I did it! Officially a PhD Candidate!

The past few weeks months have been the most stressful times of my academic career. On November 16th, I successfully defended my original proposal in front of my preliminary committee and officially became a PhD candidate!!! I was looking forward to this day all through the summer and fall. I submitted my written proposal a month before my oral defense date and received feedback from my committee about the experiments proposed and the validity of my hypothesis. I am extremely grateful for each and every one of my preliminary committee members for taking the time to review my proposal and for providing their valuable feedback and criticism. This entire process helped me grow as a scientist and helped me think and write critically. I am also grateful for my family and friends who took the time to review my proposal, attended my practice talks, and provided useful comments.

As mentioned in my previous post, our graduate program requires us to pick a topic outside our main research area and develop an NIH-style original proposal related to the chosen topic. I chose to study the role of Myeloid-Derived Suppressor Cells (MDSCs) in Type 1 Diabetes (T1D). MDSCs are a heterogeneous population of immune cells that suppress or down-regulate the effector T cell responses in various immune microenvironments. In tumor microenvironments, T cells help kill the tumor cells and prevent the tumor cells from growing. However, MDSCs suppress these T cells and prevent them from killing the tumor cells thereby causing the cancer cells to proliferate. An autoimmune microenvrionment is opposite to the tumor microenvironment. In T1D, the T cells become autoreactive i.e., the T cells start killing the innocent insulin-producing beta cells in the pancreas. This leads to reduced insulin production and increased glucose in the bloodstream in the body. Insulin is an important hormone that helps in the transfer of glucose molecules into the cells that can then serve as the energy source for the cells and tissues. The destruction of the pancreatic beta cells therefore leads to an imbalance in the glucose homeostasis in the body. In such a microenvironment, we require MDSCs to suppress the T cells and prevent them from destroying the beta cells in the pancreas. The first question to ask here is, are MDSCs induced during T1D? The answer is yes. It was shown in 2014 that T1D patients have an increased MDSC induction in their peripheral blood. As to the best of my knowledge, this is the ONLY study that focusses on the native (body’s own) MDSCs during T1D. However, not much is known about the MDSCs and the different subpopulations of these cells that exists that are responsible for interacting with T cells in the pancreas. MDSC subsets and their mechanism of action are dependent on the specific tissue or the site of inflammation. Understanding the role of MDSCs in T1D and the specific MDSC subsets involved in T1D lead to several questions. I chose to investigate a few in my proposal:

  1. If MDSCs are induced in T1D patients, why are they unable to suppress the T cell responses in the pancreas? i.e., Are MDSCs defective during T1D?
  2. What are the specific subsets of MDSCs induced during T1D that are specific to the pancreatic microenvironment? MDSCs are incredibly heterogeneous and can exhibit several phenotypic and molecular states. These subsets are unique to the local tissue microenvironment.
  3. What is an MDSC-specific immune regulatory molecule and its corresponding pathway implicated in T1D that may contribute to disease pathogenesis? 

Without going into the details of each question posed, I proposed several experiments and techniques ranging from single-cell RNA sequencing analysis of the MDSC populations in the pancreas to generating MDSC-specific conditional gene knockout experiments in mice to answer these key questions. There were a few flaws in my experiments that were brought up during the presentation and I tried to address them to the best of my ability by proposing alternative approaches. Overall, my committee members were impressed with the breadth of background knowledge and experiments presented. The most important factor was to develop a hypothesis-driven proposal with a solid premise to back my hypothesis. The presentation didn’t feel one-sided and eventually developed into a curiosity-driven discussion.

Transitioning from a PhD student to a PhD candidate is a backbreaking process. Perhaps it is meant to be this way. Even though I felt numb for a few hours after the conclusion of my presentation, I could feel the academic apocalypse building up in a cloud over my head already. Here’s hoping for more successes and vital experiences in the future!

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Grad school diaries: The preliminary exam

The past few weeks months have been terrifying, nerve-wracking, depressing, and scary. My friends and family have also been subjected to my constant irritable and grouchy behavior. I have been preparing for my preliminary examination and everything seems to be coming together (very) slowly. I have woken up to sweaty nightmares about missing deadlines, submitting a complete crap proposal to my committee, and being told that my “scientific caliber” is not up to the mark to pursue an academic career (gulp!)

The first week of November is officially my “prelim week” and I will continue to go through series of mini heart-attacks and one too many mood swings until then. What exactly is a preliminary examination, you ask? Well, also called as the “candidacy exam”, or “the OP” (short for the original proposal – mostly followed in life sciences, I think), it is an examination that PhD students are required to take (and pass) in order to officially become PhD candidates. Many schools and department do this differently, and I can only tell you what is done in my program. Here is a short excerpt about the exam from our handbook –

The purpose of the Preliminary Examination is to stimulate you to develop original research ideas and to assess your academic knowledge, preparation and ability to analyze and synthesize the literature on and surrounding your topic. In the written proposal, you are expected to provide the examination committee with adequate background and details to understand the current state of the chosen field of research and to evaluate your proposed experiments. The oral examination allows the committee the opportunity to test your knowledge of the chosen research project, your ability to formulate and address a few research questions to anticipate the types of results to be obtained, and to evaluate your understanding of its scientific foundation. The examination will not only assess the science involved in the proposal but also will evaluate the quality of the presentation and the writing.

Basically, we are required to come up with an original idea – a topic that is not our main thesis research, write a hypothesis-driven research proposal in the NIH Exploratory/Developmental Research Grant (NIH R21)-type format, and defend it in front of our prelim committee (which is different from our thesis committee and consists of new members). The proposal must be original and designed to advance the current state of knowledge in the chosen field. It cannot be based on our own (current or previous) research projects. Also, our advisor cannot critique the research proposal prior to submission of the proposal to the prelim committee. The whole process takes almost 8-9 months and I have briefly summarized the timeline of the process below –

March-April 2017: Brainstorming ideas for the topic; Reading, reading, and more reading. (My topic is about the role of myeloid-derived suppressor cells or MDSCs in mediating pancreatic beta-cell death in Type 1 Diabetes, which is an autoimmune disorder.)

May 2017: Topic approval by the program office.

June-August 2017: Literature review; Brainstorming ideas and key questions for experiments, techniques, aims, etc; Beginning to write… maybe…

August 2017: Prelim committee assigned; Serious writing and reviewing (rinse, repeat); More reading.

September 2017: First draft completion; Review by peers, friends, and colleagues; Schedule date and time for the oral defense with committee; MORE READING.

October 2017: Submission of written proposal to the program office and prelim committee (4 weeks prior to oral defense); Approval of proposal for oral defense (or, revise and resubmission of proposal aka “your proposal is indefensible at this stage and requires more work”); Practicing oral talk (aka “pre-prelim talk”).

November 2017: Defense! Drinking and crying (if pass); Drinking and crying (if fail); New sense of purpose in life.

A few weeks into this process (around May), the horror stories start – stories about seniors failing their defense and “Mastering out” (which is seen in a really bad light), stories about committee member issues, stories about inadequate writing, etc. I have heard one too many stories about people dealing with depression and constant stress during the period of writing and oral defense. There are tons of useful advice about what to do and what not to do during the process. Of course, the experience is unique and different for every student but it would certainly be easy if I could get on with it without constantly being traumatized by every little detail (like feeling guilty every minute that I’m not thinking about my OP or working on it).

However, a few things have indeed helped me so far:

  • Finding a studying/writing spot outside of work and my apartment. I have been working at WALC until wee hours of night these days. (WALC is the active learning center on campus and is always hustling and bustling with students.) Just being among other students and the white noise in the background seems to be a great environment to focus and get stuff done.
  • Biking to and from work every day (around 6.5 miles). My friend recently convinced me to buy a bike and I must say that it has helped me get around the campus faster and save a ton of time. Not to forget the kick of endorphins in the morning that helps me focus on my experiments in the lab and plan things more effectively through the day. I spend most of the mornings doing cell culture work (I get done with this the first thing in the morning in order to make time for meetings and other experiments through the day) and afternoons on tissue processing and protein work. This gives me sufficient time from evening until late night to work on my OP.
  • Eating regularly, but not fussing over cooking. Most of the time spent on cooking and cleaning can be replaced by quickly grabbing something to eat on the go. (I can hear my sister squeaming at this already!)
  • Talking Ranting to friends, especially colleagues about the OP, work, life, and everything in general to relieve all the stress. I am fortunate to be on the same boat as many folks who can relate to my situation and listen to my rambling.
  • Reading something completely un-related to my research or the OP over the weekends. I have read three books in the past few months (check out my reading list!).

Alright, I should probably get back to work now (this was some major procrastination and I am feeling guilty already). Perhaps I should talk about my topic in detail on the next post. Until then, I will try to keep calm and carry on.

Does arginase mediate immune suppression in the brain?

This week I want to talk about an interesting enzyme: Arginase-I (Arg1) and its metabolic pathway in an immune microenvironment. Arg1 is a cytosolic protein that is involved in the urea cycle. Specifically, it catalyzes the hydrolysis of L-arginine to L-ornithine and urea. It has been shown that the expression of Arg1 by macrophages has an important role in tumor growth. Macrophages that are recruited into the tumor microenvironment (tumor associated macrophages) express high levels of Arg1 resulting in the depletion of arginine – an essential nutrient required for T cell metabolism. Cytotoxic T cells, therefore, can no longer function and inhibit the tumor cells from proliferating. Arg1 is implicated in several inflammatory diseases as well as in autoimmunity. Arg1 plays a significant role in mediating immune suppression and blocking its metabolism is a novel strategy in preventing tumor growth and other inflammation-related conditions.

T cell suppression by MDSC
Myeloid-Derived Suppressor Cell suppresses cytotoxic T cell function through Arg1 metabolism. (IFNγ, IL-4, and IL-13 are cytokines that induced MDSC activation)

One hypothesis is that the immune suppressive cells in other immune microenvironments in the body must be similar to the Myeloid-Derived Suppressor Cells (MDSCs) that induce T cell suppression in the cancer microenvironment. The question is, do such immunosuppressive cells express increased levels of Arg1 and act through the Arg1 metabolic pathway? Since I am interested in the brain and neurodegeneration, this hypothesis can be extended to the brain immune microenvironment. Microglial cells in the brain also upregulate Arg1 and are neuroprotective in nature (in a healthy brain). These cells are the resident macrophages of the central nervous system and function by phagocytosing cell debris and toxic misfolded proteins (that eventually form aggregates and lead to neuronal death as seen in Alzheimer’s disease) out of the brain environment. The question now is – do the microglial cells exhibit immunosuppressive behavior by altering their Arg1 metabolism?

Kan et al., 2015, recently showed that CD11c positive microglial cells are immunosuppressive in the CVN-AD mouse model and that immune suppression is caused due to the deprivation of arginine (increased levels of extracellular Arg1 causing decreased levels of total brain arginine). What isn’t explicitly mentioned in this study is that arginine is also the substrate for nitric oxide synthase (NOS) that makes nitric oxide (NO) in an alternate L-arginine metabolic pathway. L-arginine is a substrate for both Arg1 and NOS. The Arg1 pathway polarizes the macrophages to M2 phenotype and the NOS pathway polarizes the macrophages to the M1 phenotype (Rath et al., 2014). The current model of microglial activation in the CNS is limited to these two polarized states, where, the M1 microglia are neurotoxic and the M2 microglia are neuroprotective. Arg1 is upregulated in microglia in the healthy brain and aids in phagocytosis of misfolded proteins and other cell debris. The classical microglial activation is through the M2 phenotype wherein the induced nitric oxide synthase (iNOS) is upregulated thereby accelerating inflammation in the brain (neuroinflammation is one of the hallmark characteristics of several neurological diseases such as Alzheimer’s Parkinson’s, Multiple Sclerosis, Traumatic Brain Injury, etc).

M1 and M2 microglia
The current model of microglial activation is limited to the Arg1-mediated M1 and iNOS-mediated M2 polarized states.

So, if an immunosuppressive cell exists in the brain, is it possible that the immune suppression is regulated through the M2 activation and that M1 activation is absent? In other words, L-arginine is metabolized through the Arg1 pathway and not through the NOS pathway. Other questions to consider: MDSCs upregulate both Arg1 and iNOS – so how does that fit into the two-state polarization model? How does iNOS modulate MDSC activity? We know that increased NO expression by MDSCs increases T cell suppression in the tumor microenvironment. Currently, both Arg1 and iNOS inhibitors are being developed to block the immune suppressive activity of MDSCs in the cancer microenvironment. However, understanding immunosuppression in the brain is still a long way to go and the idea is not widely accepted within the neurobiology community (my understanding from the currently available literature or published studies). Investigating this mechanism in the brain will be useful in developing potential therapeutic strategies for treating neuroinflammation and neurodegeneration.

References:

  • Kan MJ, Lee JE, Wilson JG, et al. Arginine Deprivation and Immune Suppression in a Mouse Model of Alzheimer’s Disease. The Journal of Neuroscience. 2015;35(15):5969-5982. DOI:10.1523/JNEUROSCI.4668-14.2015.
  • Rath M, Müller I, et al. Metabolism via arginase or nitric oxide synthase: two competing arginine pathways in macrophages. Front. Immunol., 27 October 2014. DOI: https://doi.org/10.3389/fimmu.2014.00532

[Almost] one year milestone – my first advisory committee meeting

Advisory committee meetings are held once every year (or twice every year, if the student or the committee chooses to do so) to asses the progress of a grad student’s PhD thesis. The meeting involves a written report that is to be submitted to the committee a week prior to the meeting and an oral presentation on the D-Day. During the presentation, the validity of the research work is thoroughly discussed along with the future direction(s) of the project(s) being undertaken. The advisory committee meetings are extremely important for the successful advancement and completion of a thesis – it is where brutal yet honest feedback is conveyed. We as grad students are forced to think critically of our work and defend our hypotheses as well as our results.

My first advisory committee meeting was an intense two-hour long session on a rather dull Tuesday afternoon. As I explained the premise of my work and my goals for the next year, my committee members brought up important questions that I had not previously ever considered. All the members of my committee, including my advisor, were supportive and encouraging. I learned some valuable lessons from the entire experience and got some great feedback from everyone. Some interesting and important points highlighted in my feedback assessment were –

  • Think carefully about how to present data and set up an argument in my presentation.
  • Work on clearly identifying the premise that sets the stage for my hypotheses.
  • Be critical about my data.
  • Continue to read literature: more reading, and reading more critically.
  • Focus on developing more robust immunological assays to answer the questions in my aims.
  • Interact more with colleagues on campus and at other schools to learn and get insight into techniques and relevant assays (wrt understanding what works and what doesn’t).
  • Explaining the experiments in detail before delving into my results (every assay is unique and has a question to be answered).
  • Think about how I want to present the previous studies done in the field that are relevant to my questions.
  • My hypotheses should be provided with a context (what is the data in support or against my hypotheses?)

These were just some of the significant parts of the feedback that I received. Now it’s time to put these into action and definitely work on continuing to build on my project more confidently. More later.

Metabolic interplay

fimmu-08-00248-g001
Renner K et al. Front Immunol. (2017)

I recently came across this figure that shows the key metabolic processes that dictates an immune cell behavior and function. Biochemists and pharmacologists sometimes focus on one or two key pathways in a disease model and forget that proteins don’t function in isolation. Protein networks are complex pathways with many overlays. A drug designed to inhibit or activate a specific protein can also affect other proteins in the connected pathways. This figure is focussed on an immune cell (natural killer cell) and its interaction with a tumor cell. The interplay between the different metabolic pathways applies to all kinds of cells in the body.

This figure is also quite interesting to me because I have been studying the arginase-1 (Arg1) pathway in microglial cells and this gives me a brief overview of where my study lies in the spectrum of key cellular metabolic pathways. Arg1 is an enzyme that metabolizes L-arginine to L-ornithine and urea in the urea cycle. With the help of ornithine decarboxylase (ODC), L-ornithine further makes polyamines that are important (? – it depends) for cell growth and survival (? – it depends). I think it is quite interesting to see how Arg1 and ODC would dictate the phenotypes of the microglial cells in the brain. Microglia are the brain’s resident immune cells – they chew up all the toxic stuff and get rid of them (this is known as phagocytosis). We have always studied these cells based on their two active states (M1 or M2). There has been evidence in the recent years to show that these cells in fact may exhibit multiple activated states (not just M1 and M2). Just like many immune cells in the body that exhibit a heterogenous phenotype, microglia in the brain may be no different. I’m curious if Arg1 and ODC may be involved in regulating a similar mechanism in microglial cells during neurodegeneration..

Source: Renner K., Singer K., et al. Metabolic Hallmarks of Tumor and Immune Cells in the Tumor Microenvironment. Front Immunol. 2017; 8: 248.

 

It’s brain awareness week!

Hello all!  I wanted to take a few minutes to write something for the brain awareness week. This is important to me because my research focusses on understanding the role of the immune system in the brain. For a very long time, the brain was thought to be an “immune privileged” organ i.e., it was thought that the brain is protected from all the peripheral insults and that it is “divorced” from the rest of the body. In 2015, it was shown that there exists certain lymphatic vessels that connect the CNS to the rest of the body (1). The lymphatic system carries immune cells through a network of vessels and tissues; it connects the bloodstream and tissues in order to remove dead cells and other debris. The discovery of the new “glymphatic system” has opened new avenues to study the connection between the brain and the rest of the body. This is especially helpful in understanding the role of the peripheral immune system on the CNS during infections, injury, and other disease insults.

glymphatic system
Old lymphatic system (left) and newly discovered lymphatic system in the CNS (right). Source: University of Virginia Health System

My work focusses on a specific cell type in the brain known as microglia which are are the resident macrophages of the CNS (they eat up and clear out the bad stuff in the brain like dead cells and mis-folded proteins). Microglia are the only known immune cells of the brain. Compared to all that’s known about the cells of our body’s immune system (B cells, T cells, NK cells, neutrophils, basophils, Treg cells, MDSCs, TH1, TH2, and many many more with several subtypes of each cell), it is safe to say that cells of the CNS are poorly understood. My efforts are focussed towards understanding the role of microglial cells in neurodegenerative diseases such as Alzheimer’s Diseases (AD) , Parkinson’s Disease (PD), Multiple Sclerosis (MS), etcetera. These diseases are characterized by mis-folded proteins that aggregate in the different regions of the brain tissues causing the neurons to degenerate and eventually die. The microglial cells in these disorders play a major role in disease progression by regulating many pathways involved in cell-cell communication, cell survival, and cell death. This is a relatively new and an exciting area of study with many missing links and questions to be answered. I will try my best to keep this space alive with updates and stories! In the meantime, here’s a fun read on Leonardo da Vinci’s contributions to neuroscience: http://www.sciencedirect.com/science/article/pii/S0166223600021214

And here’s a 1504-1506 drawing of the human brain by da Vinci:

Leonardo da Vinci's contributions to neuroscience
In the upper figure, the three ventricles are labeled imprensiva (anterior ventricle, corresponding to the paired lateral ventricles), senso comune (third ventricle), and memoria (posterior or fourth ventricle). Below the ventricles, seven pairs of cranial nerves are shown. The lower figure shows a human head in an exploded view, with the skull raised over the brain and from the head. Source: 

Sources:

  1. Louveau A, et al. Structural and functional features of central nervous system lymphatic vessels. Nature. 2015;523(7560):337–341. doi: 10.1038/nature14432.

Blots, cultures and assays concludes rotation two

This week officially concludes my second laboratory rotation in the neuropharmacology lab with research focussed on  G protein-coupled receptors and their application in several neurological disorders such as depression and anxiety. In the eight week duration of my rotation, a few things were achieved with respect to validating the activity of the newly developed M4R-DREADD (a designer M4 muscarinic receptor exclusively activated by a designer drug). Designer receptors are engineered such that they are solely activated by a synthetic ligand. This opens new avenues in the activation and control of G protein-coupled receptors’ function in vivo.

After a long break from my Master’s research, I got back to maintaining two cell lines – CHO (Chinese Hamster Ovary) and HEK293 (Human Embryonic Kidney) cells, in which the opioid receptors were expressed for all my experiments. These cells were used to characterize the receptor signaling by western blot analysis of the downstream MAPK/ERK signaling  upon stimulation by a few agonists/drugs of interest. Luckily, the lab acquired a new fluorescence microscope during this period which helped us observe the recruitment of the β-arretin2 protein by δ-opioid receptors in HEK293 cells stimulated with clozapine-n-oxide, a synthetic ligand.

mrrd-gfp barrest-cherr cno 0 min 20x_Overlay copy
HEK293 with M4R-dreadd 20x
mrrd-gfp barrest-cherr cno 10 min 20x_Overlay copy
HEK293 with M4R-dreadd 20x

This week, I had a lot of difficulty in handling the mice. Being my first experience with animal work, watching the mice anxious and struggle while we held them down was hard. I am still pretty unsure about how I feel about animal work (if I HAVE to do it to save my research in the future, I will) but I definitely need more exposure and practice with them.

Overall, this lab taught me a lot, even if some days were stressful and  tiring. I feel like I learned and enhanced many skills in the process (primer design, restriction analysis, cell culture, cloning, western blot, cAMP assay), and got a feel for the lab at the same time. Through the course of these past two rotations, I have met some really smart and dedicated people. In the end, I am grateful to have had this opportunity.