Picking my permanent lab

Almost a year has passed since I started my Ph.D. journey in the land of snow and maize. After four long lab rotations across three departments and hopping from one project to another, it is time to pick a permanent lab and a research direction.

IMG_6374.JPGI am happy to announce that I have officially joined the distinguished Department of Chemistry at my university and have begun my research at the Institute for Drug Discovery where I will work for the remaining period of my doctoral degree. I couldn’t be happier with my decision which was mainly determined by three aspects – my advisor/mentor, the research area and the lab (environment and members). It feels good to finally know where I am headed towards and not feel lost or uncertain. Every one of my rotations was unique and helped me learn the nitty-gritty of grad school. Moving forward, I will focus on brain-related disorders like Alzheimer’s Disease and work towards understanding a tiny piece of a large puzzle which may aid in finding a cure/prevention/slow down the progression of the disease. Specifically, the overarching theme of my work will be to identify and test compounds predicted for the disease by taking into account all the possible interactions between biomolecules in the protein universe (aka the proteome). Traditional drug discovery methods involve targeting a specific protein or a specific pathway and thereby limiting the possibility of finding successful leads. In reality, we know that one biomolecule interacts with several other biomolecules in several different pathways. Interactome-based drug discovery is promising because of its broader and quicker approach compared to the other mainstream pipelines that exist today.

One other major factor that helped me decide my major lab was the computation aspect involved in drug discovery research. Taking the challenging Computational Chemistry course this semester helped me take the first step towards learning about some of the components of computer-aided drug discovery. It is amazing how the two channels of research (wet lab and dry lab) finally come together in solving some of the major challenges in health and medicine. Anyway, I will continue to update here more on my day-to-day lab rat adventures. I am excited to start this new chapter of my life and see where it takes me! :)

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.

The Human Kinome

The entire set of 518 protein kinases in the human genome makes up one of the largest of all human gene families. These enzymes catalyze the phosphorylation of proteins, specifically serine/threonine and tyrosine residues – an important reaction that regulates key cellular functions like cell division, metabolism, and apoptosis in normal and disease states. This makes kinases key therapeutic targets in several diseases such as cancer, neurodegeneration, behavioral disorders, diabetes, and cardiovascular diseases. Interestingly, both the labs that I’ve been in so far are focussed on kinases involved in pancreatic/prostrate cancer and GPCR signaling in the light of alcohol/drug addiction. Leaving this nice phylogenetic tree here as a reminder and reflection of kinome research!Map of the Human Kinome

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.

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.