Depression is one of the leading causes for disability WORLDWIDE.

There are currently 121 million people worldwide who are affected by depression.

Only 25% — ¼ — of those individuals have proper access to effective treatment for the disease.

THEN, of that 25% of depressed patients, only about 60 to 80 % report to have been treated effectively.

This means that roughly 25 to 50  million people worldwide experience no relief from their systems, and receive no adequate treatment.

Despite all of these statistics that indicate that depression is nearly unbeatable in many cases, it can in fact be diagnosed and treated in primary care.

So – how is depression treated? The simple answer is that depression individuals are more often than not prescribed anti-depressant drugs. These anti-depressants, sometimes called AD’s, work by elevating levels of brain norepinephrin, which is more commonly known as serotonin.

HOWEVER, typically, the effects of AD therapy lag about 3 to 4 weeks behind this elevation in brain serotonin levels – and THIS finding suggests that long-term changes neural plasticity or neurochemistry are mediating antidepressant effects. The enzyme that is responsible for the synthesis of serotonin in the brain is called tryptophan hydroxylase 2 (TPH2), and mutations in the TPH2 gene have been strongly associated with major depression and responses to antidepressants.

Thus, this brings me to the focus of the lab that I have been working with since joining the Research Scholars Program.

We are currently investigating the effects of brain serotonin deficiency in response to AD’s.

In order to do this, my mentor and I have been comparing the cellular responses of WT mice and mice with a ‘loss of function’ mutation in their TPH2 gene to chronic treatment with antidepressants.

So far, we know that CHRONIC and NOT ACUTE treatment with fluoxetine (the active ingredient in AD’s like Prozac) for 3 weeks leads to increased neurogenesis in the dentate gyrus of the non-mutant mice, while those mice with mutant forms of TPH2 no show increased neurogenesis.

NOW, we are in the process of determining whether serotonin supplementation in mutant TPH2 mice restores the therapeutic effectiveness of fluoxetine by restoring the ability to increase neurogenesis in the dentate gyrus.

And just to clarify, serotonin supplentation simply means that we administer the neural serotonin precursor – 5-HTP— to the mice with the TPH2 loss of function mutation.

As it stands, the goal of this study is to eventually make it possible to screen depression patients for TPH2 mutations or for brain 5-HT (serotonin) deficiency, thereby allowing medical professionals to specifically treat those individuals with a combined SSRI (antidepressant) AND serotonin supplementation strategy.

In the lab, I have been growing mutant cells in YDP growth medium and comparing trehalose levels in the cells treated with trehalase with the cells treated without trehalase. I do this by preparing a glucose assay and measuring the glucose levels in each cell, which indirectly measures trehalose levels (because trehalase breaks down trehalose into glucose, higher glucose levels means less trehalose). Cells that show lower levels of trehalose in trehalase-treated cells versus non-trehalase-treated cells indicate that these cells have a mutation that affects how much trehalose is produced.

Lucy Yang

The plan is for the experimental set-up to include a rotating plate that is heated at the edges and cooled in the center. This heating and cooling creates a temperature gradient on the rotating plate so that when a droplet of liquid is added, the centripetal force spreads the liquid outwards. The temperature gradient will cause a contraction effect, canceling out the centripetal force and hold the water in place in the form of a thin film. I will be responsible for writing the Labview program to measure the temperature gradient of rotating plate. We hope that through this experimental set-up, physical properties of the thin film can be observed and recorded.

So far I have been preparing numerous PCR reactions to amplify a gene called cyclophillin A (cpa1) and to soon express this gene with a GFP tag. For the PCRs I have been using Ex Tag enzyme to catalyze the reaction and typically use 0.3 microliters per 50 microliters of reaction mixture. To make sure my PCR products are the right size, I usually run the products on 0.7 to 1.5 % TBE agarose gels. I have also been taught the process of purifying PCR products using the Bench Protocol: QIAquick PCR Purification kit.

I will be continuing this work for the remainder of the semester and into the summer, and hopefully will get some positive results J.

In the lab so far, I have done DNA Extraction and PCR with my hybrid specimen, and ran a gel with the amplified DNA last Friday with results that look good but that I still need to analyze. The final goal of this little mini-project is to get me comfortable in the Pryer Lab and hopefully get my name on a declaration of this hybrid fern species. My mentor, Amanda, has been incredibly helpful getting me oriented in the lab, teaching me new protocols, and giving me tons of background about ferns, genetics, and biological practices. She is also really fun to hang out with, and I have a blast each time I come into the lab. Recently, we have started dreaming about a field trip to South America some time in the future, but alas those dreams are a long ways out.

When I do start a project of my own, it will be related to the fruit fly Megaselia Scalaris.  This species of fruit fly has not been studied extensively, and thus much remains unknown about it.  Nevertheless, it is known that this species features some very interesting characteristics.  I am beginning to read papers on the species so that I may develop a project idea.  One interesting characteristic of Megaselia Scalaris is that there is not really a dedicated Y-chromosome.  The male-determining factor has been found on different chromosomes, and even has been found to jump from one chromosome to another when a line of Megaselia Scalaris are bred in the lab.  Another interesting characteristic of Megaselia Scalaris is that they are unusually tolerant of compounds that are toxic to similar insects.  For example, they survived chronic exposure of high levels of Manganese and Nickel in their food, and even seemed do thrive under these conditions, with a significantly increased percentage of puparia formed that successfully eclosed under the metal treatments (2009, Sorensen et al.).  Megaselia Scalaris have even been recorded as been able to survive off of paint and boot polish!  I am excited to create and begin a project with these amazing creatures!

To delete this enzyme, a specific set of procedures are followed. This set of procedures involves designing new primers to be amplified through PCR. These primers amplify the targeted region coding for the enzyme, and include a multi-cloning site that allows a drug-resistant gene (with a homologous multi-cloning site) to knockout the targeted enzyme gene. We check PCR products by running gels; we transform amplified DNA to yeast, replica plate the yeast onto YPD + drug plates, purify onto YPD + drug plates (to screen for individual colonies that are drug resistant and may have knocked out the specified gene coding for the targeted enzyme), prep the new DNA, and run gels to verify if the targeted gene (coding for the enzyme) is actually deleted and if the drug-resistant gene is in its place.

We are currently on our second attempt at deleting this targeted gene that codes for this specific enzyme. The first attempt came out negative because the drug did not knockout the targeted gene; instead the drug (gene) lied down at another location in the yeast genome. Our second attempt includes some modifications in the screening process.

I am really enjoying working in lab. I’m learning a lot of new techniques dealing with DNA, and I am beginning to feel more comfortable working in a lab environment. I hope to continue working in this lab and working on this project in the future.

I will be conducting my research with Dr. Rebekah Fleming and based on our conversations it will include using behavioral measures, molecular biology, electrophysiology, and neuroproteomics methods to investigate the effect of binge alcohol exposure during adolescence. It is believed that neurological changes will occur during this young adulthood exposure that will result in physiological hypersensitivity to the effects of alcohol on memory. We will be using rats as the animal model on which our results will be based.

I am very excited for the upcoming weeks when I think that I’ll be able to start working in the lab! It seems like a very interesting projects and I am excited for all that I will learn.

Heart defects are among the most common birth defects, and a large proportion of congenital heart disorders are neural crest-related developmental defects. The neural crest cells are a transient, migratory cell population that gives rise to diverse cell types. The cardiac neural crest cells are required for proper remodeling of the arch arteries and correct alignment of the arterial pole of the heart. Dr. Keyte’s main hypothesis is that the neural crest cells modulate levels of FGF8 in the pharynx via endocytosis, which is required for normal cardiovascular development. The primary aims of this research is to determine the effects of this endocytosis on FGF8 as well as the consequences of altering the endocytosis on development.

So far, I have been primarily working in histology where I have been preparing slides of chick embryos at different stages of development so that they can be analyzed by observing the presence of neural crest cells. I have begun to learn how to use the photographic microscope that can view the different stains that have been put on the slides. I have enjoyed working with Dr. Keyte, and she is always willing to help or explain things when I have questions. My first exposure to research has been very enlightening, and I am eager to see what the future holds!