User:Z3374392

From CellBiology

Lab attendance

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MOOSE

REDDIT

Cells

Red White Blood cells 01.jpg

Red White Blood cells 01.jpg

Red White Blood cells 01.jpg
Red White Blood cells 01.jpg
amazeballs

Red White Blood cells 01.jpg

[[1]]

File:Cat.jpg


Lab assignments

Lab 1 with reference update for Lab 2

Membrane transport capabilities.png

Venn Diagram Showing the Distribution of Transporter Families across the Three Domains of Life [1]


Copyright: © 2005 Ren and Paulsen. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


References

  1. Qinghu Ren, Ian T Paulsen Comparative analyses of fundamental differences in membrane transport capabilities in prokaryotes and eukaryotes. PLoS Comput. Biol.: 2005, 1(3);e27 PubMed 16118665

Lab 2

When researching structural specialisations in biological samples, differentiation of structures is often limited by the resolving capacity of standard light microscopy. This can be overcome by using methods like super resolution microscopy. A recent study explores 4D super resolution microscopy with conventional fluorophores and single wavelength excitation in optically active thick cells and tissues.[1] The research group aimed at studying proteins at a molecular level and the super resolution microscopy enabled them to more clearly view various biological systems and associated proteins. Prior to 4D super resolution microscopy, when thicker specimens were used, problems would arise including out-of-focus fluorescence as well as a reduction in contrast due to autofluorescence making localisation more difficult.Through the use of this new microscopy, the group was able to investigate optically thick samples, including human tissue sections, cardiac rat myocytes and densely grown neuronal cultures using readily available fluorochromes and operating operating in a wavelength range where biological autofluorescence is minimised.


References

  1. David Baddeley, David Crossman, Sabrina Rossberger, Juliette E Cheyne, Johanna M Montgomery, Isuru D Jayasinghe, Christoph Cremer, Mark B Cannell, Christian Soeller 4D super-resolution microscopy with conventional fluorophores and single wavelength excitation in optically thick cells and tissues. PLoS ONE: 2011, 6(5);e20645 PubMed 21655189

--Mark Hill (talk) 13:41, 11 April 2013 (EST) This recent article on 4D super-resolution microscopy and your explantation meet the assessment criteria. I have also fixed your lab assessment au-sub-heading format.

Lab 3

The following journal articles provide insight into the structure of the spindle apparatus:

Article 1: As reviewed in Glotzer (2009), the spindle apparatus is made from a combination of microtubules, motors and microtubule associated proteins (MAPs). [1] This review article is mainly concerned with the central spindle that coordinates cytokinesis. Microtubules that make up spindles are cylindrical polymers that are assembled from dimers of alpha-tubulin and beta-tubulin. They are polar filaments that have a fast-growing plus end and a slow-growing minus end that is often capped by the gamma-tubulin ring complex, a ring-shaped microtubule nucleator. During metaphase, the mitotic spindle is comprised of kinetochore fibres, astral microtubules and interpolar microtubules. The fusiform shape of the spindle is the result of the microtubule minus ends focusing at the poles and by cross-linking interpolar microtubules in an overlapping region situated in the midzone. At the beginning of anaphase, the kinetchore fibres shorten ( delivering sister chromatids to the poles) and astral microtubules elongate. The region between the two poles is called the spindle midzone and the microtubules that populate this region are called midzone microtubules. The term central spindle refers to the structure at the centre of the midzone, where the plus ends of the microtubules interdigitate. The microtubules of the central spindle eventually lose their interaction with the spindle poles. As the formation of the cleavage furrow progresses, the central spindle becomes compacted dense structure known as a the midbody.

Article 2: In the spindle, kinetochore microtubules have their plus ends embedded in the kinetochores of the sister chromatids and their minus ends at the spindle pole. This study shows that kinesins are important to maintain spindle bipolarity. [2] The simulataneous KinI induced disassembly at both the plus and minus ends may result in the poleward driving forces. Upon disassembly, chromosome associated kinetochore microtubules are driven back to their poles. Centromere-associated KinI proteins act to disassemble the plus end, causing the spindles to shorten during anaphase.

Article 3: In most animal cells microtubules are nucleated at the centrosomes found at the spindle poles. However, it has been observed that spindles can still form in cells lacking centrosomes. The results show that non-centrosomal microtubules contribute to to spindle formation even in cells with centrosomes. These cells expressed GFP-alpha-tubulin. It was also found that the centrosomal microtubule array can be composed of both nucleated and peripheral microtubules. Peripheral bundles were able to move laterally in order to form the spindles between the spindle poles. [3]

Article 4: For sister chromatids to be correctly segragated between daughter cells, the kinetochore forms bivalent attachments with the spindle microtubules and the kinteochores position themselves correctly with respect to the division plane of the cell. Bivalent attachment of the sister chromatids to the spindle is achieved when the plus ends of the microtubules emanating from each pole interacts with the kinetochores of each sister pair and then becomes embedded. It is well established that CLIP-170/Tip1 localizes to the kinetochore.The plus-end microtubule binding proteins ( +TIP) play a significant role in the regulation of microtubule stability and cell polarity during interphase. In this study, they investigated the role of +TIP proteins during mitotic progression and provide evidence suggesting that the +TIP protein Tip1 affects directly or indirectly the movement of the chromosomes towards to the poles during anaphase [4] .


Mitosis and spindle geometry.jpg

Diagrammatic representation of mitosis, the mitotic apparatus, and different types of kinetochore attachments [5]

Copyright: © 2012 Silkworth and Cimini; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.



References

  1. Michael Glotzer The 3Ms of central spindle assembly: microtubules, motors and MAPs. Nat. Rev. Mol. Cell Biol.: 2009, 10(1);9-20 PubMed 19197328
  2. Gregory C Rogers, Stephen L Rogers, Tamara A Schwimmer, Stephanie C Ems-McClung, Claire E Walczak, Ronald D Vale, Jonathan M Scholey, David J Sharp Two mitotic kinesins cooperate to drive sister chromatid separation during anaphase. Nature: 2004, 427(6972);364-70 PubMed 14681690
  3. U S Tulu, N M Rusan, P Wadsworth Peripheral, non-centrosome-associated microtubules contribute to spindle formation in centrosome-containing cells. Curr. Biol.: 2003, 13(21);1894-9 PubMed 14588246
  4. Sherilyn Goldstone, Céline Reyes, Guillaume Gay, Thibault Courthéoux, Marion Dubarry, Sylvie Tournier, Yannick Gachet Tip1/CLIP-170 protein is required for correct chromosome poleward movement in fission yeast. PLoS ONE: 2010, 5(5);e10634 PubMed 20498706
  5. William T Silkworth, Daniela Cimini Transient defects of mitotic spindle geometry and chromosome segregation errors. Cell Div: 2012, 7(1);19 PubMed 22883214


Good websites to look into :

http://www.biolreprod.org/content/69/6/2059.full.pdf+html

http://www.nature.com.wwwproxy0.library.unsw.edu.au/nature/journal/v432/n7017/full/nature03061.html

http://www.nature.com.wwwproxy0.library.unsw.edu.au/nrm/journal/v10/n1/full/nrm2609.html

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2200191/pdf/jc11051623.pdf

Spindle assembly and the art of regulating microtubule dynamics by MAPs and Stathmin/Op18

http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0028884

Lab 4

antibody : anti-desmocollin 2 antibody

http://www.abcam.com/desmocollin-2-antibody-ab95967.html ( The downloadable data sheet can be found on the right hand side of the page. A direct link to the data sheet is unavailable).

Desmocollin 2 is a type of desmosomal cadherin.

Junction type : desmosome

Host / Isotype: Rabbit / IgG

Class: Polyclonal

Type: antibody

Species Reactivity: Human, Dog

Recombinant fragment, corresponding to a region within amino acids 480-676 of Human Desmocollin 2 isoform 2A and 2B therefore can be used in immunohistochemistry and western blotting.

Journal article ( This study uses anti-Dsc2. Refer to 'Antibodies, Fluorescent probes and reagents' under Experimental Procedures): http://www.ncbi.nlm.nih.gov/pubmed/21071449?dopt=Abstract This study investigated association of desmosomal proteins with cholesterol-enriched membrane domains, commonly called membrane rafts, and the influence of cholesterol on desmosome assembly in epithelial Madin-Darby canine kidney cells (clone MDc-2). An association was seen with anti-Dsc2 [1].

References

  1. Natasa Resnik, Kristina Sepcic, Ana Plemenitas, Reinhard Windoffer, Rudolf Leube, Peter Veranic Desmosome assembly and cell-cell adhesion are membrane raft-dependent processes. J. Biol. Chem.: 2011, 286(2);1499-507 PubMed 21071449


Lab 6

Tm4.jpg

1. Do you see any change in phenotype between group A and B?

Group A ( Tm4 over expressing cells)

- Highest numbers of stringed and pronged

- More branching and processes

- Appears to be grater fluorescence

- Lamella fans are wider

- Some of the lamella fans have a yellow rim


Group B ( control)

-highest numbers of broken fan and stumped

- more dull in colour

-lamella fans are thinner


2. If you see a difference/ change speculate about a potential molecular mechanism that has lead to the change. if you don't see a change, speculate why that could be.

Group A ( Tm4 over-expressed) group had higher numbers of stringed and pronged cells where as the control group had higher numbers of broken fan and stumped. Research by Had et al (1994) suggests that ‘TM-4 may be involved in the motile events of neurite growth and synaptic plasticity’ which would explain the higher numbers of pronged and stringed structures [1]. Tm4 expression was concentrated in the growth cones of cultured neurons, particularly areas of active neurite growth. It was also postulated that Tm4 could be involved in the stabilisation of actin filaments and the regulation of myosin proteins and actin (which is required in growth cone motility). This correlates with the result of this lab as Tm4 overexpression was depicted in the phenotypes which had more branching and neurite growth and interaction.


  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


Effect of Tm4 expression on db cAMP induced differentiation of B35 cells

Differences in phenotypes

Tm4 over-expressing cells:

-Increased appearance of branching and neurite formation

-Indicates the presence of some form of interaction

-Predominant phenotypes - pronged and stumped

-Increased clustering of cells

-Processes appeared to be shorter and wider

Control:

-Decreased appearance of branching and processes compared to Genotype A

-Therefore less interaction observed

-Thinner and longer processes

-Predominant phenotypes - broken fan and stringed


db-CAMP induces the differentiation of B35 cells, causing more processes to form however it inhibits the expression of Tm4. This results in the decreased length and hence growth of processes in Tm4 overexpressing cells. In contrast, the predominant phenotype in the control is the stringed phenotype with longer processes, indicating that db-cAMP did not affect it as much as the Tm4 overexpressing cells.[1]


  1. R Ferrier, L Had, A Rabié, C Faivre-Sarrailh Coordinated expression of five tropomyosin isoforms and beta-actin in astrocytes treated with dibutyryl cAMP and cytochalasin D. Cell Motil. Cytoskeleton: 1994, 28(4);303-16 PubMed 7954857

Lab 8

Peer reviews:

Group 1 Peer review

Introduction

• The introduction was clear and concise. I think it summed up the topic nicely.

• An image in the introduction could be an eye catching visual stimulus to grab the attention of the audience

History

• Nice colour scheme

Entry into M phase • I think a relevant image would have really enhanced this section. E.g. e.g. image showing CDK and cyclin interaction.

• The reader may not have any previous knowledge of the topic. Terms like G2, M phase etc could be explained through an external link.

• The information was clearly well researched and presented concisely which I liked.

• The images are relevant however they have not been cited properly. Also the information this section is lacking referencing.

Metaphase to anaphase

• I feel that a few introductory sentences about the metaphase to anaphase transition would benefit the reader.

• A relevant image would help to break up the text.

• The information is well referenced.

Mitogens

• This section was written well and structured nicely. The definition also helped to quickly bring the reader ‘up to speed.’ But perhaps the definition could have acted as an introductory sentence instead of a stand alone piece of information at the top.

Disease

• Disease in table form is a great idea. It breaks the information up for the reader and prevents the page from being Current/ future research

• Current research needs more information. The information in future research was good but there are no citations present.

Further comments:

• The group appears to have done some extensive research.

• to improve on 'peer teaching' element, they could have included explanations with the images. This would show their understanding of the diagram and how it relates to their topic.

• some of the references appear multiple times in the list.

• A brief table of history would be helpful for the reader to show the evolution of knowledge on this topic

• A glossary would also help the reader


Group 2 peer review

Introduction :The information was good however I think an introduction should be a slice of what you will be covering in the wiki page. I don’t think this intro offered any insight of what to expect. The image used it eye catching and great to use for an introduction. There are no citations in the introduction.

History : I enjoy the colour scheme of the table. However I believe three entries is not enough to successfully give the reader a taste of the evolution of knowledge on this subject area. I’m sure a lot has happened after 1902.

Mechanisms : The headings, subheadings and general layout was easy to follow. I liked how the bold words take the reader to the glossary however it goes not take you to the specific word so it seems slightly pointless. But then again, it does save the reader from having to scroll to the bottom. The level of information was excellent and shows that the group has done extensive research. However, there are so many proteins involved, it makes me not want to read it. Perhaps the information should be simplified? More images are required to break up this dense body of text. This is make it more enjoyable to read.

Microfilament organization: There are no references here! I think it is essential to add an image of the microfilament organisation here. The information itself is concise and reads well.

Animal vs plant cells: Since this section is about comparison, I think it would be FAR more effective to use a table.

Cytokinesis failure: This was a well written and well structured section. The use of images helped to break up the text. That first image has no copyright information added.

Current research: I like the external links because they allow the reader to pursue more information if they want to.

Further comments: All of the images were relevant to the information discussed which is helpful to the reader. External links to videos are a great idea because it enhances the learning experience for students. Many students would rather watch a video than read if they want a quick brush up or overall impression of the topic. There was discussion on their page which included possible changes and new topics to be included after learning new material from the cell division lecture. They have acted upon these changes.


Group 3 peer review

Introduction: I like how the introduction cleared stated the purpose of the wiki page. The information was concise and clear. Structure: I think there has been an overuse of short sentences which made reading the paragraph a little awkward. The information was concise and clear. The image of the golgi apparatus could have a more detailed description so people can see how the image relates to the descriptions in the body of text. There is no copyright information under the image.

Function: I think this section on function was well written.

History: Personally, I would place the history before the structure and function. It's current position seems a bit out of place. The colour scheme is nice choice and easy to read from. It is good to see that each entry has been referenced.

Models of divisions: Diagrams would be useful for the reader in this section. Numbering the models and using dot points breaks it up for the reader so I like the way it has been structured.

Morphology and molecular mechanisms : This section is well written and well referenced which makes it clear that extensive research went into it. The images are used well to break up the text and the use of schematic diagrams is a good one because it simplifies the complex processes. However, I think the images need more detailed descriptions. The first image is not correctly referenced.

Current model for behaviour during mitosos: The image was a stand out for me and I really enjoyed it. However, I don’t think the image was references correctly. This section is structured very well and there is good use of subheadings. The information was also excellent.

Limitations of current models: Some of the terms should be explained ( perhaps in a glossary) e.g. Sar1p. I like the use of the subheadings and the explanations were good. Areas of future research: The first point definitely needs more information.

Further comments: There is evidence of group discussion on the discussion page. Group members have proposed changes and provided reasons for these changes. Most of the entries on the discussion page are by one person which shows a lack of communication between group members. However the page is looking great so has made a good recovery.Some of the references appear multiple times in the list.


Group 5 peer review

Introduction: I thought this was a great introduction. It was both clear and concise. However I don’t think it’s necessary to make direct references to Hetzer in the introduction.

History: The colour scheme was well chosen and made the words easy to read. The information in the table is very well structure and there is a reference for each entry which is good to see.

Structure of the nuclear envelope: Is it possible to get any more info on the outer membrane? If so, I would definitely add it. I think I read in another peer review that you guys shouldn’t have used review articles. However, you guys have made the proper references to them e.g. As reviewed in blah, so I think it’s fine. Images used are relevant and correctly referenced. Student explanations have been given which shows initiative.

Cell division: There is a looooot of information here. The body of text is far too dense so it requires more images to break it up a little. Also, perhaps the information needs to be summarised more? Too much detail can deter the reader. However the level of information presented shows a superior level of research skills.

Open vs Closed Mitosis: I really like the image that has been used here. It effectively summarises the information.

Current and future research: I think a subheading is required for each new research area. Further comments: Clearly reflects on editing/feedback from group peers and articulates how the Wiki could be improved based on peer comments/feedback. Some of the references appear in the list multiple times.

Group 6

Introduction: I think the introduction has too much mention of the specifics already. The introduction should instead be a broad overview of anaphase and the aspects of it that the wiki will be exploring.

Meiosis vs mitosis: I like the way this is set out. The numbering makes it easy to read and it’s a no-nonsense structure that clearly and concisely gets the information across.

History: Be careful when using dark colours. The text might be difficult to see. I think 4 entries are not enough for the history table. It does not effectively show the evolution of knowledge in this field. Each entry has been referenced which is good to see. The image with the thumbnail ‘further history’ is rather misleading. There was no historical information.

Sections which follow: The information was good. However a number of the images are not accompanied with any supporting information. Providing information with your images not only shows initiative but also complements the information in text body. I also noticed that that all the images are on the right. It would look much better if some of the images were moved to the left. There is a good use of headings and subheadings.

Defects: There has clearly been extensive research here but it’s presented as a massive chunk of writing which I don’t particularly want to read. Subheadings will enhance this section and make it easier to read.

Current research: Well researched and well set out. Maybe a table might be better?

Further comments: Upon looking at the discussion page, it is clearly evident that group members have proposed and discussed improvements to the page and then acted upon them. There’s no index at the start. Some of the references appear multiple times on the list.


Group 7

Introduction: This is a well written introduction however there were some grammatical errors. Also, there is an issue with the thumbnail that needs to be fixed. The elements that you will be exploring in the wiki page should be made clearer.

Structure: This section is well written and the information is quite good. I really like the image that has been used. It is correctly referenced.

Function: The citric acid cycle has no references and should be addressed. I like the use of headings and subheadings. The information for the complexes could be presented in a table perhaps.

History: I think the history table should be under introduction. Its current position seems out of place. I enjoy the colour scheme since it allows the reader to easily see the words.

Mitochondrial fission and fusion: The level of information is great. However, I think a more effective way of presenting this information is in table format.

Disease: This section shows extensive and in depth research.

Further comments: Some of the references repeat in the list. To further improve the element of 'teaching at peer level,' a greater description can be provided for each image to show the student's understanding of the image and how it relates to their topic.

lab 9 note

Random

[1] (16) [2] (10) [3] (11) [4] (33) [5] (33) [6] (1) [7] (21) [8] (8) [9] [10]

  1. Simone Kreth, Niklas Thon, Sabina Eigenbrod, Juergen Lutz, Carola Ledderose, Rupert Egensperger, Joerg C Tonn, Hans A Kretzschmar, Ludwig C Hinske, Friedrich W Kreth O-methylguanine-DNA methyltransferase (MGMT) mRNA expression predicts outcome in malignant glioma independent of MGMT promoter methylation. PLoS ONE: 2011, 6(2);e17156 PubMed 21365007
  2. Jörg Felsberg, Niklas Thon, Sabina Eigenbrod, Bettina Hentschel, Michael C Sabel, Manfred Westphal, Gabriele Schackert, Friedrich Wilhelm Kreth, Torsten Pietsch, Markus Löffler, Michael Weller, Guido Reifenberger, Jörg C Tonn, German Glioma Network Promoter methylation and expression of MGMT and the DNA mismatch repair genes MLH1, MSH2, MSH6 and PMS2 in paired primary and recurrent glioblastomas. Int. J. Cancer: 2011, 129(3);659-70 PubMed 21425258
  3. M Esteller Epigenetic lesions causing genetic lesions in human cancer: promoter hypermethylation of DNA repair genes. Eur. J. Cancer: 2000, 36(18);2294-300 PubMed 11094302
  4. Wei Zhang, Jing Zhang, Katherine Hoadley, Deepa Kushwaha, Valya Ramakrishnan, Shouwei Li, Chunsheng Kang, Yongping You, Chuanlu Jiang, Sonya Wei Song, Tao Jiang, Clark C Chen miR-181d: a predictive glioblastoma biomarker that downregulates MGMT expression. Neuro-oncology: 2012, 14(6);712-9 PubMed 22570426
  5. Deborah S Malley, Rifat A Hamoudi, Sylvia Kocialkowski, Danita M Pearson, Vincent Peter Collins, Koichi Ichimura A distinct region of the MGMT CpG island critical for transcriptional regulation is preferentially methylated in glioblastoma cells and xenografts. Acta Neuropathol.: 2011, 121(5);651-61 PubMed 21287394
  6. H S Friedman, T Kerby, H Calvert Temozolomide and treatment of malignant glioma. Clin. Cancer Res.: 2000, 6(7);2585-97 PubMed 10914698
  7. Simone Kreth, Elisabeth Limbeck, Ludwig C Hinske, Stefanie V Schütz, Niklas Thon, Kai Hoefig, Rupert Egensperger, Friedrich W Kreth In human glioblastomas transcript elongation by alternative polyadenylation and miRNA targeting is a potent mechanism of MGMT silencing. Acta Neuropathol.: 2013, 125(5);671-81 PubMed 23340988
  8. Simone Kreth, Elisabeth Limbeck, Ludwig C Hinske, Stefanie V Schütz, Niklas Thon, Kai Hoefig, Rupert Egensperger, Friedrich W Kreth In human glioblastomas transcript elongation by alternative polyadenylation and miRNA targeting is a potent mechanism of MGMT silencing. Acta Neuropathol.: 2013, 125(5);671-81 PubMed 23340988

Lab notes

Polyclonal antibodies : when a new protein is introduced into a new organism, it will make antibodies against it. The antibodies will however recognise different parts of the protein and these will all congregrate to recognise the protein. This allows for amplification. However if there are any other proteins that share the same characteristics as that protein, the antibodies will bind to these also therefore it is a limitation because it can increase specificty but can also decrease specificity.. Another limitation is that there there is only a certain amount of antibodies you can make form one animal.

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