Randomly self-assembling copolymers have repeatedly demonstrated great promise toward eliciting therapeutic immune responses, and specifically for autoimmune diseases. However, their mechanisms of action are not yet fully understood. Many factors such as pH and temperature can affect the nature of the self-assembly of copolymers thus influencing immunogenicity. We tested the hypothesis that factors such as pH and temperature can be manipulated to optimize the self-assembly of copolymers thus eliciting a more desirable immune response through shifting the nature of the T-cell response. This was implemented by analyzing the effect of pH, temperature, and sonication on the stability of the copolymers dissolved in solution. According to our results, increasing heat and pH were the most effective methods to optimize the formation of fibers. We next plan to use these copolymer solutions to vaccinate mice and use methods like ELISA to analyze the nature of their immune responses. This will help us gain a better understanding of the ideal steps to be taken to increase the solubility of these copolymers while simultaneously optimizing their immunomodulatory actions. Through exploring different methods to increase the solubility of copolymers in solution, we can work to better understand how to optimize the immunogenicity of copolymers in vaccinations.
A Polycyclic Aromatic Hydrocarbon Environmental Chemical Mixture Increases Growth of Inflammatory Breast Cancer Tumor Emboli
Exposure to polycyclic aromatic hydrocarbon (PAH) chemicals is widespread due to their presence in emissions from tobacco smoke, wood stoves, and organic fuel burning throughout the world. Many PAHs are classified as carcinogens, and prior studies have shown an increase proliferation in an estrogen-receptor positive human breast adenocarcinoma cell line due to exposure to low doses of a complex PAH mixture. The objective of this study was to observe the effects of the same PAH chemical mixture on an aggressive human inflammatory breast cancer cell line using a 3D tumor emboli assay. We hypothesized that greater concentrations of PAH mixture would lead to greater tumor emboli growth. Cells were seeded in ultra-low attachment well plates and once emboli were formed, they were treated with different concentrations of PAH mixture, called Elizabeth River Sediment Extract (ERSE). Microscopic and statistical analysis revealed that low nanomolar doses of ERSE result in greater tumor emboli size compared to untreated emboli, in a dose-dependent manner. There was a positive correlation between emboli size and increased ERSE concentration, although a high micromolar ERSE dose was cytotoxic to the cells. Collectively, these results suggest that low-dose exposure to this PAH mixture can enhance growth of aggressive breast cancer cells, and may have a wider and more substantial impact on cancer progression and outcome.
Self-assembling peptide nanofibers have been shown to have self-adjuvanting properties—stimulating targeted immune responses in vivo and raising long lasting antibody responses without an accompanying inflammatory response. Despite these novel properties, the mechanisms behind nanofiber induced immune responses are still not known. To investigate these mechanisms, the self assembling Q11 peptide sequence was used. Dendritic cell (DC) activation was measured in vitro using the DC2.4 dendritic cell line, which immunohistochemistry identified nanofiber localization within the lymph node in a murine model of immunization. DC activation in vitro was shown to be dependent on the presence and concentration of the Pan-DR-epitope (PADRE) within the nanofiber. Measurements made by flow cytometry showed a dose dependent upregulation of CD83 and MHCII when PADRE containing nanofibers were added to DC cell culture media. Additionally, immunohistochemical analysis of lymph nodes containing nanofibers conjugated with the fluorescent molecule Tetramethyl-6-Carboxyrhodamine (TAMRA) showed a colocalization of injected nanofibers with both macrophages and DCs in vivo. These results indicate that nanofiber induced immune responses function in both canonical pathways, involving DCs, and non-standard pathways, involving macrophages. This provides a framework to begin to understand the complex immune responses generated by self-assembled peptide nanofiber vaccines.
This first draft of my abstract is the result of careful and meticulous revisions to quite a few sentences to reduce the word count to fewer than 200 words. Who knew how tough it would be to fit so many of the ideas swirling around my brain into less than 200 words? Words fly when you’re having fun.
ABSTRACT – Both ABL and DDR kinases appear particularly hyperactive in the context of chemoresistance, in which cancer cells become resistant to chemotherapy. Furthermore, preliminary data suggest that receptor tyrosine kinases, DDR1 and DDR2, are ABL kinase targets. Thus, the hypothesis that DDR and ABL kinases form a feedback loop and that hyperactivation of the ABL-DDR network occurs during therapy resistance was tested. Due to the suggestion that DDR kinases are implicated in subsets of breast and lung cancers, DDR1/2 expression in these cancers was evaluated. It was demonstrated that increased ABL2 activity using the constitutively active form of ABL2 (ABL2-PP) in lung cancer cells induces phosphorylation of DDR1, whereas treatment with the ABL kinase allosteric inhibitor, GNF5, and the ABL and DDR kinase inhibitor, Nilotinib, decreases DDR1 phosphorylation without decreasing total DDR1 amounts. These data suggest that DDR kinases are downstream targets of ABL kinases, which supports the idea that DDR and ABL kinases constitute a novel signaling axis. If hyperactivation of the ABL-DDR network is indeed important during therapy resistance, there is potential that inhibition of the multifunctional ABL kinases through targeted therapy will resensitize cancer cells to the treatment to which they had been resistant before.
To be continued… as I add the results of the experiments I plan to perform next week, fresh from the oven!
Note: “Fresh from the oven” is solely meant to convey the idea that new and not yet acquired data are still to be incorporated into my abstract. I do not actually carry out my experiments in an oven. 😉
Update: For those of you who are interested, here is the final draft of my abstract, in all of its glory:
Novel signaling axis between ABL and DDR kinases in chemoresistance and metastasis: promise for targeted therapy in breast and lung cancers?
In the context of metastasis and chemoresistance in lung and breast cancer cells, the ABL family of tyrosine kinases and the receptor tyrosine kinases, DDR1 and DDR2, have been revealed to be hyperactive. My preliminary data suggest that the DDR kinases are ABL kinase targets. We hypothesize that DDR and ABL kinases form a feedback loop and that hyperactivation of the ABL-DDR network occurs during therapy resistance in subsets of breast and lung cancers. My data demonstrate that increased ABL2 activity using the constitutively active form of ABL2 (ABL2-PP) in lung cancer cells induces phosphorylation of DDR1; however, treatment with the ABL kinase allosteric inhibitor, GNF5, and the ABL and DDR kinase inhibitor, Nilotinib, decreases DDR1 phosphorylation without decreasing total DDR1 expression. These results suggest a novel ABL-DDR signaling axis, and preliminary data suggest that hyperactivation of this pathway may be important during metastasis and therapy resistance. Importantly, we demonstrated that pharmacological inhibition of the ABL kinases resensitizes therapy-refractory tumor cells to standard-of-care chemotherapy.
The Role of Glucagon in Insulin Secretion
Glucagon and Insulin are products of the alpha and beta cell respectively, that are essential to maintaining glucose homeostasis. It’s also known that incretins, signaling hormones secreted in our small intestine are essential insulin secretagogues. In understanding ways to mediate insulin production in the beta cell, more must be known specifically about the relationship between glucagon and the glucagon like peptide (GLP-1). In hypoglycemia conditions glucagon is secreted to increase blood glucose concentrations, whereas in hyperglycemia conditions GLP-1 stimulates insulin secretion to reduce blood glucose concentrations. In this study, we examined the relationship between glucagon and GLP-1 because we hypothesize that glucagon and GLP-1 work together to increase insulin secretion. We examined this relationship by performing glucose tolerance tests on our beta cell glucagon receptor knockout mice model(GCGR), to understand the response between glucagon and the beta cell. Then we inhibited the GLP-1 receptor to further understand the relationship between glucagon and GLP-1. We used a Perifusion machine to examine the relationship between the mice’s insulin response specifically in the beta cell to different buffers. If glucagon and the GLP-1 receptor support our hypothesis, future work can be dedicated to creating new agonists that could improve treatment for type 2 diabetic individuals.
This is a first draft of my abstract
Ecologic Factors Influencing Hormone Concentrations in Female Baboons
Although studies have shown how ecologic factors influence human female hormone concentrations and male baboon hormone concentrations, little is known about how these same factors effect female baboon hormone concentrations. This study measures correlations between estrogen, progesterone, and glucocorticoid levels and rank, weather, parity, age in the female yellow baboon population in Kenya’s Amboseli National Park. To determine the hormone concentrations, each fecal sample collected in Kenya was purified into a serum. Radioimmunoassays were then run on each sample, and hormone concentrations were correlated with field data on the individual the sample was from, time of sample, rank, and temperature. Although we do not yet have results, our hypothesis is that female baboon hormone concentrations and female human hormone concentrations will be similarly affected by parity and age, and that weather will effect both male and female baboon hormone concentrations similarly. Rank in female baboons is matrilineal, that is, a daughter baboon will be lower in rank than her mother suggesting that rank and hormone concentrations would not be correlated.
The Dzirasa lab uses chronic social defeat stress to create an ecologically relevant model for depression in mouse models. The model results in the expression of two distinct phenotypes, resilient and susceptible, similar to the phenotypes seen in human patients who have different responses to stressful events.The aim of my project was to correlate the behavioral changes associated with chronic anti-depressant treatment of socially defeated mice with biochemical changes of the level of phosphorylation of the protein MeCP2 in the lateral habenula. Previous work by the West lab studying the methyl DNA-binding protein MeCP2 has found that MeCP2 is phosphorylated by monoamine neurotransmitters. Increased levels of MeCP2 phosphorylation in the lateral habenula were found with chronic treatment of imipramine, indicating that MeCP2 may have a role in the brain’s biochemical response to chronic anti-depressant treatment. In order to correlate MeCP2 phosphorylation during anti-depressant treatment with behavioral recovery of a prosocial phenotype in socially defeated mice, mice will undergo two rounds of behavioral tests before and after imipramine treatment and then will be tested for levels of MeCP2 phosphorylation. If phosphorylation is correlated with behavioral recovery, susceptible mice that display resilient behavior after imipramine treatment will also have increased levels of MeCP2 phosphorylation in the lateral habenula.
My abstract draft:
Will they learn to court?
Studying the role of Or47b in drosophila courtship learning behavior
Behaviors are made up of both innate and plastic components and courtship is no exception. It has been discovered that courtship can be learned in drosophila melanogaster (fruit flies), given that the innate component is absent. However, we do not know the exact mechanism that enables this learning. In this study, we are focusing on the olfactory system because we hypothesize that olfaction is important in learning; more specifically that Or47b is a key olfactory receptor neuron that regulates the learning of courtship behavior. To do this, we are using fru mutant fruit flies and fru and Or47b double mutant fruit flies and group housing them with other males or females for a few days and then observing their courtship behavior. We are currently waiting for results, however if our hypothesis is correct, we expect the double mutant fruit flies to not court even after group housing them. These results could better our understanding of the mechanisms driving behavior changes in general.
The effect of serration angle on spine puncture mechanics
Spines, defined as rigid biological structures that come to a point, are physical features found across a wide range of organisms; however general relationships between spine structure and function remain unclear. This study explores a specific aspect of the form-function relationship of spines, by investigating the influence of serration angle on spine puncture mechanics. Autodesk Fusion 360 was used to 3D-model spines with differing serration angles, which were then printed out with hard resin. The models will undergo materials testing as they are punctured into and retracted from ballistics gel. The maximum force needed for both puncture and retraction will be recorded and analyzed to see whether a change in serration angle leads to a change in puncture mechanics. Maximum puncture force is expected to increase with serration angle, as serrations angled more towards the front of the spine will increase the surface area on which the ballistics gel can resist the spine’s entry. An inverse trend with maximum retraction force is also expected, as similar gel-spine interactions will occur but in the reverse direction.
*This abstract is the first draft of what will ultimately be used for the poster presentation at the end of this program*
The gut microbiome & depression: what is the best method for DNA extraction?
Our gut microbiomes play an important role in immune functioning, nutrient processing, and regulating many aspects of brain functioning. This gut-brain axis has been implicated in major depressive disorder, and inducing a depressive phenotype in mice using social defeat (SD) is one commonly used model to study the shifts in microbial diversity that occur. To do so, an effective protocol to extract and sequence the bacterial DNA from the fecal matter of subject mice is required. As it stands, methods and results in the field vary widely. This study compared two DNA extraction kits—the Maxwell RSC PureFood GMO & Authentication Kit and the MoBio PowerSoil DNA Isolation Kit. The Promega kit was hypothesized to be superior due to its high degree of automation. In order to assess the quality of the extracted DNA, spectrophotometer, fluorometer and electrophoresis were used. Results from 16S rRNA gene sequencing are awaited for phylogenetic classification of the samples. Analyses indicate that the Promega kit is more efficient, requiring less sample, but may have co-purified contaminants. Sequencing results will indicate effects of this, if any. Further work must explore microbial changes after SD and treatment, and the roles of specific bacterial species on the CNS.
Mild Traumatic Brain Injury (mTBI): Role of rotational kinematics through the mechanism of shear shock
ABSTRACT— Despite decades of research, the exact mechanism in mild Traumatic Brain Injury (mTBI) remains unknown. The purpose of this study was to determine whether rotational velocity or acceleration is the primary contributor to mTBI and explore the effect of shear shock waves generated from rotational acceleration. It was hypothesized that rotational acceleration, not rotational velocity, has a primary role in mTBI, and shear shock waves due to the nonlinear pressure waves are a mechanism for mTBI. This study was conducted with a dual modeling and experimental approach. First, strain and deformation response was observed and recorded through the finite element analysis Simulated Injury Monitor (SIMon model). These results were then compared to deformations and strains recorded during pig brain rotational impacts using three dimensional imaging analysis. So far, the simulations reflect the relatively lower strain and stresses created through rotational velocity. Results indicate that rotational acceleration through the mechanism of shear shock waves is a prevalent mechanism in mTBI and could largely alter how future head injury criterions are created and how protective gears for sports players and the military are crafted.
Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous contaminants produced from human activity, such as combustion of biofuels, often found in aquatic ecosystems. Embryonic fish have been shown to be significantly sensitive to PAH exposure. Further, PAH toxicity can be modulated by environmental factors such as hypoxia and temperature. This study investigated how sunlight exposure modifies the toxicity of carbazole, a hetereocyclic PAH. Zebrafish embryos were exposed at 6 hours post fertilization (hpf) until 96 hpf using photoactivated and parent carbazole. Photoactivation was induced using a sunlight simulator. Survival rates, deformities, CYP1A activity, and oxidative stress were examined. Additionally, chemical analysis of photoactivated carbazole is under development to identify photoproducts. Exposure to parent carbazole did not cause mortality, but did cause minor embryonic developmental effects. Contrarily, photoactivated carbazole caused significantly higher rates of mortality and deformities at 72 hpf at concentrations of 1000 µg/L and higher. Photoactivated carbazole induced higher CYP1A activity, indicating that photoproducts acted as aryl hydrocarbon receptor (AHR) agonists despite carbazole being a known AHR inhibitor. Such induction may be related to the increased toxicity, which remains to be tested. These results show that sunlight can play a role in modifying the toxicity of certain organic contaminants.
Below you will find the first draft of the abstract that summarizes my summer research project in the Kuhn lab. Please keep in mind that this abstract is very preliminary especially regarding result interpretation and conclusions. Enjoy with a grain of salt.
The neurobiology of depression remains largely unknown and has not been studied in respect to sex-differences. Rapid tryptophan depletion (RTD) is a method used to lower levels of endogenous tryptophan (TRP) and serotonin (5-HT) and was used in this study to explore possible sex differences in the way rats react to tryptophan depletion. This study administered one of three treatments to 36 rats: 1) RTD, 2) a balanced amino acid treatment (BAL), or 3) None. Plasma and brain samples were collected and later analyzed using high performance liquid chromatography (HPLC) to determine concentrations of TRP and 5-HT. While RTD had similar depletion effects in the plasma of both females and males, there were significant differences in how TRP and 5-HT levels shifted in the hippocampal brain region of males and females. Compared to female controls, RTD females experienced significantly lowered levels of 5-HT and TRP, more so than RTD males compared to male controls. This suggest that sex-mediated differences in the serotonergic systems of rats is responsible for the different responses to tryptophan depletion. Further work must explore this neurobiological distinction between male and female rats and clinical research should focus on determining whether a similar difference exists in humans.
This week, we had a chalk talk where everybody in the research fellowship gave a general overview of their projects and what they were doing in their projects. The projects in the group spanned from poop to the mechanisms of the brain to immunology and other parts of the sciences. Everyone did an amazing job during their chalk talks and many of the projects got me very excited for the future of the sciences.
One of the projects that I found the most exciting was the one that Anikka is working on that deals with the gut microbiome and its link to depression. I have always been interested in mental illness and the brain but who would have guessed that mental illness and the stomach are so closely connected? I was once watching an ASAP Science video that briefly talked about the link between our guts and who we are. I did some researching online and found that about 80-90% of our serotonin is actually in our gut. This not only makes serotonin a neurotransmitter, but it also means that it is a type of hormone. That is simply amazing. Serotonin affects our mood so that explains why I feel so grumpy when I’m hungry and why I feel so happy when I eat something good or when I’m full.
Anikka’s project was also looking on how fecal transplants could also change the anxiety in rat models. That means, if you took the poop from a rat without anxiety and put it into a rat with anxiety (in an attempt to change the gut bacteria), would the rat with anxiety no longer have anxiety? Believe it or not, these fecal transplants are actually being done on humans in present day. I heard of a project at MIT that was having students donate their fecal matter in order to use it as microbiome therapy for other people.
But if anxiety can be affected by the bacteria in our guts, what else can be affected? Can you change intelligence by changing your gut bacteria? How about the fears that you have? If you take the gut bacteria of someone who loves snakes (like Dr. Grunwald) and put it into someone who is deadly afraid of snakes (like a lot of his students), will that person’s fear of snakes and their sympathetic nervous system response to snakes be different? These are some of the thought that ran through my head as Anikka was giving her presentation. I hope that I can soon find out what some of the results say.
I thought that Iris’ research into Traumatic Brain Injury (TBI) was quite interesting. I was initially interested in the project because I am also doing research in neuroscience. What I quickly discovered through her talk, however, was that her research was much less molecular and much more mechanical. Thinking about the different kinds of acceleration that could damage the brain was a very new experience for me. The idea of thinking of the brain in as a rigid structure that could be thrown and broken was a very new idea.
Rather than repeatedly breaking our prized model subject, Iris first models these assaults in a computer program. I thought it was really cool that they had this predictive power to know what particular injury would occur given a certain type of trauma. I thought this could be of great clinical relevance as a reconstruction of the trauma, quickly ran on a computer, could be a great aid to a surgeon to know where bleeding would occur.
I was also surprised when Iris said that she would use a dead pig brain to test these forces and strains. As her questions are purely on the forces the brain was enduring, physiological responses are not necessary.
The final thing that was different about Iris’ talk was her ending it with ideas about the potential impacts of her research. While I’m sure my PI must be thinking of these things while writing grants, I had never thought of a reason for my research other than to learn more about the brain. Iris’ specific mention of applications to headgear and neurodegenerative disease, which are implicit in any neurobiological research, were an interesting consideration to make.
All the talks from this past week were incredible; I was so awed by the research everyone was working on and how relevant and meaningful these studies are.
That being said, I believe immunology is such an interesting and riveting field that’s developing so quickly these days. I learned about it briefly in my microbiology class this past semester and thought it was such so fascinating. Maddie O and Cassie’s talks this week really enlightened my understanding of it and how it’s being tackled in different fronts using molecular engineering.
Cassie’s chalk talk on dendritic cells in the mammalian immune system reminded me of the phenomena of antigen-presenting methods that cells practice once faced with a pathogen. Her project’s take on the topic using fluorescent peptides is such a fresh way to tackle the issues of the immunocompromised. Synthesizing these nanofibers from scratch sounds so exciting.
Maddie O’s presentation on epitopes and the immune response with T cells and inflammation really opened my eyes to the way her project can benefit the field. Her study of the major histocompatibility complex really highlighted the importance of testing for these pro-inflammatory or anti-inflammatory responses.
These two chalk talks were very intriguing and gave me some more insight into immunology and where more research can take us into the future.
This past week I have enjoyed listening to everyone’s research. Since all the fellows are in a lab conducting research most of the time, we don’t have that much time to talk about our research, so it was great learning about what others are doing. I enjoyed learning how some of the projects being conducted intertwine into mines and it made me rethink the way I am tackling my research project. I saw some interconnections between my research and others but I enjoyed Iris’s project since it is completely different than mines.
Iris Chang works in Dr. Dale Bass lab in the department of Biomedical Engineering. Iris main goal for her project is to uncover the mechanism of Traumatic Brain Injury (TBI), and discovering what causes such thing to occur. The two schools of thought for TBI is between linear and angular kinematics. Iris is more focus on exploring the role of rotational kinematics via the mechanism of shear shock waves. Studies have shown that injuries can send shock waves through the brain in an undesired direction which causes such tragic brain injury. However, how rotational acceleration works through shear waves is still not well understood.
The way she is tackling such project is by using a computer model that allows her to input rotational kinematics and analyze the strain in the model brain. Her second method is analyzing the damage done to the brain by conducting a pig brain impact testing. Her methods in her project involve methods I have never conducted and it is exciting knowing Iris is doing it. Her research made me wonder if the results from both methods be similar or will they differ? And if they differ, why? Can’t wait to hear Iris explain her lab’s data to us at the poster presentation in a few weeks. The best is yet to come.
When Georgia started presenting her chalk talk on that particularly humid Tuesday morning, I felt myself leave the room for just a few seconds. I was no longer in our room in the LSRC but on the ground level of Bell Tower house in a small projection room with eleven other students and one red-haired professor. It took only a microsecond for me to realize what was going on: I was having a flashback to my first semester Writing 101 class, “Can chimps have culture?”. A class I ultimately got stuck with due to scheduling difficulties and grew to despise, it was an experience I had tried to obliterate from memory yet I was here, dreaming about it. But just like that, I was back to reality and listening to Georgia talk about environmentally-induced stress in yellow baboons. Her chalk talk brought up some old memories and it made me think critically about how I view science outside of my own.
I grew to hate Writing 101 not because of my professor or her teaching quality but the students and the course content. It tore me to shreds and killed me internally every single time I had to force myself to read a research paper about chimpanzees, gorillas, and a number of assorted monkeys and their propensity for culture. I lost more and more hope for my intellectual future as a Blue Devil when I would have to sit in a room full of closed mouths in the middle of a class discussion we were supposed to have. But more than anything it was the primates. I just couldn’t stand reading about them. All the research began to blend together and paper after paper seemed to be talking about the exact same thing. I thought they would never be a part of my life again but they came back with Georgia.
She was describing her research project: analyzing the glucocortisol levels in the fecal matter of yellow baboons in different environmental contexts to assess stress levels. She was talking about primates again and I really thought my eyes were going to roll to the back of my head and not come back until she was finished. But just the opposite was happening: I was interested. I cared about how food conditions and hierarchy could affect yellow baboons in such a way as to induce stress. I was engaged and even asked how this research might pertain to humans: due to the fact that the two species share more than 90% of their DNA, understanding stress responses in yellow baboons might allow us to understand them more in humans. At the end of it I realized that other than that memory of Writing 101 at the beginning, I had managed to pay attention to the whole talk.
Why was that? Why was I suddenly engaged and interested in an area of research that I despised just a few months ago? I didn’t know it but the answer was simple: I had been engaged effectively in this research for the first time. While my professor had taken us through stacks of research articles in the study of primates, I had always felt detached from the research. I knew about all these studies and their impacts but I was struggling to see the “So what?” in what I was studying. Georgia changed that for me. She had contextualized interesting research in primates for me and made me understand that it was a real, living scientific field with important implications in the real world. I couldn’t feel detached from this research anymore because it was staring me in the face. Someone was finally talking about it with excitement in their voice and vigor in their conviction. For this, I thank Georgia. In the future, I now know that I can avoid the pitfall of detaching and even resenting a field of scientific research just because I think it isn’t interesting. It’s a matter of going to people that know how to communicate their science and have them show you just why their work is interesting, a lesson for all.