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Laboratory 1

beutiful

Reference

This is about Prokaryote.[1]

Benjamin Mielich-Süss, Rabea M Wagner, Nicole Mietrach, Tobias Hertlein, Gabriella Marincola, Knut Ohlsen, Sebastian Geibel, Daniel Lopez Flotillin scaffold activity contributes to type VII secretion system assembly in Staphylococcus aureus. PLoS Pathog.: 2017, 13(11);e1006728 PubMed 29166667

Lars Witting The natural selection of metabolism and mass selects lifeforms from viruses to multicellular animals. Ecol Evol: 2017, 7(21);9098-9118 PubMed 29152201

Conte Antonella, Papale Maria, Amalfitano Stefano, Mikkonen Anu, Rizzo Carmen, De Domenico Emilio, Michaud Luigi, Lo Giudice Angelina Bacterial community structure along the subtidal sandy sediment belt of a high Arctic fjord (Kongsfjorden, Svalbard Islands). Sci. Total Environ.: 2017, 619-620;203-211 PubMed 29149744

Messaouda Khallef, Süleyman Cenkci, Dilek Akyil, Arzu Özkara, Muhsin Konuk, Djamel Eddine Benouareth Ames and random amplified polymorphic DNA tests for the validation of the mutagenic and/or genotoxic potential of the drinking water disinfection by-products chloroform and bromoform. J Environ Sci Health A Tox Hazard Subst Environ Eng: 2017;1-6 PubMed 29148923

Ulrich Omasits, Adithi R Varadarajan, Michael Schmid, Sandra Goetze, Damianos Melidis, Marc Bourqui, Olga Nikolayeva, Maxime Québatte, Andrea Patrignani, Christoph Dehio, Juerg E Frey, Mark D Robinson, Bernd Wollscheid, Christian H Ahrens An integrative strategy to identify the entire protein coding potential of prokaryotic genomes by proteogenomics. Genome Res.: 2017; PubMed 29141959


  1. Tomohiro Shimada, Yukiko Yamazaki, Kan Tanaka, Akira Ishihama The whole set of constitutive promoters recognized by RNA polymerase RpoD holoenzyme of Escherichia coli. PLoS ONE: 2014, 9(3);e90447 PubMed 24603758


Test Image

Chlamydomonas cells in neon.png

Chlamydomonas cells in neon[1]

  1. Richard Robinson Centrioles position the nucleus and one another. PLoS Biol.: 2007, 5(6);e161 PubMed 20076674 | PLoS One.

Individual Assessment - Lab 2

Part 1

Progeria Syndrome.png

Hutchinson-Gilford Progeria Syndrome and nucleus[1]

  1. Paola Scaffidi, Leslie Gordon, Tom Misteli The cell nucleus and aging: tantalizing clues and hopeful promises. PLoS Biol.: 2005, 3(11);e395 PubMed 16277559 |PLoS One.

--Mark Hill (talk) 14:54, 3 April 2014 (EST) As I mentioned in the lab, you have uploaded an image already on the site and in fact I used in the lecture. I was expecting you to find your own (new) image).

Part 2

Stimulates Emission Depletion (STED) microscopy is a super-resolution microscopy that was used to measure the size of mitochondrial nucleoids. An important feature of this device was that it enabled a resolution of 40 to 50 nm and thus could give a reliable picture of the nucleoid diameter (~100nm - antibody decorated).This form of microscopy is advantageous as no tissue sectioning is necessary which allows the capacity to produce a more realistic image. Using STED microscopy, mitochondrial nucleoids from several mammalian species were analysed and the results showed that all mitochondrial nucleoids had uniform sizes and were often organized in clusters. It was also observed that there was only a single copy of mtDNA present within most mitochondria nucleoids.[1]

  1. Christian Kukat, Christian A Wurm, Henrik Spåhr, Maria Falkenberg, Nils-Göran Larsson, Stefan Jakobs Super-resolution microscopy reveals that mammalian mitochondrial nucleoids have a uniform size and frequently contain a single copy of mtDNA. Proc. Natl. Acad. Sci. U.S.A.: 2011, 108(33);13534-9 PubMed 21808029

--Mark Hill (talk) 14:56, 3 April 2014 (EST) Very good. Now that we have had the mitochondria lecture, what do you think this paper means?

Individual Assessment - Lab 3

Part 1

ARTICLE 1

I went for a search after old articles that wrote about Nuclear Pore Complex (NPC) so I could grasp some simple concepts about it. “Architecture and Design of Nuclear Pore Complex” talks about its structure and they figured the approximate diameter of the nuclear pore by coating cargo-receptor with gold particles. Division of the nuclear pore into Peripheral and Central channels was also noticed by observing dissected macronuclei from Xenopus laevis oocytes on electron microscopy. In addition, the article tells you about the role these channels play in passive and active transports through nuclear membrane, as well as a picture of their structure.[1]


ARTICLE 2

This article explore the wide role of Nuclear Pore Complex (NPC) on passive and facilitated transports, once this is the only route material can go from nucleus to cytoplasm and vice-versa. The experiment used HeLa cells, where marked molecules were injected. The range of molecules included ones that could go through NPC either by passive diffusion or receptor-mediated routes. It was shown that ions, metabolites and intermediated-sized macromolecules crossed the nuclear membrane by diffusion, whereas proteins, RNA and ribonucleoprotein complexes needed the facilitated transport. It was noticed that though both transports occurred in the central part of NPC they didn't interfere with each other. However, molecules utilizing the same mode of transport affected each other pathways. Hence it was concluded that passive and facilitated transports don't share the same route.[2]


ARTICLE 3

This article investigates the impact of hormones on nuclear permeability. The experiment was made with hepatic cells and several marked molecules were injected in it. It was observed that Ca+ plays an important role when it comes to increase nuclear permeability. They linked this information to the fact that some hormones, such as vasopressin and angiotensin, increase the amount of Ca+ within the nucleus, therefore they concluded that hormones may have a short-term control over the permeability of nucleus membrane.[3]


ARTICLE 4

This interesting article compares the traffic of molecules in health and disease, such as tumour and viral infections. It mentions oncoproteins and tumour suppressor genes and how their balance within the nucleus is important to keep a normal function of the cell, once these genes regulate cell growth and DNA division. Another important point is the role of Nucleuporine 98 (Nup98) and its relation with disease when affected by mutations. Regarding to viral infections, it’s shown that virus can take over export machineries and hinder mRNA from going to cytoplasm and explains why this is beneficial to the virus.[4]


  1. J E Hinshaw, B O Carragher, R A Milligan Architecture and design of the nuclear pore complex. Cell: 1992, 69(7);1133-41 PubMed 1617726
  2. Bracha Naim, Vlad Brumfeld, Ruti Kapon, Vladimir Kiss, Reinat Nevo, Ziv Reich Passive and facilitated transport in nuclear pore complexes is largely uncoupled. J. Biol. Chem.: 2007, 282(6);3881-8 PubMed 17164246
  3. Elizabeth M O'Brien, Dawidson A Gomes, Sona Sehgal, Michael H Nathanson Hormonal regulation of nuclear permeability. J. Biol. Chem.: 2007, 282(6);4210-7 PubMed 17158097
  4. Amir Mor, Michael A White, Beatriz M A Fontoura Nuclear trafficking in health and disease. Curr. Opin. Cell Biol.: 2014, 28;28-35 PubMed 24530809

Part 2

Structures of Caldedrin and Jacob.png

Calderin and Jacob, proteins involved in nucleus signaling[1]

  1. Daniela C Dieterich, Anna Karpova, Marina Mikhaylova, Irina Zdobnova, Imbritt König, Marco Landwehr, Martin Kreutz, Karl-Heinz Smalla, Karin Richter, Peter Landgraf, Carsten Reissner, Tobias M Boeckers, Werner Zuschratter, Christina Spilker, Constanze I Seidenbecher, Craig C Garner, Eckart D Gundelfinger, Michael R Kreutz Caldendrin-Jacob: a protein liaison that couples NMDA receptor signalling to the nucleus. PLoS Biol.: 2008, 6(2);e34 PubMed 18303947 |PLOS Biology.


Individual Assessment - Lab 4

-Anti-Nuclear Pore Complex Proteins antibody (ab73291)

-The antibody derives from Rabbit

-working concentration depends on the different types of application

Application

WB------>Use a concentration of 1 - 2 µg/ml

ICC/IF-->Use a concentration of 20 µg/ml.

ICC----->Use a concentration of 2.5 µg/ml.

-paper that uses this antibody

[1]

  1. Theodore A Sarafian, Christopher M Ryan, Puneet Souda, Eliezer Masliah, Upendra K Kar, Harry V Vinters, Gary W Mathern, Kym F Faull, Julian P Whitelegge, Joseph B Watson Impairment of mitochondria in adult mouse brain overexpressing predominantly full-length, N-terminally acetylated human α-synuclein. PLoS ONE: 2013, 8(5);e63557 PubMed 23667637

Lab 9 Assessment

1. Write a hypothesis that you are going to test.

Large quantities of cytochrome C will be detected in the cytoplasm of the cell which can only be released via the intrinsic pathway during apoptosis.

2. Write aims of your experiment.

To measure the amount of cytochrome C within the cytoplasm.

3. Identify key techniques and procedures used in your investigation (Spell these out in some detail).

Centrifugation – To separate the supernatant from the tissue culture. Western blotting – analytical technique used in this experiment to detect cytochrome C.

4. Identify suppliers that have resources that you will need for your study (create links to the supplier resource pages, kits, antibodies etc).

CytoPainter Mitochondrial Staining Kit - Green Fluorescence

Cytochrome c Antibody

Cytochrome C ELISA Kit


5. Now prepare a flow diagram of how the experiment will be carried out and analysed.

Tissue -> Add 1ml of mitochondria Extraction Buffer and 1 ml of Cytosol Extraction Buffer individually -> Centrifuge -> Wash cells with PBS Incubate on ice -> Homogenize cells in an ice-cold dounce tissue grinder -> Collect supernatant as Cytosolic Fraction and Mitochondrial Fraction -> Load 10 μg of each cytosolic and mitochondrial fraction on a 12% SDS-PAGE Western blotting and probe with cytochrome c antibody.

6. What will different experimental results (outcomes) mean.

After using the Assay kit we will see a large amount of Cytochrome C within the Cytoplasm, and by analysing the Mitchondria, we will see pores formed on the outer membrane. Thus, proving that Apoptosis occurred due to the Intrinsic Pathway.