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lecture 4

External Link


Identification of a new widely expressed STIM1 isoform.gif

Lab Homework Week 2

Identify a reference article that uses the "superresolution" microscopy technique.

Brown TA, Tkachuk AN, Shtengel G, Kopek BG, Bogenhagen DF, Hess HF, Clayton DA. Superresolution fluorescence imaging of mitochondrial nucleoids reveals their spatial range, limits, and membrane interaction. Mol Cell Biol. 2011 Dec;31(24):4994-5010. Epub 2011 Oct 17.

PMID: 22006021

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

What did the paper show that normal microscopy could not show?

Under normal microscopy, as demonstrated by Brown et al, there is no clear line of demarcation between mitochondrial nucleoids. As a result of this, many questions about their exact locations in relation to one another, as well as their functions and constituents, etc are left unknown.

Through the use of PALM and iPALM, superresolution fluorescence microscopy was shown to be far superior to normal imaging. They were then able to observe “the core dimensions and relative locations of mitochondrial nucleoides at an unprecedented resolution.” Brown et al, has illustrated through both two- and three-dimensional views that the nucleoids vary in size and shape, that the mitochondrial DNA located inside the nucleoids are “extraordinarily condensed” in ellipsoidal nucleoids and that they are “closely associated” with the inner membrane. This information in itself is enough to testify the advantages of superresolution fluorescence microscopy.

Lab Homework Week 3

Locate a current SDS for one of the fixatives described in today's lab. Identify the properties and hazards associated with that chemical.

Formalin (Formaldehyde)

Chemical and Physical Properties

Appearance: Clear Liquid.

Odour: Pungent Odour.

pH: Not Available.

Vapour pressure: Not Available.

Vapour density: Not Available.

Boiling point/range: BP ~ 100°C. MP ~ 0°C.

Freezing/melting point: Not Available.

Solubility: Soluble in all proportions.

Specific gravity or density: About 1.02.

Flash Point: 72°C.

Flammable (explosive) limits: Not Available.

Ignition temperature: Not Available

Hazards Identification

Hazard Classification: Hazardous according to criteria of NOHSC.

Risk Phrases: R40, Possible risk of irreversible effects. R43, May cause skin sensitisation by skin contact.

Safety Phrases: S1/2 Keep locked up and out of reach of children. S16 Keep away from sources of ignition - No smoking. S24/25 Avoid contact with skin and eyes. S26 In case of contact with eyes, rinse immediately with plenty of water and contact a doctor or poisons information centre. S36/37 Wear suitable protective clothing and gloves. S45 In case of an accident or if you feel unwell, contact a doctor or poisons information centre and show this container or label. S51 Use only in well ventilated areas.

Identify 4 papers required for your group work project.

1. Yuan JS, Kousis PC, Suliman S, Visan I, Guidos CJ. Functions of notch signaling in the immune system: consensus and controversies. Annu Rev Immunol. 2010 Mar;28:343-65. PMID: 20192807

Julie S Yuan, Philaretos C Kousis, Sara Suliman, Ioana Visan, Cynthia J Guidos Functions of notch signaling in the immune system: consensus and controversies. Annu. Rev. Immunol.: 2010, 28;343-65 PubMed 20192807

This article reviews recent advances in terms of Notch signaling as well as tries to clearly identify Notch functions within immunological processes. It is useful in terms of getting an overview into the history of the gene as well as gaining more of an understanding into the actual pathway itself and its normal function.

2. Liu J, Sato C, Cerletti M, Wagers A. Notch signaling in the regulation of stem cell self-renewal and differentiation. Curr Top Dev Biol. 2010;92:367-409. PMID: 20816402

Jianing Liu, Chihiro Sato, Massimiliano Cerletti, Amy Wagers Notch signaling in the regulation of stem cell self-renewal and differentiation. Curr. Top. Dev. Biol.: 2010, 92;367-409 PubMed 20816402

This article reviews notch signaling pathways at multiple stages of organismal development. It would be useful to gain a broader understanding of the pathway and how it is considered useful within the human body.

3. Radtke F, Fasnacht N, Macdonald HR. Notch signaling in the immune system. Immunity. 2010 Jan 29;32(1):14-27. PMID: 20152168

Freddy Radtke, Nicolas Fasnacht, H Robson Macdonald Notch signaling in the immune system. Immunity: 2010, 32(1);14-27 PubMed 20152168

This article talks about the abnormal funtionings of notch signaling as well as current research as to how these are associated with several human disorders including hematopoiesis, lymphocyte development and T cell acute lymphoblastic leukemia. It would be useful to gain more of an insight into current research and how abnormalities in the pathway can lead to dysfunctions within the body.

4. Carlson ME, O'Connor MS, Hsu M, Conboy IM. Notch signaling pathway and tissue engineering. Front Biosci. 2007 Sep 1;12:5143-56. PMID: 17569636

Morgan E Carlson, Matthew S O'Connor, Michael Hsu, Irina M Conboy Notch signaling pathway and tissue engineering. Front. Biosci.: 2007, 12;5143-56 PubMed 17569636

This article talks about the dysfunctions of Notch signaling pathway and how it has been linked to the pathogenesis of several inherited human diseases. It describes the components within signal tranductions plus outlines the role of the pathway and finally summarises current research within the area.

Lab Week 4

Okano H, Kawahara H, Toriya M, Nakao K, Shibata S, Imai T.Function of RNA-binding protein Musashi-1 in stem cells.Exp Cell Res. 2005 Jun 10;306(2):349-56. Epub 2005 Mar 24. PMID:15925591

Hideyuki Okano, Hironori Kawahara, Masako Toriya, Keio Nakao, Shinsuke Shibata, Takao Imai Function of RNA-binding protein Musashi-1 in stem cells. Exp. Cell Res.: 2005, 306(2);349-56 PubMed 15925591

Both Musashi 1 and 2 are RNA-binding proteins which is significant in asymmetric cell division of ectodermal precursor cells. It does this by regulating the translation of target mRNA. Both types contribute to the maintenance of neural stem cells.

Musashi Antibody identifies endogenous levels of both proteins. Polyclonal antibodies are produced by immunising animals in resopnse to human Musashi.

Description: immunologically called Rabbit IgG H&L antibody and has a molecular weight 35kDa.

Target Species: Rabbit

Recommended Antibody Dilutions taken from Cell Signaling:

Western blotting = 1:1000

Immunofluorescence = (IF-F) 1:25

The following information has been taken from Cell Signaling:

Secondary antibody: Goat anti-Rabbit IgG H&L (Chromeo™ 488) secondary antibody

Description: molecular weight of 39 kDa

Specificity: ab60314 reacts with whole molecular rabbit IgG and the light chains of other rabbit immunoglobulins. No cross-reactivity with non-immunoglobulin serum proteins observed.

Recommended Antibody Dilutions taken from Cell Signaling:

Immunofluorescence = 1:5000

Lab Homework Week 7

Analysis of morphological phenotypes in Tm4 overexpressing B35 vs control.JPG

Describe if there is a difference in the phenotype

Pygnotic - cell count of Tm4 overexpressing B35 3% less than control

Stringed - cell count of Tm4 overexpressing B35 16% less than control

Pronged - cell count of Tm4 overexpressing B35 7% more than control

Stumped - cell count of Tm4 overexpressing B35 18% more than control

Broken Fan - cell count of Tm4 overexpressing B35 13% less than control

Fan - cell count of Tm4 overexpressing B35 8% more than control

How did tropomyosin lead to a change in the phenotype?

Tropomyosin is an actin-binding protein with the primary function of regulating actin activity. Tropomyosin is said to be involved in "the motile events of neurite growth and synaptic plasticity" which would explain why pygnotic, stringed and broken fan phenotypes were more common in the control, whereas pronged and stumped were found in abundance in the Tm4 overexpressing B35 neuroepithelial cells.

L Had, C Faivre-Sarrailh, C Legrand, J Méry, J Brugidou, A Rabié Tropomyosin isoforms in rat neurons: the different developmental profiles and distributions of TM-4 and TMBr-3 are consistent with different functions. J. Cell. Sci.: 1994, 107 ( Pt 10);2961-73 PubMed 7876361

Lab Homework Week 8

still researching and collaborating through pathway and trying to understand more about the different receptors and proteins involved in the process.

Lab Homework Week 9

  1. Identify a mammalian cell line in the ATCC catalogue (and add a link)

CCL-147™ from the common house mouse. Organism called Mus musculus.

  2. Identify the original tissue of origin of that cell line.

From the Mus musculus, the original tissue of origin of the cell line is the brain.

  3. Identify the original paper that characterised the properties of that cell line.

Buonassisi V, et al. Hormone-producing cultures of adrenal and pituitary tumor origin. Proc. Natl. Acad. Sci. USA 48: 1184-1190, 1962. PubMed: 13874682 V BUONASSISI, G SATO, A I COHEN Hormone-producing cultures of adrenal and pituitary tumor origin. Proc. Natl. Acad. Sci. U.S.A.: 1962, 48;1184-90 PubMed 13874682

Burn JH, Rand MJ. Acetylcholine in adrenergic transmission. Annu. Rev. Pharmacol. 5: 163-182, 1965. PubMed: 14290806 J H BURN, M J RAND ACETYLCHOLINE IN ADRENERGIC TRANSMISSION. Annu Rev Pharmacol: 1965, 5;163-82 PubMed 14290806

Lab Homework Week 10 (Peer Assessment)

GROUP 1 – Testosterone

Introduction – articulate intro but there needs to be copyright details for the image

History – the table was a very great idea it broke everything down and the colour made it a little more fun + well researched

Biosynthesis – liked the correlation between the table and picture, correctly referenced however maybe put this subheading under something else, im not sure it needs its own section?

Regulation – coherent and easy to understand as well as correctly referenced, not as much information but I don’t think it needs it

Signalling Pathway – liked the use of subheadings

Normal Function – I think you need a picture or a table to break the writing up make it a little more visual

Abnormal function – liked the use of subheadings

Clinical uses – the table was a very good idea, information was succinct and well referenced, the video link was a good idea made it more exciting for the reader

Current and ongoing research – informative and easy to understand

Glossary – a very good idea

Images – that one image (same image as before) must have copyright details

References – an impressive list

Overall - an extremely succinct and well-researched project, be a little careful about grammar and in-text citations

GROUP 2 – Vascular Endothelial Growth Factor

Introduction – colourful picture draws me in instantly

History – use of the table was a good idea, made the information easy to follow, perhaps consider the use of colour? Well referenced

Normal Function – subheadings made text relatively simple to follow; only concern is the bullet points? Not sure if its intentional

Signalling pathway – this section was very good, the table and picture helped make the entire pathway clear; the information was succinct, really good job

Abnormal function – very well researched; the use of images in the table drew me in; the gruesome pictures were a weird and fun way of dragging the reader in

Research – again well-researched, use of table and photos keeps reader engaged and correctly referenced

External Links – maybe consider finding a few more helpful external links, the sheading seems a little random with just one link and one line describing it

References – list isn’t too shabby at all

Overall – this assignment has been well-planned well-researched and very neat. Good work guys

GROUP 3 – Extrinsic Apoptosis

Introduction – succinct introduction and good length however consider putting an image or table in, something to attract readers’ eye, great definition

History – well researched, think about putting a table in though, just makes the information a little easier to follow

Signaling pathway – for this section I can see you have done an impressive amount of research but what about an image like a flow chart or a diagram just so we can understand the pathway a little better

Function – still very well researched however lacking a little visual appeal

Current research – well-referenced diagram easy to follow research I like that you’ve broken down review articles and summarized them paper by paper

References – ?

Overall – really well researched but is lacking something to pull the reader in

GROUP 5 – Wnt/beta-catenin Signaling Pathway

Introduction – witty intro

History – really good picture, well-researched extensive information

Mechanism of Action – liked use of sub-headings and figure, broke everything down and very simple to follow

Diseases associated with WNt-beta-catenin signaling – figures were good and table was excellent really good way to follow information, only thing is maybe consider putting in more visuals in another column next to mutations

Key players – really good section, well referenced, pictures look great and very easy to follow; there are some unfilled sections though; is that intentional?

Embryonic development – great photo, concise text

Future directions – needs more information, maybe talk about recent research articles you’ve come across and summarise them or put more in-text refernces into the other bullet points

Reference list – very extensive list

Overall – very well researched, looks really good guys

GROUP 6 – Insulin

Introduction – really good intro but maybe consider putting in more citations

Structure of Insulin – like the use of picture very succinct doesn’t really need many words though very self explanatory

History – well researched maybe consider expanding on some of the experiments

Insulin receptor – great student drawn image, very simple and easy to follow

Signaling pathway – was able to correlate the diagram with the information + correctly referenced

Normal function – very concise but I think it works, it has all the information any reader could possibly need

Abnormal function – very well researched and the information is good only thing is it’s a bit all over the place, perhaps if you actually put the definitions etc underneath the actual conditions when you list them in the beginning, I wouldn’t want to keep scrolling up and down

Current research – the Wilson research institute I think you should maybe add an external link to their website if they have one?

Overall – a lot of effort has gone into this, keep up the good work and you guys are on the way to an excellent project

GROUP 7– G-protein coupled receptors

Introduction – very well researched but too much information for an introduction, its purpose is to just briefly introduce

History of the pathway – well covered

Gene description – very concise maybe consider putting the information in a diagram

Receptor Agonists – good use of visuals

Receptor structure – very clear

Pathway and normal function – maybe find or draw a few more diagrams just to further highlight and contrast things like the two regulatory mechanisms

Abnormal function, diseases and treatments – a very well referenced and informative table, toward of the end the amount of information is a little overwhelming maybe consider putting in another visual or so or put more colourful diagrams?

Overall – really great work, a lot of time and hard effort has gone into this

GROUP 8 – Leukocyte extravasation

Introduction– really clear succinct intro

Pathway – good description but try and make the diagram bigger for convenience

History – absent

Normal function – very well researched + easy to comprehend

Abnormal function – a lot of information and well researched too with ample citations however maybe consider putting in a few visuals

Proteins – really good use of diagrams and they correlate with information nicely

Overall – really good work, lots of references, more intext references needed

GROUP 9 – p53 Signalling Pathway

Introduction – good intro perfect size + well referenced however I don’t think student signatures are necessary

Pathway – well described perhaps make diagram bigger though?

History – history is very well researched and very visually appealing

Current research – good you have a good start but it needs a little more to it

Normal function – very well broken down into subheadings and very easy to understand

Abnormal function – id still love to know more about the abnormal function

Overall – quite a bit of research has been accomplished, good presentation now just add in a bit more text and a few more visuals, you guys are almost there

--Mark Hill 12:33, 17 May 2012 (EST) We'll structured review of each project using the same criteria, and internally consistent. I would have liked some information around the assessment criteria in addition to your overview of the projects structure.

Lab Homework Week 12

  1. Identify a current technique used in gene sequencing.

Next Generation Gene Sequencing

  2. Identify a recent cell biology research paper that has used microarray technology.

Kathryn M Munro, Victoria M Perreau, Ann M Turnley Differential gene expression in the EphA4 knockout spinal cord and analysis of the inflammatory response following spinal cord injury. PLoS ONE: 2012, 7(5);e37635 PubMed 22629434

  3. What aspect of the research findings were contributed by the microarray technique.

A microarray was performed on spinal cord tissue from mice with spinal cord injury to try and understand the mechanisms by which EphA4 modified the response to neural injury.