Author Archives: Aaliyah Davy

I am elated!!

This summer has been so great, it’s unbelievable. I went in knowing that I wanted to further my interest in microbiology, and I’m glad to confirm my interest! I will be continuing to work in Dr Heitman’s lab this school year, which I am very grateful for and ecstatic about! This summer has really lived up to my expectations and then some. The members of the lab are so nice, helpful, and funny. And I’m so happy they all showed up to the poster session to see my work. It has been great to start working in the lab, and I am so happy with how the summer turned out!

As for the future, the faculty talks added to my original interest of pursuing an MD/PhD. And I look forward to wherever this journey will take me ūüôā

 

Abstract Draft (199 words)

Identifying the endoreplication pathway in Cryptococcus neoformans

Aaliyah Davy                                                                                                                     Mentors: Ci Fu, PhD., Joseph Heitman, MD., PhD.                                             Department of Molecular Genetics and Microbiology

Cryptococcus neoformans is an opportunistic fungal pathogen that affects the immunocompromised and rarely, the immunocompetent. The fungus has two different sexual cycles- that is, bisexual and unisexual. Central to bisexual reproduction is cell and nuclear fusions that are indicative of ploidy duplication and the yeast-hyphal morphology transition. In this study, we investigated the currently unidentified endoreplication cycles that these cells go through to attain ploidy replication in the unisexual cycle. We hypothesized that one of six cell cycle genes we have chosen, that have differential expression throughout unisexual reproduction, will have some impact on the pathway. To test this, we generated gene deletion constructs with dominant selectable marker neomycin (G418), and conducted biolistic transformation on the unisexual strain XL280őĪ. Transformants were streaked on additional G418 plates and verified by PCR. We tested whether deletion of these genes impact unisexual reproduction. This endoreplication pathway has also been linked to the formation of titan cells (greatly enlarged versions of the pathogen) in host infection, so we tested the significance of these genes in titan cell formation as well.

It’s about two engineers

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.

It’s about time…management

My day usually starts with me trying to drag myself out of bed…which takes a while.
Then I go to the program meeting or have breakfast. Before work or at least sometimes during, I usually have some sort of coffee, which is very unlike me. But ever since I came here, I’ve hopped on the coffee train and can’t seem to get off.

My time in the lab is usually spent planning tests, coming up with ways to change variables, and checking for transformants. Recently I’ve been repeating experiments to see if we get faster/better results. For instance, it took a while for us to find definite transformants in the last set of plates we shot with DNA, so I redid the experiment with twice the amount of DNA and different goldbeads.

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I’ve also developed improved techniques for transferring cells to drug plates. I’ve wrapped up the last week by streaking more drug plates in different sections to test for growth. If they grow, it confirms that we actually do have transformants. I’m very excited to check today if we have any!

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After work, I usually rush to buy food before everywhere closes (I leave work around 7 or 8, or sometimes 9pm). And I spend the rest of the day relaxing in prep for tomorrow so that my mind is clear. Sometime I get to leave work early and I go to the gym ūüôā But I’ve recently been going to the gym on the weekends, after I’ve run a few errands.

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My days are usually longer than I anticipate, so I think what I would love to accomplish before I leave is better time management.

 

It’s about this fungus and its titan form

Cryptococcus neoformans is a cryptic pathogenic yeast that is able to elude macrophages by replicating its chromosome set (among other cellular components) until it’s too big to be engulfed. The result are huge polyploidal cells, called titan cells, that are amazingly able to bud into haploid progeny. Just how this occurs is what my postdoc, Ci Fu, Dr., and I are currently researching.

The primary focus of this project are six cyclin genes that my postdoc chose as possible candidates associated with the C neoformans endoreplucation pathway. In the first week, I worked on creating gene deletion constructs (with a drug at the center) for the project, and by the end of the first week, I was able to create them all (shutout to PCR). In fact, by the middle of the second week, I had to make 8 copies of each gene which resulted in 48 PCR tubes.

Yes, I took pictures of my conquest….

Next we worked on cell culturing for two strains in order for us to do biolistic transformation. My postdoc has been working on a different strain, doing the same thing I am. But the absolute best part of the third week has been shooting a gun- a gene gun, that it is. This beautiful contraption allows me to create pressure in a chamber and literally shoot an aliquot of my gene (mixed with goldbeads) with helium.

…It’s not your average gun…but it does ‘fire’.

After at least 3 hours of recovery, I transferred each shot plate onto two drug plates, which resulted in 48 drug plates (each gene deletion construct was shot four times onto different plates). The idea here is that the genes constructs I made will have stuck to the goldbeads (using a washing process I completed beforehand). Once shot, it will kill the cells in the middle of the plate but transform the ones on the periphery. Since we don’t know which cells were transformed, we have to carry ALL the cells from the shot plate and select for them on a drug plate (I used water, a cell spreader, and a pipet to transfer).

The final part of the project is to see whether these drugs impact titan cell formation. So the third week and during this week, we have to determine conditions for titan cells. Last week, we found conditions for a larger version of my postdoc’s strain, but it wasn’t technically a titan. C neoformans becomes a titan¬†in vivo when they fear being engulfed by macrophages in the lung of the affected individual. Getting these titan cells in vitro has rarely been done before. So my postdoc and I are essentially using the only protocol known so far for getting these titan cells (sent by another scientist). But, we weren’t exactly successful this past week, so this week we have to change the ingredients for incubation in 96 well plates- we limit nutrients so that these cells can hoard it into large vacuoles, a common characteristic of a titan cell. We plan on relying on the studies of a professor who started out in our lab- under my PI Joe Heitman, MD., PhD. – who is now revolutionizing¬†in vivo¬†studies of the pathogenic fungus: Kirsten Nielsen, PhD.

Dr Nielson and her team found this titan cell (in the red) in vivo. The macrophages are clear/gray, to the right and the normal C neoformans cells are in the blue. 

Nonetheless, whether we get an actually solid answer from this project, I am so grateful for the opportunity to be here and learn so much. It’s been fun and exciting to learn new techniques and use incredible technology like a biolistic gun and a multichannel pipet. Everything has been amazing.

It’s about making connections

Often times, one would envision ‘the scientist’ as an individual with specific plans, designs, and goals, all culminating into this profoundly ambitious dream. And that’s true. But, there’s something missing.

Dr Joe Heitman, MD., PhD.- my PI- stressed the importance of making connections in science, whether it be with other people, between different concepts, or among methods of inquiry. Working on these things, even little at a time, can get you so far.

Science is social. This doesn’t mean that ‘the scientist’ wouldn’t get anywhere with talking to anyone. But, communication is at the center of what makes great papers and discoveries. Dr Heitman emphasized the importance of having an open dialogue in the lab, and between labs that may even be a few states or nations apart. This work is isn’t about being another star in the night, a well-known ball of energy adding to the light of this sky. It’s about sending signals, working with others in order to leave some permanent or influential knowledge that could potentially shape current efforts or inspire new ones.

Dr Heitman told me stories about the people he’s met in his career that took him on paths he’d otherwise never consider. With subtle advice, some have even inadvertently led him to address questions in his research that ultimately led to significant breakthroughs. The key thing to take away is that although your mind should be focused, your ears should always be open. In fact, Dr Heitman’s primary interest has always been about molecular structures, specifically nucleic acids. Yet, here he is- in a microbiology lab instead of genetics. It’s amazing where a path can take you.

I definitely enjoyed speaking with him and getting to know his many transitions from a chemist to biologist to medical doctor to principal investigator.

 

 

 

It’s about comfort and a feeling

I’m not sure what exactly to expect from this summer, but I do know what I would like to get from it. I’d like to be able to follow lab protocols independently, and gain experience that will contribute to a degree of comfort and confidence in a lab.

Going into this program, I thought that I would only be in this lab (Molecular Genetics and Microbiology department) for the summer because I planned on returning to my previous workplace, Duke Molecular Physiology Institute, during the school year. Little did I realize that it is possible to work in two labs. Thank goodness! I am in love with both places and I’m very excited for what they will teach me about lab technique and procedures, and the opportunities they can provide for my own research interests. I feel completely honored to be able to actively engage in revolutionary research.

There is so much that DMPI has taught me that facilitated my transition into my new lab this week. I find it so beneficial and reassuring that across two different yet closely related fields- microbiology and molecular bio- there are protocols and technology that are parallel in structure. And I am so appreciative of the tools my introductory microbiology class has given me this past semester, especially with such a short timeframe.

Even during my very first meeting with my mentor, postdoc, and grad student, I was introduced to the possibility of one day going to grad school, and using lab work as a means of getting a degree. It’s a thought that reccured in my mind throughout the week, when I felt at ease and almost a giddy feeling while working. My experience so far already has me considering applying to Program II so that I can major in Microbio and Molecular Cell Bio. And maybe I will pursue a PhD in microbiology….

So for this summer, I expect, overall, to start shaping the way I view research and the career choices it could mean for my future, wherever I may be.