Tag Archives: RF2016-Week3

The Nicolelis Lab is well known for researching brain-machine interfaces (BMI) in an effort to develop brain controlled prosthetic limbs to be used by patients suffering from quadriplegia. Although the lab has multiple ongoing projects, my research falls within the BMI project.

For my project, I am using data that has already been collected by the lab. Using this data, I am calculating lag times between velocity models and neural firing rate models. My hypothesis is that the lag time will increase when a monkey is completing multiple tasks simultaneously, rather than just one task. Knowing and understanding these lag times is integral to the application and construction of brain-machine interfaces to be used by humans.

Although I am unable to go into greater detail of my project on this blog, due to the sensitive nature of my data, I will be sure to edit this blog entry after the data has been published.

The Pickup lab studies poxviruses and their interactions with host cells, as well as their potential to be used as vectors in immunizations. Previous studies using a poxvirus known as Modified Vaccinia Ankara (MVA) were conducted to try and elicit a mucosal immune response in rabbits against the HIV protein GP120. However, MVA administered intranasally failed to elicit a strong, mucosal immune response.

The project I have been working on is crafting a different poxvirus to be used in this study. We are working towards modifying the genome of Rabbitpox Virus to make it replication-defective and capable of expressing GP120. Rabbitpox is very pathogenic in rabbits and infects the cells of rabbit mucosa well; therefore, in theory it should be a more effective vaccine vector than MVA. However, due to its ability to cause serious disease in rabbits, we must modify its genome so that it cannot replicate and produce progeny virus following infection. It is our hypothesis that a replication-defective strain of rabbitpox will elicit a stronger mucosal immunity than MVA against GP120 without the consequence of morbidity in rabbits.

I’ve really enjoyed working on this project thus far and have learned so much. It’s been a lot of work with plasmids and gel electrophoresis, as well as learning to use restriction endonucleases to cleave DNA into pieces of interest. I’m very excited to see this project through, and am excited to see what results it will provide in the end. If our hypothesis is correct, then we may have several opportunities to craft new research questions from our results. This project has been very insightful into the world of research and virology, and I’ve loved every moment of it.

Hello everyone! This week I wrote about the overall research project in Kuo Lab and describe my particular role in it. I hope you enjoy it!

Ependymal cells located in the walls of brain ventricles play an important role in neurogenesis (the growth and development of nervous tissue) and maintenance of brain homeostasis through regulation of cerebrospinal fluid. They are derived from radial glial cells which also give rise to the neural stem cells responsible for adult neurogenesis.  The neural stem cells located in the subventricular zone of the brain are surrounded by ependymal cells and need these cells to differentiate into new neurons, astrocytes and other type of neural cells. Although some studies have claimed to show that ependymal cells may also give rise to new neurons under certain conditions such as concussions or brain injuries, this subject is fairly controversial and need to be studied further to provide a clear answer. Recently, my lab discovered a gene that is critical for maintaining ependymal cell stability. I am conducting mutagenesis experiments to identify functional residues of this protein to make it non-degradable and further investigate the role of it in ependymal cells.

Hopefully, I will be able to find some answer to my questions by the end of the summer and continue to develop this project in the foreseeable future.

When discussing the development of any type of organism, the topics of both genetics and the environment are strongly considered, which has contributed to the debate known as “nature vs. nurture.” At the Donohue Lab, both heredity and the environment are studied as contributors to the germination patterns of Arabidopsis thaliana seeds, but the two factors aren’t as black and white as some “nature vs. nurture” lectures may make them seem. While a human development class may ask the question “Is the parents’ genetics or the environment of the offspring a higher indicator of gene expression?” the Donohue Lab is currently addressing the question “Is the maternal environment or offspring environment a higher indicator of gene expression and germination patterns?” In this research, genetics and the environment are considered together while their interconnection is being further explored.

It was originally expected that the seed’s environment would be a stronger predictor of its germination patterns that the maternal environment. After all, it’s nearly impossible for the mother plant to know what conditions the seed will face when it germinates due to the varying length of dormancy. What if the maternal plant lived in a dense canopy, but a fire comes and wipes out most of the seed’s possible competition? Unexpectedly (as researched by lab members Lindsay Leverett and Gaby Auge in a paper that’s currently in press), the maternal environment seemed to have a much more significant impact on the proportion of seeds that germinated. Furthermore, when the maternal plants lived (while the seeds were maturing pre-dispersal) under a simulated canopy, meaning greater competition when the seeds dispersed and germinated, the seeds were more likely to germinate. Which is really strange time to germinate, as you’d might expect that the seed will be less likely to survive when there were more plants around competing for the same resources.

The maternal environments used in the previous study were simulated with filters. A clear filter allowed white light to pass through, mimicking little to no competition. A green filter both lowered the irradiance of light that passed through and altered the red to far red light ratio, mimicking a dense canopy. My project introduces a neutral filter, which lowers the irradiance of light but does not change the light ratio, which could help explain the results of the previous experiment. This will answer the first question of the project: is light acting as a source of information (light ratio) or a source of energy (level of irradiance)?

The second objective is to determine what role FLC (FLOWERING LOCUS C) plays in the germination patterns. Four genotypes will be used: Ler (nonfunctional FLC), LFC (high FLC expression), 145, and 146 (both of which have high FLC expression that has been knocked down through RNA interference).

Therefore, this project has a lot of different variables that will be analyzed to determine their impact on germination. We’ve already prepared 2304 plates of agar (!) and put 20 seeds in each according to genotype, maternal light treatment, germinant light treatment, and temperatures. We’ve also seeded them, so the plates now look like this:

We’ve also done the first week germination census, so some data has started to come in! After another week, there will hopefully be enough data to begin analyzing the results. All in all, the goal of this project is to explore the variety of information sources that can have an impact on germination and how genetics and the environment play a role together.

The Poss lab focuses on the concepts and mechanisms of regeneration in zebrafish. Zebrafish are a known animal model system for studying regeneration because of their remarkable ability to regenerate lost tissue after injury. Adult zebrafish are capable of regenerating tissue in the heart, as well as fin, spinal cord, and retina.

Poss, Kenneth D. “Getting to the Heart of Regeneration in Zebrafish.” Seminars in Cell & Developmental Biology 18.1 (2007): 36-45.

In fact, it has been shown that zebrafish are able to regenerate their hearts, not due to stem cells, but the proliferation of spared cardiomyocytes (Poss, Wilson, & Keating 2002).

Poss, Kenneth D., Lindsay G. Wilson, and Mark T. Keating. “Heart Regeneration in Zebrafish.” Science 298.5601 (2002): 2188-190. 1 July 2016.

This picture shows the regeneration of ventricular myocardium in the resected zebrafish heart. After 20% of the ventricle was amputated off, by day 60, no sign of injury remained and the heart is fully repaired.

In contrast, mammals do not have this capacity to regenerate and repair its tissue efficiently.

Poss, Kenneth D. “Getting to the Heart of Regeneration in Zebrafish.” Seminars in Cell & Developmental Biology 18.1 (2007): 36-45.

Thus, injury in the heart often leads to scarring and fibrosis. This makes diseases such as heart disease a significant threat to human health. Studying the mechanisms of regeneration in zebrafish can illuminate factors that may be applicable to the stimulation of human heart regeneration.

My research project focuses on heart development. Specifically, I am looking at 4 different genes and the roles they play in cardiac development and regeneration. The zebrafish heart is divided into two main parts – the atrium and the ventricle. The overall concept of this project is to examine what factors in heart development are responsible in pushing some cells into differentiating into the atrium while pushing other cells into becoming the ventricle. In order to examine these genes’ influence in heart development, I look at whether these 4 genes are atrial specific, meaning they are uniquely expressed in the atrium, or whether there are ventricle specific, meaning they are only present in the ventricle. Two of the genes I am examining were hypothesized to be atrial specific while the other two genes were hypothesized to be ventricle specific.

In order to examine where in the heart these genes are being expressed, I am performing in situ hybridization with adult zebrafish hearts. In situ hybridization is a method that uses a labeled complementary piece of DNA or RNA to locate a segment of DNA or RNA of the gene of interest in tissue (Wikipedia). I will first make a RNA probe that is a segment of RNA complementary to one of the genes of interest. This RNA probe will contain digoxygenin tags that will then be used to immunohistochemically label cells expressing the complementary mRNA. The areas of the heart where the gene is being actively expressed will turn blue. With this method, I will be able to see exactly where in the heart the genes of interest are being expressed. I will also perform in situ hybridization on 6 week old zebrafish to see where the genes are active during the earlier stages of zebrafish life. Furthermore, I will perform in situ hybridization on injured adult zebrafish hearts in order to see where in the heart these genes are being expressed during regeneration and if there are any changes in levels of expression compared to uninjured adult zebrafish hearts. The next step in this project would be to identify possible atrial or ventricle specific enhancers of these genes, clone them in front of a GFP gene, inject into zebrafish embryos, and observe where in the heart these enhancers are active (turns green). Then, CRISPR/Cas9 gene editing would be used to delete the enhancers of these genes and to examine the effects of the deletion on the cardiac development of zebrafish. Overall, with this project I am hoping to find out what roles these four specific genes play in heart development and regeneration.

Unlike the majority of the Poss Lab that focuses on regeneration, my project looks more at heart development. However, development and regeneration are closely linked, so I hope I will be able to contribute information on the roles of these genes in the heart in terms of both cardiac development and regeneration.