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!

Advertisements

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.

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.

 

Thoughts on lab rotations

The thing with first-year rotations in a Ph.D. program is that anxiety starts kicking in somewhere along the way when you consciously identify the lab that you want to join and want to get started right away. Having realized that this is going to be a long journey and rushing into things may not help, I am now gaining patience and perspective, and hope to make the most of the remaining time of my first year.

Rotations are a great way to learn about a lab and get involved in the nitty-gritty of research. I was warned at the beginning by a few seniors that I would either love a lab or reject it within the first few weeks of the rotation. Mind you – this has nothing to do with the science pursued in the lab (one wouldn’t decide to rotate in a lab if they didn’t find the research interesting in the first place). This is more about getting comfortable with the way a lab functions and deciding if the environment is a good fit for you. An eight-week lab rotation is really like an eight-week long interview with a potential PI and the lab! It is essential to identify the kind of relationship you foresee having with your advisor for the next couple of years (and beyond). This is perhaps one of the most important aspects of a rotation for me, next to the research work. A good mentor-mentee relationship can go a long way and can be extremely beneficial to one’s academic/professional career. I prefer having an open channel of communication with my mentor and learn as much as possible from him/her.

Not all graduate programs require laboratory rotations. Many departments or programs accept or reject students simply based on their application and/or an interview. In the UK for example, students are recruited to work on specific projects and grants as a part of their Ph.D. for the time period of around 3 years. This may not benefit the candidates who wish to propose their own ideas and develop their own thesis based on their individual research interests. In the US, for most graduate programs in the life sciences (mainly biology and chemistry), the average time for graduation is around 5-6 years. I believe that the freedom and independence of this system trump the short graduation time of the other systems. Although I am certain that both sides have their set of merits and demerits, at the end of the day, the journey is unique to each one of us and what we make of the experience matters the most.

Orientation week: What do I want out of grad school?

The first week of grad school was intense and exhaustive with all kinds of information being tossed at us from all directions. We started off with a formal introduction to the school, the department, and all the resources available at our disposal like the libraries, mentors, health benefits, and so on. Besides all this, a main objective of the orientation week was to decide the first two labs that we are interested to rotate in. The process involved meeting with several professors, going on lab tours, meeting other grad students and evaluating if a lab was a good fit for us or not. Although I knew the direction of research I wanted to pursue, discovering so many options and learning about cool new research areas left me wondering if I really knew what I wanted to be doing for the next five years! Right now, I feel like a first grader starting school for the first time and constantly being exposed to many things I never knew existed.

Grad school 101 - What I don't know
Grad school 101 – What I don’t know

Being in a big umbrella program, there are ten different training groups to choose from. First year graduate students pick four labs within any of the groups to rotate in during their first year. This is very different from a departmental graduate program where a student can only rotate in labs within that respective department. After all the decisions and evaluations, I have chosen my first two labs for the semester and I am looking forward to be officially starting next week.

This process has made me question some decisions that I’ve taken in the last couple of years. “What do I want out of grad school?” seems to be the most significant one. Before beginning my journey, I knew that I wanted to train to be a good scientist, learn how to think, develop skills unique to my field, master techniques that will make me employable, learn how to learn, and be an overall well rounded researcher. Now I’m not sure if there is a definite answer to the question. It is something that I’d have to figure out on-the-go.

G protein-coupled inwardly-rectifying potassium (GIRK) channels

My current Master’s thesis research is focussed on understanding the structural and functional properties of G protein-coupled inwardly-rectifying potassium (GIRK or Kir 3.x) channels. This class of potassium ion channels are responsible for regulating the heart rate and modulating the neuronal excitability of certain neurons.

GIRK channels are activated by G-protein coupled receptors (GPCRs) including the muscarinic, dopamine, serotonin, GABA, opiod, and acetylcholine receptors, which are involved in many signal transduction pathways in the cell. The activation of a GPCR by its ligand (neurotransmitter or hormone) results in the release of Gα and Gβγ, two intracellular effector molecules. The activated Gβγ binds to the GIRK channel and opens it up to potassium ions resulting in the hyperpolarization of the cell (increased negative charge due to efflux of K+ ions).

Activation of GIRK channel
GIRK channels are activated upon GPCR stimulation by direct interaction with Gβγ.

Molecular cloning techniques have led to the discovery of four channel subunits – GIRK1 (Kir 3.1), GIRK2 (Kir 3.2), GIRK3 (Kir 3.3) and GIRK 4 (Kir 3.4). GIRK1 through 3 can be found in the central nervous system and GIRK4 is primarily found in the heart. Four of these subunits assemble either as homomers or heteromers (in 1:1 subunit ratio) to form a tetrameric functional channel.

Structurally, the channel is divided into cytoplasmic and transmembrane domains. The amino- (NH2) terminus and the carboxyl- (COOH) terminus are present in the cytoplasm and contribute to the formation of the intracellular/cytoplasmic domain. Each subunit is composed of two transmembrane domains separated by a P-loop containing the “ion-selectivity filter”. This type of channel assembly results in significant interactions between the cytoplasmic domains of the four subunits.

Crystal Structure of GIRK channel. Left - Front view of four GIRK2 subunits (color coded) channel assembly. Right - Top view of the cytoplasmic domain forming the selectivity filter.
Crystal Structure of GIRK channel. Left – Front view of the channel comprised of 4 subunits (color coded). Right – Top view of the cytoplasmic domain forming the selectivity filter. Protein Data Base ID – 3SYQ and 2QKS (Whorton, M. R., Mckinnon, R., 2011)

I have been specifically involved in understanding how certain amino acid residues residing in the hydrophobic pockets of the subunits influence channel activation and function. I use multiple experimental methods to investigate the interaction between the N- and C- termini of the GIRK1 and GIRK4 channel subunits to analyse protein expression and domain association. Previous research (Sarac et al, 2005) has revealed that certain mutations in the amino acid residues of these two subunits alters channel function.

Understanding how the interaction between the different GIRK channel subunits influences the channel formation and activity is critical for the elucidation of certain cellular mechanisms involved in cell physiology as well as in various channelopathies. New studies also suggest that ethanol binds to a hydrophobic pocket in the channel and activates it. (Bodhinathan, K., Slesinger, P. A., 2013 and Aryal, P., et al, 2009) Ethanol activation of the channel can be utilised for developing selective therapeutics to treat alcohol-related disorders like alcohol addiction and abuse.

Selected Resources:

Stress – it’s all in your head!

Stress is an interesting body response that is stimulated by our brain due to incoming auditory, visual and/or somatosensory signals. It is how we feel and how our body reacts when we encounter an imbalance in the normal rhythm of life. Watching a horror movie, coming face to face with a deadly creature or simply feeling overwhelmed due to daily tasks may all evoke stress. How does our brain respond to a stimulus that elicits fear and anxiety?

The key areas of the brain that are involved in stress are the thalamus, hippocampus, amygdala, and the prefrontal cortex. The thalamus located in the forebrain processes the incoming visual and auditory signals and relays them to the prefrontal cortex and the amygdala. The prefrontal cortex is the hub for executive function. With respect to stress, it gives meaning to the relayed signals and makes us conscious of what we see and hear. This part of the brain is also critical for ‘turning off’ the stress response once the condition is passed.

The Neurobiology of Stress - Brain regions involved in stress response
The Neurobiology of Stress – Brain regions involved in stress response

The amygdala is the emotional center of the brain and is responsible for triggering the stressful response. It is a part of the limbic system and is located deep within the temporal lobes of the brain. The amygdala also drives the body’s sympathetic nervous system to initiate anxiety that is associated with stress. This includes increasing the heart rate, blood pressure, hyperventilation of the lungs and increasing perspiration.

Finally, the hippocampus located in the medial temporal lobe stores the memory linked to a particular stress response and allows the brain to access these memories when the same visual and auditory triggers of stress are encountered later on.

It is also essential to mention the role of the hypothalamus and the linked pituitary gland that pumps out high levels of cortisol – “the stress hormone”. Recent studies suggest that cortisol can damage and kill brain cells, especially that in the hippocampus. (The hormonal response of stress is in fact a huge area of study with lots of factors involved.)

A critical question in this area of study that interests me is, “How much stress is bad for us? Can a little stress actually be helpful?” It turns out that acute stress (short-lived, unlike chronic stress) may actually be good for us. New research suggests that it conditions the brain for improved performance by inducing an increased level of alertness, behavioural and cognitive performance. This may explain why we get most of work done when we’re under pressure!