Difference between revisions of "User:Z3289738"
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* correct referencing (eg. copyright)
* correct referencing (eg. copyright)
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* brief paragraph of my assessment
* brief paragraph of my assessment
* paste all the assessments onto your personal page.
* paste all the assessments onto your personal page.
Revision as of 07:10, 13 May 2012
- 1 Lab Attendance
- 2 Lab 1 - Introduction
- 3 Lab 2 - Microscopy
- 4 Lab 3 - Fixation
- 5 Lab 4 - Immunochemistry
- 6 References
- 7 Lab 5 - Cell Knockout Methods
- 8 Lab 6 - Cytoskeleton Exercise
- 9 Lab 7 - Confocal Microscopy
- 10 Contributions to Group 5 Project
- 11 Lab 9 - Tissue Culture 1
- 12 Lab 10 - Tissue Culture 2
- 13 Peer Assessment
- Lab 1: --Z3289738 15:29, 8 March 2012 (EST)
- Lab 2: --Z3289738 14:10, 15 March 2012 (EST)
- Lab 3: --Z3289738 14:04, 22 March 2012 (EST)
- Lab 4: --Z3289738 14:51, 29 March 2012 (EST)
- Lab 5: --Z3289738 13:58, 5 April 2012 (EST)
- Lab 6: --Z3289738 14:24, 19 April 2012 (EST)
- Lab 7 - excursion
- Lab 9: --Z3289738 14:32, 3 May 2012 (EST)
- Lab 10: --Z3289738 15:20, 10 May 2012 (EST)
Lab 1 - Introduction
- 'External link' has one [ ]
- 'Internal link' has two [[ ]]
Lab 2 - Microscopy
Uploading a picture onto the wiki:
- Save picture to desktop
- 'Toolbox' - 'Upload file'
- Copy and paste the title into the wiki, put [[__]]
Dear Mark, I realized that although my original image was very pretty, it may be subject to copyright so I've updated my picture to this one.
Homework Lab 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. (2011). Superresolution fluorescence imaging of mitochondrial nucleoids reveals their spatial range, limits, and membrane interaction. Molecular Cell Biology. PMID: 22006021
What did the paper show that normal microscopy could not show?
Superresolution fluorescence microscopy exceeded previous imaging techniques by allowing scientists to see within the small and highly compartmentalised mitochondria. Using PAML and iPALM techniques, scientists were able to visualize core dimensions and relative locations of mitochondrial nucleoids, discovering that they are much larger than previously anticipated.
Lab 3 - Fixation
* Tissue preservation for slides
Homework Lab 3
Locate a current SDS for one of the fixatives described in today's lab. Identify the properties and hazards associated with that chemical.
The SDS for Chloroform can be found at the following website: http://www.jmloveridge.com/cosh/Chloroform%20Spirit.pdf
Properties and hazards associated with Chloroform
- Colourless mobile liquid
- Chloroform odour
- Miscible with water and ethanol
- Highly flammable
- Harmful if swallowed
- Harmful: danger of serious damage to health by prolonged exposure through inhalation and if swallowed.
- Limited evidence of a carcinogenic effect.
- Carcinogen Category 3
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.
Taketo, M Mark. "Shutting down Wnt signal-activated cancer." Nature Genetics 36. (2004): 320-22. DOI: 10.1038/ng0404-320
In this article, New evidence suggests that Wnt signaling can be suppressed or further activated by upstream signals, even though the pathway seems to be constitutively activated by downstream mutations in cancer cells.
Macdonald, Bryan. "Wnt/β-catenin Signaling: Components, Mechanisms, and Diseases." Developmental Cell 17.1 (2009): 9-26. PMID: 19619488
This article highlights some key aspects of Wnt/β-catenin signaling in human diseases including congenital malformations, cancer, and osteoporosis, and discuss potential therapeutic implications
Luu, Hue. "Wnt/β-catenin Signaling Pathway as Novel Cancer Drug Targets." Current Cancer Drug Targets 4. (2004): 653-71. Doi: 1568-0096/04
This review discusses some of the strategies that are being used or can be explored to target key components of the Wnt/β-catenin signaling pathway in rational cancer drug discover.
Giles, R., Johan, H., Clevers, H. “Cought up in a Wnt storm: Wnt signalling in cancer”. Biochimica et Biophysica Acta, (2003). 1653 1-24. http://dx.doi.org/10.1016/S0304-419X(03)00005-2
This review considers the spectra of tumors arising from active Wnt signaling and attempts to place perspective on recent data that begin to elucidate the mechanisms prompting uncontrolled cell growth following induction of Wnt signaling.
Lab 4 - Immunochemistry
A researcher has a project for a student on a newly discovered protein called by the discoverer "Musashi".
Musashi-1 and Musashi-2 are RNA-binding proteins which play a role in asymmetric cell division of ectodermal precursor cells by regulating the translation of target mRNA.  Musashi contributes to the maintenance of neural stem cells. While Musashi-1 is frequently used as a marker for proliferating neural precursor cells, it is also expressed in epithelial stem cells including intestinal and mammary gland stem cells. Database: NCBI - Musashi
Musashi antibody #2541
- Database: Cell Signaling Technology - Musashi Antibody
- Source: rabbit
- Applications: Western Blot, Immunoflourescence (IF-F) Endogenous
- Species cross reactivity: human and mouse (determined by western blot)
- Molecular weight: 30kDa
- Anti-rabbit secondary antibodies must be used to detect this antibody
- Recommended Antibody Dilution: Wesetern blotting 1:1000; Immunofluorescence (IF-F) 1:25.
- Specificity/Sensitivity: Musashi Antibody detects endogenous levels of total Musashi 1 and 2 protein.
Anti-rabit secondary antibody:
- Database: Cell Signaling Technology - Alexa Fluor® 488 Conjugate
- Alexa Fluor 488 goat anti—rabbit IgG.
- Supplied by Life Technology
- Size 0.5mL for $440.
- It is a green flourescence.
- Formulated at 2 mg/ml.
- Specificity/Sensitivity: F(ab’)2 fragments are prepared from goat antibodies that have been adsorbed against pooled human serum, mouse serum, plasmacytoma/hybridoma proteins and purified human paraproteins.
Lab 5 - Cell Knockout Methods
See: 2011 Lab 5
The actin cytoskeleton together with actin binding proteins, such as trypomyosin, is an important regulator of cell function. Transgenic and knockout mice play a key role in studying the mechanism by which these structural proteins function.
List the key differences in the generation of transgenic and gene knock-out mice.
- Transgenic mice are used to study over-expression of a gene product, and mainly focus on dominant alleles. Mice are generated by DNA microinjection of fertilized oocytes, which are then transfered to a pseudo-pregnant female. This results in random integration of the DNA, and the gene product is over-expressed. The advantages inculde a relative high rate of insertion of the injected gene into the genome, however the random insertion can lead to position effects.
- Knock-out mice are used to look at recessive disorders by deleting the existing gene. Knockout mice are generated by the injection into a blastocyst of genetically modified ES cells. The blastocytes are transfered to a pseudo-pregnant female, producing Chimeric mice with the gene product missing or mutated. The advantages include specific insertion of a gene at specific location or removal of specific genes (KO) as well as the ability to mimic recessive disorders (loss of function mutations). The disadvantages are the low level of ES cells with wanted gene inserted, and that further breeding is necessary to obtain non-chimeric homozygous animal.
Describe how genetically modified mice have been used to study the biological function of tropomysin. In your answer include:
i. The type of cell used
Fat cell (adipocyte)
ii. The measurements used to determine whether any phenotypic changes were observed
- Organ weights (fat, brain, kidneys)
- Adipocyte proliferation
- Glucose uptake & insulin secretion was observed to have increased in the transgenic mice, which was measured by using a glucose intolerance test.
iii. A model to explain how actin changes in the levels of tropomysin can lead to the observed phenotypic changes.
Tm5NM1 increases filamentous actin in adipose tissue (evidence: F-actin staining). The over-expression of Tm in transgenic mice results in increased levels of actin filaments. This increase results in more GLUT4 transporters reaching the membrane surface, resulting in an increase in glucose uptake.
Lab 6 - Cytoskeleton Exercise
1) Do you see a difference in phenotype (morphology) between Tm4 overexpressing and control cells?
- Fan: Slightly more cell numbers in the TM4 group.
- Broken Fan: The number of cells in the TM4 group is reduced by about 15%, indicating that TM4 may inhibit this cell phenotype.
- Stumped: There is a slight increase in TM4 group of about 6% which may not indicate the role that TM4 played in this particular phenotype.
- Pronged: The TM4 group expressed an increase about 6%, which may indicate that TM4 is actively involved in the events of neurites growth for this phenotypes.
- Stringed: There is a slight decrease in TM4 group of about 2%. This may not indicate the role that TM4 played in this particular phenotype.
- Pygnotic: Very small percentage of cells found for the TM4 group. This may indicate that TM4 entirely inhibits this phenotype.
2) If so, how could Tm4 over-expression lead to this difference?
TM4 is a striated muscle isomer of Tropomyosin, which is involved in the motile events of neurite growth and synaptic plasticity. This can be observed in the 'Over expression of Tm4' group, whereby the neurites involved have more processes branching than in the control group. These processes are necessary for cell-cell interaction and communication.
Lab 7 - Confocal Microscopy
Contributions to Group 5 Project
Hey guys, If we decide to go with notch signaling here's a really great diagram & description: http://www.cellsignal.com/reference/pathway/Notch.html
--Z3289738 14:28, 22 March 2012 (EST)
Hey guys, I found four articles relating to abnormal function of the pathway:
Taketo, M Mark. "Shutting down Wnt signal-activated cancer." Nature Genetics 36. (2004): 320-22. DOI: 10.1038/ng0404-320 <pubmed>15054482</pubmed> In this article, New evidence suggests that Wnt signaling can be suppressed or further activated by upstream signals, even though the pathway seems to be constitutively activated by downstream mutations in cancer cells.
<pubmed>19619488</pubmed> This article highlights some key aspects of Wnt/β-catenin signaling in human diseases including congenital malformations, cancer, and osteoporosis, and discuss potential therapeutic implications
<pubmed>15578921</pubmed> This review discusses some of the strategies that are being used or can be explored to target key components of the Wnt/β-catenin signaling pathway in rational cancer drug discover.
<pubmed>12781368</pubmed> This review considers the spectra of tumors arising from active Wnt signaling and attempts to place perspective on recent data that begin to elucidate the mechanisms prompting uncontrolled cell growth following induction of Wnt signaling.
--Z3289738 10:48, 29 March 2012 (EST)
Notes: Diseases associated with Wnt/β-catenin signalling
Wnt/β-Catenin Signaling: Components, Mechanisms, and Diseases 
- Signaling by the Wnt family of secreted glycolipoproteins is one of the fundamental mechanisms that direct cell proliferation, cell polarity, and cell fate determination during embryonic development and tissue homeostasis (Logan and Nusse, 2004). As a result, mutations in the Wnt pathway are often linked to human birth defects, cancer, and other diseases (Clevers, 2006). A critical and heavily studied Wnt pathway is the canonical Wnt pathway, which functions by regulating the amount of the transcriptional coactivator β-catenin, which controls key developmental gene expression programs.
- Given the critical roles of Wnt/b-catenin signaling in development and homeostasis, it is no surprise that mutations of the Wnt pathway components are associated with many hereditary disorders, cancer, and other diseases (Table 1).
- Association of deregulated Wnt/β-catenin signaling with cancer has been well documented, particularly with colorectal cancer (Polakis, 2007) (Table 1). Constitutively activated β-catenin signaling, due to APC deficiency or β-catenin mutations that prevent its degradation, leads to excessive stem cell renewal/proliferation that predisposes cells to tumorigenesis.
- Mutations of β-catenin at and surrounding these serine and threonine residues are frequently found in cancers, generating mutant β-catenin that escapes phosphorylation and degradation (Table 1).
Caught up in a Wnt storm: Wnt signaling in cancer 
- The Wnt signaling pathway, named for its most upstream ligands, the Wnts, is involved in various differentiation events during embryonic development and leads to tumor formation when aberrantly activated. Molecular studies have pinpointed activating mutations of the Wnt signaling pathway as the cause of approximately 90% of colorectal cancer (CRC), and somewhat less frequently in cancers at other sites, such as hepatocellular carcinoma (HCC).
- Greater than 90% of all CRCs will have an activating mutation of the canonical Wnt signaling pathway, ultimately leading to the stabilization and accumulation of β-catenin in the nucleus of a cell.
- Fig. Schematic representation of a colon crypt and proposed model for polyp formation. At the bottom third of the crypt, the progenitor proliferating cells accumulate nuclear β-catenin. Consequently, they express β-catenin/TCF target genes. An uncharacterized source of WNT factors likely resides in the mesenchymal cells surrounding the bottom of the crypt, depicted in red. As the cells reach the mid-crypt region, β-catenin/TCF activity is downregulated and this results in cell cycle arrest and differentiation. Cells undergoing mutation in APC or β-catenin become independent of the physiological signals controlling β-catenin/TCF activity. As a consequence, they continue to behave as crypt progenitor cells in the surface epithelium giving rise to aberrant crypt foci.
Lab 9 - Tissue Culture 1
1. Identify a mammalian cell line in the ATCC catalogue (and add a link) 2. Identify the original tissue of origin of that cell line. 3. Identify the original paper that characterised the properties of that cell line.
- Mouse cell line: ATCC: Mus musculus
- Tissue: subcutaneous connective tissue; areolar and adipose
- Earle WR, et al. Production of malignancy in vitro. IV. The mouse fibroblast cultures and changes seen in the living cells. J. Natl. Cancer Inst. 4: 165-212, 1943.
Lab 10 - Tissue Culture 2
3 major types of contamination in tissue cultures:
- bacteria. appearance: black dots. visible under phase microscope. Precipitation will be present at the bottom of the dish. - fungal. filamentous expansion through media (strings of fungus) or yeast looks like circular structures. Precipitation will be present at the bottom of the dish. - Microplasma = micro bacteria. Infect cells (live inside the cell cytoplasm). Not easy to identify because they are smaller than bacteria & grow inside the cell as opposed to the media. How to avoid? Regularly screen and check for microplasms in the cell line via PCR analysis or DNA screen or external Microplasm testing.
- correct referencing (eg. copyright)
- brief paragraph of my assessment
- paste all the assessments onto your personal page.
The content in this page is well researched and demonstrates a lot of initiative with the hand-drawn images. The tables are bright and easy to read, and the images are well selected and are strongly linked to the written content. The amount of content shows the extend of research and it is clear that a lot of effort has been put into it.
Area of improvement: the structure and organisation of content makes the information overwhelming and exhausting to read. The long paragraphs could be summarized by the use of tables, diagrams (a picture says a thousand words!), bolded text to highlight key points and more subheadings to break up the paragraphs.
- Introduction: Needs a sentence at the beginning to explain how the 3 receptors are related to each other.
- History: Well researched and I like how you have included links to the research articles.
- The Gene Description and Receptor Agonist sections could be merged into one table.
- Intro: Well written and easy to understand. A picture would make this section more eye-catching. Needs references!
- Pathway: The use of numbering and bolded text makes the information very appealing and easy to read. The diagram is also clear and easy to understand. Try to connect the text and diagram by matching the numbers with the information (ie. 'point 1' in the text correlates to 'point 1' in the diagram). A video would also be great for this section, I remember seeing one in a biology lecture once and it was very interesting! This section needs references.
- Normal Function: Excellent introductory paragraph, shows a good understanding of the topic area. You need to edit the spacing within the paragraphs, for example you have numbers listed within the paragraph which could be better read as a list going down the page. Needs references.
- Abnormal Function: All the information is relevant, however could be better represented in a table format.
- Proteins: The paragraphs are too long. They need to be broken up by subheadings, tables and lists to highlight key information. The first image is excellent, the only thing missing is a caption.
- Overall impression: The choice of content shows a good understanding of the topic area. For the most part the information is very well written and easy to understand, however the use of lists, tables and diagrams could make the page easier for the reader to navigate around key information. More images would make the page more eye-catching and appealing to read.
- The first paragraph is very clear and well explained.
- The second and third paragraph seem to be irrelevant for the introduction, however would be useful in other sections such as 'Abnormal Function' for the third paragraph.
- A picture in the introduction would make the page more eye-catching
- The information is well written, however there is too much writing in this section. The information would be clearer and easier to read with the use of subheadings and perhaps bullet points to highlight key information.
- Relating the information to the flowchart would also make the information clearer.
- The last paragraph provides a clear summary of the pathway.
- The table format makes the information clear and easy to read.
- The short sentences are clear and straight to the point. This highlights the key points and makes the information easier to read.
- You need to go through and edit the grammar, especially for the use of sentence case.
- Where possible, include the names of the researchers and/or project groups that made each discovery or breakthrough.
- I like that you added a link to the article
- Rather than writing "a group of scientists", include the names of the researchers and the name of their research article
- This section has the best use of subheadings, which immediately makes the information clearer and easier to navigate.
- To make the information even clearer, try relate it to an image of a pathway
- I think this section should be more towards the top of the page.
- Really good diagram
- I can see this section is incomplete, however some ideas would be to add information about the effects of mutations and associated diseases.
Overall Impression: Although the page seems largely incomplete, the completed sections are well written and could be improved with the use of subheadings and images to illustrate information.