User:Z3331812

From CellBiology

Lab Attendance

--Z3331812 15:16, 8 March 2012 (EST)

--Z3331812 14:12, 15 March 2012 (EST)

--Z3331812 14:08, 22 March 2012 (EST)

--Z3331812 14:05, 29 March 2012 (EST)

--Z3331812 16:05, 5 April 2012 (EST)

--Z3331812 14:16, 19 April 2012 (EST)

--Z3331812 14:16, 26 April 2012 (EST)

--Z3331812 14:02, 3 May 2012 (EST)

--Z3331812 14:08, 10 May 2012 (EST)

--Z3331812 14:17, 17 May 2012 (EST)

--Z3331812 15:54, 24 May 2012 (EST)

--Z3331812 14:08, 31 May 2012 (EST)

Group 9

2012 Group 9 Project

User:Z3272558

User:Z3272212

User:Z3293029

Useful Links

Textbook

The Cell

Lectures

Lecture 1 - Cell Biology Introduction

Lecture 2 - Cells Eukaryotes and Prokaryotes

Lecture 3 - Cell Membranes and Compartments

Lecture 4 - Cell Nucleus

Lecture 5 - Cell Export (Exocytosis)

Lecture 6 - Cell Import (Endocytosis)

Lecture 7 - Cell Mitochondria

Lecture 8 - Cell Junctions

Lecture 9 - Cytoskeleton Introduction

Lecture 10 - Cytoskeleton (Intermediate Filaments)

Images

Autophagy of Mitochondria .jpg

A double membrane surrounds organelles such as mitochondria (A) during autophagy.

Reference

<pubmed>13862833</pubmed>


Rockefeller University Press Copyright Policy This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.jcb.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).

Lab 2 Homework

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

<pubmed>18077327</pubmed>

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

Shroff et al demonstrated that when using supperresolution microscopy techniques, particularly two-colour photoactivated localization microscopy (PALM), some molecular interactions inside cells are seen more clearly and are therefore able to be correctly identified. Normal microscopy showed that several pairs of proteins were “colocalized”. This means that there are two or more different fluorescent labels that were partly covering each other, preventing a clear image. Superresolution, however, showed “distinct interlocking nano-aggregates” of proteins, allowing us to see the spatial relationship between two proteins in whole, fixed cells. For example, Shroff et al demonstrated that superresolution clearly showed one type of protein (α-actinin) to be denser in a specific area but vinculin was spread out. Distinguishing these two proteins to observe their functions cannot be done using normal microscopy due to “colocalization”.

--Mark Hill 12:41, 20 March 2012 (EST) Well done, good summary of this papers findings.

Lab 3 Homework

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

Formalin

Properties

Appearance CLEAR COLOURLESS LIQUID

Solubility (water) SOLUBLE

Odour PUNGENT IRRITATING ODOUR

Specific gravity 1.1 (Approximately)

pH 2.4 to 4.0 % Volatiles > 44 %

Vapour pressure NOT AVAILABLE

Flammability CLASS C1 COMBUSTIBLE

Vapour density 1.04 (Air = 1)

Flash Point 85°C (Approximately)

Boiling point < 100°C

Upper Explosion Limit 73 %

Melting point NOT AVAILABLE

Lower Explosion Limit 7 %

Evaporation rate NOT AVAILABLE

Autoignition temperature 430°C

Decomposition temperature NOT AVAILABLE

Partition coefficient NOT AVAILABLE

Viscosity NOT AVAILABLE

Hazards

Health Hazard Summary: Toxic - corrosive: This product has the potential to cause adverse health effects. Use safe work practices to avoid eye or skin contact and inhalation. Contact may result in burns with possible tissue damage. May cause sensitisation by skin contact. Formaldehyde is classified as a confirmed human carcinogen (IARC Group 1). Chronic exposure may result in cell mutations, reproductive system effects, liver damage and insomnia. Chronic exposure to methanol may result in optic nerve damage.

Eye: Corrosive - irritant: Contact may result in irritation, lacrimation, pain, redness, corneal burns and possible permanent damage.

Inhalation: Toxic - corrosive: Over exposure may result in mucous membrane irritation of the respiratory tract, coughing, chest pain and sensitisation with asthma-like symptoms, breathing difficulties, pulmonary oedema and convulsions at high levels. Chronic exposure may result in liver damage and fertility effects (sperm count and viability, increase in spontaneous abortions).

Skin: Corrosive: Contact may result in irritation, redness, pain, rash, dermatitis and possible burns. May cause sensitisation by skin contact.

Ingestion: Toxic - corrosive: Ingestion may result in gastrointestinal ulceration, nausea, vomiting, abdominal pain, acidosis and diarrhoea (bloody). Ingestion of large quantities may result in liver and kidney damage, pulmonary oedema, unconsciousness and death which may be delayed.


2. Identify 4 papers required for your group work project. Cite on the Group Project discussion page and also on your own Individual page. Add one sentence for each as too why they are relevant to your group topic.


<pubmed>22410433</pubmed> This review discusses the regulation of p53 and how possible anti-cancer treatments could be developed using proteins.


<pubmed>14534297</pubmed> This article explains the structure of p53 and how it is mutated, particularly in carcinomas.


<pubmed>22085928</pubmed> This article highlights the importance of a specific enzyme in the regulation of p53 during and after stress to a gene.


<pubmed>22454691</pubmed> An interesting experiment on proposed pancreatic carcinoma treatment, involving the upregulation of p53 protein.

Lab 4

Musashi - Protein?

Musashi is an RNA binding protein which regulates the asymmetric cell division of ectodermal precursor cells through the translational regulation of target mRNA.

<pubmed>8043282</pubmed> It has a sequence length of 362 AA. UniProtKB

Antibody

Primary Antibody Msi1 antibody: Rabbit polyclonal to Musashi 1 / Msi1

Concentration: 100 µg at 1-1.4mg/ml

Applications: ICC/IF: Use at a concentration of 1 µg/ml.

WB: Use at a concentration of 1 µg/ml. Detects a band of approximately 39 kDa (predicted molecular weight: 39 kDa). Can be blocked with Musashi1 peptide (ab23870).

Is unsuitable for IHC-P. abcam

Secondary Antibody Musashi-1 (D46A8) XP® Rabbit mAb

Monoclonal antibody with a dilution of 1:200.

Labome

Lab 6

Analysis of morphological phenotypes in Tm4 over-expressing B35 neuro-epithelial cells - Group 1

Z Analysis of morphological phenotypes in Tm4 over-expressing B35 neuro- epithelial cells.JPG

Do you see a difference in phenotype (morphology) between Tm4 over-expressing and control cells? If so, how could Tm4 over-expression lead to this difference?

The Tm4 over-expressing cells had larger somas and more branching processes, compared to the control cells with no branching and longer processes extending from the soma. TM4 may be involved in the stages of neuritis growth in the last phenotypic stages.

Effect of Tm4 expression on db cAMP induced differentiation of B35 cells; Groups 1-3

Do you see a difference in phenotype (morphology) between Tm4 over-expressing and control cells? If so, how could Tm4 over-expression lead to this difference?

In the Tm4 over-expressing cells there are less stringed and pronged phenotypes when compared to the control. When there is dB cAMP in the cells, there is less neuritis growth because dB cAMP reduces Tm4 transcription.

Lab 7

To date, my contributions to the group project (9) include:

  • uploading an image for the p53 signalling pathway
Different gene expression in p53 signalling pathway
  • researching, creating and uploading a timeline for the history of the p53 molecule

1979: p53 was first described in studies on the SV40 virus [1][2]

1982-83: TP53 gene first cloned at the Russian Academy of Sciences [3] followed by further cloning of the human tumour antigen p53 [4]

1984: cloned p53 were tested for oncogenic properties [5][6]; p53 was inactivated in tumor cells [7][8]; and p53 was reactive to UV radiation [9]

1988: the murine (rodent) wild-type p53 sequence was confirmed [10][11]

1989: research shows that p53 has tumor suppressor gene characteristics, rather than oncogenic, and is assumed a tumor suppressor gene [12]

1990: p53 germline mutations (those that occur during meiosis) found in Li-Fraumeni syndrome [13][14] and cell proliferation studies show that p53 stimulates cell cycle arrest [15][16]

1990-1992: p53 is found to be a transcription factor (sequence-specific DNA-binding factor) in vitro [17][18][19][20]

1991: studies show that wild-type p53 induces apoptosis [21][22]

1992: the oncogene MDM2 acts as a negative regulator, preventing transcription [23]; p53 knock-out mice are found to be cancer-prone [24]; and p53 appears to maintain genome stability via cell cycle control and gene amplification [25][26]

1993: the p53/MDM2 feedback loop is established [27] and p21 is described as a potential mediator of p53 tumor suppression [28]. p53 is voted ‘Molecule of the Year’ by Science magazine [29]

1994: the first p53-DNA complex crystal structure is described in order to understand tumorigenic mutations [30]

1997: MDM2 is found to drive p53 ubiquitination and degradation [31][32]; p63 and p73 are described [33][34]; a connection between ARF and p53 is made [35]; and p53 is found to implicate senescence [36]

1998: in response to DNA damage, ATM phosphorylates p53 [37]

2000-2001: p53 is cloned in Drosophila [38] and C. elegans [39]

2002: p53’s role in organismal aging is established [40]

2003: apoptosis is induced via p53 acting on mitochondria [41]

2004: MDM2 polymorphism is found to accelerate cancer [42]; small-molecule antagonists of MDM2 are believed to activate the p53 pathway in cancer cells [43]; and p53 gene therapy is approved in China [44]

2005: multiple isoforms of p53 are identified[45]; an antioxidant function is discovered in p53 [46]; and p53 is found to regulate metabolism [47]

2007: p53’s importance in reproduction is revealed as it is required for embryo implantation [48]; p53-induced senescence prevents cancer in vivo [49][50]; p53 is found to regulate miRNA expression [51]; and p53 is found to inhibit the IGF-1/mTOR pathway [52]

2008: small molecule p53 activators discovered in vivo with potential for therapeutic interest [53]

2010: new approaches to cancer drug discovery as structures of inhibitors of MDM2 and MDMX are revealed [54]

  • added terms to the glossary of the group page which relate to the 'History' section I wrote

ARF: ADP Ribosylation Factor (ARF) is a member of the GTP-binding proteins responsible for regulating both COPI coat assembly and clathrin coat assembly at Golgi membranes. [55]

ATM: a protein that regulates several cellular responses to DNA breaks. [56]

C. elegans: Caenorhabditis elegans is a nematode (unsegmented) worm with very simple anatomy. [57]

Drosophila: Species of small fly, commonly called a fruit fly, much used in genetic studies of development. [58]

Gene therapy: The correction of a genetic deficiency in a cell by the addition of new DNA and its insertion into the genome. [59]

Germline mutation: present constitutionally in an individual (ie, in all cells of the body) as opposed to somatic mutations, which affect only a proportion of cells. [60]

IGF-1/mTOR pathway: sense the availability of nutrients and mitogens and respond by signaling for cell growth and division. [61]

Li-Fraumeni syndrome: A rare, inherited predisposition to multiple cancers, caused by an alteration in the p53 tumor suppressor gene. [62]

MDM2: a protein that normally inhibits the ability of p53 to restrain the cell cycle or kill the cell, is overexpressed in several cancers. [63]

miRNA: microRNA is a type of RNA found in cells and in blood. They are smaller than many other types of RNA and can bind to messenger RNAs (mRNAs) to block them from making proteins. [64]

Murine: rodent family, including rats and mice. [65]

Oncogenic: typically an oncogene is a mutant from a normal gene involved in the control of cell growth and division – it will make the cell act more cancerous. [66]

Senescence: as primary cell structures age, cell proliferation slows and terminates. [67]

SV40 virus: Simian virus 40, a polyoma virus of monkeys, which has been a model for the basic studies of viral pathogenesis and for cell transformation and neoplasia. [68]

Ubiquitination: protein inactivation which involves the attachment of ubiquitin to the protein. [69]

Lab 8

Cell line identified from ATTC:Catalog Search

Cell Line: CRL-1832™ - HIG-82

This cell line was derived from a synoviocyte from a young female rabbit's intrarticular soft tissue from the knee joint. The cells have retained many of the features of normal rabbit synoviocytes including production of cytokines that activate primary cultures of normal chondrocytes.

The article which originally characterized the properties of the HIG-82 cell line: Georgescu HI, et al. HIG-82: an established cell line from rabbit periarticular soft tissue, which retains the "activatable" phenotype. In Vitro Cell. Dev. Biol. 24: 1015-1022, 1988. <pubmed>2846503</pubmed>

Lab 9

Peer Review

Group 1: Order of headings makes it easy to follow, and the text is well referenced throughout. Not sure what ‘steriodogenesis’ diagram is for, it appears rather complicated. A few punctuation and grammatical errors were observed in the normal function section but that can easily be fixed before the due date. Also, there was no student drawn image on this page, which is required. The use of subheadings breaks up the large sections of text – effective way of keeping the reader interested. Displaying the history and clinical uses in tables is also an effective way of breaking up large sections of text.

Group 2: Text is short and succinct, supported by appropriate images. There is no evidence of a student drawn diagram – perhaps someone could draw the structure by hand, or draw another image for the signalling pathway section. Good idea to add the therapeutic applications section, it is very interesting. The abnormal function section is very comprehensive. Great idea to create links to the glossary, this saves the reader having to scroll and search for a word they may not understand.

Group 3: In terms of referencing, the introduction appears to be plagiarised because there are no references. Also, the referencing throughout the remainder of the project should be altered so that there are in text citations and a reference list. The instructions on how to do this can be found under the ‘Project Referencing’ link. The history section should really be a combination of various sources of your own finding, rather than taken from a timeline in a single journal article. There is only one image on your page, so perhaps to enhance its appeal, add some more images such as diagrams or structures and don’t forget you need a student drawn image. The existing text is interesting.

Group 4: Well researched, however some sections do not appear to be referenced, such as the ‘Protein and Receptors’ section and some of the ‘Pathway’ section. The page needs a title to make it clear what the topic is. Headings are placed in a ‘flowing’ order and the use of subheadings helps to break up large chunks of text. Perhaps more sections could be added, for example abnormal function? The videos are an effective way of engaging the audience and ‘teaching’ them, as the marking criteria for this project require.

Group 5: Overall, an extremely well researched project. Interesting use of the EMBO Conference to make the topic relevant to the audience. The history is comprehensive and the image in the mechanism section supports the text. Clever naming of ‘Diseases’ section rather than abnormal function like most other groups. Only faults I noticed were an incomplete ‘key players’ table and one or two of the references doubled up in the list – the instructions on how to change it so a reference only appears once are on the ‘Project Referencing’ page. Also, if time permits, maybe some of the abbreviations could be explained like the glossary to enhance the readers understanding.

Group 6: The project needs a title, even though it becomes obvious that the project is on Insulin Signalling, it helps to have it stated at the beginning. The ‘Introduction’, ‘Normal functioning’, the diabetes part of ‘Abnormal function’ and the Wilton Research Institute part of ‘Current Research’ needs more referencing, leaving it as it is might lead to plagiarism. In the ‘Glossary’, rather than just listing abbreviations, add a description to enhance the readers knowledge on the subject. It is more engaging when you understand what you are reading about. Good use of diagrams, they support the text and provide useful information.

Group 7: References in the introduction need to be put with the text they refer to. Remove the ‘signatures’ within the text and leave the signatures for the discussion page. ‘Beta-adrenergic receptors’ isn’t finished. Large sections of text are difficult to read and understand, and even though it is evident a lot of research has gone into this project, perhaps some sections should be summarised a bit more. Creative use of student images.

Group 8: The ‘History’ is not started, either is ‘Current research’. Possibly add an image to the ‘Abnormal pathway’ section to break up the large paragraphs of text. Also, there needs to be a student drawn image on your project. The ‘Pathway’ section is very easy to understand, well summarised and supported by an appropriate diagram. There are no references in the first half of the project; however the referencing in the remainder of the project is complete.


--Mark Hill 13:44, 17 May 2012 (EST) These are concise reviews of each project. You may consider also using the project assessment criteria when applying your own peer assessment.


Lab 12 - Microarray

1. Identify a current technique used in gene sequencing.

Next Generation gene sequencing. Next-generation DNA sequencing methods


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

<pubmed>21818335</pubmed>


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

The micro-array technique was used to display the control cells vs cells with altered gene expression. The cells displayed genes controlled by RORα in liver cells. After the miro-array technique, qRT-PCR analysis was used to amplify and quantify the target DNA molecule. This technique allowed opening of "new routes on the roles of RORα in glucose metabolism and carcinogenesis within cells of hepatic origin".

  1. <pubmed>218111</pubmed>
  2. <pubmed>222475</pubmed>
  3. <pubmed>6295732</pubmed>
  4. <pubmed>6296874</pubmed>
  5. <pubmed>6095116</pubmed>
  6. <pubmed>6088057</pubmed>
  7. <pubmed>6095069</pubmed>
  8. <pubmed>2858093</pubmed>
  9. <pubmed>6092932</pubmed>
  10. <pubmed>2832726</pubmed>
  11. <pubmed>3060794</pubmed>
  12. <pubmed>2649981</pubmed>
  13. <pubmed>1978757</pubmed>
  14. <pubmed>2259385</pubmed>
  15. <pubmed>2143581</pubmed>
  16. <pubmed>2143698</pubmed>
  17. <pubmed>2047879</pubmed>
  18. <pubmed>1301998</pubmed>
  19. <pubmed>1588974</pubmed>
  20. <pubmed>1614538</pubmed>
  21. <pubmed>1852210</pubmed>
  22. <pubmed>1584781</pubmed>
  23. <pubmed>1535557</pubmed>
  24. <pubmed>1552940</pubmed>
  25. <pubmed>1356076</pubmed>
  26. <pubmed>1525830</pubmed>
  27. <pubmed>8319905</pubmed>
  28. <pubmed>8242752</pubmed>
  29. <pubmed>8266084</pubmed>
  30. <pubmed>8023157</pubmed>
  31. <pubmed>9153395</pubmed>
  32. <pubmed>9450543</pubmed>
  33. <pubmed>9288759</pubmed>
  34. <pubmed>9774969</pubmed>
  35. <pubmed>9393858</pubmed>
  36. <pubmed>9054499</pubmed>
  37. <pubmed>9733514</pubmed>
  38. <pubmed>10778859</pubmed>
  39. <pubmed>11557844</pubmed>
  40. <pubmed>11780111</pubmed>
  41. <pubmed>12667443</pubmed>
  42. <pubmed>15550242</pubmed>
  43. <pubmed>14704432</pubmed>
  44. <pubmed>16149900</pubmed>
  45. <pubmed>16131611</pubmed>
  46. <pubmed>16286925</pubmed>
  47. <pubmed>15866171</pubmed>
  48. <pubmed>18046411</pubmed>
  49. <pubmed>17251933</pubmed>
  50. <pubmed>17251932</pubmed>
  51. <pubmed>17554337</pubmed>
  52. <pubmed>17409411</pubmed>
  53. <pubmed>18455128</pubmed>
  54. <pubmed>20237429</pubmed>
  55. Molecular Biology of the Cell. 4th edition. Alberts B, Johnson A, Lewis J, et al. New York: Garland Science; 2002.
  56. <pubmed>9733514</pubmed>
  57. Molecular Biology of the Cell. 4th edition. Alberts B, Johnson A, Lewis J, et al. New York: Garland Science; 2002.
  58. Molecular Biology of the Cell. 4th edition. Alberts B, Johnson A, Lewis J, et al. New York: Garland Science; 2002.
  59. Griffiths AJF, Gelbart WM, Miller JH, et al. Modern Genetic Analysis. New York: W. H. Freeman; 1999. Glossary.
  60. Morrison PJ, Spence RAJ. Genetics for Surgeons. London: Remedica; 2005. Glossary.
  61. <pubmed>17409411</pubmed>
  62. PDQ Cancer Information Summaries [Internet]. Bethesda (MD): National Cancer Institute (US); 2002-. Dictionary of Cancer Terms.
  63. Molecular Cell Biology. 4th edition. Lodish H, Berk A, Zipursky SL, et al. New York: W. H. Freeman; 2000.
  64. PDQ Cancer Information Summaries [Internet]. Bethesda (MD): National Cancer Institute (US); 2002-. Dictionary of Cancer Terms.
  65. mouse. (n.d.). Collins English Dictionary - Complete & Unabridged 10th Edition. Retrieved May 01, 2012, from Dictionary.com website: http://dictionary.reference.com/browse/mouse
  66. Molecular Biology of the Cell. 4th edition. Alberts B, Johnson A, Lewis J, et al. New York: Garland Science; 2002.
  67. Alberts B, Johnson A, Lewis J, et al. Molecular Biology of the Cell. 4th edition. New York: Garland Science; 2002. Glossary.
  68. <pubmed>9776239</pubmed>
  69. Ubiquitination. (n.d.) The American Heritage® Medical Dictionary. (2007). Retrieved May 1 2012 from http://medical-dictionary.thefreedictionary.com/Ubiquitination