4 DNA molecules by end.

Repeat cycle 30-35 times.

Primers attach within and copies a small section.

CONVENTIONAL PCR.

189

What is Real-time quantitative PCR?

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Monitors PCR product generation as it occurs, using fluorescent labels.

SYBR-green-binds to double stranded DNA – fluorescence proportional to quantity of DNA.

Can be used for many applications including diagnostics, validating gene expression differences, SNP detection.

Threshold level ----> positive

''After 20 cycles hits threshold" CT=20

  • Collect DNA sample.
  • Extract DNA sample.
  • Run PCR (90 – 120 minutes).
  • If real-time PCR, analyse computer data, If not, clean up sample (+/- restriction digest).
  • Run on gel (couple of hours).
  • View gel.

Eventually, enough amplified product accumulates to yield a detectable fluorescence signal. The cycle number at which this occurs is called the quantification cycle, or Cq.

190

What is Fluorescence recovery after Photobleaching? (Microscopy)

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191

What is Fluorescence Loss in Photobleaching? (Microscopy)

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192

What is Cell sorting?

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Separate into cell types, but they look similar?

=> FACS - labelled antibodies, fluorescent tags.

Detector detects tag and adds a charge to the cell which therefore separates them.

193

Three major class of lipids found in the plasma membrane?

Phospholipids

(a polar head group, consisting of the glycerol backbone + phosphate group + a base e.g. choline. This is hydrophilic (water soluble) • a tail, of two long hydrocarbon chains. This is hydrophobic (water insoluble))

Glycolipids

Cholesterol

194

Phospholipids

PC – phosphatidyl choline

PE – phosphatidyl ethanolamine

PS - phosphatidyl serine

PI – phosphatidyl inositol

most of the PC and all of the glycolipids are found in the external (extracellular) half of the bilayer.

most of the PS, PE and PI are in the internal (cytoplasmic) half of the bilayer.

cholesterol is distributed evenly between the two halves.

195

Channels and Carriers?

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Channels form a water-filled pore in the core of the protein, and allow the passage of molecules or ions on the basis of their size and charge.

Carriers are similar to enzymes (often called permeases) in that they bind a particular class of molecules, e.g. sugars, with high specificity. They then undergo a conformational change which moves the bound molecule across the membrane (this is equivalent to the catalytic step of an enzyme).

196

What happens when membrane proteins “go wrong”?

SNARE - soluble NSF attachment protein receptor

  • v-SNAREs are incorporated into the membranes of transport vesicles during budding.
  • t-SNAREs are located in the membranes of target compartments.
  • Toxin blocks neuromuscular transmission - freezes.
  • Snares hold vesicle for transfer, block snares = vesicles cant bind therefore no transmitters are released therefore no movement is possible.

Diabetes -

  • Type 1 – Insulin secretion affected.
  • Type 2 - GLUT4 trafficking - receptors dont have signal to go to surface and breakdown sugar.

hypercholesterolemia -

  • Very high low-density lipoprotein ("bad cholesterol") levels.
  • Mutations in the LDL receptor protein, which normally removes LDL from the circulation.
  • Mutations in apolipoprotein B, which is the part of LDL that binds with the receptor.
197

5 examples of proteins in cellular membranes?

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ION CHANNELS - (K leak channel).

TRANSPORTERS - (Na pump). Uniport, symport, antiport.

ANCHORS - Inside component anchored to outside. (Integrins).

RECEPTORS - Platelet derived growth factor receptor (PDGF) receptor.

ENZYMES - Bind outside, trigger inside. (Adenylyl cyclase).

Some fixed, some move through cytoplasm (FRAP).

198

Endosymbiotic Theory?

Prior to the formation of eukaryotic cells there were probably three major groups of cells:

1) prokaryotic cells which depended on nonoxidative metabolism.

2) prokaryotic cells which depended on oxidative metabolism

3) photosynthetic prokaryotic cells.

The main theory which explain how eukaryotic cells arose from these cells is called the Endosymbiotic Theory.

Endocytosis occurs when cells take in particles or even other cells.

On some occasions a symbiotic association might be set up between a non-oxidative cell and an oxidative or a photosynthetic cell (symbiotic part of theory).

Many bacteria cannot survive in an O2 environment (anaerobes).

New metabolisms evolved in cells to deal with O2 . This increase in O2 level also led to a formation of an ozone layer protecting organisms from UV irradiation.

This allowed cells to move into a wider range of environments.

Margulis proposed that the newly formed cells would phagocytose and digest other cells in their environment. Symbiosis is an association between two organisms in which both organisms gain benefits, usually a long-term selective advantage.

The endosymbiotic theory describes how a large host cell and ingested bacteria could easily become dependent on one another for survival, resulting in a permanent relationship. Over millions of years of evolution, mitochondria and chloroplasts have become more specialized and today they cannot live outside the cell.

199

What is the Evidence for endosymbiotic theory?

1) Size of Mitochondria / chloroplasts vs prokaryotic cells.

2) Mitochondria / chloroplasts / prokaryotic cells have circular DNA.

3) Mitochondria / chloroplasts have ribosomes and synthesise some proteins.

4) Protein synthesis inhibitors – affect Mitochondria / chloroplasts / prokaryotic cells.

5) Mitochondria / chloroplasts / prokaryotic cells have similar membrane composition (double membrane).

200

Function of cellular organelles?

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NUCLEUS - large, pores in membrane for travel.

MITOCHONDRIA - Cristae/matrix/cytoplasm, double membrane, huge surface area, ATP production.

ER -

Rough: Ribosomes on cytoplasmic membrane surface, protein synthesis, membrane synthesis.

Smooth: Lacks ribosomes, lipid synthesis , steroid synthesis, carbohydrate metabolism, drug detoxification.

GOLGI - Transport, layers, modify and mature proteins ready for secretion.

LYSOSOMES - Vesicles,

Clean: damaged organelles taken up, enzymes break them down and parts are reused.

Collect: take up particles, break them down and get rid of rest.

Destroy: dying cell, primary lysosomes rupture and dygestive enzymes are produced. Programmed cell death.

201

Lysosomal storage disease?

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It interferes with the body's ability to break down and recycle specific mucopolysaccharides, also known as glycosaminoglycans or GAG. Hunter’s syndrome is one of several related lysosomal storage diseases.

Hunter syndrome MPS II MPS VI

Short Stature and Flexed-Knee Stance. Seen with coarse facies in a 16-year old male with rapidly advancing disease. A 3-year-old with slowly advancing disease does not exhibit such obvious features.

MPS patients exhibit poor exercise capacity, probably from a variety of causes, including cardiopulmonary involvement, respiratory compromise, and musculoskeletal constraints.

202

How does protein transport through nuclear pores work?

Proteins move between the cytosol and nucleus through nuclear pores.

The proteins are transported in their folded conformation.

The nuclear pores act as selective gates.

Proteins destined for the nucleus have a “sorting signal”, the nuclear localisation signal.

This NLS binds to the cytosolic nuclear transport receptor.

The NLS of the folded protein binds to the cytosolic nuclear transport receptor.

203

How does protein translocation into mitochondria, chloroplasts and peroxisomes work?

These organelles make some of their own proteins and import the remainder.

Proteins move from the cytosol into these organelles via Protein Translocators in the membrane.

TOM (Translocase in Outer Membrane)

204

How does protein transport of secretory proteins destined for ER and onwards work?

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These use a substantially different transport mechanism from the other proteins synthesised in the cell.

They are carried by transport vesicles from one compartment to another.

All secretory proteins enter the ER, as well as those proteins for the ER itself.

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2 types of proteins transferred into the ER?

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Water-soluble proteins which pass into the lumen of the ER and are secreted.

Prospective trans-membrane proteins.

https://www.youtube.com/watch?v=4qf1BSXn_tk

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What are single and double pass membrane proteins?

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For membrane proteins, single pass means that the polypeptide chain goes through the membrane once. Double pass means that the polypeptide chain goes through the membrane twice.

Therefore, a single pass membrane protein has its C terminus and its N terminus on opposite sides of the membrane. A double pass membrane protein has its N terminus and C terminus on the same side of the membrane (since the protein has to loop to go through the membrane a second time).

207

Familial isolated hypoparathyroidism (FIH)?

Symptoms

  • Low calcium and high phosphorus levels in blood serum
  • Low/undetectable parathyroid hormone (PTH)  Symptoms of neuromuscular irritability, tingling and tetany

Cause

  • Starts out as preproparathyroid hormone (PPTH)
  • Signal peptide removed in ER – proparathyroid hormone (pro PTH)
  • After processing - parathyroid hormone (PTH) released into blood (calcium regulator)
  • Mutations in signal peptide may lead to FIH (one known example is a T to C point mutation)
208

Autosomal dominant retinitis pigmentosa.?

Carbonic Anhydrase IV (CA4) normally expressed in choriocapillaris of the eye.

Mutation close to signal sequence cleavage site leads to apoptosis, then ischemia in retina and autosomal dominant retinitis pigmentosa.

209

What is the cytoskeleton?

In the cytoplasm there is an extensive network of filamentous structures.

These filaments are known collectively as the cytoskeleton (cell's skeleton).

Provides an organism with a supporting framework, protection and levers to which muscles attach, bringing about body movements.

210

How do filaments conducts cellular motility?

Motility is usually generated by the interaction between the filament and another protein termed a motor protein.

All of these filaments are formed by the aggregation or polymerisation of large numbers of identical, protein molecules.

In the filaments, the monomers or protomers (also known as sub-units,) bind to each other by hydrophobic association

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Microfilaments (Thin filaments/Actin)

Found in all eukaryotic cells.

In the EM they are seen as solid filaments with a diameter of about 7nm.

Actin is a globular protein, hence it is referred to as G-actin.

It has a M.Wt 42k and consists of a single polypeptide chain of 375 amino acids.

Actin is the most abundant intracellular protein (approx 5%) and highly conserved (80% identity between humans and yeast.)

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How do thin filaments form?

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G-actin polymerises into actin filaments, or F-actin.

Each G-actin binds very strongly to its four nearest neighbours (one above, one below and two to the side).

G-actin and F-actin filament have polarity.

In the diagram protomer 3 is shown binding to four other protomers, one above and below and two to the side.

The polarity of the filaments is confirmed by a technique which uses a fragment of the myosin molecule.

Myosin binds to actin, generating movement.

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How does polymerisation of actin work?

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This can be studied in the test-tube, in vitro.

G-actin can only be added or released from F-actin at the ends of the filaments

If a fragment of myosin containing the actin-binding site (S1 myosin) is added to actin filaments, an arrowhead (directional) pattern is seen with EM.

If a short section of F-actin is decorated with S1 myosin and G-actin is added, the G-actin polymerises onto both ends.

Protomers are added much faster at the barbed (+ve) end than at the pointed end therefore the length of new Factin is much longer at the barbed end.

These studies emphasise that the polymerisation of actin is reversible

214

What controls the polymerisation of actin?

Whether a filament grows or shrinks is controlled by local conditions and by a large number of actin-binding proteins.

There are a number of fungal toxins which alter polymerisation. The best known are phalloidin which stabilises filaments, reducing depolymerisation, and cytochalasin which prevents polymerisation

215

What is the role of actin filaments?

Cellular motility

In most examples of motility, the movement is generated by the interaction of actin filaments with myosin.

It is also known as striated muscle because it has a prominent pattern of alternating dark and light bands or striations.

The muscle cells or muscle fibres are very unusual; they are long cylindrical cells from 1 to 200 mm long and have a diameter of 10-100 µm. They contain many myofibrils.

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Actin and myosin

Thin filaments also contain two other proteins, tropomyosin and troponin.

The thick filaments (myosin) make up 44% of the total protein of muscle cells.

217

Skeletal muscle activity?

The contraction of a muscle cell results from the contraction of the myofibrils, which results from the combined contraction of all the sarcomeres along the length of the myofibril.

The sliding-filament model can be elaborated to suggest that the myosin head groups (cross-bridges) walk along the thin filaments.

The head group binds to actin, ATP is hydrolysed and there is a conformational change which pushes the bound actin and hence the thin filament towards the centre of the sarcomere in each half of the filament.

218

Components of the sliding filament model?

Myofibril: A cylindrical organelle running the length of the muscle fibre, containing Actin and Myosin filaments.

Sarcomere: The functional unit of the Myofibril, divided into I, A and H bands.

Actin: A thin, contractile protein filament, containing 'active' or 'binding' sites.

Myosin: A thick, contractile protein filament, with protusions known as Myosin Heads.

Tropomyosin: An actin-binding protein which regulates muscle contraction.

Troponin: A complex of three proteins, attached to Tropomyosin.

219

What is the sliding filament model?

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1) A nervous impulse arrives at the neuromuscular junction, which causes a release of a chemical called Acetylcholine. The presence of Acetylcholine causes the depolarisation of the motor end plate which travels throughout the muscle by the transverse tubules, causing Calcium (Ca+) to be released from the sarcoplasmic reticulum.

2) In the presence of high concentrations of Ca+, the Ca+ binds to Troponin, changing its shape and so moving Tropomyosin from the active site of the Actin. The Myosin filaments can now attach to the Actin, forming a cross-bridge.

3) The breakdown of ATP releases energy which enables the Myosin to pull the Actin filaments inwards and so shortening the muscle. This occurs along the entire length of every myofibril in the muscle cell.

4) The Myosin detaches from the Actin and the cross-bridge is broken when an ATP molecule binds to the Myosin head. When the ATP is then broken down the Myosin head can again attach to an Actin binding site further along the Actin filament and repeat the 'power stroke'. This repeated pulling of the Actin over the myosin is often known as the ratchet mechanism.

5) This process of muscular contraction can last for as long as there is adequate ATP and Ca+ stores. Once the impulse stops the Ca+ is pumped back to the Sarcoplasmic Reticulum and the Actin returns to its resting position causing the muscle to lengthen and relax.

During maximal contraction the sarcomere length decreases from about 3 µm to 2 µm (~33% contraction).

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Which parts of the sliding filament model change length?

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M-line

The line at the center of a sarcomere to which myosin myofilaments bind.

Z-line

Neighbouring, parallel lines that define a sarcomere.

H-band

The area adjacent to the M-line, where myosin myofilaments are not superimposed by actin myofilaments.

I-band

The area adjacent to the Z-line, where actin myofilaments are not superimposed by myosin myofilaments.

A-band

The length of a myosin myofilament within a sarcomere.

At the level of the sliding filament model, expansion and contraction only occurs within the I and H-bands. The myofilaments themselves do not contract or expand and so the A-band remains constant.

221

What are Intermediate Filaments?

Network through the cytoplasm, surrounding the nucleus and extending to the cell periphery.

Adjacent cells attach to each other by desmosomes, and intermediate filaments anchor to the plasma membranes at these junctions.

Also in nucleus.

The nuclear lamina is a mesh of intermediate filaments which underlies and strengthen the nuclear envelope in all eukaryotic cells.

222

Where are intermediate filaments found?

Keratin filaments in epithelial tissue.

Vimentin (and related) in connective/muscle tissue and glial cells.

Neurofilaments in nerve cells.

Nuclear lamins in all animal cells. - The intermediate filaments in the nucleus disassemble and reform at each cell division.

223

What are keratin filaments?

Most diverse family

Found in epithelial cells.

Different sets of keratins can be found in different epithelia such as gut lining or epidermal layers of the skin.

The filaments usually span the epithelial cells and distribute stress (i.e when skin is stretched.)

Epidermolysis bullosa is caused by a mutation of keratin genes.

Blistering of the skin caused by a mutant keratin gene.

224

What are microtubules?

Part of the cytoskeleton and are found in all eukaryotic cells.

They are long hollow tubes of about 25nm diameter and several µms long.

225

Formation of microtubules?

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In vitro , tubulin dimers will add to either end of a growing microtubule, although they add more rapidly to one end, the plus (+) end than the other, minus (-) end.

Evidence suggests that the +end is the end with B-tubulin exposed and the -end is that with A-tubulin exposed.

226

Microtubule organisation?

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Microtubules grow from specialised centres called microtubule organising centres, MTOCs.

These control the number, position, and orientation of microtubules in the cytoplasm.

In animal cells, the major MTOC is the centrosome, usually found at one side of the cell nucleus.

227

Drugs affecting microtubules?

Vinblastine, Vincristine and Colchicine (from the autumn crocus). These bind tightly to free tubulin and prevent polymerisation into microtubules.

Taxol (from yew) binds tightly to microtubules and prevents them from losing subunits. Since new subunits can still be added, the microtubules can grow but cannot shrink.

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What is dynamic Instability of some Microtubules?

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Coexistence of assembly and disassembly at the ends of a microtubule.

The microtubule can dynamically switch between growing and shrinking phases in this region.

Tubulin dimers can bind two molecules of GTP, one of which can be hydrolyzed subsequent to assembly.

During polymerization, the tubulin dimers are in the GTP-bound state.

The GTP bound to α-tubulin is stable and it plays a structural function in this bound state.

The GTP bound to β-tubulin may be hydrolyzed to GDP shortly after assembly.

The assembly properties of GDP-tubulin are different from those of GTP-tubulin, as GDP-tubulin is more prone to depolymerization.

A GDP-bound tubulin subunit at the tip of a microtubule will tend to fall off, although a GDP-bound tubulin in the middle of a microtubule cannot spontaneously pop out of the polymer.

Since tubulin adds onto the end of the microtubule in the GTP-bound state, a cap of GTP-bound tubulin is proposed to exist at the tip of the microtubule, protecting it from disassembly. When hydrolysis catches up to the tip of the microtubule, it begins a rapid depolymerization and shrinkage.

This switch from growth to shrinking is called a catastrophe.

GTP-bound tubulin can begin adding to the tip of the microtubule again, providing a new cap and protecting the microtubule from shrinking. This is referred to as "rescue".

229

Microtubule function?

Determining the polarity of animal cells.

Microtubules grow out from the centrosome and can be stabilised by capture in different parts of the cell.

If many microtubules are orientated in a single direction the cell will be polarised into a distinct shape.

Also play a part in cell motility.

They do this by interaction with two classes of motor protein.

The motor proteins move along microtubules carrying some cellular component, or cargo, along the microtubule.

  1. Kinesins.
  2. Dyneins.
230

What are kinesins and dyneins?

Two heavy chains with two head groups with ATPase activity.

Use their head groups to walk along microtubules in opposite directions.

Most kinesins move along microtubules towards the + end.

All dyneins move along microtubules towards the - end.

Different types of cargoes are transported by different kinesins and dyneins.

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How do kinesins and dyneins move?

KINESINS - co-ordinated fashion leading to a “hand-over-hand” - processive movement.

Play a role in axonal transport and in mitosis.

DYNEINS - form complexes with several proteins creating the receptor for their cargo.

Responsible for mitochondrial movement, endosomal and lysosomal trafficking, transporting mis-folded proteins bound for degradation, nuclear positioning, and mitosis.

Vesicles are carried along microtubules from the endoplasmic reticulum to the Golgi apparatus.

The Golgi apparatus is located near the centrosome and thus these vesicles are transported towards the - end by dyneins.

232

Microscopy HeLa cells (LAB)

HeLa cells = larger and spiky - 30µm

Yeast cells = attached circles - 4µm

Bacterial cell = small rods - 1.5µm

233

What type of cell is Leishmania?

Eukaryote as double membrane nucleus.

234

Liver hepatocyte

Large amount RER for energy production.

235

Freeze fractured electron micrograph - plasma membrane mouse liver cells

Direction of shaddowing = hollows have darker shading, light on the other side, goes over top of other side. (As if its a ''beam of shaddow''.

Information not gained from sectioned material = which intramembranous particles are static vs moving. Just see the distribution of the individual particles.

236

Sizes of light and electron microscopes?

LIGHT 500nm ---> 5mm.

ELECTRON 0.3nm ---->100µm.

237

Why stain and what to heavy metals appear as?

To see non-coloured organelles and detail.

Lighter and darker areas.