Category Archives: Week 3

BRET and Schizophrenia

Research Question: To what extent do the D3 and D4 receptors play a role in mediating a response to a schizophrenia drug? What implications would this have for the need for more highly specific drugs in the future?

Metabotropic (7-TM GPCRs) receptors are a major family of membrane receptors (the largest, most diverse group in eukaryotes comprising the largest class in the human genome). GPCRs are also the target for ~50% of drugs on the market, and yet only a small portion of GPCRs have been tested, leaving the possibility for great potential in the field of receptor pharmacology. All 5 dopamine receptors are metabotropic, with the D2/D3/D4 receptors exhibiting close homology and the D1/D5 receptors exhibiting a close homology. While the D2 receptor is often implicated in contributing to reward in the human midbrain, the question of the effects of drugs on the D3 and D4 receptor suddenly becomes relevant. Many of the dopaminergic pathways originate in the midbrain (like the substantia nigra) and one of the hallmarks of schizophrenia is dysregulated doapminergic pathways (like excess dopamine in the midbrain). Traditional antipsychotics are antagonists of the D2 receptor which can alleviate the symptoms associated with the midbrain, but not with the forebrain where the dopamine is already depleted.

Using BRET assay, it is my hope that I will be successful in seeing how a new schizophrenia drug may act on the Dopamine 3 and 4 receptors which previously have been essentially disregarded. Due to recent publications commenting on the widespread distribution of the D3 and D4 receptors in the cortex, it is now important to not only observe the effects of biased signaling but also of signaling associated with receptor specificity.

Where is PTPRZ1?

My project comes out of the work of my mentor Katie Baldwin. Katie used data collected from the Barres lab at Stanford to compile a list of genes that were highly expressed in astrocytes relative to other cells in the brain. She then took this list and looked into the identity of each gene to see what was known about it and if it could be interacting with synapses (the focus of the lab being astrocytes’ influence upon synapses). She then looked at the effect of knocking out some of these genes on astrocytes to see if there were any clear differences in the shape of the cells. Once she had made this list of research subjects, Katie, as well as some other members of the lab, began to investigate what these genes are doing in astrocytes.

When I came to the lab, Katie told me about a few genes from this list that hadn’t been investigated yet and would be good projects for me to work on. One such gene she was particularly interested in was PTPRZ1. PTPRZ1 is a membrane-bound receptor that removes phosphate modifications from proteins. This sort of process can be important for controlling existing proteins, so I was very intrigued by this research target. Katie had also already seen that losing this gene caused astrocytes to look strange.

This summer I have been working on trying to figure out where this protein is expressed and developing techniques to be able to see it both in cells and in dead tissues. I hope to have good protocols for microscopy and western blotting to learn more about this protein’s function. I also plan to return to the lab when the semester starts to further our understanding of PTPRZ1 role in astrocytes.

It’s about this fungus and its titan form

Cryptococcus neoformans is a cryptic pathogenic yeast that is able to elude macrophages by replicating its chromosome set (among other cellular components) until it’s too big to be engulfed. The result are huge polyploidal cells, called titan cells, that are amazingly able to bud into haploid progeny. Just how this occurs is what my postdoc, Ci Fu, Dr., and I are currently researching.

The primary focus of this project are six cyclin genes that my postdoc chose as possible candidates associated with the C neoformans endoreplucation pathway. In the first week, I worked on creating gene deletion constructs (with a drug at the center) for the project, and by the end of the first week, I was able to create them all (shutout to PCR). In fact, by the middle of the second week, I had to make 8 copies of each gene which resulted in 48 PCR tubes.

Yes, I took pictures of my conquest….

Next we worked on cell culturing for two strains in order for us to do biolistic transformation. My postdoc has been working on a different strain, doing the same thing I am. But the absolute best part of the third week has been shooting a gun- a gene gun, that it is. This beautiful contraption allows me to create pressure in a chamber and literally shoot an aliquot of my gene (mixed with goldbeads) with helium.

…It’s not your average gun…but it does ‘fire’.

After at least 3 hours of recovery, I transferred each shot plate onto two drug plates, which resulted in 48 drug plates (each gene deletion construct was shot four times onto different plates). The idea here is that the genes constructs I made will have stuck to the goldbeads (using a washing process I completed beforehand). Once shot, it will kill the cells in the middle of the plate but transform the ones on the periphery. Since we don’t know which cells were transformed, we have to carry ALL the cells from the shot plate and select for them on a drug plate (I used water, a cell spreader, and a pipet to transfer).

The final part of the project is to see whether these drugs impact titan cell formation. So the third week and during this week, we have to determine conditions for titan cells. Last week, we found conditions for a larger version of my postdoc’s strain, but it wasn’t technically a titan. C neoformans becomes a titan in vivo when they fear being engulfed by macrophages in the lung of the affected individual. Getting these titan cells in vitro has rarely been done before. So my postdoc and I are essentially using the only protocol known so far for getting these titan cells (sent by another scientist). But, we weren’t exactly successful this past week, so this week we have to change the ingredients for incubation in 96 well plates- we limit nutrients so that these cells can hoard it into large vacuoles, a common characteristic of a titan cell. We plan on relying on the studies of a professor who started out in our lab- under my PI Joe Heitman, MD., PhD. – who is now revolutionizing in vivo studies of the pathogenic fungus: Kirsten Nielsen, PhD.

Dr Nielson and her team found this titan cell (in the red) in vivo. The macrophages are clear/gray, to the right and the normal C neoformans cells are in the blue. 

Nonetheless, whether we get an actually solid answer from this project, I am so grateful for the opportunity to be here and learn so much. It’s been fun and exciting to learn new techniques and use incredible technology like a biolistic gun and a multichannel pipet. Everything has been amazing.

Before you take that Puff, think about the children

Weed. Marijuana. Mary Jane. J.

There are many names for the drug that most of us know so well. Many people all over the country have been pushing for the legalization of marijuana with places like Colorado having succeeded. Though not completely legal everywhere, there has been so much scientific evidence as to the medical benefits of marijuana it is used to treat certain illnesses. With all its positive medical benefits, it is not without health risks. There have been studies that show that women who use cannabis during pregnancy are at high risk for having babies with abnormal neurobehavioral functioning in addition to other abnormal biological traits (Huizink, Anja C., and Eduard JH Mulder, 2006). But we all know that there are a lot of things that mothers shouldn’t do because it would affect their children. But what about fathers? They play half the part in making the baby too (in terms of DNA that is). What the father does could also affect the baby as well right?

As most of you may know, this summer I am working in the Neurotoxicity/Neuropharmacology lab of Dr. Edward Levin. The specific project that I am involved in looks at THC and how it affects epigenetics in the offspring when it is administered to the father. Of course, it would be a little unethical to use humans for this study so instead, we are using rats. We are administering THC to male rats, mating them with female rats that have never been high in their life, then putting the offspring through a few tests while also looking at the brain functioning and structure of the offspring. Theses tests include attention tasks, maze tasks, and other assessments that look at cognitive functioning. After those tasks, we will be comparing the brains of those rats and normal rats to see if there is a difference.

I find this study so interesting because as an aspiring neuroscientist\physician it is very imperative to look at how lifestyle choices could affect the neurocircuitry, cognitive functioning, and epigenetics of future offspring. Abnormalities in neurocircuitry could ultimately lead to different types of mental illness. Of course prevention is better than a cure so since I also want to have a focus on mental illness, this working on this study is a great way for me to play a hand in the field that I want to go into in addition to making discoveries that could impact how I help my patients in the future.

References

Huizink, A. C., & Mulder, E. J. (2006). Maternal smoking, drinking or cannabis use during pregnancy and neurobehavioral and cognitive functioning in human offspring. Neuroscience & Biobehavioral Reviews, 30(1), 24-41.

All Things Poop

Our gut microbiomes have long been known to be critical for immunity, nutrient processing, etc. More recently, research has suggested that gut bacteria play an important bidirectional role in brain development & function, and the modulation of stress response. Major Depressive Disorder (MDD) patients have altered microbial compositions and many metabolites which play a role in depression are byproducts of gut microbiota.

My lab is focusing on the effects of chronic social defeat on the microbiomes of mice (as a pre-clinical model). In short, the social defeat (SD) paradigm involves placing the subject mice into the same cages as aggressive mice, allowing them to fight, and then separating the mice using a divider while but keeping them in close proximity to each other for a day. After the process is repeated 10 times with different aggressive mice, the subjects present with anxiety and depressive symptoms.

The aim is to study changes in microbial richness and diversity, and differences in the relative abundances of gut bacteria at the family and phylum levels between depressed and healthy subjects. This is measured through fecal samples which are collected from the subjects’ cages before the paradigm (a baseline) and after SD. The mice are then treated with electroconvulsive shocks (ECS), and post-treatment samples are taken to examine whether reductions in symptoms are also accompanied by a stabilizing microbiome. Many people don’t realize that ECS is still used today to treat severe depression in humans!

The aggregate sequencing results from a pilot study by Kara McGaughey. Each color represents a bacterial phylum, and the shifts in the microbiomes of the depressed group are easily visible.

I’m involved with comparing two DNA extraction kits using the pre-SD fecal samples to choose the more suitable one for the rest of the experiment. I was initially surprised to learn that there is no ‘gold standard’ in DNA extraction and that the various kits in the market all detect varying species & proportions of bacteria. Although my project has ended up being more about microbiology than neuroscience, learning about the different bacterial DNA extraction and sequencing protocols is definitely cool. I’ve also learnt a ton about the measures used to assess the purity (microplate spectrophotometer), concentration (fluorometer) & quality (TapeStation) of extracted DNA. I’m really looking forward to seeing & comparing the sequencing results!

Tyrosine Kinases!

Ever heard of the Philadelphia chromosome? How about imatinib or Gleevec, the highly successful miracle drug most famously used to treat CML (chronic myelogenous leukemia)?

If so, you may have heard of my lab’s focus: the Abl family of protein tyrosine kinases.

When I searched through labs in the Department of Pharmacology and Cancer Biology and came across one with the focus of researching the functions the Abl family of tyrosine kinases, the faintly familiar ideas of the Philadelphia chromosome and Gleevec, which I had associated with the Abl gene, caught my attention. The Philadelphia chromosome represents the abnormal translocation in chromosome 22 found in leukemia cancer cells that results in the Bcr-Abl fusion gene. Imatinib, a chemotherapy medication, hinders the Bcr-Abl tyrosine kinase. Thus, I knew that research in the Pendergast lab, which centers on the exploration of Abl kinases, was sure to be interesting.

Each lab member has an individual project that stems from the Abl kinase focus of the Pendergast lab. Several lab members are utilizing mouse models for their projects. Others, actually all members minus me, continually learn and incorporate creative, new techniques to advance their projects. As for me, I am also learning many new techniques, but ones that have been around for much longer… Nevertheless, I am very grateful to my two lab mentors for taking time off of their own individual research projects to guide me and provide me with the tools I need to conduct research in this lab.

My research project builds on the project of the previous undergrad student in the Pendergast lab, who graduated in May. She focused on triple-negative breast cancer cells, in particular. The goal of my project is to define what role Abl kinases have in the signaling of a specific type of receptor tyrosine kinase (protein) in breast and lung cancer cells. Specifically, I am looking into the role of the interaction between Abl kinases and this particular protein in factors such as cell growth, EMT (epithelial-mesenchymal transition), migration, and invasion.

Now, you may be wondering what exactly is a tyrosine kinase? I will start by explaining one of the roles of phosphate groups. When transferred to a specific protein, a phosphate group can activate that protein. Enzymes (proteins) that add phosphate groups to other molecules, thereby activating the molecules, are called kinases. Tyrosine kinases are a subclass of protein kinases in that they possess the amino acid, tyrosine, to which the phosphate group attaches. Abl genes encode protein tyrosine kinases that activate proteins that are involved in factors such as cell growth and development. A continuous activation of proteins that are involved in important cell processes can lead to cancer, which is caused by an abnormal and uncontrolled division of cells.

Now that you hopefully understand some potentially fatal implications of the interactions of the Abl family of tyrosine kinases with other molecules, you may be wondering, how does one choose which proteins to explore in their interaction with Abl kinases? As my PI, Dr. Pendergast, explained to me, a former postdoc in the lab, along with the previous undergrad, who I mentioned earlier, conducted an unbiased screen for Abl2-induced tyrosine phosphorylated targets, which identified a specific set of kinases to be highly phosphorylated Abl targets. Moreover, it has been shown that these particular kinases are upregulated in breast cancer patients who develop resistance to diverse therapies. Thus, it is worthwhile to investigate and analyze the effects of the signaling axis between Abl kinases and these particular proteins.

Question: how many times can one say, “kinase” in a post? Read the above carefully to find out. 🙂

My research project has involved techniques that include culturing of various breast cancer and lung cancer cell lines, viral transductions, loss- and gain-of-function experiments, immunoprecipitations, and of course, western blots, the bread and butter of our research. I can’t wait to learn even more techniques and practice, practice, practice!

What’s in a Rat Brain Smoothie?

Even though my summer research project can be summarized as simply running a seemingly endless amount of HPLC samples, such a description would not do service to the lab I’m a part of. What I will do is part of a larger project with a much wider scope.  My summer project falls under the umbrella of a Tryptophan depletion study conducted in rat models which is a continuation of work done by my lab in the pas

Of particular interest in the etiology of depression and related affective disorders is the role of the serotonergic neurotransmitter system, the system responsible for the transport of serotonin/5-hydroxytryptamine (5-HT). Even though the exact underpinnings of serotonin’s role in mental disease are unknown, it has been confirmed to be involved with the onset of depression. The theory that depression involves a serotonin deficiency is still widely circulated but study results have sometimes produced contradictory and confounding results. Other neurotransmitter systems have been speculated in playing a role in affective disorder including the dopaminergic system. However, the serotonin deficiency theory is the most accepted and makes up the basis of Tryptophan depletion as a method of studying depression.

Due to its clinical significance, effective methods of studying depression in animal models have been the object of scrutiny in the past. One of the more relevant methods involves implementing a social defeat model: a small male mouse is put in an enclosure with a bigger male mouse which then proceeds to “beat up” the smaller mouse. This method leads to depressive behavior in the small mouse but such a model is not effective in female mice and thus excludes the gender that suffers the most from depression. To correct this problem, rapid tryptophan depletion (RTD) has been suggested as a method to induce depressive behavior in both male and female animals to study it.

Based on the serotonin deficiency theory of depression, RTD involves feeding animals (rats in our lab’s study) a mixture of several large neutral amino acids (LNAAs) which reduces the level of endogenous tryptophan (TRP) in the animal and subsequently the levels of 5-HT. TRP depletion relies on the fact that only a limited amount of LNAAs can pass through the blood brain barrier (BBB). The ingested LNAAs do two things to lower endogenous TRP and 5-HT levels: they stimulate protein synthesis and compete with endogenous TRP for entrance into the brain through the BBB. Because TRP is the amino acid precursor to 5-HT, reduced TRP levels theoretically lead to reduced 5-HT synthesis and depressive behavior.

My lab has done work with RTD in the past and right now they are focusing on studying possible differences of RTD between adolescents and adults. I have joined the lab just as they are in the middle of another RTD study and I will contribute the most in two different was: HPLC analysis and rat sacrifice and dissection. High performance liquid chromatography (HPLC) is an advanced analytical chemistry technique used to detect the presence of certain analytes in a sample. It relies on an electrode system to detect analytes as they pass through the column of the apparatus. Analytes take the form of different peaks in an HPLC program and the area is indicative of the concentration. My summer work deals mostly with the analysis of tryptophan, serotonin, dopamine, and the respective metabolites of these neurotransmitters.

Running samples through the HPLC apparatus makes of the bulk of my work but I also help in harvesting blood and brain samples from the rats used in the study. On my first day of brain dissection, my PhD supervisor even said I was a natural for rat brain dissection and that I knew how to chop a rat head just perfectly! What I think is hilarious, however, about analyzing rat brains using HPLC is that one must “homogenize” them. Rat brain matter on its own is too large for the apparatus so we liquefy them and essentially turn them into rat brain smoothies.

I hope to have analyzed enough samples to complete an interesting poster at the end of the summer. Even though this project has elements that might not align exactly with the research I want to do in the future, I hope to continue learning and enjoying this project like I already am.

Mice, Injections, Proteins, Oh My!

One of the core approaches my lab uses to model depressive-like symptoms in mouse models is the social defeat paradigm. This paradigm allows my lab to simulate behavioral conditions that lead to the onset of depressive-like symptoms which we can then use to study and/or treat. My lab uses this method because it allows us to study the onset of depressive-like symptoms in as close to a real world setting as possible, while not relying on the monoamine hypothesis that states that depression is largely caused by a lack of serotonin or norepinephrine in the brain. As the focus of my summer research work, I have been assisting various lab members on different projects that use the paradigm to study the effectiveness of different treatment methods for depression.

Currently, I am helping a fellow undergraduate student study the relationship between behavior and microbiology in the lateral habenula on the brain. Scientists have found that over excitability in the lateral habenula is correlated with depressive-like symptoms while a smaller region in the lateral habenula has been shown to inhibit these symptoms. Previous data has suggested that a protein, MeCP2, has an antidepressant like effect when phosphorylated in the small region of the lateral habenula. The project that I am assisting with seeks to understand whether MeCP2 is phosphorylated in the brains of mice that have been treated with an anti-depressant called imipramine.

The experimental set up involves 10 days of social defeat followed by 28 days of imipramine injections, or 27 days of saline injections and 1 day of imipramine injections. We will then test 6 mice out of each experimental group and 6 mice of the control group that receives 28 saline injections to isolate and measure MeCP2 levels in the lateral habenula. Our hypothesis is that the mice that have had 28 days of imipramine injections will have the strongest anti-depressant effects and will have the highest levels of MeCP2.

The Dark Side of Light

The Di Giulio Lab focuses on ecotoxicology, the study of contaminants in the environment and their effects on organisms. Most of his work investigates how polycyclic aromatic hydrocarbons (PAHs) and nanoparticles affect the development of zebrafish and killifish.

My project investigates a heterocyclic PAH. Many PAHs have increased toxicity when exposed to UV light; however, this toxicity is usually attributed to photosensitization rather than photomodification (production of more toxic products as the PAH degrades). In my project, I activate a PAH to see if it will be more embryotoxic to zebrafish. Some measures that I assess are pericardial edema, yolk sac edema, bent notochord, and basic survivability.

Pericardial edema can be seen in the bottom photo. The pericardium surrounds the heart, and certain chemicals will cause it to be filled with fluid. Yolk sac edema can also be seen in the bottom photo. Bent notochord can be seen in the middle photo. The skeletal rod of the embryo is not in alignment like in the top photo.

It fascinates me how toxic chemicals can become more toxic when exposed to light, which occurs in nature. Photosensitisation and photomodification can cause contaminants to be more toxic than we actually think. It’s important to explore this to understand the extent of toxicity.

While I’m interested in my research, it involves a lot of tedious tasks. Having to count over 200 fertilized embryos and pipetting them one by one requires patience. Yet in the end, being enlightened by this topic is better than living in the dark of the potential harms of light.

 

Engineering the Immune System

The focus of my lab is developing and characterizing self-assembling, self-adjuvanting nanofibers and other materials to induce certain immune responses to provide another possible platform for vaccine design and other medical applications. I have been paired with Lucas Shores, a rising second year graduate student, and I am helping him to run his experiments. The project we are working on builds on past projects by Collier Group in order to better understand these peptide nanofibers. We are trying to figure out exactly how the nanofibers interact with the immune system and induce an immune response, and how to engineer this response to me more catered toward a particular disease (by, for example, engaging B cells and Tfh cells, rather than a Th1/Th2 response if you want the vaccine to activate the humoral side of the adaptive immune system).

Lucas is looking specifically at using nanofibers to target the IL-17 cytokine in order to possibly create another treatment for inflammatory diseases, like autoimmune diseases such as Rheumatoid Arthritis. IL-17 is an inflammatory molecule, and so the theory is that if a vaccine can induce an immune response to destroy or deactivate IL-17, then inflammatory symptoms would be reduced. Lucas has designed and synthesized a few peptide epitopes targeting IL-17 and is testing their effectiveness in creating an immune response in mouse models.

In addition to these mouse experiments, Lucas and I are culturing dendritic cells from a cell line in order to do experiments that investigate antigen uptake by dendritic cells and are doing lymph node immunohistochemistry in attempt to further understand which cells the nanofibers interact with when they enter the mouse.

Peptides Galore

Autoimmune diseases are very challenging to treat. They lead a person’s body to go into a self-destruction mode, killing many perfectly healthy cells in the process. Furthermore it is often times difficult to pinpoint the reasons for the onset of these illnesses consequentially causing immense difficulties in treatment. Several decades ago, researchers created a synthetic randomly self-assembling peptide, glatiramer acetate (GA), which was soon discovered to aid in the reversal of multiple sclerosis (MS).

Decades later, researchers are still trying to pinpoint the exact mechanisms in which GA is able to help MS patients. By discovering these mechanisms it can lead to the development of more drugs of this nature to increasingly lead to improved therapeutic immune responses.

In the Collier Lab, I have gotten a chance to learn about synthetic randomly self-assembling peptides, from how they are created in a peptide synthesizer to techniques (such as ELISA) to test the immune responses these peptides elicit.

However, one of the challenges of working with creating vaccines with these peptides is that due to the nature of their structure it is difficult to stop these peptides from precipitating out of their solvent, which can lead to challenges in ensuring the reliability of the administration of the vaccine. Therefore, one of the goals I will be tackling will be to devise different methods to optimize the solubility of these peptides.

I am excited for the coming weeks and am eager to keep learning more about these self-assembling peptides. I have learned so much these past three weeks and can’t wait for what else is in store.

Insulin Secretion

The overall theme of my lab concerns looking at the pathways of insulin secretion so that we can better understand type 2 diabetes. One of my post doc focuses for her project involves the relationship between GLP-1 and GIP. In previous studies done at the DMPI, data has suggested that there is a compensatory effect when cognate G-protein coupled receptor (GCCR) was eliminated, leading to an increase for alpha cell GLP-1 content. This information is important for future studies because GLP-1 levels are important for insulin secretion and glucose tolerance.

Right now, our lab is focusing on knocking out different mechanisms in that pathway, for example dipeptidyl peptidase (DPP-4), glucagon-like-peptide-1 (GLP-1), and gastric inhibitory peptide (GIP) to try to understand the critical keys in the pathway for insulin secretion. We’re using different strands of mice to knockout these genes. From these mice, we perform different tests to determine insulin secretion levels. Our main test are glucose tolerance tests and our second test involves using a Perfiusion machine. This machine allows us to measure insulin secretion in islet cells we obtain from our mice. From here we hope to better understand the pathway to insulin secretion, to better aid type 2 diabetic individuals.

Stress and Reproduction

The research project I am working on in the Alberts Lab examines how drought, and the stress caused by drought, can affect pregnancy and conception in wild baboons. We are specifically studying the wild baboon population in Amboseli National Park in Kenya. Like humans, baboons are fertile and mate year-round, rather than having a breeding season. This, then, begs the question: Why do female baboons not become pregnant each cycle? One potential reason could be stress-level, caused by environmental factors such as drought.

In the lab, I am using radioimmunoassays to detect the level of glucocorticoid, a stress hormone, in fecal samples collected from the baboon population in Amboseli. Radioimmunoassay, developed by Rosalyn Yallow, uses competitive binding between antibodies and antigens to determine the concentration of antigens (in this case, glucocorticoid). A set amount of radioactively-tagged glucocorticoid is mixed with an antibody and the two bind. Then,  a small amount of the fecal sample, which has been purified and concentrated into a liquid which contains the glucocorticoid, is added to the mixture. The radioactively tagged glucorticoid and the non-radioactive glucocorticoid from the sample compete to bind to the antibody. The antigens that have not bound to the antibody precipitate out of the solution, and the supernatant is removed. Then, the amount of radioactivity in the precipitate is counted by a gamma counter. The more radioactive the precipitate is, the less glucocorticoid hormone from the sample is present, meaning that this sample had less glucocorticoid originally. From this, we can extrapolate how much of the stress hormone was in the sample. Fecal samples collected in Amboseli are labeled with the name of the individual and the time and date of the sample. Using this information, data collected in the field about the reproductive cycle phase of the female baboon, and the glucocorticoid levels determined in the lab, we can look at the relationship between stress and reproduction.

Wild Baboons in Amboseli National Park (Source: http://amboselibaboons.nd.edu/publications/)

Spines: what’s the point of it all?

When you hear the word “spine,” your first thought is probably a backbone: that familiar stack of vertebrae running from the base of your skull to your tailbone. At least, that’s what popped into my head when I first discussed my project with my mentor Jacob Harrison, a PhD student in the Patek Lab. However, there’s another type of spine that is often overlooked, one that is far more prevalent in nature than you might think.

Note: drawings are not realistic depictions of species

My research focuses on spines in the spiky sense. For my project, spines are defined as rigid biological structures that come to a point (J. Harrison). Barbs, quills, thorns, spines… these are all different names used across the literature for fundamentally similar structures (J. Harrison). Many of us are aware that spines exist in nature, because we’ve experienced (or tried to avoid) painful run-ins with them. However, until now I never appreciated just how diverse spines are across biology. Some organisms such as sea urchins have conical toothpick-like spines, while other species like stingrays have flattened barbs reminiscent of knife blades. Some spines are smooth, like the stingers of scorpions, while other spines display serrations of varying size, number, and orientation. For instance, while both the sea urchin and stingray have many small serrations on their spines, these serrations run in opposite directions (see Fig. 1)! Furthermore some structures, such as the raptorial appendages of spearing mantis shrimp, contain several spines at once (see Fig. 1).

Aside from being diverse in structure, spines vary widely in their function. Stingrays use their barbs defensively, embedding their spines in the bodies of predators (and sometimes, the feet of unwary beachgoers!). Meanwhile, spearing mantis shrimp use their spines for predation, skewering prey that swim above their sand burrows. This large difference in function occurs, despite the fact that both species utilize the same underlying tool of the spine.   This suggests that small changes to the structure of a spine play a role in how it is used, and ultimately begs the question: how do changes in spine morphology (or structure) influence spine function?

Fig. 1 – The structures of a sea urchin spine, stingray barb, and a spearing mantis shrimp dactyl (foreclaw)

To better understand the relationship between spine form and function, we’ll be investigating how spine structure affects puncture and draw mechanics. We decided to use 3D modeling for this project, because this will allow us to perform more controlled comparisons of changes in spine structure. First, we’ll design a basic underlying spine shape as a control, and then manipulate different aspects of that spine’s morphology (ex. serration number, size and angle) in set increments. After printing the resulting variations using the Patek lab’s 3D printer, we’ll then record the force required for each of the spines to pierce ballistics gel using a Material Testing System (MTS), which measures forces in tension and compression. This will allow us to see whether/how changes in the spine’s morphology affect its puncture/draw mechanics (i.e. how it pierces or retracts from the gel).

Rough idea of a base spine and resulting variations. We chose to model the spine after a stingray barb because 1) it’s an easily replicable shape, and 2) we know that it is definitely used to puncture things that the stingray views as a threat.

Currently I’m in the process of designing prototype spines using the 3D-modeling software 3Ds Max. Below are some printed models!

Feelin a bit like Tony Stark looking at his Hall of Armors

Because these spines aren’t precise replicas of ones found in nature, we have to be careful about what conclusions we can draw about ecological/evolutionary functions. However, the effects we observe with our basic models can still give us insight into the fundamental influences that spine structure can have on function.

The Patek Lab focuses their research on the intersection between physics and evolution, which is an inherent part of my project. I’m really excited to see what we might learn, not only because I am curious about the nature of spines and the organisms that wield them, but because I think our findings could have practical applications to people’s lives. After all, wouldn’t you want to know the structure of a stingray barb if it revealed an easier way to get it out of your foot?

Of course, there’s a lot more to explore, prepare, and test before I can say anything for sure. But still, I’m excited to take a stab at this investigation and see how it goes!

Will they mate?

Because of my research project, I’ve begun to think fruit flies are kind of cute.

ok, I admit…they look cuter in person (credit: http://theconversation.com/animals-in-research-drosophila-the-fruit-fly-13571)

My research project revolves around connecting a particular pathway in the brain to a particular behavior. Here is some background information: in the research realm of drosophila (fruit flies), researchers have identified this gene called fruitless (fru). Researchers rely on this gene as a marker that identifies sex-specific behavior pathways in the nervous system. Interestingly enough, only males have a working copy of this gene. We are particularly interested in olfactory system and olfactory receptor neurons which have a large part in the fru pathway (and also have a huge impact in just the daily life of a fruit fly). Researchers have found that fru mutant males (basically males with a nonfunctional version of fru) usually do not court or there’s very diminished courtship, unless they learn to court (similar to conditioning in a way). This learning is not limited to only other female fruit flies of the same species though. And this is where my research project begins.

A previous research paper was able to identify key players in this sex-specific behavior pathway. Even more importantly, they found that courtship behavior can be adaptable – which makes sense. As said by my mentor, if a species can’t adapt then it’s dead (essentially). That is what I’m focusing on. I am currently trying to see if an olfactory receptor neuron known as Or47b is an essential key factor in this adaptable behavior pathway.

Pretty picture just to show where the olfactory receptor neurons go (credit: http://menuz.lab.uconn.edu/)

As of now, researchers know Or47b to be important in socializing for fruit flies. It correlates to courtship, however it attracts a fly to both females and males (it is not gender specific). We are hypothesizing that olfactory senses and thus Or47b is significant to learning courtship. To test this, I am using many different mutants of fruit flies (and oh my goodness are there a lot for me to handle) and then putting them through different mating tests to track any patterns. For example, in one scenario I put one male fruit fly from each mutant line into its own little vial and leave him there for about 4-6 days and then put him and a wildtype virgin female (up until this research project I never realized how important virgin flies were) into a mating chamber – yes, a mating chamber and a very exquisite one at that.

Similar mating chamber to what I use (but mine’s fancier-ish) (credit: https://www.youtube.com/watch?v=xSBalKckyDM)

Then I just watch them creepily for about 10 minutes and record which pairs mated and which didn’t. I am also planning to take isolated male flies and group them with other female flies (and maybe other male flies) for about a week and then test them everyday in the mating chamber with one other female virgin to see what happens. Hopefully, I’ve explained the gist of what I’m doing.

So currently my mentor is studying many things in her lab including how neuronal circuits interact with each other and produce a particular behavior. She is also studying how these behaviors are regulated and how it sometimes leads to adaptation. She is very interested in the development aspect of fruit flies, especially in the olfactory circuitry. This all directly correlates with my project because my project makes use of an already identified neural circuitry and tries to see how one particular neuron receptor can affect the whole thing, including the end result (the behavior of the fruit flies). This is all to explore more into the complex nervous system and try to understand what’s going on and how behavior is what it is.

The Game Plan

Anybody who knows me relatively well will know that I am a huge pro-football fan (33 days till Hall of Fame Game Cowboys vs. Cardinals, but who’s counting?) But many fanatics, myself included, often severely overlook the risks that athletes take when they play sports: traumatic brain injury one of many not yet fully understood. Brain injury extends far from sports, however, including military implications and even normal day life—surprisingly, motor vehicles are only the third leading cause of traumatic brain injury (TBI), landing behind ‘falls’ and ‘individual being struck by another object’ (Meaney 2). 

Dr. Bass’ Injury and Orthopaedic Biomechanics Laboratory seeks to dig deeper into different aspects of brain injury; my mentor Chris and I hope to investigate the key mechanism of TBI. Currently, two branches of ideology exist in regards to how mild traumatic brain injury arises; the first believes that mainly direct impacts to the head, linear velocities and accelerations, are the key mechanisms of head injury and the second school conjectures that rotational velocities and accelerations cause head injury. A plethora of experiments (Gennarelli, Euckerhave concluded that head rotation is the greater cause of mild traumatic brain injury, but the exact mechanism of TBI, whether angular velocity or acceleration and whether parameters to measure concussion include shear strain, relative displacement, shear stress, pressure waves, etc. remains to be confirmed. Currently, some of the main parameters used to determine and assess level of brain injury are cumulative strain damage measurements (CSDM), maximum principal strain (MPS), and maximum pressure.

This summer, a twofold process will be used to analyze the true mechanism that is causing shear and strain in the brain. First, a program called LS-DYNA/LS-PrePost will be used to analyze the finite element analysis SIMon model. The SIMon (Simulated Injury Monitor) was made to evaluate injury potential by directly imposing measured responses on a finite element model, which allows deformation and predicts how a product reacts to real world forces.

Using finite element analysis to model reactions to real world forces, such as a ball hitting a plate with a set velocity.

SIMon Model to analyze effects of rotational velocity on the brain

Second, a gel will be used for hands-on experimentation. A built device will allow different accelerations and constant velocities to be manually created and the resulting strain will be reflected in the gel—different colors (similar to figure 4) will appear and can be compared to the strains found from the SIMon model. Although the materials are different (SIMon model set brain material versus brain-like gel), the strains should reflect relatively the same values.

Finally, an experiment will be conducted to attempt to understand the effect of shear shock waves on the brain. Currently, the exact effect of instantaneous pressure waves and energy mounts from shear shock waves (hemorrhages, microcavitations, etc.) is unknown. In order to visualize injury that MRI scans usually cannot pick up on, the same device as the gel experiment will be used to give impact to a pig’s brain. Different boundary conditions will be placed; the pig’s skull will be replaced with a clear, transparent, skull cap in order to visualize the interior of the brain during the impact. Analyzing the effects of shear shock will allow better understanding of how these waves truly contribute to traumatic brain injury. Although the ideas are still preliminary, by the time NFL season rolls around, I hope to have delved deeper into my research project and reaped some interesting results!

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Drugs versus IBC: who will win?

Dr. Devi gave me some freedom during the beginning of the program to explore the work of other scientists in the lab to see what kind of research I would be interested in conducting. My interest lies in pharmacy; thus, I am interested in drug therapy and how certain diseases will react with certain drugs. I used my interest of pharmacy to design a project that suited my interest but at the same time would help the lab gain new knowledge.

One of the focuses of Dr. Devi’s lab is inflammatory breast cancer (IBC). IBC is a very aggressive form of breast cancer that forms no one solid tumor mass, but instead, forms characteristic diffuse tumor cell clusters which spread along the breast parenchyma and block lymph vessels. Furthermore, IBC is also known to affect minority populations and younger populations disproportionately and is thus an important health disparity issue. Polycyclic aromatic hydrocarbons (PAHs) are a class of chemicals emitted from incomplete organic fuel combustion, many of which are carcinogenic and linked to increased cancer risk at certain human exposure levels. In vitro studies with PAH chemical exposure in Dr. Devi’s lab have seen morphological and signaling pathway changes in breast cancer cells due to this exposure, wherein low-doses of PAHs can ultimately increase growth and survival of a panel of breast cancer cells.

The research I will be focusing on this summer is to validate the Devi lab 3D tumor emboli model for inflammatory breast cancer and to test the effects of an environmental chemical mixture on tumor emboli formation and/or pre-existing morphology using this model. Concurrent chemotherapeutic treatments may also be added in addition to this environmental chemical mixture to scratch the surface of understanding therapeutic resistance in these aggressive IBC emboli. The environmental chemical mixture is comprised of thirty-six PAHs characterized by mass spectrometry, and different concentrations of the mixture will be administered to an IBC cell line. Potential chemotherapeutic drug therapies to be used concurrently are U0126 (a lab-grade ERK inhibitor), Eloxatin (a topoisomerase inhibitor), and Trastuzumab/Herceptin (a HER2 inhibitor). These three different drugs all have different inhibition targets, but their overall goal is to restrict cancer cell proliferation and promote apoptosis. I can’t wait to see the results I will get from these experiments, and to share them with the BSRUF and Duke community.