Learning Journal 5

1.       Finding a classmate that agree to let you see their wordpress blog/portfolio…

Find a classmate who is comfortable sharing their portfolio with you, and whom you are you are comfortable sharing yours with.

Name of the classmate whose portfolio you will be viewing: Ardalan Hendi

2.      Commenting on the portfolio …

Browse through all the viewable parts of your classmate’s portfolio.

• Imagine that you did not know this person at all, and you had to evaluate their skills, knowledge and learning using only the content of their portfolio. What would you say the biggest strengths of this person are, and why (what evidence did you use to come up with the answer)?

One of Ardalan’s biggest strengths is his ability to hook the audience in to what he is presenting and really catch their attention. Especially in the field of science, it can be extremely beneficial to be able to communicate ideas effectively to all audiences (including those with limited knowledge in the science field). Ardalan’s Lay Person Summary effectively demonstrates this ability from beginning to end. He uses the analogy of sick leave at work to help communicate the idea of neuron’s taking over the innervation area of another neighboring neuron that was destroyed.

• After answering these questions as part of your own LJ5, post them as a comment on the classmate’s portfolio.

See: https://blogs.ubc.ca/biol463463/author/ardalan-hendi/

3.      Your final project and your classmate’s final project

• What is the most valuable thing that you learned from doing your Final Project (mini-research proposal)?

The most valuable thing that I learned from my own final project would probably be the importance of thorough background research and planning before actually beginning an experiment. As I was researching my gene of interest, Pho88, I found that as I read more and more literature on it and the rest of the genes in the phosphate transport pathway, the better of an idea I was able to build about where Pho88 might fit in the pathway, and which experiments would actually give me novel information. In addition, as I researched more and more, I found that gene regulation was not the only mechanism that could effect my results, other cellular processes such as intracellular trafficking and post-translational modifications could affect them as well.

• Look at what your classmate has posted under their Final Project section. What do you think your classmate may have learned, that you have not (and/or what do you think you have learned, and they may not have)? After the end of term, feel free to check your classmate’s portfolio again to see if your answers match.

Ardalan seems to have taken a lot of what he learned from this class and applied it to the research field specifically. This is evident in posts such as his “research funding one”, as well as his final project which is in the area of neuroscience research in C. elegans (which we study in a lab together). I feel that I have taken what I’ve learned from this course in parallel with my other big courses this term, BIOL 441 and BIOL 340, and applied my knowledge to every day interactions and how to communicate science to those without an extensive background in the field.

lay-person-summary

biol-463-annotated-bibliography

biol-463-final

In-class assignment: Fukuda et al. (2016) paper

While the main claim of the paper is related to the maintenance of Xist imprinting, we will focus more on three aspects of the mechanism of this imprinting: chromatin ‘relaxation’, the effects of Kdm4b and TSA, and the real of Oct 4.

You are welcome to work on these questions individually or as part of a group. If you work in a group, your group will submit one set of answers for the whole group. Please ensure that all group members participate in the development of the answers.

The answers are to be submitted by email (as an attachment) by the end of class.

PLEASE FOCUS ON ANSWERING THE QUESTIONS IN PURPLE (2, 4, 6, 8, 9).

Chromatin ‘relaxation’ and Xist

2. What can be said about the chromatin of and around the Xist locus in bimaternal, very early embryos that express Kdm4b and are treated with TSA? How do Kdm4b  and TSA affect the levels of chromatin ‘relaxation’? How does this relate to Xist expression and XCI?

It was found that loss of H3K9me3 via Kdm4b (a H3K9me3 demethylase), or gain of histone acetylation by TSA induced Xm-Xist expression. In an investigation of whether chromatin decondensation is associated with Xist expression through Kdm4b and TSA, Fukuda et al. found that Kdm4b/TSA-XmXm embryos showed significantly relaxed chromatin states in both stages compared to Egfp/DMSO (control) – XmXm embryos. This shows that Kdm4b and TSA affect chromatin by promoting the relaxed state, and therefore increasing expression of Xist. This increased expression of Xist induces X chromosome inactivation.

Lethality of embryos without paternal Xist.

4. How do you think Kdm4b and TSA can rescue the lethality of a missing paternal Xist in Xm Xp embryos?

Since Kdm4b and TSA promote chromatin relaxation at the Xist region of the chromosome, they increase the expression of Xist. Once the chromatin is decondensed in early preimplantation phase, Xm-Xist can be derepressed and result in the rescue of lethality by Xist paternal deletion (XmXpΔ). This indicates that genetic lethality can be overcome without direct gene manipulation. Since Xp lacks Xist, it will continue to be active, but decondensation of the Xist region on Xm induces XCI on Xm. This prevents two X chromosomes from being active, and thus is able to rescue lethality.

Roles of the pluripotency factor Oct 4

6. What can you directly conclude from Figure 5a?

From Figure 5, we see that in Oct4 knockdown Xist is expressed at the same levels as in the Scramble-treated cells, but Tsix is expressed at significantly lower levels. From this, we can conclude that Oct 4 is required for the expression of Tsix (at normal levels).

Mega-model

9. Now, try to fit everything you have learned about XCI into a sensible model that explains how XCI occurs in mouse at the pre-implantation and post-implantation stages. Your model should include at least Xist, Tsix, Jpx, CTCF, Oct 4, Nanog, Cdx2, H3K27me3 and H3K(various)ac. If you feel adventurous, make a note of which part of the model corresponds to which hypothetical mechanism seen at the start of the unit (i.e. blocking factor, symmetry-breaking factor, alternate states of the two Xs, etc).

Start with Xist expressed on paternal and Tsix expressed on maternal. Cdx2 comes with the paternal X – it is what silences Tsix and keeps Xist expressed on the paternal X. Oct 4 is turned on and activates the transcription of Xm Tsix in cis. Nanog comes in and dimerizes with Oct 4 having no effect on Xm as its function is repressing Xist. Cdx2 expression is turned off at Xp. This allows Nanog Oct 4 dimers to bind and cause Xp silencing by repressing Xist and promoting Tsix expression in the embryo. One of the chromosomes (paternal or maternal) is then inactivated randomly by dissociation of dimers. On the other hand, Cdx2 remains bound  to Xp in the extraembryonic tissues, thus Xp is kept inactive. Expression of Nanog and Oct 4 is turned off – thus there is no expression of Xist or Tsix on the Xm.

In cases where no Nanog Oct 4 dimers are bound. Acetylation and methylation of H3 play a role in determining whether Tsix/Xist are/aren´t expressed.

Furthermore, expression and binding of CTCF close to Xist promoter prevents Xist transcription. However Jpx can bind CTCF and drag it away – allowing expression of Xist.

See short video:

http://mashable.com/2016/11/29/skingun/#E83TWOtke5qL

I recently came across this video on facebook and was quite amazed. The video highlights a new treatment being used by burn victims to help them grow new skin where theirs has been badly burned. The treatment involves a gun type apparatus called the SkinGun that sprays stem cells of the burn victim directly onto their wound. The stem cells can help the body regenerate new skin on the wounded area without leaving visible scars behind in the healing process. The SkinGun has yet to be approved for commercial use in the US, but hopefully will be soon. If I were to experience a severe burn on part of my body such as my arms or legs, I would definitely consider using the SkinGun as a treatment. One of the reasons I would be so eager to try this treatment is because it would use my own stem cells to help regenerate my burned skin. Having someone else’s tissue or cells used in this type of treatment can sometimes result in the tissue or cells being recognized by your body as foreign and therefore be rejected. By using a victim’s own cells, this type of rejection is very unlikely to occur. In addition, this method may be a great alternative to skin graft transplantation where a patch of skin from some other part of the body is taken to help repair the burned area.  The SkinGun would not require excision of any large patches of skin on the victim’s body to heal the burn, only iPS cells!

Genetically engineered foods are greatly criticized, and often feared, by many people. Initially people feared that exogenous DNA from the food item could then transfer to the consumer’s cells and cause harmful effects. More recently (and with the advent of siRNA technologies) there is a worry that ‘transgenic RNA’ from the food could get into the consumer’s cells and cause misregulation of the consumer’s genes. What is your view on this, and how would you present it to someone who has no biology background?

Although some people still have fears about genetically engineered or modified foods, I believe that they are safe for consumers. There are a couple of reasons that I believe GMO foods are safe for consumers, the first being that research shows that GMO foods are safe. My boyfriend’s mother is very health conscious and her own concerns inspired me to do some research on the subject of GMO produce a few months ago. Through my research I discovered that no significant differences have been found between nations that consume large quantities of GMO produce (ex: USA), and those who don’t (ex: Britain), in the ratings of cancer prevalence, obesity, diabetes, and many other illnesses feared to be associated with GMOs. In addition, I could’t find any studies  that found significant causal or correlational relationships between GMO food consumption and any disease or illness. The second reason that I believe GMO foods are safe is that the digestive system and cells of the body do not take up and use DNA or RNA in food being consumed. Foods that are taken in and processed by the digestive system are broken down into very small particles and molecules. DNA and RNA molecules of other organisms do not have the required signals to pass through the plasma membrane of the cell AND the nuclear membrane, which would be required in order to enter the nucleus of cells and be transcribed. As a result, these molecules are broken down into very basic components that can be utilized by the body to construct other molecules. People should not have a fear that the human body could use or be disrupted by foreign DNA or RNA since the cell highly regulates what enters and leaves its membrane. Even if RNA or DNA were to somehow find its way into the cell, it would need to have very good homology or sequence consistency with the DNA of humans in order to function and induce some sort of change. This is not usually the case since many differences exist between human and plant or animal DNA. In order to diminish some of the fears people have about GMO foods, I would try to present this information to them in a conversation. I would explain how the human body works and why RNA or DNA from a different organism would not be able to induce a change in gene expression in humans if consumed. Educating people on the basics of genetics and the body would probably be the most affective avenue for alleviating fear of GMOs.

One concept about gene regulation that I have learned through this course.

One concept about gene regulation that I have learned through this course as well as BIOL 441 (Cell Biology of Intracellular Trafficking) this year, is that gene regulation can be very difficult to study because it occurs at so many different levels. These different levels of control can exist within intracellular processes, but can also differ between cells depending upon signals they receive from the environment. The first, and arguably the most significant level of control is that achieved at the transcriptional level within the nucleus. Even at the transcriptional level alone, there are many ways that a gene can be regulated. From altering the number of copies of RNA that are transcribed, to the variation in timing of when the gene is actually transcribed. The next level of regulation is at translation or when the gene transcript is converted into a protein. Here, the ribosome complex “reads” the information encoded by the mRNA transcript, and it builds an entire protein out of the amino acids in the cytoplasm. Regulation through the number of ribosomes working on each transcript can be achieved at this level.

In BIOL 463, we focussed a lot on epigenetic regulation of genes. Epigenetic regulation is any functionally relevant changes to the genome that occur, but do not involve a change in the nucleotide sequence. Examples of epigenetic mechanisms are DNA methylation and histone modification, which alter how genes are expressed without altering the DNA sequence itself. Gene expression can be controlled through the action of repressor proteins that attach to silencer regions of the DNA. In BIOL 441, I learned about another mechanism that can indirectly regulate gene expression by affecting the amount of transcript or protein present in a certain cell compartment. This mechanism is intracellular trafficking. For example, if a transcript or protein acts in the extracellular environment, up regulating its secretion and release from vesicles could increase its effect without directly changing gene transcription or translation levels. Another way gene expression can be regulated is by having any protein or regulatory molecule involved be turned on or off by the activity of an enzyme.

This is why its important to test for levels of your molecule of interest at each level. Ex: You may notice that the phenotype of two individuals is the same, but there is a possibility that transcription differs greatly between the two and that the trafficking of the molecule is simply being held up at one point, in one individual.

If I could develop anything through an iPS cell experiment, what would it be? Why?

If I could develop anything through in iPS cell experiment, I would love to find a way to generate egg cells for females. Specifically, infertile females or those with low fertility. Personally, having problems with fertility and being able to have a child of my own has always been one of my greatest fears. Being able to have a child who shares much of my genetics would be incredible. In addition, one of my best friends is unable to produce fertile eggs because of the treatment she received for cancer when she was 11, and she has experienced a lot of hardship in accepting the idea that she will never be able to carry a baby and have a child who shares her own genetics. These are a couple of reasons I, and I think many people would be supportive of the generation of egg cells from a woman iPS cells. Right now, one of the common options for women who cannot produce viable eggs is to purchase eggs that have been donated by another woman. In this case, the egg can be fertilized by your partner’s sperm in vitro (in a laboratory), and then later implanted into your own, or a carrier’s uterus. Although this method works, it still does not solve the problem of women who cannot produce viable eggs being able to share their genetic material with their child. For their case, only the DNA of their partner can be transferred to the child, but DNA in the egg cell will come from the donor female. I brought this idea up in class when Dr. Kalas asked students what we would do if we could conduct any experiment with iPS cells. One of the problems with my idea that was mentioned was that it is very difficult to generate reproductive cells or gametes from iPS cells. Hopefully one day this technique can be possible so that women can always have the opportunity to have children that share their DNA.

PS: This was achieved successfully in mice for the first time this year. Study published just over a month ago, check it out!:

http://www.sciencemag.org/news/2016/10/mouse-egg-cells-made-entirely-lab-give-rise-healthy-offspring

Egg cells derived in the lab from embryonic stem cells.

O. Hikabe et. al., Nature 538, 7625 (20 October 2016) © MacMillian Publisher Ltd.

You have been working on developing a novel, testable question on a gene regulation-related topic. What is the most challenging aspect of it and why?

For my final project, I have been working on developing a novel testable question about a gene called pho88. When choosing a topic, I wanted to select a gene that very little was known about so that I could have a very wide array of experiments and options to choose from. Pho88 is gene related to the phosphate transport pathway in yeast, but as no research was done specifically on this gene, and therefore it is definitely poorly understood. Unexpectedly, this aspect of it has made it quite difficult for me to narrow my focus down to one testable question about it because so much could still be discovered about it. Specifically, I have found that the pho88 mutant has many phenotypic similarities to pho86, another phosphate transport gene. Both of these proteins have proven to localize in the ER membrane of yeast, suggesting possible interaction or interaction in the same pathway. More importantly however, an up regulation in certain phosphate transport genes was seen in the double mutant of pho88 and pho86 under high phosphate conditions. This affect on gene regulation is the opposite of what occurs in wild-type yeast, and therefore it suggests that pho88 and/or pho86 could influence gene regulation. This is the knowledge gap I will be investigating in my project. However, since it is known that pho86 interacts with other phosphate transport proteins in yeast (such as pho84), I also want to test interactions between pho88 and these proteins, as well as pho88 with pho86. All of these observations have sparked my interest in pho88 and what its role is in the phosphate transport pathway, but they have also made it difficult for me to choose only one or two experiments to “conduct” in my project.