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.

[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.

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.

Chasing cancer cells

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Glioblastoma (astrocytoma) WHO grade IV – MRI sagittal view, post contrast. 15-year-old boy. Image courtesy: Wikimedia commons

Glioblastoma multiforme (GBM) is one of the most invasive forms of malignant brain tumors. By the time the tumor is removed from a region in the brain, the cancer cells rapidly metastasize and spread throughout the brain. Common treatment procedures such as chemotherapy and radiotherapy are not completely effective due to the aggressive nature of the tumor invasion. Therefore, many treatments also target the migratory properties of the tumor cells. Several proteins (focal adhesion kinase, paxillin, vinculin) are over-expressed in the extracellular matrix of the tumor microenvironment and help the GBM cells proliferate through the brain tissues. A treatment approach is to target these proteins and hopefully prevent -or at least reduce- the tumor cell invasion.

Studying this type of a cancer model is tricky. Most of the work that has been done in the traditional 2-dimensional cell culture environment cannot be translated into the 3-dimensional environment of the brain. We need a system that mimics the brain to realistically model the tumor cell growth and migration. As a part of my third rotation, I have been investigating the migration characteristics of GBM cells in tissue-engineered, 3-dimensional cell culture matrices that mimic the brain environment. The cells are grown in a collagen matrix containing components of the brain extracellular matrix (hyaluronan, astrocytes, etcetera) and the tumor cell migration is studied by tracking the focal adhesion proteins. However, this is not easy. Cancer cells have shown to modify their migratory patterns based on the physical conditions of the tumor microenvironment (Herrera-Perrez et al. 2015 Tissue Engineering Part A). This makes it more difficult to target the adhesion receptors to ultimately inhibit tumor invasion. It is also challenging to prevent the disruption of the cross-talk between the targeted receptor protein and other important signaling molecules during evaluation of the treatment procedures. Overall, new innovative strategies are required that focus on the diversity and adaptability of tumor cell invasion and migration.

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.

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HEK293 with M4R-dreadd 20x
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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.

Two months in: Last day in cancer lab

Today marks the last day of my first laboratory rotation. I want to pen down a few things that I learned and experienced during my time in the cancer lab:

  • Starting fresh in a new field of research was challenging at first, but got interesting once the different pieces of the puzzle were pieced together along the way.
  • Understanding the nitty-gritty of the investigation entails failures, failures, failures, followed by lots of optimizations and practice. Patience and perseverance is the key.
  • Staying positive and motivated throughout the journey can go a long way. My mentor/ senior grad student in the lab is one of the most optimistic people I’ve met in recent times.
  • Almost always, grad students manage multiple projects at the same time. It is essential to have a main project (or two) and a few side projects to keep the lab active, and research moving forward.
  • Taking a computing course along with the Biochemistry course has kept my study diverse and helped me broaden my thought bubble. At the same time, it has contributed to an additional pressure of having to take exams and submit assignments frequently (which I don’t want to be doing a lot of at grad school).
  • I nearly broke my arm while working with the french press for cell lysis (it really is a workout in itself!)
  • Having flexible working hours in the lab was good, but there were days when I took this for granted and ended up being awfully lazy. I am still learning to implement a fixed schedule for the weekdays to increase my productivity through the week.
  • Caffeine addiction is a real thing. I now even have a brew preference to satisfy my taste buds and more importantly, my brain.