Additional Cancer Lecture Slides

8/14/2019 Additional Cancer Lecture Slides

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CANCER

Cancer is a group of diseases characterized by uncontrolledgrowth and spread of abnormal cells. If the spread is notcontrolled, it can result in death.

(American Cancer Society)


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TUMOUR CLASSIFICATION

BENIGN TUMOURS

Develop in any tissue

grow locally

May cause problems by pressure (brain) or obstruction ( colon)

Histologically resemble the tissue of origin

Covering or lining tissues of skin, intestine, bladder etc may producewart-like outgrowths containing all cell types

In other situations only one cell type may be present- may produce anexcess of particular hormone

Benign does not mean completely harmless

Do not spread to distant sites


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IN SITU TUMOURS

Usually develop in the epithelium

Usually small

Have altered histological appearance

Loss of normal arrangement of cells

Variations in cell size and shape, increase in nucleus size andstaining ( increased DNA ), presence of abnormal chromosomes

Do not invade basement membrane and supporting mesenchyme


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CANCERS

Fully developed malignant tumours with the specific capacityto invade and destroy the underlying mesenchyme.

Metastasise

Stimulate angiogenesis and development of bloodsupply

difficult to treat.


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Two Broad Classes of Genes are Involved in the Onset of Cancer

1). Proto-oncogenes

activated by mutation to become oncogenes,- excessivelyactive in growth promotion.

2). Tumour Suppressor genes

normally restrain cell growth- damage to these genes allowsinappropriate growth

Many of the genes in both classes code for proteins involved in

entry into, and passage through, the cell cycle cell death by apoptosis

repair of damaged DNA


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Chemical Carcinogens

-Earliest example 1775 coal tar and skin cancer

-Later, 2 naphthylamine as a bladder carcinogen

-Wide chemical diversity and many ( eg polycyclic aromatichydrocarbons) show great chemical stability.

- Now known to be converted to highly reactive compoundsby detoxification enzymes in the liver.

-Guanine is often converted to methyl guanine, acts likeadenine and pairs with thymidine in the copied strand-hence G-C pair is converted to A-T pair as point mutation.


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3 ways in which cancer cells become growth signal autonomous.

- modulation of growth factor provision

- modulation of growth factor receptor activity

- modulation of intracellular signalling pathways


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Self Sufficiency in Growth Signals

ii). Modulation of growth factor receptor activity

Many growth factor receptors are protein tyrosine kinases

Overexpression allows tumours to respond to low levels of growthfactor that would not normally produce a growth response.

EGF-R ( the receptor for EGF) and Erb-B ( the receptor for hereglulin )are upregulated in stomach, brain and breast tumours.

HER2/neu is overexpressed in stomach and breast tumours

Overexpression of GF receptors may result in ligand independentsignalling.


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ii). Modulation of growth factor receptor activity(cont)

Receptors may become structurally altered- ligand independent as aresult.

Truncated versions of the EGF receptor lacking the cytoplasmicdomain are constitutively active

Alteration of integrins expression (ECM receptors) to those favouringgrowth.

Ligand activated GF receptors and pro-growth integrins attached toECM often activate the SOS-Ras-Raf-MAP Kinase pathway.


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iii). Modulation of intracellular signallingpathways

Frequently involves the SOS-Ras Raf- MAPK cascade

About 25% of human tumours have a mutated Ras protein.(90% pancreas, 50% colon, 30% lung -- the first oncogenediscovered in human tumours))

--- mitogenic signals are transmitted without any upstreamactivation of the pathway

Ras also interacts with PI3 kinase

This enables growth signals to simultaneously generate survival signalsie. Signals which protect against apoptosis


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Insensitivity to Antigrowth Signals

Tumours have developed several ways to block TGF action

Expression of TGF receptor is down regulated

The receptor is mutated to a less active form

Intra-cellular signalling is disrupted by-

Mutation of Smad

Loss of p15

Mutation of CDK4 to be less p15 sensitive

Mutation of Rb


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Tumours have developed several ways to block TGF acting through Rb

In some DNA virus induced tumours ( cervical carcinomas) Rb isinactivated by being complexed with a viral protein .

In human cervical tumours this is the E7 protein of human papilloma virus


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Cancer cells can also

Turn off the expression of cell adhesion molecules that

transmit antigrowth signalsThese probably act through Rb also


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Some tumours have developed mechanisms for differentiation

One such mechanism involves the c-myc oncogene


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Evasion of Apoptosis

Hormone dependent tumours undergo massive apoptosis if thehormones were removed.

Suggested that increased cell growth and apoptosis occurred at

the same time

Apoptosis may be switched on by oncogene overexpression

Elimination of cells with activated oncogenes by apoptosis maybe the primary means by which mutant cells are continuallyremoved from the bodys tissues.

For a tumour to progress it has to inactivate the apoptopic machinery


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In 50% of lymphomas, there is a mutation in c-myc

and a mutation in bcl-2

Further evidence for a myc-bcl-2 interaction

Fibroblasts overexpressing myc grown in culture

In low serum the c-myc expressing cells show high apoptosis

Increased apoptosis could be abolished by

Addition of survival factors such as IGF-1 to the medium

Overexpression of Bcl-2 or Bcl-XL

Disruption of the FAS pathway


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In transgenic mice,

Inactivation of Rb ( expected to increase cell proliferation)produced slow growing microscopic tumours with a high rate ofapoptosis

Additional inactivation of p53( a key mediator of apoptosis) in thesame cells produced rapidly growing tumours

Mutation of p53 and the presence of a mutated p53 protein isextremely common in human tumours ( greater than 50%)

Some lung and colon cancers produce a decoy non-signalling receptor for the FAS ligand


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Limitless Replicative Potential

Why should tumour cells need to become immortalised?

Normal human cell types have the capacity for 6070 doublings.

This should enable clones of tumor cells to expand to numbersthat vastly exceed the number of cells in the human body.

There seems to be no sense in the idea that the tumour cells haveto become immortal in order for malignant tumour growth to occur

But

During tumour development there is widespread apoptosis along side the

increased cell division.

The number of cells in a tumour greatly under represents the celldivisions required to produce it.

Thus the generational limit of normal somatic cells may be a barrier tocancer development.


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Telomeres

Telomeres are simple-sequence DNA repeats found at the end of chromosomes

Human telomeres contain 250-1500 copies (6-12 kb) of the sequence TTAGGG

At each cell division, 50100 bp of telomeric DNA are lost from theends of every chromosome

DNA polymerases are unable to completely replicate the 3ends.

Progressive shortening of the telomeres occurs with each division.

Eventually the telomeres lose the ability to protect the ends of thechromosomes

This results in end to end chromosomal fusion and the death of theaffected cell.


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Telomeres

..and Telomerase

Telomere maintenance occurs in just about all malignant cells

The majority (85%90%) upregulated expression of an enzyme calledtelomerase

This adds hexanucleotide repeats onto the ends of telomeric DNA.

The telomeres are thus kept at a length above a critical thresholdwhich allows for unlimited multiplication of descendant cells.


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Sustained Angiogenesis

Cells in a tissue need to be within 100 of a capillary blood vessel

This closeness is achieved during organ development and once a tissue isformed, the growth of new blood vesselsthe process of angiogenesisis

transitory and carefully regulated.

Normal Cells and Tissues

Cancer Cells

Cancer cells initially lack angiogenic capacity

this limits initial expansion of the tumour.

To develop to a clinically detectable size the tumours usually developangiogenic ability.


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Cancer Cells

A cancer cell mass of about 2 millimeters emits signals that recruitsurrounding connective tissue and vascular cells to the tumor andinduce them to grow into blood vessels.

The blood supply to the tumour provides nutrients and oxygen,and provides a route to the rest of the body,

ie. A route to metastasis.


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Angiogenesis is controlled by both positive and negative signals.

vascular endothelial growth factor (VEGF)

acidic and basic fibroblast growth factors (FGF1 and FGF2)

thrombospondin-1

binds to a transmembrane receptor( CD36) on endothelial cells.

Important ones are


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Tumors activate angiogenesis by changing the balance of inducers andinhibitors.

Many tumors increase expression of VEGF and/or FGFs.

Some down regulate expression of inhibitors such asthrombospondin-1

Some do both.

The mechanisms remain largely incompletely understood.

- activation of rasoncogene may upregulate VEGF expression.

-thrombospondin-1 is to positively regulated by the p53.

-Loss of p53 function, which occurs in most human tumors, causesthrombospondin-1 levels to fall, releasing the endothelial cells frominhibition.


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Metastasis

An exception to the immobilised format of tissue structure is the whiteblood cell.

This can migrate out of the blood stream and enter into other tissues.

For tumour cells to metastasise, they must acquire the migratoryproperties usually restricted to white-blood cells.


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Metastasis

Spread may occur via the blood or the lymphatics.

Localised spread to lymph nodes is a sign of poor prognosis.

Certain tumors spread to particular organs.

- prostate cancer to the bones.

- colon cancer to the liver.

-stomach cancer to the ovary.

-occular melanoma to the liver.


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Metastasis

Metastatic cells must loose connections with theother cells and ECM of the primary tumour

Proteases used to degrade the basal membrane

Plasminogen activators ( serine proteases)

- leads to conversion of plasminogen to plasmin

Cathepsin B ( cysteine protease)

Matrix metalloproteinases ( MMPs)

- often converted from a pro- (inactive ) form byplasmin


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Metastasis

2 mechanisms for surviving in the bloodstream.

Travel in clusters, increases the possibility that at least one will survive.

Surround themselves with blood cells such as platelets, -- masks thecancer cells from immune surveillance.

Cancer cells in the blood stream


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Metastasis

The secondary site

The secondary site is often specific.

Specific interactions between the cancer-cell surface and the endothelialcells that line the blood vessels in the new host tissue.

Carbohydrates on the cancer-cell surface bind to a specific receptor on the

endothelial cells called a selectin.

Different selectins recognise different carbohydrates on the cancer cellsurface.

Normally, the carbohydrate-selectin interactions are used by white bloodcells to identify particular tissues to combat local infection.

Each cancer-cell type expresses a different set of carbohydrates on itssurface, attracted to different selectin molecules.

The specificity of these interactions helps account for the differentialhoming specificities of different types of cancer cells.


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Metastasis

The secondary site

The cancer cell contacts a surface where the cells express the appropriateselectins.

More bonds, mediated by integrins, form between the cells.

The cancer cell migrates through the blood-vessel wall, degrading theconnective-tissue matrix with proteases.

The cancer cell is now ready to proliferate and form a new tumor in its newhost tissue


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Cancer Therapy

Surgery if possible

some cancer cells remain at the original site

others may have already started to metastasise to distant organs

So the patient is given Radiation

eradicates cells by inducing apoptosiscan be directed very specifically to where the primary tumor waslocated in order to destroy any remaining cancer cells. However,undetected metastases elsewhere go untreated

Radiation is often given in conjunction with Chemotherapy

designed to curtail division and proliferation

many normal cells with high turnover rates, such as skin, hair andblood cells, are affected along with the cancer cells

Sometimes cancer cells develop resistance to chemotherapy

Current therapeutic approaches are thus fairly 'blunderbus-like'

Current Therapy is fairly crude


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Cancer Therapy

Potential Improved Therapeutic Approaches

Stimulate the patient's immune system

Boost the patient's immune system, and direct it against molecules expressed onthe cancer cells but not on healthy cells.

Tumor-specific antigens have been hard to identify

Many of the immune agents now in use target healthy cells as well

In the test tube, immune cells are effective in killing the cancer cells.

Unfortunately, in vivo once the immune cells meet the cancer cells, nothing furtherseems to happen.

The immune cells fail to mediate any attack.


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Cancer Therapy



Cancer cells ward off possible attack by secreting large amounts ofimmunosuppressive messenger molecules, such as interleukin-10

transforming growth factor b

and prostaglandin E2.

They also secrete molecules such as 2-macroglobulin, which inhibit cancer-cell-destroying proteases.

May travel through the circulation in clusters, (increasing the possibility ofsurvival) or may surround themselves with platelets to escape immunesurveillance.


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Cancer Therapy



The cancer cells may also attack the immune cells using the Fas/ Fas-L system.


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Cancer Therapy

Doxorubicin induces the expression of both Fas and FasL on cancercells

- causes cancer cells to kill themselves by inducing apoptosis.

Doxorubicin ( Adriamycin)



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Cancer Therapy


Some modest successes with immunostimulants.

Injecting the bacterial preparation BCG seems to be effective in

early stage bladder tumours.

Interleukin 2 (IL-2) or with alpha-interferon (IFN-) has beenbeneficial in some instances.

Generally attempts to stimulate the host immune systemsufficiently to beat off the cancer have been disappointing.


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Cancer Therapy


Monoclonal Antibodies as therapeutic agents

The receptor Her2 is overexpressed in a subset of breast tumours.

Anti-Her2 monoclonal antibody (Herceptin) has proved highly successful.

The antibody combines with the receptor and blocks it so that growthfactors ( EGF) no longer bind.

May also cause the receptor it to be internalised

This leads to the selective death of the cancer cell


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Cancer Therapy


Immunotoxins

Cancer Therapy


Immunotoxins

Monoclonal antibodies coupled to a cytotoxic drug.

Mylotarg.

MAb against a cell-surface molecule (called CD33), found on acutemyelogenous leukemia cells coupled to a complex oligosaccharide calledcalicheamicin

Makes double stranded breaks in DNA.

BL22, a MAb against CD22 (found on some leukemias and lymphomas) joinedto a bacterial endotoxin that blocks protein synthesis.


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Cancer Therapy


Monoclonal antibodies against tumor antigens can also becoupled to radioactive atoms such as indium -111 , yttrium -90 oriodine-131.

Radioimmunotherapy


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Cancer Therapy


Treat leukemia with massive doses of chemotherapy and radiation

- the leukemic cells are killed.

- also kills the patient's own bone marrow.

The patient must be then given a transplant of donor bone marrow

containing the stem cells from which all blood cells are derived.

Allografts of T cells


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Cancer Therapy


The idea here is that T cells isolated from within solid tumours are

specific for tumour antigens.

T cells are isolated from the solid tumour,- grown to large numbers outside the body ( in culture)-activated- and reintroduced back into the patient.

Has been successful with malignant melanomas of the skin.

Successful in about 77% of patients, and worked against metastases aswell as the primary tumours.

Autografts of T cells: Tumor-Infiltrating Lymphocytes (TIL)


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Cancer Therapy


1). Dendritic-cell Vaccines

Dendritic cells are the most potent of the antigen-presenting cells.-harvest dendritic cells from the patient-expose these in vitro to antigens associated with the typeof tumour in the patient-inject the "pulsed" dendritic cells back into the patient-hope they kick the immune system into action.

So far have shown promise against melanomas, prostate cancer andlymphoma.

Cancer vaccines


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Cancer Therapy


Cancer vaccines

2). Tumour-specific Antigen Vaccines

Tumour cells are removed from the patient,- treated with heat or chemicals so they are not viable,- mixed with an adjuvant (such as BCG)- injected back into the patient,-hopefully to now stimulate the patient's immune system.

Such vaccines are currently in clinical trials for use against chronicmyelogenous leukemia CML).


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Cancer Therapy


Two angiogenesis inhibitors with potential clinical applications

1) angiostatin (an amino-terminal fragment of plasminogen)

2) endostatin (a 20 kD protein derived from the carboxyl-terminaldomain of collagen XVIII)

Both inhibit angiogenesis in laboratory animals

Inhibit Tumour Angiogenesis


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Cancer Therapy



Much of the research has been centered on VEGF.

Antisense constructs against VEGF inhibit experimentalangiogenesis.

Monoclonal antibodies against VEGF receptors have also beensuccessful in stopping angiogenesis.

Genetically engineered cells secreting a soluble form of the VEGF

receptor have been shown capable of inhibiting angiogenesis atdistant sites.


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Cancer Therapy



Small molecule inhibitor of receptor tyrosine phosphorylation- inhibits the tyrosine phosphorylation of VEGF receptors

- and PDGF receptor and thus inhibits signalling .

Has potent anti-angiogenic effects in pre-clinical models and is

undergoing clinical trial.


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Cancer Therapy



Various interferons also are reported to inhibit angiogenesis

Interferon alfa-2a given to a 5-year-old girl with a large rapidlygrowing tumor of the mandible.

Resulted in dramatic decreases in urinary excretion of FGF,and complete tumor regression over a three year treatment-free

interval


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Cancer Therapy


Recent experimental strategies attempt to compensate for orcorrect defective genes.

Eg with ras

Correct Mutated Signalling Pathways

The ras protein has to be post-translationally modified.

This requires that ras has a lipid molecule attached to it.

This is catalyzed by an enzyme called farnesyl transferase.

Inhibitors of farneslyl transferase are being tested in clinical trialsas anti -cancer agents.


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Cancer Therapy


Another (theoretical) approach is to restore the function ofgrowth-inhibiting genes inactivated through mutation.

Gene therapy, -- replace the mutated gene with a functioning one.

To date the clinical results have been disappointing.

Restore Growth Inhibitory Pathways


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Cancer Therapy


Restore adhesion-molecule synthesis

Gene-replacement therapy has worked in thelaboratory,

but it is still a long way from being useful clinically.

Halt Metastasis


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Cancer Therapy


Other potential targets are the proteases that metastatic cells use.

Marimastat, an inhibitor of matrix metalloproteinases (MMPs) causedgreat excitement.

Marimastat is water-soluble.

Marimastat inhibited tumour development and metastasis in animals.

Treated animals showed increased amounts of connective tissuearound the tumors, consistent with the drug inhibiting MMPs.

Early clinical trials were promising.

Unfortunately the early results were not borne out by more extensivestudies.

Despite the initial optimism, the usefulness of marimastat may belimited to a small subset of patients.

Halt Metastasis


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Cancer Therapy


Another approach involves a specific small, water-soluble inhibitor of

cathepsin-B.

- claimed to inhibit only the cathepsin-B on the cell membrane- not the internal lysosomal enzyme- and to have minimal effect on normal cells.

Inhibition of extracellular, but not intracellular, activity suggests

therapeutic promise, because it blocks only the pathologicallyexpressed cathepsin-B and not the physiologically required stores.

Halt Metastasis


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Cancer Therapy


Given orally to rats with colon cancer that had metastasized to the liver

the number of tumors was reduced by one-third

and the size of the tumors was reduced by two-thirds.

Strongly suggests that cathepsin-B is involved in colon-cancermetastasis to the liver.

It has yet to be shown that this drug has any use therapeutically,

but it seems possible.

Halt Metastasis


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Cancer Therapy


Attempts to stimulate apoptosis specifically in tumour cells seem towork in the laboratory, but have not yet been useful clinically

So-

Overexpression of BAX (apoptosis stimulator) in tumor cellsincreases the sensitivity to conventional chemotherapy and todecrease the growth rate when implanted into mice.

Another approach has been to stimulate apoptosis by transfection ofwild type p53 with the addition of a construct expressing FAS-L.

This approach led to dramatically enhanced apoptosis in transfectedglioma cells in culture

Yet another approach has been to inhibit the activity of bcl-2 by theuse of drugs, which seems to work in cells overexpressing Bcl-2

Induce Apoptosis in the Tumour


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Cancer Therapy


A second approach assumes that tumor cells which strongly

express telomerase should drive a telomerase promoter.

Transfection with a cytotoxic transgene in which the telomerasepromoter is attached to the gene for Bax (to promote apoptosis)should produce tumour-specific killing.

Telomerase as a Potential Target

This was found to be so.

Induction of Bax elicited tumor-specific apoptosis in vitro andsuppressed tumor growth in nude mice.

Normal, telomerase negative, cells were not affected

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Additional Cancer Lecture Slides