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Lab Attendance

--Z3332227 14:33, 15 March 2012 (EST)

--Z3332227 14:27, 22 March 2012 (EST)

--Z3332227 15:48, 29 March 2012 (EST)

--Z3332227 15:08, 5 April 2012 (EST)

--Z3332227 16:01, 19 April 2012 (EST)

--Z3332227 11:04, 28 April 2012 (EST) (Lab 7)

--Z3332227 14:41, 3 May 2012 (EST)

--Z3332227 14:07, 10 May 2012 (EST)

--Z3332227 22:56, 24 May 2012 (EST) (Lab 10)

--Z3332227 22:56, 24 May 2012 (EST)

--Z3332227 14:48, 31 May 2012 (EST)

Lab 1

Internal Link to ANAT3231 Glossary

External Link to the Journal of Cell Biology


Lab 2

Image Upload

Abnormal NMJs in muscle from wild-type and AD-EDMD mouse models.jpg [1]


Reference

  1. Alexandre Méjat, Valérie Decostre, Juan Li, Laure Renou, Akanchha Kesari, Daniel Hantaï, Colin L Stewart, Xiao Xiao, Eric Hoffman, Gisèle Bonne, Tom Misteli Lamin A/C-mediated neuromuscular junction defects in Emery-Dreifuss muscular dystrophy. J. Cell Biol.: 2009, 184(1);31-44 PubMed 19124654


Super-resolution Microscopy Reference

By overcoming the diffraction limit of light, super-resolution microscopy produces a higher resolution image and thus allows the observation of otherwise unviewable (by traditional light microscopy) intracellular structures. Super-resolution microscopy combined with EM immunochemistry allowed visualisation of kinesin-1 showing preferential binding to GTP-tubulin–rich axonal microtubules involved in polarised vesicle transport.

Takao Nakata, Shinsuke Niwa, Yasushi Okada, Franck Perez, Nobutaka Hirokawa Preferential binding of a kinesin-1 motor to GTP-tubulin-rich microtubules underlies polarized vesicle transport. J. Cell Biol.: 2011, 194(2);245-55 PubMed 21768290


Lab 3

Current SDS for Formalin

Formalin (Formaldehyde) SDS

Properties:

  • Colourless gas
  • Volatile, pungent odour
  • Flammable
  • Formalin is an aqueous solution that is 37% formaldehyde by weight


Hazards:

  • Inhalation of formaldehyde can produce bronchospasm and pulmonary edema.
  • Possible hypersensitivity (allergic) reactions; Asthma, Dermatitis and Urticaria
  • Toxic Pneumonitis
  • Dermatotoxin (skin burns)
  • Known Carcinogen


References for Wnt Beta-Catenin Group Project - Normal Functioning

Bryan T MacDonald, Keiko Tamai, Xi He Wnt/beta-catenin signaling: components, mechanisms, and diseases. Dev. Cell: 2009, 17(1);9-26 PubMed 19619488

Overview of the Wnt signaling pathway; ligands, agonists and antagonists and their interactions with Wnt receptors. Also mentions its implication in the development of human disease.


Yoshiaki Kawano, Robert Kypta Secreted antagonists of the Wnt signalling pathway. J. Cell. Sci.: 2003, 116(Pt 13);2627-34 PubMed 12775774

Main extracellular antagonists of the Wnt signalling pathway - regulation of cell growth/differentiation.


Poongodi Geetha-Loganathan, Suresh Nimmagadda, Martin Scaal Wnt signaling in limb organogenesis. Organogenesis: 2008, 4(2);109-15 PubMed 19279722

Involvement of Wnt signalling molecules in the control of embryonic development.


Roel Nusse Wnt signaling and stem cell control. Cell Res.: 2008, 18(5);523-7 PubMed 18392048

Role of the Wnt pathway in proliferation, differentiation and apoptosis in adult tissues (and therefore oncogenesis).


Lab 4

Musashi Protein

Musashi is a neural RNA-binding protein first identified in 1998 which plays an important role in regulating cell differentiation in precursor cells. There are two isoforms of the protein (Musashi-1 and Musashi-2) which are responsible for regulating cell fate via Notch signalling. Musashi genes are expressed in a range of tissues including pancreatic beta-cells and the CNS.

M Szabat, T B Kalynyak, G E Lim, K Y Chu, Y H Yang, A Asadi, B K Gage, Z Ao, G L Warnock, J M Piret, T J Kieffer, J D Johnson Musashi expression in β-cells coordinates insulin expression, apoptosis and proliferation in response to endoplasmic reticulum stress in diabetes. Cell Death Dis: 2011, 2;e232 PubMed 22113197

Human Musashi Protein Information Sheet


Many antibodies are available against the protein, one being Musashi-1 Antibody from Novus Biologicals. This is a polyclonal IgG antibody raised in a rabbit host. It acts as a Neuronal Stem Cell Marker by reacting with residues 5-21 [APQPGLASPDSPHDPCK] of the human, mouse and rat Musashi-1 protein. The antibody is sold at a concentration of 1.0 mg/ml and should be kept within a dilution range of 1:100-200 in order to reduce non-specific binding and ensure optimum fluorescence.

Musashi 1 Antibody 0.025 mg

P E B Nickerson, T Myers, D B Clarke, R L Chow Changes in Musashi-1 subcellular localization correlate with cell cycle exit during postnatal retinal development. Exp. Eye Res.: 2011, 92(5);344-52 PubMed 21320487


An anti-rabbit IgG secondary antibody conjugated with a green fluorescent Alexa dye (Catalogue Number A11034) is available from Invitrogen Molecular Probes. It is raised in goats and reacts with IgG heavy chains and all classes of immunoglobulin light chains from rabbit. Immunofluorescence is best observed at a dilution of 1:500.

Alexa Fluor® 488 Goat Anti-Rabbit IgG (H+L) *2 mg⁄mL

Sunshin Kim, Dong Hwa Jun, Hye Jin Kim, Kyung-Chae Jeong, Chang-Hun Lee Development of a high-content screening method for chemicals modulating DNA damage response. J Biomol Screen: 2011, 16(2);259-65 PubMed 21233308


Lab 6

Cell Phenotype Graph.jpg


Fan: Insignificant change in cell numbers between groups

Broken Fan: Tm4 over-expressers showed a reduction of approximately 20% in cell numbers of this phenotype

Stumped: Tm4 over-expressers showed a smaller reduction of around 10% in cells possessing this phenotype

Pronged: Tm4 over-expressers had an increase of nearly 20% in cells of this phenotype

Stringed: Tm4 over-expressers showed a modest increase of about 5% in cells of this phenotype

Pygnotic: No cells found in the Tm4, very small number of cells found in the control group


Tm4 Over-expressers:

  • The majority of cells (over 70%) were of the stumped or pronged phenotype, with fewer numbers of broken fan and stringed cells (8% and 12% respectively)
  • Significant increase in the percentage of total cells belonging to the pronged and stringed phenotypes when compared to the control group
  • There appeared to be an increase in branching and extension of processes of cells belonging to this group
  • The cells of this group also seemed to fluoresce to a higher degree
  • Lighter (pinker) cytoskeleton
  • Lamella clearly stained a yellow colour at the periphery of the cell


Control Group:

  • The majority of cells belonged to the broken fan, stumped or pronged phenotype (over 90% of cells)
  • Larger percentage of cells were of the fan, broken fan and stumped phenotype
  • Less abundant branching of cells
  • Lower degree of fluorescence
  • Darker (redder) cytoskeleton
  • Lamella stained red at periphery of the cell


Explanation:

The results depicted above suggest Tm4 over-expression is associated with an increase in cell motility and the extension of cellular processes. Tropomyosins are alpha-helical dimers that bind to and stabilize actin microfilaments. Tropomyosin has a high affinity for actin and plays a role in preventing its disassembly, as well as regulating the accessibility of actin filaments to other actin-associated proteins such as myosins. Tm4, an isoform of tropomyosin, has been shown to be found in particularly high concentrations in both the dendritic and axonal growth cones of cultured neurons, and in areas where neurites are growing during development. It is believed that Tm4 could be involved in stabilising the spatial organisation of the actin filaments, and in turn controlling the level of interaction of actin binding proteins such as myosin I and II.[1] The myosin proteins act as molecular motors for growth cone motility, and thus explain why a higher level of cell motility is seen in Tm4 over-expressing cells.


  1. 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 7

Wnt Beta-Catenin Signalling - Mechanism of Action

"Off" and "On" Sates of the Wnt/Beta-catenin Signalling Pathway
  • Wnt proteins encompass a network of secreted glycolipoproteins
  • Wnt signalling best known for playing a variety roles in embryogenesis, control of cellular proliferation and the resulting birth defects, cancers and other diseases arising from mutations in the pathway
  • Beta-catenin commonly exists as a subunit making up the cadherin protein complex
  • Cadherin proteins play an integral role in the formation of adhesion junctions between cells
  • Beta-catenin also acts to help anchor the actin cytoskeleton within the cell
  • Beta-catenin plays a vital role in the Wnt signalling pathway by directly affecting the control of protein synthesis within the cell. The interaction of beta-catenin with the TCF/LEF family of transcription factors on the template DNA strand converts them from repressors to activators, triggering downstream transcription of Wnt target genes and synthesis of their encoded proteins.
  • This interaction is reliant on an intracellular cascade which ultimately dictates the amount of Beta-catenin present in the cytoplasm which is then able to reach the nucleus and effect transcription.
  • In the absence of Wnt (“off state”), beta-catenin is targeted for proteasomal degradation. Intracellular Axin, GSK-3β (glycogen synthase kinase 3β) and APC (Adenomatous polyposis coli – coded for by the APC tumour suppressor gene) form a “destruction complex” leading to phosphorylation of β-catenin by the coordinated action of CK1 and GSK-3β and subsequent ubiquination.
  • In the presence of Wnt (“on state”) an extracellular “Wnt signal” binds to a cell surface G-protein coupled receptor of the “Frizzled” (FRZ) family. This results in the activation (phosphorylation and poly-ubiquination) of proteins of the “Dishevelled” (DSH) family implicated in the inactivation of the “destruction complex” by the recruitment of GSK-3β away from the complex. With the complex now interrupted, an increased amount of beta-catenin is able to reach the nucleus where it can promote transcription of Wnt target genes.
  • Low-density lipoprotein receptor-related protein (LRP) family functions as a transmembrane co-receptor for Frizzled. It functions to recruit axin to the membrane, leading to axin degradation upon the initiation of the Wnt signalling cascade and the dissociation of beta-catenin into the cytoplasm without degradation by the “destruction complex”.
  • The Beta-catenin gene can be said to function as an oncogene given that the promotion of transcription of Wnt target genes by beta-catenin has been shown to be involved in the development of basal cell carcinoma, colorectal cancer and breast cancer.


Lab 8

Mammalian Cell Line - Detroit 551

ATCC® Number: CCL-110™

Growth Properties: Adherent

Organism: Homo sapiens

Organ: Skin

Disease: Normal

Cell Type: Fibroblast

Isolation date: 1965


  • Finite lifespan of about 25 serial passages from the tissue of origin.
  • Can be transformed by SV40.
  • Growth of the cells is enhanced by addition of tumor necrosis factor alpha (TNF alpha) to the medium.


Original paper characterising the properties of the cell line:

B J Sugarman, B B Aggarwal, P E Hass, I S Figari, M A Palladino, H M Shepard Recombinant human tumor necrosis factor-alpha: effects on proliferation of normal and transformed cells in vitro. Science: 1985, 230(4728);943-5 PubMed 3933111



Lab 9

Group Assignment Peer Evaluations

Group 1 – Testosterone

  • Pros
    • Well formatted tables
    • Comprehensive “Current and Ongoing Research” section
    • Content correctly cited and referenced
  • Cons
    • No copyright information included for testosterone structure image under “Introduction”
    • Two Wikipedia images used
    • No student-drawn image
    • Needs proof-reading (e.g. “1994 – History” “...also identified testosterone and a risk factor for hypotension and obesity”
    • Image titled “Steroidogenesis” very complex, maybe draw own simple flow diagrams for each of the steps of the biosynthesis pathways?
    • Also could draw simple flow diagrams to describe signalling pathways (e.g. “Classical” and “Non-classical”) to make the information easier to read and understand


Group 2 – VEGF

  • Pros
    • Well formatted tables and general page organisation
    • Content correctly cited and referenced
    • Good use of internal links to the glossary
    • Content easily readable due to the use of subheadings, dot points and tables
    • I liked the use of pictures to visually represent the diseases resulting from abnormal function in the pathway
    • “Research: Therapeutic Applications” section showed extensive research and was interesting
  • Cons
    • “Normal Function” section needs expanding; further research needed
    • No student-drawn image
    • “Normal Function” section is very dense; maybe add subheadings or use dot points?


Group 3 – Extrinsic Apoptosis

  • Pros
    • Large amount of information included but hard to ascertain whether it is a result of extensive research or plagiarism due to lack of citations and referencing
  • Cons
    • No references for “Introduction”, “History” or “Function” sections (aware this was due to technical difficulties)
    • References for “Signalling Pathway” and “Current Research” sections incorrectly formatted
    • No reference list or glossary
    • Page needs more images/diagrams and reorganisation of text into a more readable format (Tables?)(Dot Points?)
    • Flow charts (possibly student drawn) would aid in understanding the pathways described in the text
    • “Signalling Pathway” section lists proteins involved in extrinsic apoptosis pathway but there is no description of their specific roles
    • No student-drawn image
    • No abnormal function/implications in disease section


Group 4 - Notch

  • Pros
    • Content correctly cited and referenced
    • Well formatted table in “History” section; makes information easily readable
    • Good use of dot points and subheadings in “Proteins and Receptors” section
    • Subheadings in “Normal Function” section made information easier to follow
  • Cons
    • “History” section lacking a bit of information and research (only 3 sources referenced)
    • “Pathway” section hard to follow (maybe include flow diagrams of signalling pathway? Possibly student-drawn?)
    • Images under “Proteins and Receptors” and “Normal Function” sections lack copyright information
    • Greater breadth of research needs to be conducted, reference list is not very comprehensive
    • “Normal Function” section needs proof-reading (For example, the opening three lines read: “Notch signalling pathway plays a vital role in metazoan development. Notch protein activates a signalling pathway that controls the expression of genes that are responsible for cell division, growth, migration and apoptosis. Utilization of the Notch signalling pathway determines the fate of cells in the embryonic phase.”). These sentences sound more like dot points; add “The” to the start of them or format them in dot point form.
    • No abnormal function/implications in disease section
    • No student-drawn image


Group 6 – Insulin

  • Pros
    • Content correctly cited and referenced
    • Good use of subheadings; makes information more easily readable
  • Cons
    • Needs a main heading for the actual page titled “Insulin Signalling”
    • No copyright information for images under “Structure of Insulin” or “Signalling Pathway” sections
    • “History” section is lacking citations
    • Page needs more breadth of research, especially the “Introduction” and “Normal Function” sections
    • No student-drawn image
    • Maybe add text to the external link to YouTube under “Insulin” section of the Introduction so readers know it’s a video (could be confused for just a citation)


Group 7 - G-Protein Coupled Receptors: Beta-adrenergic receptors with a focus on Beta1-adrenoceptors

  • Pros
    • Clear and simple student-drawn images
    • Lots of information included, reflects amount of research done
    • Content correctly cited and referenced
    • Found the “Abnormal Function, Diseases and Treatments” sections very interesting and well-written
  • Cons
    • Maybe consider using more tables/dot points or summarising information a bit more; some of the sections are very dense


Group 8 – Leukocyte Extravasation

  • Pros
    • Content correctly cited and referenced
  • Cons
    • “History” section incomplete
    • No references for “Introduction”, “pathway” or “normal function” sections
    • No copyright information for image under “Pathway” section
    • No student-drawn image
    • Text needs to be reorganised into a more readable format instead of just large chunks of writing (Tables?)(Dot Points?), for example, “Leucocyte Activation” sections under “Normal Function” outlines the steps involved in phagocytosis. These would be much more easily readable as dot points, and possibly even supplemented with a few diagrams, flow charts or images.


Group 9 – p53

  • Pros
    • History section well referenced and formatted
    • Content correctly cited and referenced
    • “Normal function” section interesting, well-phrased and formatted (due to use of subheadings)
  • Cons
    • “Pathway” and “Normal function” sections lacking breadth of research
    • “Receptor”, “Proteins” and “Abnormal Function” sections unfinished
    • No student-drawn image
    • Image included under “Pathway” section very complex and hard to understand (find simpler image or draw own?)
    • Maybe the “History” section would be better put directly after the introduction as a general background of the development of knowledge concerning the pathway, then go into describing it
    • Apart from the “History” section, all other sections need further research


--Mark Hill 12:43, 17 May 2012 (EST) You have made a good pro/con format and your comments support the classification used in each project assessment.

Lab 12

1. Identify a current technique used in gene sequencing.

Comparitive genomic hybridisation or CGH (used to detect specific DNA sequences of a genome)

  • Subject DNA is tagged (for later analysis by fluorescence) and hybridised to reference DNA to detect chromosomal changes ("DNA copy number" differences)
  • Utlises current technologies such as gene chips offered by affymetrix


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

Ricardo J Flores, Yiting Li, Alexander Yu, Jianhe Shen, Pulivarthi H Rao, Serrine S Lau, Marina Vannucci, Ching C Lau, Tsz-Kwong Man A systems biology approach reveals common metastatic pathways in osteosarcoma. BMC Syst Biol: 2012, 6;50 PubMed 22640921



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

In this study, Flores et al (2012) utilise microarray analysis to identify common metastatic pathways expressed in osteosarcoma (OS), the most common malignant bone tumor in children and adolsecents. Two human metastatic OS models were studied to identify common pathways implicated, jointly supported by both mRNA and protein expression data retrieved from microarray analysis. The current survival rate for patients with OS is very low, although characteristaion of genes implicated in metastasis and a worse prognosis yields potential for therapeutic intervention of these pathways and a viable treatment for OS.