User:Z3378280

From CellBiology

LAB 1 WORK

Ingested hrp.jpg

--Z3378280 15:37, 22 March 2012 (EST)

LAB 2 WORK

Qu.1) Timothy A Brown, Ariana N Tkachuk, Gleb Shtengel, Benjamin G Kopek, Daniel F Bogenhagen, Harald F Hess, David A Clayton Superresolution fluorescence imaging of mitochondrial nucleoids reveals their spatial range, limits, and membrane interaction. Mol. Cell. Biol.: 2011, 31(24);4994-5010 PubMed 22006021


Qu.2)This journal article details a study done on mitochondrial DNA in which the primary imaging method was superresolution microscopy. Confocal microscopy was used for preliminary cell viewing but its uses are limited in this study of nucleoids due to its inability to 'break' the diffraction barrier, meaning cell structures separated by a distance of less than half the wavelength of the light used by the particular microscope can not be clearly viewed (Abbe's law). However Superresolution microscopy enabled discernemnt of basic nucleiod characteristics such as size and shape, as well as location and approximate numbers contained within the mitochondria, as well as a whole host of other details that a confocal light microscope simply couldn't provide.


LAB 3 WORK

  1.Fixative= Acetone        SDS from REDOX CHEMICALS PTY LTD
 PROPERTIES(physical and chemical)    appearance-clear colourless liquid    odour-pungent and sweet  ph- 5-6                     vapour presure- 307 974 hPa @ 25 degrees C
                                      vapour density- 2.0                  boiling and melting points- 56.2 and -95.3 degrees C  autoignition temp- 465 degrees C 
                                      solubility (water)-soluble           specific gravity- 0.79                                flammability-highly flammable
                                      flash point- -20 degrees C           upper and lower explosion limit- 12.8% and 2.6%       

 

HAZARDS- Summary; Low to moderate toxicity- irritant. This product has the potential to cause adverse health effects with over exposure. Use safe work practices to avoid eye or skin contact and inhalation. Over exposure may result in central nervous system (CNS) depression, nausea, dizziness and unconsciousness at high levels.

Eye-irritant. contact may result in irritation, lacrimation, pain and redness.

Inhalation- irritant.over exposure may result in irritation of nose and throat, coughing, nausea, vomiting, weakness and headache.

Skin- irritant. contact may result in dryness and defatting of the skin, rash and dermatitis. may be absorbed through skin with harmful affects.

Ingestion- low to moderate toxicity. ingestion may result in gastrointestinal irritation, nausea, vomiting, headache and weakness. ingestion of large quantities may result in dizziness, drowsiness, kidney damage, unconsciousness and coma.



2. 4 articles/ journals etc relevant to out project on extrinsic apoptosis.


    

H Bantel, T Brüning, K Schulze-Osthoff Activation of caspases by death receptors. Eur. Cytokine Netw.: 1998, 9(4);681-4 PubMed 9889414

     

A H Stegh, M E Peter Apoptosis and caspases. Cardiol Clin: 2001, 19(1);13-29 PubMed 11787807

      

D L Vaux Apoptosis timeline. Cell Death Differ.: 2002, 9(4);349-54 PubMed 11965486

        

Susan Elmore Apoptosis: a review of programmed cell death. Toxicol Pathol: 2007, 35(4);495-516 PubMed 17562483


LAB 4

--Z3378280 14:07, 29 March 2012 (EST)

PROTEIN; Musashi Hideyuki Okano, Takao Imai, Masataka Okabe Musashi: a translational regulator of cell fate. J. Cell. Sci.: 2002, 115(Pt 7);1355-9 PubMed 11896183

RNA binding protein that regulates the expression of target mRNAs at the translation level. Regulates expression of the NOTCH1 antagonist NUMB.May play a role in the proliferation and maintenance of stem cells in the central nervous system. Molecular weight of 65.8 kDa.

ANTIBODY;Anti-Musashi 1 (ab21628)

         Description-Rabbit polyclonal to Musashi 1 / Msi1
         Supplier- abcam  [1]
         Polycloneal. Reacts with mouse and human Musashi 1 (Msi1)

ANTI-ANTIBODY;Alexa Fluor® 488 Goat Anti-Rabbit IgG (H+L)

             http://products.invitrogen.com/ivgn/product/A11008
             Price; 347 AUD for 0.5 ml
             Host; Goat       Reactivity; Rabbit  Supplier; Invitrogen


LAB 5

--Z3378280 14:21, 5 April 2012 (EST)

Transgenic- random chromosomal integration of foreign DNA into germ line Homologous Recombination-knock out (KO) or knock in(KI)

                       -conditional KO/KI; tissue specific and inducible

Creation of transgenic mice; fertilized egg is inserted with DNA via a micro-injection pipette- then this fertilised egg is planted in the oviducts of the ready mother

Types of transgenics- 1)target protein may be overexpressed

                     2)target protein may be ectopically expressed              
                     3)mutated protein is expressed to produce constitutively active (gain of function)  or dominant negative (loss of function)form of a protein

Knockout-modification in which activity of gene is eliminated Knockin-modification in which a specific mutation is introduced and the gene remains functional

Why usw a KO/KI? -to understand the function of genes -to study pathophysiology of disease and test therapeutic approaches -most useful to mimic recessive disorders

Making a KO mouse requires- a pluripotent embryonic stem cell


LAB 6

--Z3378280 15:41, 19 April 2012 (EST)

Tropomyosin- actin associated protein. binds to actin and regulates actin formations and structure

If we want to upregulate tropomysoin production in a neuroblastoma, we introduce a plastid containing the gene Tm4 which is then expressed by the neuroblastoma in its corresponding protein form of tropomyosin 4.

In this same plastid we include the resistant gene for antibiotic geneticin so that neuroblastomas become resisant to geneticin. Hence our neuroblastomas produce tropomysoin 4 and are resistant to the antibiotic geneticin- therefore we can separate all neuroblastomas which were sucessful in uptake of the plastid from those that were not sucessful by treating them with the geneticin and killing off the unsucessful cells.

questions;1) do you see a difference in phenotype (morphology) between Tm4 overexpressing and control cells?ANS: Yes, there is a significant difference which can be put down to the fact that the Tm4 overexprssing cells were inserted with the TPm4gene and hence influenced to overproduce tropomyosin. The control group demonstrated a wider variety of phenotypes. All phenotypes were represented, including fan and pygnotic. With the Tm4 overexpressing however, there was much less variety. Almost no pygnotic and fan phenotypes were recorded, but there were predominantly stringed, pronged and stumped. The differences in phenotyes between the control and Tm4 overexpressing can be clearly seen in the table below. 2) if so, how could Tm4 overexpression lead to this difference? Tm4 overexpression leads to tropomysoin overexpression. Tropomyosin, being an actin associated protein, favours phenotypes demonstrating higher numbers of neurites and other 'appendages' and processes.


Lab 6 (percentage).png


Lab 7

What i have done for my group project: -written an introduction to the projects page - written notes on the caspase cascade -collected articles on the morphology and pathology of our topic


MY NOTES on microscopy methods at lowy cancer institute

Tirf- total internal reflective fluorescence

-Exciting molecules that almost in direct contact with the cover glass -must find optimum angle at which light strikes the slide for optimum viewing -layer by layer viewing -capture the whole cell but just illuminate one layer WTF


Live cell imaging - keep cells happy - 37 degrees, oxygen etc, CO2 for buffering ph - heat the whole microscope or chamber so u don't get focus drift - tropomyosin tagged with flurophore -


Wide field you see all fluorescence coming from cell where as confocal you are viewing the light coming from just one optical plane Confocal uses a pinhole to eliminate out of focus light. Only light in focus on the desired area is viewed.

Resolution-classic exam question- the ability to see two different things as different things. Look it up. 1) wavelength 2) numerical aperture. / the number after the slash gives an indication as to how well the microscope lens resolves light. The higher the # the better.

Don't like exposing cells to light for too long because you get photo toxicity and bleaching of the cell.

Z stack is x-y plane stacked axially/layered for as many stacks as u ask-giving us 3D imaging up to the thickness of the z stack

Disadvantage of confocal is the time factor because the whole image is not scanned at once- only but by piece- so u have to wait.


LAB 8

--Z3378280 15:06, 3 May 2012 (EST) my notes two types of tissue culture 1)cell line- same properties, never changing, stored in banks (USA, Europe), grow continuously given right conditions- able to buy samples(price depends on type of cell), shipped frozen

2) primary culture- cells are derived from rat, human etc, eg neurons- which dont divide so must be taken directly from the tissue

Culture method

-adherent cultures, separated from subtrate 1. mechanically 2. enzymatic eg trypsin which digests adherence proteins that the cells use to stick to the dish with. another example is EDTA which removes calcium which is necessary for the cell to bind to the substrate -semi-adherent - not very common eg monocytes (replicative of how they travel in blood) -suspension culture

Cell media

-must be right conditions for cell eg pH of 7.4 (usual pH in the body) - use bicarbonate as buffer in incubator -incubator provides darkness, 5% CO2, at 37 degrees, water for humidity, (BE CAREFUL OF FUNGI GROWING IN INCUBATOR), different metabolic requirements for different cells -serum -antibiotics, doesnt kill cells, just stops them from dividing, covers sloppy work and method

Lab 8 questions

1)Rhabdomyosarcoma RD (CCL-136)

Rhabdomyosarcoma RD (CCL-136)

2)Tissue:Muscle Rhabdomyosarcoma from 7 year old female caucasian

3) T R Chen, C Dorotinsky, M Macy, R Hay Cell identity resolved. Nature: 1989, 340(6229);106 PubMed 2739733


LAB 9

--Z3378280 15:38, 10 May 2012 (EST)

my notes

-indications of bacterial contamination include cloudiness, change in pH (perhaps indicated by change in colour), slight whiteish films -fungal contaminations may demonstrate spores or fuzzy growth -mycoplasma infections can be checked for by PCR or by DNA screening or commercial group mycoplasm contamination screening

-to maintain a good cell line you must continually check for any contaminations

- neurons need a high glucose media, as in the body where the brain uses 25% of all glucose

-antibiotics have a limited cell life so must be changed every few days

-to count cells, use a grid. but dont count every cell, count one square and then multiply by the total number of squares

-dont want to count dead cells, only live cells, so add trypan blue to tell which cells are alive

-machine is used to count cells; tells you how many are alive and how many are dead

-cell storage is important so that we can work to our own timeline and not that of the cell -store shorterm using -80 degrees celcius - you dont want ice crystals in the cells because they will puncture the membrane and kill the cell

LAB 9 asessment is to make comments on the other projects; paste comments here

Disease Aetiology/Pathogenesis Signs and Symptoms Treatment Image
Autoimmune Lypmphoproliferative Syndrome (ALPS) Fas germline or somatic mutation at the intracellular death domain results in defective lyphocyte apoptosis and consequent imbalance in lyphocyte homeostasis Enlargement of lymphoid organs such as slpeen and lymph nodes. Organ specific symptoms include dermatitis, thyroiditis and hepatitis Splenectomy, Immunosuppressants eg corticosteroids
Non-small cell lung cancer Two mechanisms:1) downregulation of Fas receptor expression in tumor cells due to Fas gene polymorphisms (anti-apoptotic effect)

2)Upregulation of Fas ligand expression by tumor cells, triggering apoptosis of tumor-targeting immune cells (protective for tumor cells and due to FasL polymorphisms)

Non-small cell lung cancers include squamous cell carcinomas, adenocarcinomas and large cell carcinomas. Often silent till late stage progression,but possible symptoms are persistent cough or pneumonia, haemoptysis and airway obstruction. Chemotherapy and infrequently surgical resection
Parkinson's Disease (PD) Apoptosis of neurons via extrinsic pathway due to increased production of TNF-alpha and TNF-alpha receptor (one of many implicated pathways in (PD)) 4 cardinal symptoms: Tremor, postutal instability, muscle rigidity, brady kinesia. other possible symptoms include anxiety, constipation, depression, anosmia, fatigue There is no cure for PD but it can be managed. Prevention of dopamine metabolism (MAO type B inhibitors), dopamine replacement therapy (Levodopa). Surgery is an option but only on a case by case basis, it is not appropriate for everyone.
Gastric Carcinomas Gastric carcinomas have a complex, multifactorial aetiology and pathogenesis, and it has been shown that mutations in the gene coding for pro-caspase-8 play a central role in advanced stomach cancer. Defective caspase-8 → dysfunctional extrinsic apoptosis → tumour cell proliferation. Very similar to those of chronic gastritis- epigastric pain, heartburn, chest pain, nausea and vomiting. Surgical resection, chemotherapy, eradication therapy for H. Pylori
Leprosy (Hansen's Disease) Infection with Mycobacterium lepra and mycobacterial agonist production leads to an acute inflammatory response accompanied with wide spread and accelerated cell death due primarily to TNF-alpha release by monocytes Numbing of hands and feet due to nerve damage which leads to infection due to inability to react to painful stimuli. These consequent infections can culminate in finger and toe loss or necessitate amputation. Paralysis may cause the fingers and toes to curl up permanently. Leprosy is curable but if untreated can lead to severe deformities. Although a know teratogen, Thaliomide was approved and recommended by WHO for treatment of leprosy in 1998. Thaliomide inhibits production of TNF-alpha by accelerated degradation of the mRNA which codes for the pro-apoptotic ligand. Many other treatments exist.


Lab 9

--Mark Hill 12:54, 17 May 2012 (EST) You have not completed the peer assessment process yet. If you have made comments on each project page they need also to be pasted here today for me to include in your individual assessment.

Peer assessment

Group 1

Since most peer assessments before me have given orderly and insightful feedback for each subheading and section of the project, I wish to do my feedback differently. Unlike most before me, I am not going to go through each section and comment(which has been helpful), but rather I am going to see how the whole project scales up against the marking criteria provided by Mark at the start of semester. This different style to all the other peer assessments will hopefully allow the groups to align their projects with the criteria and will provide a new viewpoint and perspective for them to work from. Thanks.

• Firstly, good choice of topic, especially with the London Olympics coming up and all the cheating that goes on now days. Maybe, just for the readers interest and curiosity, include a brief segment on advantages and disadvantages of getting on the ‘roids’ and how cheats use it these days

• Key points and their descriptions are all present and explained clearly. Since the focus is cell signalling, it would enhance the projects quality if you included a little extra on the intracellular signalling and pathway which results from testosterone binding to its receptor.

• Citation and referencing is done extensively, appropriately and correctly, with a good range of articles from recent years 1990s-2011(therefore up to date information is guaranteed)

• Good images/text ratio and an appropriate level of depth and detail is employed, except in the signalling pathway

• Follows Wiki guidelines in format, editing, structuring, referencing and has that ‘element of teaching’ that Mark has asked for

• Overall, well rounded Wiki page which to adheres to the learning aims of Cell Biology , but it will need to be run through a fine tooth comb (grammar, structure) before submission, just to polish it up to the same level of effort that seems to have gone into its making.


Group 2


• Key points are all present but still need touching up. Normal function is not exactly lucid. “Vascular endothelial growth factor plays a crucial role in angiogenesis and vasculogenesis as well as the proliferation, migration, and survival of endothelial cells” --mentioned during intro but not again in normal function—probably should be.

• Choice of content, headings and subheadings etc seems to reflect broad knowledge on topic however this knowledge needs to be conveyed in a manner that peers will understand. For example, clearer explanations of the signalling pathway and the relevance of the different types of VEGF.

• Good range of references is used and they all come in a decent date range, so it’s up to date.

• Use of images and diagrams is effective and is to be complemented

• Evidence that the group is able to conduct suitable research is present. Has a informative approach but probably needs more of that teaching element to peers: remember we haven’t spent the time researching the subject, but still want to know all about it


Group 4


• Not all key points are described or present eg abnormal functioning. More detail is needed on proteins and receptors and history and future research need more body

• Detail of normal functioning must be commended- if the entire project delved into the same level of detail as the normal functioning section does, then it would be comprehensive to say the least.

• Inclusion of relevant and interesting videos is an absolute winner idea but more images and diagrams are needed

• More information is the key. This will come good if more research articles are identified and used- this will also lead to improvement of your glossary and reference list. Remember that the length of your reference list is usually a good indicator as to how detailed and deep your research has been, proving you didn’t find one article and copy all its good points

• The evidence of the capability to conduct in depth research and information gathering is evident, so it just seems that time is the key- give it the required time and the project will come together



Group 5

• This project has adhered closely to the marking criteria and learning aims of cell biology

• For FAP in abnormal fucntion you have mentioned that one mutated allele is inherited at birth. It would be a good idea to mention that this is therefore heterozygous at this genetic locus, and therefore there one inherited bad copy, and one inherited good copy. For the disease process to start, the individual undergoes “loss of heterozygosity” which means the good copy of the allele is sporadically mutated (caused by radiation etc) and therefore two mutated copies are present and polyps grow out of control. But I am being nit-picky.

• All key points relating to the topic are present

• The choice of content, headings and sub-headings, diagrams, tables, graphs show a good understanding of the topic area

• The project is full of proof that the topic has been researched to a suitable depth

• Contains and element of teaching and does this through dot points, text, diagrams, images and videos

• Correct and citation and referencing


Group 6

• Key points, titles, subheadings are all present, except future research

• More diagrams, tables and images. For example, maybe a slide of normal histology of islet of Langerhan compared to a slide of the histology present in Type 1 diabetes. This is a just a quick example and this sort of thing will aid you in satisfying the marking criteria

• If the diagram that represents the structure of insulin is your ‘hand drawn’ image, I politely suggest you do it again by hand or find another one to draw, because it a bit to basic for this level of assessement. I am sure Mark is not expecting a work of Picasso, but I think he’d rather something more substantial. If this image is not your ‘hand drawn’ image, I still suggest that you replace it, its too basic. Then the next image goes to the opposite extreme and is probably too detailed for this level. All that being said, more images etc will add to the aesthetics of your project and more importantly to the “teaching element” that is aimed at your peers

• Correct citations and referencing is present which demonstrate that the research is being done.Seems to have developed and edited the Wiki entries in accordance with the sites guidlines


Group 7


• Most key points are present except for current and future research which are important, we’re all keen to know what direction this topic is going in the future

• The choice of content, headings and sub-headings, diagrams, tables, graphs demonstrates appropriate level of understanding of the signalling pathway which is the focus and it suitably provides a teaching element- if i wanted to get a quick detailed grasp on beta-1-adrenoreceptors, then this page would provide just that

• Student drawn diagram is excellent, showing that care, time and detail are going into this projects compilation. This is also demonstrated by

• Relates the topics and content of the Wiki entry to learning aims of cell biology

• If possible it’d be good to have macroscopic images for the diseases involved with you pathway, for example, show us a hypertophic heart or a dilated heart along side a normal, just to hit home the severity of the effects that can happen when a normal cellular signalling pathway has turned defective

• Well done on the drug inclusion to treat the diseases you mentioned. The drugs are most often aimed at the signalling pathways.That way on your page we get the physiological pathway, the pathological pathway when the normal pathway is disrupted and then the pharmalogical pathway which is when the drugs get involved. You know students know there stuff when they can talk about the physiological, pathological and pharmalogical pathways involved in a disease. This is evidence of great research.

Group 8

• Key points headings etc are all used but the project lacks images, you could include some electron micrographs and possible macroscopic images

• Individual proteins are described at length, evidence of solid research. Compilation of the text is weel done- reads well and easy to follow. Includes that teaching aspect which peers enjoy

• Citations and references are done correctly as well as editing in line with Wiki guidelines

• This project contains its material within the learning aims of cell biology


Group 9

• Lacking in some key points. Introduction provides a good back ground, but should also outline the basic aims of the project

• Pathway is non-cohesive and hard to follow. Due to the crucial importance of p53 (guardian of the genome) physiologically and oncogenically, I am sure you can find plenty of info that would allow you to map out an detailed but understandable pathway. Remeber the focus of this project is cell signalling, so if there is anything that you should absolutely hit the mark with, it is the signalling pathway.

• Information regarding receptors and proteins is lacking

• Again, due to its physiological importance, p53 mutations also have a huge pathological role and so i am sure that you can find more information on abnormal functioning

• Images are needed to enhance, learning, curiosity, and aesthetics

• Referencing and history are good


• You need to develop more material, expand on some key points, add images and i am sure the project will come together



LAB 10

in class

--Z3378280 14:34, 17 May 2012 (EST)


Lab 11

Brief summary of key improvements suggested by our peers for our project (extrinsic apoptosis, group 3)

• Referencing and Citations- at the moment non-existent- MUST be done

• Introduction needs to be simplified and less complex. Also maybe a little too long. Add an image. Everything outlined in the intro should be expanded on in the project


• Tabulate the history? Overall was good but still needs references (more than one). Needs summarising, too detailed here and there.

• Signalling pathway needs to have more flow: where does it start and where does it finish. Use a diagram. Include more proteins


• Function needs to move away from the signalling pathway and talk about what extrinsic apoptosis is actually used for.

• Current research is good. Include image of possible. Do we have future research?


• Need glossary, abnormal functioning, morphology, images, diagrams, tables, references, inhibitors

• Need to get a better structure happening, and improve aesthetics (diagrams, tables, images)


Final Lab

--Z3378280 16:05, 31 May 2012 (EST)

1)Next generation gene sequencing

2) Sebastian Treusch, Susan Lindquist An intrinsically disordered yeast prion arrests the cell cycle by sequestering a spindle pole body component. J. Cell Biol.: 2012, 197(3);369-79 PubMed 22529103


3)microarray gene expression analysis was performed on the gene Rnq1 to assess toxicity. When there is Rnq1 overexpression, there is resultant elevated transcription of several chaperones and stress-related proteins.