Category Archives: Week 2

In Dr. Volkan’s lab, the primary focus of my project is to look at the genes that underlie the neural circuits that deal with sexual behavior in flies, specifically in Drosophila melanogaster. We already know about a gene named Fruitless or Fru in males that controls all male courtship behavior, and another gene named Doublesex that controls experience-dependent sexual behavior. In other words, the level of courtship these flies maintain controls whether or not doublesex is expressed. Additionaly, ChAT (choline acetyltransferase), a gene that encodes an enzyme to make the neurotransmitter acetylcholine, is known to be involved in the neural circuits that deal with courtship behavior. If ChAT is being used, then that means that neural circuits are being used as well.

Furthermore, in my project we are putting male flies (some with mutations in the genes previously mentioned) in different social environments where they are either group housed or isolated and look at the expression of those different genes. Studies have shown that group housed male flies that are mutant for Fru do in fact court one another and form a chain link (Villella et al.), while isolated male flies, when rejoined with other flies after isolation, fail to court alltogether most times (Pan and Baker). Consequently, if we separate male flies by their social experience, we can see what genes are turned on and off by protein markers such as RNA polymerase, H3K27ac (a histone epitope that deals with acetylation and is associated with the higher activation of transcription) and others.

In my project, we are using a method named ChIP, which is “a type of immunoprecipitation experimental technique used to investigate the interaction between proteins and DNA in the cell”. In this method, antibodies made specifically for proteins like RNA polymerase will attach to the proteins, which are also attached to genes, and will be taken out of solution by magnetic beads so that the RNA can be isolated and sequenced into DNA, giving us the DNA sequence related to the behavior we witnessed!

Overall, with this project our lab hopes to find out what genes in the brain let the fly know when to mate and who to mate with, or simply how the fly makes decisions when presented with a mate. This information will give us more insight on the circuit for sexual behavior in flies and can even be used to understand the human brain in the future!

Heads of the flies!

More fly heads because practice makes perfect!

Simeon Holmes-What I am working on this summer!

Cryptococcus deneoformans is an opportunistic human fungal pathogen that mainly infects immunocompromised patients, including AIDs patients and patients with organ transplant. It infects approximately 280 thousand people and contributes to about 180 thousand deaths per year. Though C. neoformans species complex poses a huge threat to global human health, few strategies are available in treating Cryptococcosis.

The purpose of my research in Dr. Heitman’s lab is to study the functions of two essential cell cycle regulating genes involved in the endoreplication pathway during Cryptococcus deneoformans unisexual reproduction. To study these essential genes, we plan to use the copper repressive promoter pCTR4 and the galactose inducible promoter pUGE2 to modulate gene expression. By up- and down-regulating these genes, we would like to examine how perturbation of gene expression could impact C. deneoformans unisexual reproduction and other cellular processes.

Study of essential genes also provides opportunity in discovery of novel drug targets that are essential for cell growth. We are interested in testing how these genes impact the virulence of the pathogen by using DAmP alleles of these genes with disrupted terminators. Based on these work, we could potentially apply these techniques in studying essential genes in other fungal pathogens and provide hope in novel anti-fungal target discoveries

The Murphy Lab focuses on studying heritable epigenetic effects that are induced by various environmental stimuli. One of the latest projects is  Cannabis-Induced Potential Heritability of Epigenetic Revisions in Sperm, or CIPHERS, which focuses on the heritable epigenetic changes associated with cannabis use in males.

For my project this summer, I will be analyzing genes that are critical to certain aspects of development in order to see if there is differential methylation in the progenitor (F0) and offspring(F1) generations. While I cannot go into specifics at this project, I can provide a general overview of expectations and  potential significance of this project. This work is essential to the overall focus of CIPHERS as it could provide evidence for cannabis use inducing transgenerational epigenetic changes that affect the viability of both progenitor and offspring. The data generated by CIPHERS could in turn be used to guide policymakers and the general public in the epigenetic effects of cannabis use. In the future, I will likely be working on the F2 generation for these genes in order to solidify that cannabis exposure is in fact a transgenerational stimulus.

The Pendergast Lab, in which I am working this summer, broadly studies the Abelson family of non-receptor tyrosine kinases. Tyrosine kinases are a subclass of protein kinase, molecules which phosphorylate proteins. In the case of tyrosine kinases, the phosphate group is attached to a tyrosine amino acid in the target protein’s chain. This phosphorylation activates or deactivates the target protein, enabling or preventing its function.

Abelson kinases, encoded by the genes Abl1 and Abl2, are important molecules in a wide range of cell signaling pathways. Abl kinases are highly expressed in a range of cancers cell lines, suggesting that the pathways in which they are involved play a significant role in tumor development. The Pendergast Lab’s research focuses on mapping out these pathways, and the way in which Abl kinases function within them. My mentor is currently researching a number of metabolic pathways in which Abl kinases are potentially involved, and my project focuses on a protein involved in one of these pathways.

The protein, encoded by the gene SLC7A11, is called system xCT. It is an antiporter, a membrane transporter protein which simultaneously imports cystine and exports glutamate. Cystine is an important molecule in the cell’s response to oxidative stress, the cellular damage caused by reactive oxygen species. Recent studies have suggested that oxidative stress inhibits tumor growth, and cancer cells therefore depend on antioxidant response pathways such as the one involving xCT. Blocking this pathway and thereby leaving cancer cells exposed to the tumor-inhibiting influence of oxidative stress may be therapeutically useful. I will investigate the hypothesis that Abl kinases play a role in the activation of xCT expression.

I will begin by inhibiting Abl kinases, using the allosteric inhibitor GNF5, and observing the effect on cellular levels of xCT. I will measure the effect at the protein level using Western Blotting, and also at the mRNA level using RTPCR. A decrease in xCT upon Abl inhibition would suggest that the kinases are indeed somehow involved in increasing xCT expression or activation. Further experiments would be required to determine the pathway through which Abl kinases increase the expression or function of xCT, and the other molecules involved in this pathway.

As I have started to learn, scientific hypothesis are not always validated. If early results do not correspond to our hypothesis regarding Abl and xCT, I may have to change the focus of my project. However, the process of designing models and experiments to test one’s hypotheses is itself incredibly rewarding, and a good understanding of this process is ultimately more important than individual results.

This summer, I am working with my lab on an ongoing effort to answer the broad questions of how stable are transgenerational environmental effects and how do different genotypes affect this stability? We are studying the environmental effect of temperature on the development of Arabidopsis thaliana. 

To answer the question of how stable transgenerational environmental effects are (a.k.a does or to what extent does the effect of a plant’s ancestral environment depend on the present environment of the plant), we are monitoring 3 generations grown in two temperatures. To elaborate, the grandparental generation is grown in a warm and cool temperature, and offsprings of each respective environment are then also grown in warm and cool temperature to give 4 scenarios. Finally, for the last generation, we take the 4 different parentals and self-pollinate them to give 8 possible scenarios (CCC, CCW, CWC, CWW, WCC, WCW, WWC, WWW). Using data collected of key developmental marks such as seed germination, seedling traits, time and size of bolt, flowering time etc., we will be able to analyze if the effects of ancestral environments depends on the present environment of the plant. We answer the next question of the effects of genotype on transgenerational environmental effects by growing Arabidopsis Thaliana of various genotypes in strictly all warm or all cool climates for 3 generations (CCC or WWW).

In both experiments we are testing plants that require vernalization (a cold period before they flower) versus those that do not require vernalization.

This is a project that was started during the school year, so my portion is conducting the last generation for the two different experiments. I do germination assays (counting the number of germinants from each of the scenarios) and record specific phenotypes. Because plants take a while to grow, there is time where I’m not directly working on my portion of the project. During this time, I am learning about data analysis techniques for when the data becomes available. My mentor has recommended that I start to learn a bit of computer programming, so that we can analyze all aspects of the data. This portion is very statistic heavy, but I am very excited to explore biology through a quantitative lense.

(The name of the project is Epistability, because there are factors beyond genetics (environment) that affect these plants. )

What happens when you remove cells essential for development? How can cells detect changes around them in order to change their gene expression. Why are these questions even important?

Ettensohn and McClay show that the removal of skeletal cells from the sea urchin embryo causes non-skeletogenic mesoderm (NSM) cells to transfate in order to take on the role of the missing skeletal cells. Ettensohn et al. showed that a skeletal cell specific gene is expressed during the process of transfating because it plays an important role in the gene regulatory network in the skeletal cell lineage.

However, we still do not understand exactly how the skeletal cells suppress NSM transfating. Therefore, in the McClay lab, I work on a project to explore the question of how the embryo recognizes and regulates cell loss in the sea urchin, Lytechinus variagatus. In order to do so, I will be performing in situ hybridization experiments on a list of candidate genes expressed in skeletal cells in order to verify their expression. I have already designed and ordered primers that will be used to clone genes through polymerase chain reaction (PCR) of the sea urchin’s cDNA. The genes will then be used to synthesize RNA probes which will be used for the in situ hybridization experiments.

I will also be using drug inhibitors to see if there is ectopic (abnormal) cell reprogramming in the sea urchin embryo. This will be done by treating developing embryos at different time points with a drug inhibitor and performing in situ hybridization experiment. By inhibiting certain signaling molecules, I would be able to see whether or not the signaling molecule plays a role in the embryos recognition of skeletal cell presence.

These are my current aims for the summer, but I already have so many more questions popping up from preliminary data and reading the literature that I hope will guide me in the coming weeks and into the future.

For anyone who is confused about my title, here is a meme for your reference.

Studying the wild population of yellow baboons (Papio cynocephalus) in the Amboseli National Park of southern Kenya, the Alberts Lab focuses on how animals respond behaviorally to their social environment. As a matrilocal species, female baboons stay within the group they are born into while males will disperse to another group. Male baboons compete with each other for access to resources, such as food and reproductive opportunities, therefore leading to a dominance social hierarchy.

With on-site, documented interactions between baboons and the ability to analyze hormone concentrations from collected fecal samples, my mentor explored the relationship between dominance rank and stress in male baboons. Gesquiere et al. (2011) found that the concentration of the stress hormone glucocorticoid in fecal samples was high in alpha and low-ranking males. However, beta males had significantly lower levels of glucocorticoid compared to alpha males, with the concentration of glucocorticoid increasing the lower in rank an individual is.

This summer, I am continuing Dr. Laurence Gesquiere’s research. In order to determine potential differences in stress experienced by alpha and low-ranking male baboons, I will determine the thyroid hormone concentration in fecal samples. Thyroid hormone is being analyzed because the thyroid gland secretes more or less of the hormone based on changes in metabolism, therefore allowing the hormone to be used as a measurement of energetic stress. Energetic stress caused by agonistic interactions and mating activities is believed to be the major source of stress for alpha males. In contrast, it is hypothesized that low-ranking males experience energetic stress from limited access to food and psychosocial stress from harassment from higher-ranking males.

Therefore, by looking at the thyroid concentration present in fecal samples from male baboons, I will help elucidate different sources of stress between low-ranking male baboons and the alpha males.

And as promised last week, here’s a picture of me working in the lab:

Me aspirating samples before they go into the gamma counter. Picture courtesy of my mentor!

The Gersbach lab focuses on developing innovative methods in molecular and genetic engineering for applications in regenerative medicine, treating genetic disease, and enhancing understanding of fundamental biological processes. More specifically, many members of the lab aim to treat Duchenne’s muscular dystrophy with CRISPR-Cas9 genetic engineering tools by creating an insertion or deletion mutation that restores the reading frame of the DMD gene.

So far, their methods have been successful in skipping an exon to allow the production of dystrophin, though the deletion efficiency has been close to 2%. Though this is satisfactory for restoring muscle strength in mice, 2% is too low for humans and the methods would not be applicable to other genetic diseases. This inefficiency may be caused by the choice in guide RNAs that lead the Cas9 to the desired deletion site, as some gRNAs have been shown to have higher mutation rates than other. However, there are thousands of possible gRNAs and gRNA combinations for each exon and testing each one is tedious and time-consuming. My mentor’s project is developing a high-throughput strategy for screening pairs of gRNAs for hDMD exon deletions. For my project this summer, I will be testing 5 gRNAs on each side of exon 51 to determine which is most efficient to use as a positive control for my mentor’s screening techniques. By targeting both sides, my designed Cas9 will delete the exon and restore the reading frame shift that was caused by a mutation in an earlier exon. I can then sequence the DNA to see how many of the cells I transfected have exon 51 deleted and how well each gRNA worked.

In the future, this will hopefully lead to more frequent deletions with the use of CRISPR and Cas9 so that these genetic engineering tools can be used to treat those born with genetic diseases.

As I mentioned before, I am currently working in the Brennan Lab, a Biochemistry lab that has previously done extensive research into the trehalose pathway within fungi. In order to lead into my project, it is important to understand more about certain research in the Brennan lab and what exact implications it has in the large scheme of things.

Throughout recent times, immunocompromised host populations, whether transplant recipients, cancer victims, and other ICU patients (Intensive Care Unit), have continuously been invaded and killed by fungal diseases. In a search to identify antifungal targets, the Brennan lab investigated the trehalose pathway due to its fungicidal characteristics, broad relevance to many different types of fungi, and its low toxic consequences in mammalian species.

Looking a bit closer into this research, the trehalose pathway in fungi is regulated by two important enzymes that basically form the backbone of trehalose formation: Tps1 (trehalose-6-phosphate synthase) and Tps2 (trehalose-6-phosphate phosphatase). Tps1 creates trehalose-6-phosphate (t6p) from glucose-6-phosphate while Tps2 ultimately forms trehalose from t6p. Trehalose is extremely important in fungi survival as it has implications to energy reserves during times of stress and is crucial to carbon metabolism and regulation. Tps2 has been identified, due to structural research and animal tests, to be an ideal fungicidal target as mutant Tps2 fungi cannot survive in high stress environments. However, recent research has also uncovered that Tps1 and Tps2 seem to serve other functions within fungi than simply the trehalose path way, signaling that these are possible “moonlighting” proteins.

Well, you’re probably wondering, how does all of this relate to plants and my project? Recently, collaborators of the Brennan lab have studied a protein in maize, RAMOSA3 (RA3), that is part of the trehalose pathway in plants. RA3 serves the purpose of converting trehalose-6-phosphate into trehalose, an analogous function to that of Tps2 in fungi. Yet, when RA3 was mutated in maize, the phenotype of the maize changed and at the stem of the maize, and weird abnormally long branches would begin to form. This gives some indication that RA3 is much more than just a regulator within the trehalose pathway. Once again, this RA3 enzyme is suspected to have possible transcription regulation implications. My project is focused on taking the plasmid containing the RA3 protein and materials sent by collaborators to express the protein, purify it, confirm its function, and ultimately create a structure of it. In order to better understand the functions of RA3 and its implication or analogous functions to the possible fungi trehalose path, we need a literal structure of the enzyme to study substrates and other similar patterns. The moonlighting similarity is yet another thing to be studied. As RA3 is relatively still not understood, my project is focused on create a better familiarity with this enzyme.

Till next time,

Luke Sang

Before you wonder about the title, no, my project does not involve a study on the popular 1960’s dance (I just thought it was a fun title). The project I’m actually working on in the Wray lab is to use CRISPR/Cas9 to knockout particular genes in sea urchin embryos and observe their development. As of right now, I’m soon going to start trying to knockout endo16, a gene in sea urchins that necessary for the development of their gut (link).

The reason that I am first testing that particular gene is because its function is already known, so I should be able to easily identify if the knockout was successful. Once I’m able to get the results that are expected, I’ll be able to move on to working on genes whose functions are not yet known.

Interestingly enough, though sea urchins are considered to be great model organisms to study early development since their embryos are clear and easy to maintain, there are still many regions of the genome that have not been fully defined, as far as their function and interaction with other genes goes. However, understanding how genes within organisms produce changes at different points in development goes beyond looking at them as individuals, but through understanding them as pieces within a far more complex system, which is referred to as the genomic regulatory network (GRN) (link). This idea is what the sea urchin research in the Wray lab revolves around.

Essentially, by experimenting with individual genes and observing what developmental pathway they effect and when they effect it, those genes can be placed within the overall GRN. The interactions between that gene and other genes that regulate that pathway can be identified, creating a much richer understanding of how genomes operate. This, in conjunction with how these interactions are mediated by cis-reulatory systems, can provide us a greater understanding into how changes in development occur and how these drive evolution of species (link).

Though I haven’t gotten particularly far in my project so far as I’m still practicing the skills required to ensure the embryos are able to develop normally post micro-injection, I hope to get started on CRISPR/Cas9 by the end of this week or the beginning of next week. I’m quite excited to see what I might discover!

The Silva lab researches how cells respond to stressful environments. When cells are put in a stressful environment, many of their proteins are damaged. Damaged proteins can result in many adverse consequences for the cell, so in times of stress the cell works to remove these proteins. Eukaryotes have evolved special machinery to recognize and tag damaged proteins using ubiquitin. Ubiquitin is a short protein used to post-translationally modify other proteins and can signal many different things such as degradation of the tagged protein.  Ubiquitin accumulate in the cell, possibly resulting in negative outcomes. This accumulation and disruption of the proteasome degradation system is thought to be related to neurodegenerative diseases like Parkinson’s and Alzheimer’s disease. However, very little is understood about how the ubiquitin proteasome system is related to disease and many ubiquitin pathways and targets remain to be characterized. Additionally, stress intensity can also impact the efficiency of the proteasome.  At low levels of stress, efficiency can increase whereas at highs levels, the proteasome’s functionality is reduced. However, the causes for this response are not well understood. The Silva lab’s research focuses on characterizing ubiquitination pathways and their corresponding cellular responses as a result of oxidative stress.

My project relates to one specific kind of ubiquitin modification, Lysine 48 (K48) ubiquitination. This tag serves as a signal for protein degradation. My project focuses on characterizing K48 ubiquitination levels in cells experiencing oxidative stress. Some of the big questions of my research project are what enzymes are involved, specifically the E2 and corresponding E3 enzymes that ubiquitinate proteins. As well as what are the targets for K48, how K48 ubiquitination of ribosomes vary in stressful environments, and how these modifications impact translation. My current project is taking many different approaches to characterizing the usage of K48 ubiquitin in response to stress. I’ve started out this research experience looking at general levels of K48 ubiquitination in wild type yeast cells under different levels of oxidative stress. After this initial experiment, I’m studying K48 levels in mutant yeast stains that lack E2 enzyme genes in order to test which enzymes are important to the K48 ubiquitination process. After this, I will explore how the proteasome activity and deubiquitinating enzymes in the cell are affected by oxidative stress and then how the frequency of K48 modified ribosomes changes in a stressful environment. Characterizing this pathway could provide greater insight into the biological underpinnings of serious neurodegenerative diseases and help this area of research be one step closer to finding a cure.

During my interview with Dr. Perfect, we discussed numerous topics related to his education, science/research, medicine, his kids, etc. My most memorable moments in life are the times when I give people the oppurtunity to speak freely and simply tell their story.

In school, a guidance counselor told him he could be anything he wanted to be. So he decided to become a doctor. He continued along the pre-med course in his undergradute career and revelled in the complexity and ability to help people as a doctor. Further, he continued to specialize in infectious disease in his mdecial career because he appreciated the simplicity of infectious disease: it normally requires a diagnosis and treatment. He added his conscern that this fact is changing given the surge of antibiotic-resistant bacteria but it has not affected his work immensely (his top world coscerns were also nuclear warfare and global warming). However, he would find himself falling in deep interest in a particular yeast involved in causing meningoencephalitis.

On February 14, 1978 at 11pm, Dr. Perfect recieved a severley ill patient infected with Cryptococcus neoformans. At the time, he found the situation very intriguing and decided to study the fungus thoroughly. As a result, his lab and research over the years has contributed to the understanding of meningitus and mycology. Unfortunately, he did have many conscerns about how research is conducted even today. He believes funding for research is minimal and that PhD research and MD research is too polarized. Moreover, he attributes this to the public’s and government’s attitude toward science. People don’t fully understand the importance of the “silly little experiments” that take place in the lab and how they directly affect the people. He proposed one example in relation to molecular biology. Without the establishment of basic molecular biology data and techniques, doctors/scientists would be unable to even identify the cause of an individual illness, let alone treat it.

Also in my discussion with him, he gave me advice on considering a medical degree and his personal experiences. Dr. Perfect feels that the field of medicine is a rewarding career and appreciates that he is able to help people everyday. Upon asking him how he deals with a patient he is unable to cure, he said you must first distinguish between sympathy and empathy when in medicine. Essentially, doctors have to learn to only bring empathy to work in order to protect their own mental health. He also added “There will be some deaths but everyone does that.” Hence, you have to accept the fact that you did all you could do and that it was that person’s time. Moreover, Dr.Perfect referred to the AIDS epidemic in the 80’s and know one understood what or how to address AIDS at the time. Consequently, there were several deaths that took place because of this lack of information. What also bothered him was not just that he couldn’t help these vicitms at the time but that these victims were were forced to die alone while disowned from the families because of their sexuality (AIDS epidemic affected gay men predominantly). He advised me to prepare and not worry about how I was to obtain a medical degree but discern whether or not I had a gift for it.

Overall, this is a man who can say he influenced the future in a positive manner. He helped buid a field of molecular mycology, help construct antifungals, made friends globally, developed a productive lab that publishes regularly, as well as  taught and trained 60-70 students. What I admired most was how he used his position to bridge a connection between medincine and scientific research thus shortening the  intellecutal divide between PhD’s and MD’s. He expands his medical degree from simply seeing patients to research and teaching. He was also able put his kids through school and see them develop to influence the world, too. Lastly, he hopes that I develop an excitement in discovery and truly experience what it is like to be in a research project this summer.

Imagine you were an adult male mouse. Do you vocalize when you are in a safe environment with a female? (Duh of course) Do you vocalize when you face a male intruder mouse? (Well, gotta vocalize when appropriate to get the intruder away) What if there is a cat present? (Hmm…better off staying quiet) How would you weigh things out if there is a female and a cat present? (Ughh…Let me check with my brain)

The Mooney Lab is particularly interested in the neurobiology of hearing and communication. This summer, the two projects that I’m involved with focus on the production and perception of vocalization in mice.

Vocalization is crucial to communication and social functioning for many mammalian species. Humans, for instance, rely on complex speech to convey emotions and various information; mice, for another example, produce ultrasonic vocalizations (USVs) for purposes such as courtship and defense (Holy et Guo, 2005; Portfos, 2007). In our society, producing speech appropriately in different social contexts is essential for individuals’ social survival. Similarly, to survive and reproduce, mice vocalize in a context-specific fashion, and thus serve as a model system for studying mammalian vocal control (Chabout et al., 2012; Weiner et al., 2016). In mammals, the neural circuitry for vocalization production is a complex network across the forebrain and brainstem, obligated to integrate cues from social contexts, determine the appropriateness of vocalization and finally signal motor neurons to generate sounds through larynx (Tschida et al., unpublished). However, despite its critical role, understanding of the vocalization network has remained limited at the neuronal level.

Previously, Katie and Valerie (my mentor, wohoo!!) have worked extensively on an area within the midbrain called periaqueductal gray (PAG). PAG is a highly heterogeneous structure, pertaining to nociception, defensive, reproductive and maternal behaviors as well as autonomic regulation (Basbaum et Fields, 1978; Carrive, 1993; Tovote et al., 2016; Tschida et al., unpublished). It has also been shown to be an essential node in the vocalization circuitry, since when PAG is lesioned, individuals exhibit mutism (Esposito et al., 1999; Jurgens, 1994; Jurgens, 2002). Using CANE (capturing activated neuronal ensembles) (Sakurai et al., 2016), a viral genetic tagging method, the lab has been able to characterize distinct subpopulations of PAG neurons that are activated during USVs of male mice to females. Nevertheless, these selective subsets were only characterized in the context of male-female interactions. My first project thus examines two social contexts––courtship with females and defensive behaviors against intruding juvenile males––, and studies whether the same subpopulations of PAG-USV neurons are activated for vocalization in both scenarios.

In the last two weeks, we have performed CANE tagging brain surgery on two mice. I’ve also been practicing doing histology. I’m still very slow carrying out each step during the surgery (special thanks to my mentor for being so patient), but I’ll try my best to getting better at it with every more step I do (and to ALWAYS ALWAYS remember to check how fast the mouse is breathing).

The second project studies mouse’s auditory perception of his own vocalization. Such acoustic feedback has a significant role in vocal production as well, as changes in this feedback could elicit compensatory alterations in what the animal vocalizes (Eliades et Wang, 2002; Houde et Jordan, 2002). A previous study on marmosets showed that cortical neural responses in these primates are suppressed as a result of vocalization, and that this suppression takes place hundreds of milliseconds before the vocalization onset (Eliades et Wang, 2002). To investigate how the auditory neurons in mice respond to the animals’ vocalizations, I was tasked to habituate mice to a head-fixation device in order to obtain clearer electrophysiological recordings.

Working with mice this week, I had quite some philosophical moments, and thought more deeply about biology and my relationship with living things. While more will come in next week’s post on the interview with my PI, I want to thank all in the lab and in BSURF who discussed this big topic of life with me. I’ve learned so much so far, and I can’t wait to find out what comes next!

Mice Love! (weekend sketches)

In the Derbyshire Lab, my project focuses on studying the interaction between a Plasmodium falciparum parasite protein, PfHsp70-1 and a phospholipid, PI3P.

Plasmodium falciparum is a unicellular, protozoan, parasite species that infects humans. It is transmitted though mosquito bites and results in falciparum malaria—the most malignant form of this disease. When a bite occurs, sporozoites (forms of Plasmodium falciparum that result from a division of the cyst containing the parasite zygotes) that have clustered in a mosquito’s salivary glands enter into the bloodstream. From there, they enter and infect liver cells and multiply, rupturing to produce merozoites. Merozoites invade and infect red blood cells initiating the symptoms of malaria.

Heat shock proteins within P. falciparum act as molecular chaperones, helping in the folding/unfolding of other macromolecules. The Hsp70 family is believed to be essential for the life cycle of this parasite, as proteins in the family help mitigate the effects of changing temperature (and other physiological stressors) when the parasite is passing from the cold-blooded mosquito to the warm-blooded human host. It also facilitates invasion of host cells (by potentially trafficking parasite proteins), and increases heat resistance/resilience in the blood stage.

Within the Hsp70 family is PfHsp70-1, a protein mainly found in the cytosol of the parasite cell. PfHsp70-1 is expressed in the blood stages of malaria and is believed to be a potential exporter of parasite proteins into red blood cells during infection. It has been proposed that PfHsp70-1 works with Hsp90 for parasite growth and development, possibly helps regulate the parasite’s protein translation, and trafficking proteins into the apicoplast, a parasite organelle. The linker interface on PfHsp70-1 is believed to be responsible for communication and interaction with Hsp40 for parasite development. Due to these properties, PfHSp70-1 has been studied closely in vaccine research.

PI(3)P is a phospholipid that regulates cellular functions and assists in vesicular trafficking of proteins. Plasmodium PI3-kinase has been shown to be essential for P. falciparum growth. During red blood cell infection, it is believed that P. falciparum forms a protein and lipid trafficking system by potentially synthesizing PI(3)P. According to Mbengue et al, PI3P works to transport parasite proteins from the Plasmodium endoplasmic reticulum, into the red blood cell during blood-stage infection. However, not much is known about the actual mechanism for trafficking, thus making it a target for vaccine research and development. It is important to study PI3P as it is a key component in the blood stage of falciparum malaria.

The Derbyshire lab has previously identified that the C-terminal domain of PfHsp70-1 may mediate its interaction with PI3P, which could play an important role in parasite survival. My project deals with the potential interaction between PfHsp70-1 and PI(3)P and the part of the structure involved with binding between the two. I will be determining the binding quantitatively via isothermal titration calorimetry.

So far, I have worked to induce protein expression (developed via yeast cell culture) and protein purification of Hsp70-1 WT. I am currently being trained on ITC so fingers crossed that I will be able to get some data on the binding affinity!

Lab has been an amazing experience these past two weeks, and I’ve been learning a lot under the guidance of some pretty great teachers. I’m looking forward to learning more and diving deeper into this project!

Due Sunday, 17 June, 5 pm