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CELL BIOLOGY

Lab Attendence

Z3287603 15:13, 8 March 2012 (EST)

Z3287603 16:03, 15 March 2012 (EST)

Z3287603 14:37, 22 March 2012 (EST)

Z3287603 13:58, 29 March 2012 (EST)

Z3287603 15:00, 5 April 2012 (EST)

Z3287603 14:15, 26 April 2012 (EST)

Z3287603 16:19, 3 May 2012 (EST)

Z3287603 14:03, 10 May 2012 (EST)

Z3287603 14:26, 17 May 2012 (EST)

Z3287603 12:27, 24 May 2012 (EST)

Z3287603 14:07, 24 May 2012 (EST)

Z3287603 14:15, 31 May 2012 (EST)

Lab 1

External Link to JCB

Cell Biology Movies

Lab 2: Uploading Image to Webpage

Apoptosis of Frog Eggs.gif

Home work

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

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


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

In this article, Brown et al displays that through the usage of superresolution microscopy techniques they were able to locate the “mitochondrial nucleoids” within the “highly compartmentalized mitochondria” at an exceptional resolution. This was achieved by the use of two-colour photoactivated localization microscopy (PALM). Traditionally the use of normal microscopes did not reveal the “dimensions, composition” or their functions clearly. By employing this newly developed technique Brown et al were able to reveal the different size and shape of the nucleoids, which embarks further research into the functions of mitochondrial DNA.

Lab 3: Fixation 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.

The data below was taken from the Material Safety Data Sheet (http://proscitech.com.au/cataloguex/msds/anbfj-200.pdf)

Formalin

PHYSICAL & CHEMICAL PROPERTIES OF FORMALIN

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 OF FORMALIN

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.

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.

1. In this article, the authors utilize the notch signalling pathway to demonstrate its importance in the development stage of Drosphila Flies. They indicate that a single mutation in notch can lead to wingless Drosophila highlighting its importance in the embryonic development stage. This article may be helpful in identifying the significance of Notch signalling.

H K Hing, X Sun, S Artavanis-Tsakonas Modulation of wingless signaling by Notch in Drosophila. Mech. Dev.: 1994, 47(3);261-8 PubMed 7848873


2. This review article highlights the function and structure of notch proteins. This article gives an overview of the specific proteins and its signalling pathways which is useful in our group project.

I Greenwald Structure/function studies of lin-12/Notch proteins. Curr. Opin. Genet. Dev.: 1994, 4(4);556-62 PubMed 7950324


3. In this review article, the authors examine the importance notch activity in the ”development of different organisms”. This is very useful as it allows us to understand the function of the signalling pathway not only in invertebrates but also vertebrates.

M Lardelli, R Williams, U Lendahl Notch-related genes in animal development. Int. J. Dev. Biol.: 1995, 39(5);769-80 PubMed 8645561


4. For many years notch signalling has been associated with the fate cell differentiation during the development stage of organisms however in this study the authors indicate its importance in cell proliferation and apoptosis. This is useful as it represents a fatal role in the cell cycle and mutation in the notch signalling pathway may lead to many diseases such as cancer.

L Miele, B Osborne Arbiter of differentiation and death: Notch signaling meets apoptosis. J. Cell. Physiol.: 1999, 181(3);393-409 PubMed 10528225


Lab 4: Exercise

Musashi Proteins

Background

Musashi-1 and Musashi-2 are RNA-binding protein that is involved in regulating the maintenance of stem cells in the nervous system. This protein was first discovered in 1998. Notch signaling is closely related to this protein as Musashi-1 and Musashi-2 helps in the development pathways.

Musashi antibody (Primary Antibody)

Source: Rabbit, Polyclonal Molecular weight: 35 kDa

Recommended Antibody Dilutions: Western blotting 1:1000 Immunofluorescence (IF-F) 1:25

Methodology: Western Blotting & Immunofluorescence

External Link to Musashi Antibody Data

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

Target species: Rabbit

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.

Source:Goat, Polyclonal

Molecular weight: 39 kDa

Dilution: 1/5000

Methodology: Alexas Immunofluorescence.

External Link to Secondary Antibody Data

References

Y Kaneko, S Sakakibara, T Imai, A Suzuki, Y Nakamura, K Sawamoto, Y Ogawa, Y Toyama, T Miyata, H Okano Musashi1: an evolutionally conserved marker for CNS progenitor cells including neural stem cells. Dev. Neurosci.: 2000, 22(1-2);139-53 PubMed 10657706

Kara M Gunter, Eileen A McLaughlin Translational control in germ cell development: A role for the RNA-binding proteins Musashi-1 and Musashi-2. IUBMB Life: 2011, 63(9);678-85 PubMed 21766416

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Lab 6 (Week 7)

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


Questions:

1) Do you see a difference in phenotype (morphology) between Tm4 overexpressing and control cells? There are definitely changes in the phenotype between Tm4 overexpression and control cells. There is somewhat more number of cell in the Tm4 Group in the Fan section. In Broken Fan the presence of Tm4 may inactivate this cell type, this is prevalent as the number of cells present in the Tm4 group is decreased by 15 %. Tm4 group increase is shown in Stumped and indicates the insignificance of Tm4 in this phenotype due to the increase. There may be evidence of neuritis growth in this particular phenotypes in Pronged. This is due to the Tm4 increase. There is a slight 2% decrease in the Tm4 group in Stringed, which may support the insignificant involvement of Tm4 in this phenotype (also seen in stumped). In terms of Pygnotic, Tm4 may inactivate the expression of this particular phenotype as only small percentage of cells is involved in the Tm4 group.

Lab 7

My contribution:-

Normal Function

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. As cells begin to divide in the developmental stages, they utilize the Notch receptor protein which magnifies and combines the “molecular differences between adjacent cells”[7]. That is, Notch provides a scaffold in the embryo that guides cells to follow a certain pattern in accordance with its neighbouring cells. The correct Notch signalling pathway is important in the development of organisms as incorrect expression or mutations in the pathway can lead the progression of tumours. The importance of Notch signalling pathway can further be explored in the developmental stages of Central Nervous System in Vertebrates, Cardiovascular Development, Pancreas Development, Kidney Development and Gut Development. [edit]Role of Notch in the development of Central Nervous System


Role of Notch in the development of CNS. The development of the central nervous system (CNS) in vertebrates results in the separation of primitive neuroepithelium into two main roots, neurons and glia. Neurons originate in embryonic life from multipotent progenitors near the ventricle. They are then transferred to their desired endpoint where they “integrate into the brain circuitry”[8]. In terms of Glia cells, they are produced in the subventricular zone during “late embryonic and early postnatal stages”[8]. [edit] Notch in Cardiovascular Development The cardiovascular organ is one of the first systems to arise during vertebrate embryogenesis. The heart evolves from the cardiogenic mesoderm to form the double-walled primary heart tube. This takes place when blood islands are organised in the yolk sac of the embryo from the formation of hemangioblastic cells. These cells further differentiate into hematopoietic and angioblastic cells. The hematopoietic cells are responsible for producing blood cells and vascular endothelial cells (ECs) are generated from angioblastic cells[9]. In the process of forming new blood vessels, smooth muscle cells and pericytes work together to synthesis various sizes of vessels. Large amount of Notch signaling is conducted through transactivation. This is done by ligand-expressing cell (the signaling cell) stimulating its neighbouring receptor-expressing cell (the receiving cell). In studies conducted on mouse embryos that lacked Notch receptor[9], Notch 1, led to heart defects, further highlighting its importance in vascular development. [edit] Notch in Pancreatic Development The pancreas is derived from the endoderm and are formed from three main branches, the acinar cells responsible for producing digestive enzymes, the islets of Langerhans (α-and β-endocrine cells) regulates blood-glucose level through hormones such as insulin and glucagon and finally the ductal tree which attaches the pancreas to the duodenum. The behaviour of Notch in the development of the pancreas is not well defined. Therefore the role of the members of Notch signalling, Notch 1, Notch 2 and Rbpi were further tested. In vivo studies,Rbpjand combined Notch1/Notch2 knockout mice using Ptf1a+/Cre(ex1) mice were crossed with floxed Rbpj or Notch1/Notch2 mice [10]. The mice were examined at various stages of their embryonic development for the formation of exocrine and endocrine pancreatic progression. The study revealed that the lack of Rbpi in pancreatic progenitor cells delayed the development of exocrine pancreas up to embryonic day 18.5 and resulted in premature differentiation of endocrine pancreas cells. Hence the role of Rbpi is essential in the early developmental stages but not necessarily important in the later phases[10]. Subsequently the absence of Notch1 and Notch2 only distressed the proliferation of the pancreatic epithelial cells in the early stages and did not cause the inhibition of pancreatic development[10]. Furthermore unlike Rbpi, Notch1 and Notch2 are not crucial for the development of the pancreas. [edit] Notch in Kidney Development The development of human kidney begins with a segmentation process in which nephrons mold into an S-shape body [11]. Each region of the S-shaped body is responsible for the formation of nephron components, such as the lower segment differentiates into podocytes, the middle region forms proximal epithelial cells and the upper region of the S-shaped body gives rise to the ascending limb of the loop of Henle and distal convoluted tubule[11]. Notch plays an essential role in the developmental stages of the nephron. This was established by the expression of Notch1 in the potential mesangium, distal tubule and collecting ducts of during nephrogenesis[12].Notch2 was also expressed in the primitive proximal tubule with very limited co-localization with podocyte progenitors. Notch ligand, Jagged1 revealed limited expression in primary vesicles, comma-shaped bodies, and S-shaped bodies and later in developing distal tubules and the prospective loop of Henle[11].

  • Videos
  • Student drawn image of the development of CNS
  • Glossary words
  • Further Research

Lab 8

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

  • MDA-MB-231 [1]

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

  • Organism: Homo sapiens , mammary gland; breast

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

E V Chandrasekaran, E A Davidson Glycosaminoglycans of normal and malignant cultured human mammary cells. Cancer Res.: 1979, 39(3);870-80 PubMed 427776



<PMCID>150972</PMCID>

Lab 9: Peer reviews

1. Group 1: Testosterone Signaling

The overall group work is constructed very well as it is easy to understand the content. The project page demonstrates a good structure as well as satisfying the requirement of the assessment. The use of purple in the history section adds brightness to the page and draws attention to the text. In the biosynthesis section however, I do not see the point of clustering the information into a table as adds messiness to the page. You can simply present the information as dot points. Although the project page obeys the given criteria, one aspect is not followed; Mark clearly indicated that only one wiki image could be referenced in our projects however the images of Testosterone binding to androgen receptor and Steroidogenesis, are both referenced from Wiki. There was no student drawn image on the page.

2. Group 2: Vascular Endothelial Growth Factor The use of the image in the beginning of metastasis immediately draws your attention to the group page. Structurally the group satisfies most of the criteria of the assessment. The use of table to demonstrate the abnormal function is a clever as this allows you to understand the content represented more clearly. Although the group project is pleasing some components may need to be changed. For example the use of the colour grey in the tables dulls your text. The glossary is quite short and needs more content. Someone without a scientific background may not know words such as “edema”, “microaneurysms” etc. The most important point raised by Mark was referencing only ONE wiki image, images of rheumatoid arthritis and breast cancer are both from Wiki. No student drawn image. Overall well done!

3. Group 3: Extrinsic Apoptosis The overall impression of this project page is quite bland compared to other projects as it lacked a lot of required components ( No references, glossary or abnormal function). Firstly there is no reference list, there are references within the actual sections but this made it quite messy to look at. Glossary section was also missing from the page; people without a scientific background may find it difficult to understand the content as they may not understand scientific jargon. Signalling pathway and the function sections were easy to understand so well done! My suggestions are use images and tables to bring your page to life and not heavily focus on text.

4. Group 5:

The placement of the image in the beginning brightens up the page. The use of dot point through out the page as well not having the project heavily text based makes the content easy to understand so well done! The distinction between abbreviations and terminology in the glossary page is quite impressive. The group has done an excellent job in satisfying all of the assessment criteria (including a student drawn image and use of one wiki image). However, there are several components that may need improvement. These include; use of table for the history section and the key players in wnt/B-catenin signalling as it’s too large and chunky and you can reduce the size of the thumb images to avoid this problem.

5. Group 6: Insulin The group project is structurally pleasing overall. The three images used in the project complemented its section. The simple use of texts as well as use of subheadings made it easy to understand the content more clearly. Most of the criteria of the assessment is satisfied however there are a lot of room for improvement. There is no title for the project page, at first I was quite confused as to the main purpose of the project. In the introduction, there should be a paragraph to some up the entire project. In each of the section there is no need for the subheading “introduction”. In the glossary section you should have two sections; one for the abbreviation and another section for the meanings of certain terminologies. The image ‘Structure of insulin’ has no copyright information and this should be fixed immediately and Mark emphasised the importance of having this.

6. Group 7: G-protein coupled receptors

Overall an impressive project page which has satisfied all the components of the assessment. The use of colour in the tables in sections such as ‘Receptor Agonists’ and ‘Abnormal function’ etc really draws the readers attention so well done! Some minor changes can be done in order to create a useful wiki page, such things as tabling the history section can achieve an appeal to the section and removing the signatures of each students underneath some sections as it looks really messy.

7. Group 8: Leukocyte Extravasation The project page demonstrates an overall understanding of the signalling pathway and fills most of the assessment criteria. Reading through the page, I found it was heavily text based and could of used more images to breakup the text instead of placing them in the right hand side. Perhaps, you can integrate tables in your project page to avoid the aforementioned issue. There are no student drawn images in the project and New or Current Research section is incomplete.

8. Group 9: p53 Signalling Pathway Some components of the project page is incomplete therefore this project did not meet the assessment criteria. These include Receptor, Protein and Abnormal Function section. The history section and the colour scheme is quite impressive and you should incorporate such style in your other sections like abnormal functions as well proteins and receptors. I do like the simplified text in each sections as it is easy to read however, Most of the sections requires more information as p53 is one of the most important genes in our genome (guardian of the genome). In order to improve your project page use more images to break up texts and make the thumb images bigger (eg. 500X). If you complete the sections and make these minor changes you can improve your page by miles, Goodluck!

--Mark Hill 12:31, 17 May 2012 (EST) This has some good critical feedback, a little general in some instances and could have done with a little more than just 2 lines in some cases.


Lab 12: Microarray

1. Identify a current technique used in gene sequencing.

Next generation sequencing is the current technique used in gene sequencing. This method produces ton of sequencing data at once.In Australia it costs approximately $10000 to sequence a human genome. However, there is constant push to reduce this price to $1000.

2. Identify a recent cell biology research paper that has used microarray technology. Xu Jia, Weizhi He, Alastair I H Murchie, Dongrong Chen The global transcriptional response of fission yeast to hydrogen sulfide. PLoS ONE: 2011, 6(12);e28275 PubMed 22164259


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

Hydrogen sulfide is an important member of the gasotransmitters family that are responsible for the gaseous signals in humans. Analyzing the activity of hydrogen sulfide can give a clear understanding in certain diseases. In this experiment, Jia et al performed microarray technique on fission yeast in order to study the role of hydrogen sulfide. The microarray data showed that there were overlap between gene affected by hydrogen sulfide and stress response. Also there were evidence that exposure of hydrogen sulfide on mitorchondrial genes can reduce the mitochondrial oxygen consumption. Therefore, the importance of using microarray allows potential research into targeting molecules that regulate hydrogen sulfide. This can be harnessed in developing treatment for particular diseases.