infection, immunity and forensics

About 2-4 hours after death, the muscles start to contract and become stiff. This is rigor mortis and needs between 6-8 hours to take full effect. Joints become fixed and their position, whether bent or straight, will depend on the body position at the time of death.

• After death, musles become starved of oxygen, and oxygen-dependent reactions stop
• Respiration in cells becomes anaerobic and produces lactic acid
• The pH of cells falls, inhibiting enzymes and thus inhibiting anerobic respiration
• The ATP needed for contraction is not produced so bonds between muscle proteins become fixed
• Proteins can no longer move over one another to shorten the muscle, fixing the muscle and joints

Rigor mortis will set in more quickly and last a shorter period if environmental temperature is high or if the person has been physically active before death

• All mammals produce heat for metabolic reactions like respiration

• From the time of death, the metabolic reactions slow down and eventually stop causing body temperature to fall until it equals the temperature of its surroundings - algor mortis

• The human body cools at a rate of around 1.5C to 2.0C per hour, so from the temperature of the body they can work out an approximate time of death 

• Conditions such as air temperature, clothing, body position, humidity and body weight can affect the cooling rate of a body

• First stage involves the colonisers- e.g. anaerobic bacteria which thrive in the lactic-acid rich environment of the muscles after death
• As enzymes break down cells, the bacteria spread
• Bacteria joined by many spcies of fly, usually blowflies which are attracted to the moisture and smell of the dead body
• These feed on the tissues of the body when thy liquefy and lay eggs
• Maggots hatch and feed on the tissues, breaking them down
• Maggots pupate, turn into flies and mate and start the cycle again
• Conditions become favourable for beetles
• As a dead body dries out conditions become less favourable for flies - they leave the body and beetles remain
• When no tissue remains, conditions are no longer favourable for most organisms
• Temperature is important as the warmer the body, the faster the rate of decay
• Level of exposure of the body is important, as a buried body will decay more slowly than a body in open air as it is more available to flies and other decomposers

• When someone dies the body is quickly colonised by a variety of different insects

• TOD can be estimated by identifying they type of insect present on the body

• TOD can be identified by identifying the life cycle the insect is in

• Temperature of the air, ground, body and maggot mass can be measured to measure the rate of maggot development

• Different conditions will affect the insects life cycle, such as; drugs, humidity, oxygen levels and temperature

• After death, tissues start to break down due to the action of enzymes
• Autolysis occurs frist (body's enzymes break down cells)
• Bacteria from the gut and gaseous exchange system rapidly invade the tissues after death, releasing enzymes that result in decomposition
• The loss of oxygen favours the growth of anerobic bacteria
• Different conditions affect the rate of decomposition e.g. environmental temperature as the rate is highest between 21 and 38 degrees, but intense heat delays decay as enzymes in autolysis denature
• Injuries allow entry of bacteria that aid decomposition
  • Hours-few days after death - cells and tissues are being broken down by the bodies enzymes and bacteria that were present before death and the skin turns greenish
  • Few days-few weeks - Micro-organisms decompose tissues and organs. This produces gasses which cause the body to become bloated. The skin begins to blister and fall off
  • A few weeks - Tissues begin to liquefy and seep out into the area around the body
  • A few months-years - Only skeleton remains
  • Decades-centuries - The skeleton begins to disintegrate until there'e nothing left of the body

• DNA has a double helix structure which contains chains of nucleotide bases joined by phosphodiester bonds which contain deoxyribose (sugar) a phosphate and a base
• Bases are joined by complimentary base pairing,  A ->T G -> C (At The Golf Course) 
• The sequence of the base pairs in the molecule is used as the genetic code
• The genetic code determines which amino acids are joined together to form proteins
• Each amino acid is coded for by a codon in a gene
• In the genetic code, each codon is read in sequence, seperate from the codon before and after it. Base codons don't share bases - the code in non-overlapping
• The genetic code is also degenerate - there are more possible combinations of codon than there are amino acids. This means that some are coded for by more than one codon
• Some codons are used to tell the cell when to start and stop the production of proteins. These are called start and stop codons

mRNA copy made in the nucleus

• RNA polymerase (enzyme) attaches to the DNA at the beginning of the gene
• The hydrogen bonds between the DNA strands break, uncoiling DNA
• 1 strand becomes an antisense strand to make a mRNA copy (template strand)
• RNA polymerase lines up free RNA nucleotides alongside the antisense strand. Complementary base paring = mRNA strand being a reversed copy of DNA (except T --> U in RNA)
• Paired RNA nucleotides join together forming a mRNA strand
• RNA polymerase moves along the DNA, separating the strands and assembling the mRNA strand
• RNA polymerase move on, hydrogen bonds in DNA reform and DNA strands coil back into double helix
• When RNA polymerase reaches a stop codon, it stops making mRNA and detaches from DNA
• mRNA moves out of the nucleus through a nuclear pore and attaches to a ribosome in the cytoplasm ready for translation

• mRNA often modified before translation.
• Genes contain both introns(non-coding) and exons(coding)
• During transcription both the introns and exons are copied into mRNA
• The introns are removed by a process called splicing
• Splicing is the removal of introns and joining up exons 
• Splicing takes place in the nucleus
• Exons can be joined together in different orders to form mRNA strands
• This allows more than one amino acid sequence (and so more than 1 protein) to be produced from one gene
• After splicing the mRNA leaves the nucleus for translation

Occurs on ribosomes in the cytoplasm

• mRNA attaches to the smaller subunit of the ribosome so two mRNA codons face the two binding sites of the larger subunit
• Transfer RNA (tRNA) molecules carry amino acids to the ribosome
• A tRNA with a complimentary anticodon and corresponding amino acids to the first codon, attaches itself to the first codon by complimentary base paring
• Each amino acid has its own specific tRNA that carries it to the ribosome
• The free binding site on the ribosome is now available for another tRNA to attach to so a second tRNA molecule attaches itself to the next codon as the ribosome moves along the mRNA 
• The 2 amino acids join together via a peptide bond and the first tRNA molecule moves away leaving its amino acid behind
• This process continues, forming a polypeptide chain, until a stop codon on the mRNA strand
• The polypeptide chain (protein) moves away from the ribosome

Identification in Forensic Science

• Used to link criminals to a scene of crime
• DNA is isolated
• Each sample is amplified using PCR
• PCR products run using electrophoresis. DNA profiles are then compared.
• If samples matched, suspect linked to crime scene

Determining human genetic relationships

• DNA is inherited from our parents
• Therefore the more bands the two DNA profiles match, the more genetically similar the two people are.

DNA profiling of plants and animals

• Used to prevent interbreeding
• DNA profiling can be used to identify how closely related individuals are, the least related individuals will be bread together.

• The original DNA profiling technique involved treating the DNA sample with restriction enzymes
• These are found naturally in bacteria, where their function is to cut up the invading viral DNA
• They only cut DNA at specific base sequences 
• If the restriction sites are either side of a short tandem repeat sequence, that fragment will remain intact but will be cut away from the rest of the genome

• Used to make millions of copies of specific regions of the DNA 
• A reaction mixture is set up containing; DNA sample, primers, free nucleotides and DNA polymerase
• Primers= short pieces of DNA complementary to bases at start of fragment
• The DNA mixture is heated to 95C to break the H bonds between the DNA strands
• Mixture is then cooled to between 50C and 65C so primers can bind to the strands
• The reaction mixture is then heated to 72C so the DNA polymerase will work
• DNA polymerase lines up free DNA nucleotides alongside each template strand. Complimentary base paring means new complementary strands are formed
• Two new copies of the fragment of DNA are formed and one cycle of PCR is completed
• The cycle is repeated and 4 strands of DNA (2 origninal, 2 new) are used as templates
• Each PCR cycle doubles the amount of DNA. By the end of 32 cycles the DNA sample is almost pure
• Products of PCR are viewed using gel electrophoresis or shown as peaks on a graph

Gel electrophoresis is used to separate DNA fragments according to their length

The DNA fragments are dyed using a florescent die before gel electrophoresis

• DNA sample is placed into a well in a slab of gel covered in buffer solution which conducts electricity
• An electrical current is passed through the gel. DNA fragments are negatively  charged so they move towards the positively charged electrode at the other end 
• Short fragments of DNA travel faster and further down the gel. This separates the fragments according to length
• The DNA fragments appear as bands under UV light; this is a DNA profile
• Two DNA profiles can be compared - a match can help identify a genetic relationship
• If samples from crime scene match samples from suspects, it links a person to the crime scene

• Smallest of all microorganisms
• Not cells- arrangements of genetic material & protein that invade other living cells 
• No plasma membrane, no cytoplasm, no ribosomes
• Genetic material DNA- direct template/RNA- directs synthesis of reverse transcriptase
• Some carry proteins inside capsid e.g. HIV carries reverse transcriptase
• Protein coat around the core is called the plasmid
• Some have an extra outer layer, called an envelope, stolen from the cell membrane of a previous host cell
• Attach via viral attachment proteins (VAPs)- antigen found on cell surface membrane

Replicates in cells of body. Attacks host cells in different ways. Plasmid forms from viral DNA within bacterium.

Lysogenic pathway- many viruses are non-virulent when they get into host cell. They insert their DNA into host cell so its replicated every time the host cell divides- provirus

Lytic pathway- sometimes viral genetic material is replicated independently- mature viruses made and eventually host cell bursts- releasing new virus particles to invade other cells= virulent virus (disease-causing) and process of killing cells= lytic pathway

• Prokaryotic, most aren't pathogenic
• Cell walls made from peptidoglycan- parallel polysaccharide chains
• Gram staining= method of distinguishing between 2 different types of bacterial cell wall
• Gram positive= thick layer of peptidoglycan containing chemicals e.g. teichoic acid
• Gram negative= thinner layer of peptidoglycan, no teichoic acid
• Shape- spherical (cocci), rod (bacilli), twisted (spirilla), comma shaped (vibrios)
• Respiratory requirements- obligate aerobes (need O2), fuculative anaerobes (use O2 if present)

Structure

• Main circular DNA
• Mesosome - infolding of the cell surface membrane and site of respiration
• Ribosomes - site of protein synthesis; they occur free in the cytoplasm
• Cell surface membrane
• Pilus (plural pili) - protein tubes that allow bacteria to attach to surfaces
• Plasmids - small circles of DNA
• Cell wall - does not contain cellulose; made of peptidoglcan
• Flagellum - used for cell movement
• Capsule - a mucus layer for protection and allow bacteria to form colonies

• Most common method is asexual reproduction- splitting in two (binary fission)
• DNA replicated and old cell wall begins o break down around middle of cell
• DNA associated with cell surface membrane and may be held in position by mesosome
• Enzymes break open circular DNA strands- unwind and replicate
• New cross-walls laid down between the 2 new daughter cells
• New cell material and cell wall material extend outwards, forming 2 new daughter cells with circular chromosome
• Plasmids often divide, so daughter cells contain original genome and plasmids
• Generation time= time between cell divisions, 20 mins if favourable conditions

Sexual reproduction

• Transformation- short piece of DNA released by a donor and taken up by a recipient where it replaces a similar piece of DNA
• Transduction- small amount of DNA included by mistake- incorporated into hosts DNA
• Conjugation- genetic info transferred by direct contact. Donor cell similar to male cell and produces sex pilus cytoplasmic bridge between 2 cells through which DNA is transferred to recipient similar to female cell. Male cell contains fertitlity factor- plasmid with DNA coding for formation of sex pilus

 Chemical barriers

• Stomach acid- acidic conditions (less than 2.0) kill pathogens but optimum pH for pepsin (disgestive enzyme)
• Lysozyme- found on mucosal membranes, good at destroying gram-positive bacteria- breaks down cross-links in peptidoglycan
• Ears- contain antibacterial wax which is bacteriocidal
• Large intestine, urethra and vagina- resident bacteria out-compete e.g. nutrients
• Gut flora- bacteria aid digestive process and may competitively exclude pathogenic bacteria by competing and produce lactic acid which is useful for defence

Physical barriers

• Skin- keratin outer layer is effective in stopping entry, skin flora live on the surface and prevent colonisation by other bacteria, sebum is salty and skin impermeable to water and pathogens
•  Mucous membranes- cilia and mucus traps microbes and carry it up to the throat where it is swallowed

Inflammation

• Any cells damaged by pathogen release alarm (mostly cells that release histamine)
• Histamine causes vasodilation 'leaky' cells- can be caused by oedema (swelling)
• Response is fever- pathogen causes hypothalamus to reset to a higher body temp.

Phagocytosis

• Include macropharges (types of white blood cell) and engulf pathogens
• Phagocytes recognise the antigen on a pathogen
• Cytoplasm of phagocyte engulfs pathogen
• Pathogen contained in phagocytic vacuole in cytoplasm of phagocyte
• A lysosome fuses with the phagocytic vacuole breaking down the pathogen
• Phagocyte presents pathogen's antigens activating the specific immune response

Production of anti-viral proteins (interferons)- help prevent spreading by:

• Inhibiting the production of viral proteins preventing viral replication
• Activating specific immune response to kill infected cells
• Activate other mechanisms in the non-specific immune response

T cells- made in bone marrow, mature in thymus

T helper cells- when activated, stimulate B cells to divide and become cells capable of producing antibodies. Enhance activity of phagocytes

T killer cells- destroy any cellswith antigns on their surface membrane that are recognised as 'foreign' or 'non-self' e.g. body cells infected with pathogens

T-helper activation

• Macrophage presents antigens from bacterium on multihistocompatibility complexes (MHCs)- becomes an antigen presenting cell (APC)
• A CD4 macrophage APC binds to T-helper with complementary CD4 receptors
• When T-helper activated, it divides by meiosis to form clone of T memory cells (circulated in blood until needed) and clone of active T-helpers (help with rest of immune system)

B cells- made in bone marrow, mature in bone marrow, free in body in lymph nodes, have a membrane bound receptor (immunogloblins) and can respond to millions of pathogens

Clonal selection

• Antigens from bacterium binds to B cell with complementary receptor- becomes APC
• Activated T-helper with complementary receptor binds to APC and produces cytokines (proteins) to stimulate the B cell
• B cell divides to give clone of B memory cells, which are long-lived and enable an individual to respond more rapidly to the same antigen and clone of B-effector cells which differentiate into plasma cells which secrete antibodies that bind to antigens, identifying them for easier destruction

Sometimes pathogen inside host cell- humoral response not effective

• Bacterium infects host cell
• Host cell presents antigens on MHCs and becomes APC
• T-killer with complementary receptors binds to APC
• Cytokines from activated T-helper signal to kill the cell
• T-killer divides and differentiates to form clone of active T-killer memory and clone of active T-killer
• T-killer clones bind to infected cells presenting antigens on MHCs
• T-killer releases chemicals that cause lysis as pores form in the infected cell
• Infected cell dies (apoptosis)

• When a pathogen enters the body for the first time the antigens on its surface activate the immune system (primary response)

• The primary response is slow because there aren't many B-cells that can make the antibody needed to bind to the antigen

• Eventually the body will produce enough of the right antibody to overcome the infection. Meanwhile the infected person will show symptoms of the disease.

• After being exposed to an antigen, both T and B cells produce memory cells. These remain in the body for a long time. Memory T-cells remember the specific antigen and will recognise it the second time around. Memory B-cells record the specific antibodies needed to bind to the antigen.

• The person is now immune - their immune system will produce a quicker, stronger immune response (secondary response)

Active immunity

• This is the type of immunity achieved when the immune system makes its own antibodies after being stimulated by an antigen.
• Consists of;
  • Natural - acquiring immunity through catching a disease 
  • Artificial - acquiring immunity through vaccination
• This produces long term protection but takes a while to acquire

Passive immunity

• This type of immunity is acquired through antibodies made by a different organism
• It consists of;
  • Natural - immunity acquired due to antibodies received through the placenta and breast milk
  • Artificial - immunity acquired through injection of antibodies e.g. to stop a disease

• M. tuberculosis causes TB and is spread by droplet infection
• When inhaled into lungs, multiply slowly in primary infection, causing no obvious symptoms
• Tubercule formed by inflammatry response- mass of tissue with dead tissue and macrophages
• But can avoid immune system by producing thick waxy outer layer which prevents lysosome fusing with phagocytic vacuole
• So bacteria not broken down and can multiply undetected in phagocytes
• Most effective bacteria are selected and passed on
• Causes fever, night sweats, loss of appetite, blood coughed up in sputum
• Bacterium disrupts antigen presentation in infected cells; preventing immune system from recognising and killing infected phagocytes
• Cocktail of antibiotics treats TB, but BCG vacccine stopped due to herd immunity
• Number of TB cases increasing- deteriorating social conditions and immigration

• HIV attacks any cells with CD4 receptors (T-helpers and macrophages)
• gp120 glycoproteins on the virus surface bind to CD4 receptors
• Virus binds to co-receptors which enables virus to fuse with host's cell membrane
• HIV= retrovirus- contains RNA not DNA
• Viral genome and reverse transcriptase enter cell
• DNA copy synthesised by reverse transcriptase
• RNA degraded; second DNA strand synthesised
• DNA cicularises and intergrase functions to incorporate DNA into host cell genome
• Viral DNA inserts into hosts DNA by intergrase
• Viral DNA transcribed, yielding messenger RNAs and viral genome RNA
• Viral RNAs translated, yielding viral enzymes
• Viral membrane proteins transported to host cell membrane- more viruses made
• Final viral assembly and budding take place

Acute phase

• HIV antibodies appear in the blood
• The infected person may experience symptoms such as fever, sweats, headache, sore throat and swolen lymph nodes, or they may have no symptoms
• There is a rapid replication of the virus and loss of T helper cells
• After a few weeks, infected T kelper cells recognised by T killer cells, which start to destroy them, reducing the rate of virus replication but does not totally eliminate it

Chronic phase

• Virus continues to produce rapidly but numbers kept in check by immune system
• May be no symptoms, but increasing tendency to suffer colds or other infections which are slow to go away
• Dormant diseases like TB and shingles can reactivate

Disease phase

• Increased number of viruses and declining number of T helper cells indicates onset of AIDS, so the decrease in T helper cells makes immune system vulnerable

• Antibiotics are chemicals that kill or inhibit the growth of micro-organisms
  • Bactericidal antibiotics kill bacteria
  • Bacterostatic antibiotics prevent bacteria growing
• Antibiotics are used in the form of drugs to treat bacterial infections.
  •  
    • Some interrupt metabolic pathways causing death by blocking nucleic acid synthesis e.g. sulphonamides
    • Cell wall agents prevent formation of cross-linking in cell walls so bacteria killed by lysis (burtsting) e.g. penicillin
    • Cell membrane agents damage cell membrane, so metabolities leak out or water moves in, killing the bacteria e.g. cephalosporins
    • Protein synthesis inhibitors interrupt or prevent transcription and/or translation or microbial genes, so protein production is affected e.g. tetracyclines
    • DNA gyrase inhibitors stop bacterial DNA coiling up so it no longer fits within the bacterium e.g. quinolone
• Bacterial cells are different from mammalian cells- mammalian cells are eukaryotic, they don't have cell walls, they have different enzymes and have different, larger ribosomes. This means antibiotics can be designed to only target bacterial cells, so they don't damage mammalian cells. Viruses don't have their one enzymes and ribosomes - they use the ones in the host cell, so antibiotics don't affect them.

• Bacteria to be tested are spread on an agar plate e.g. M luteus
• IV= antibiotic used, DV= zone of inhibition
• Soak paper discs in different antibiotics and place on to plate using aseptic techniques e.g. using a Bunsen burner to sterilise instruments
• Incubate plate at 25C for 24 hours
• Measure zone of inhibition (where bacteria can't grow) with a ruler to measure radius
• Bacteria that are unaffected by antibiotics are said to be antibiotic resistant