Project Hungry Hearts & Minds – Creating a Food Exchange Platform at Pace – Final Report

“Project Hungry Hearts & Minds – Creating a Food Exchange Platform at Pace” has developed profoundly in the last three months. This summer has consisted primarily of finding and collecting data from various sources into one inclusive format. I combed through scholarly articles and databases on food insecurity, food waste, and food management on a four-year undergraduate campus and found supporting evidence to propose to Pace University administrators. Now I will finalize a two-page proposal which Erin Furey and I will present to Dean for Students, Marijo Russell O’Grady and Assistant Dean to Students and Director of Student Development and Campus Activities, Todd Smith-Bergollo, this upcoming September. Once approved, we can expedite our idea of creating an online platform or alert system with the purpose to connect food insecure students with leftover food from events; thus, preventing food waste. Our future steps also include building a relationship with Pace’s current food catering company Chartwells. We plan to carefully investigate specific food safety laws and regulations. In October, we will glove up and visit the end of student and staff events to weigh leftover food so we can gather numerical evidence. Eventually, if approved for human testimonials, we wish to conduct interviews with students at Pace University who are acquainted with food insecurity.

We have contacted other school officials that have a functioning food exchange platform portal but have not received any responses. In the upcoming year, we hope to receive word back and visit a university ourselves to personally ask them questions regarding how they created a successful system. In addition, the research this summer sparked a litany of questions leading Erin and me to do some basic Google searches regarding sustainability, composting, food conservation, restaurant donation programs, discounted food platforms and more.

My overall experience from this program has been very life changing. As a polymath and an artist, I have always tried to connect different ideas together. Despite this, science and math have been and still are my worst subjects. Since both are used in research projects, I pursued higher education never thinking twice of acquiring a research grant after my first year. My negative association with research has dramatically changed this summer because I learned that art majors can have a place setting at the research department table too. Due to this funded research program, I have experienced how interdisciplinary skills work. As a student with spatial and emotional intelligence, the results I yield in my research is very unique and creative.

My mentor has helped me grow as a researcher over the course of my project because she understands the emotional strain it has on me. Because I have personally experienced some of the problems I am researching, it makes my passion and results even more substantive. However, at times when it becomes too much, she is there for me to lean on. My mentor has provided encouragement when it becomes difficult but has also celebrated our successes when they occur. I’ve learned now that it is okay if I can’t end all hunger at Pace, but the fact that I am trying is what matters. I’ve learned it’s okay to cry and it’s okay to ask for help.

Furthermore, I discovered housing insecurity was also an issue with college students. When I met a stranger and he later confided in me that he had been homeless for over a year in college, it solidated my research. Now, I was able to visualize a human attesting to this particular social issue. The more I shared my research with others, the more emotional it became. This summer, someone suggested I apply to a homeless shelter as a helpful suggestion when I was having housing issues in August. This really hit a nerve. How could I take someone else’s space who needs it more? Can I be a researcher on the topics that affect me so personally? Am I the right person for this job? Can I handle it? Am I doing enough?

Research and statistics pinpoint food insecurity to be a huge issue on college campuses, especially with low-income students. When young undergraduates have no control of their financial situation, are first-generation students, or have experienced economically difficult times, they often do not prioritize health and food. How could I, as a first-generation, low-income student myself qualify for this research when I am included in this demographic?   

As a result of this final reflection report, I do believe now that I can surmount the challenges. All in all, I hope the three months of summer research inspires others to keep trying. In the end, all we can really do is try the best we can with what we have and hope it works out. I am curious to see what the quantitative data the academic year will bring. In my spare time, I tried to spread the research to others by discussing it and getting their ideas. This sparked many conversations relating to agricultural sustainability and environmentally friendly lifestyles. This summer, I have been consciously thinking of the waste I produce and have been cutting back on environmentally destructive habits. My research has branched out from one idea to a series of new plans and goals. I’d like to officially thank all readers who have read the progress of my team, consisting of myself and Erin Furey, Associate Director of the LGBTQA & Social Justice Center, this summer. Specifically, I appreciate all the respondents who approached me in person or through digital communications; thank you for taking the time to validate our work.

 

Ucuuba, Tamanu, and Aloe Vera Butter As A New Formulation to Enhance Bacterial Resistance And UV Protection- Final Report

In the past 3 months, I have been working diligently under my mentor, Dr. Jaimelee Rizzo, to investigate various ways to synthesize natural antimicrobial surfaces that provide protection from UV rays. Antimicrobial surfaces have the potential to prevent the growth of microorganisms, i.e. bacteria. The recipe for my surfaces is a combination of exotic butters, natural plant-based oils, and powdered supplements. In my experiments, the fixed dependent variable is ucuuba butter, aloe vera butter, and tamanu butter and the independent variables are the different plant-based oils and powdered supplements-which are infused to enhance the properties of the butter. Naturally, the butters have known restorative properties that aboriginal civilizations used for centuries to help with varying dermatological conditions; by infusing different oils and powdered supplements, we can fortify these butters to have increased wellness abilities. The antidotal effect of our starting materials is attributed to fatty acids naturally produced in their respective flowering plants. Various studies suggest essential fatty acids and their metabolites are effective in reducing the epidermal burdens caused by microbes. Common small chain fatty acids found in the starting material are trimyristin, lactins, salicylic acid, glycerols, and glycolipids- these are known to inhibit the growth of bacteria. Inherently these biological constituents are the first line defense in warding off infections in the body.

Using all-natural ingredients is favorable in this project because the skincare industry is straying away from the use of chemical products. In general, natural skin products are earth-friendly, biodegradable, reduce any uncomfortable chemical irritation, nutrient-rich with natural fatty acids, and can prevent adverse chemical imbalances throughout the body. In concluding this project, it is anticipated that we will be able to provide a naturopathic remedy for sterile wound healing while working to prevent sun-induced cancer.The formulation of antimicrobial surfaces can be divided into two protocols: the first is to generate and test the antimicrobial surfaces followed by demonstrating ample resistance towards UV radiation.

To being, individual butters are liquefied through via a hot water bath. About 4 mL of each sample butter is aliquoted into separate beakers followed by the addition of 2 mL of an essential oil is infused, and no more than 1 gram of powdered supplement. The mixture is then plated in a petri dish and left to solidify at room temperature. These samples are then transported to a partner university, LIU Post, to be tested for bacterial resistance.

Using a tryptic soy agar plate, 0.8 cm plugs of agar were removed. 175 µL of each surface was liquefied and placed in different agar holes, which were left to solidify again. 100 µL of 106 S. aureus was spread on the agar using a sterile spreader and each plate was left to incubate overnight at 37 °C. Antimicrobial activity was assessed the next day based on the diameter with which bacteria did not grow around the antimicrobial surface- this area is considered the zone of clearance, where a diameter greater than 1 cm is classified as antimicrobial.

In a transparent ziplock bag filled with UV detecting color beads, each antimicrobial surface is applied evenly over the bag- in a manner similar to the application of sunscreen. UV radiation is then induced over each ziplock bag for about 20 seconds. Samples were then ranked on a 1-10 scale based on the drastic color change in relation to a blank sample of individual butters and a comparative sunscreen.

So far, 14 different essential oils were incorporated in each of the three butters. Overall, 94 different samples were concocted, and about 45 different samples provided ample microbial resistance when tested against S. aureus. When tested for UV protection, about 40 samples demonstrated some degree of UV protection. About 35 samples were able to demonstrate both UV protection and antimicrobial resistance. The materials most commonly combined to get desirable results incorporate black cumin oil, marine powder, and ginseng powder. The most favorable results recorded to date are samples RP 65-69, 73-77, 83-85. Specifically, RP 65 had the widest margin to inhibit bacterial growth at greater than 4 cm and no UV light penetration- this sample was made with a mixture of 3.85 mL of ucuuba butter, 3 mL of black cumin oil, and 0.2 grams of marine powder. Looking forward to the continuation of this project I hope to discover more combinations that offer a zone of clearance greater than 4 cm while advancing this study to test our samples on live living cells.

As I am to graduate in spring 2019, weighing my options for a career is a pressing matter. Having spent time in Dr. Rizzo’s lab studying a cosmetic chemistry based project, this seems like a potential field of work. Understanding that the hardest part of research is failure and that patience in making samples work is the only drawback; striving to help out others is the reward and motivating force. Having Dr. Rizzo as a mentor is really beneficial because she gives me enough space to explore my research personally in order to figure out what works and what doesn’t, but she is opened enough where she welcomes questions to further my understanding of what is happening in the lab.

Components and Results -1vu56hy

 

Final Report

Over this summer, professor Eric Chang, Hayley Besser, and I have worked on our goal of developing a protein expression system in order to produce cathepsin-L that mimics the catalytic properties of silicatein-alpha. In order to accomplish this, we first tested different DNA constructs and cell types, such as E. coli, in order to determine which expressed our protein the best. To more properly understand the fundamentals of this process, since neither myself or Hayley had made cathepsin-L before, we first expressed a protein that was previously made by professor Chang, lactate dehydrogenase from the ice fish mackerel, or cgLDH. We first purchased a pUC57 vector from a company known as GeneWiz, which contained the cgLDH gene, and tried to express the protein in DH5-ɑ E. coli cells, which were purchased from New England Biolabs. While the vector was effectively compatible with the DH5ɑ E. coli cells, this was ultimately not a beneficial system for producing and isolating the protein of interest. Because of this issue, we were initially not able to isolate cgLDH protein during affinity chromatography, which raised many questions. During our second attempt, we used a pET11a vector with the cgLDH gene, which was purchased from GeneScript, and T7 express E. coli cells, which were purchased from New England Biolabs. This new setup produced the cgLDH protein on the first attempt, however we noticed that we only produced a very small amount when analyzing the samples using a spectrophotometer. In the future, we can optimize the expression conditions in order to increase the amount of cgLDH produced. Now that we have an established system that works, we can apply this to research with cathepsin-L and see if this protein can be expressed using the T7 E. coli cells and the pET11a vector. If so, we can additionally optimize the growth conditions to produce this protein.

Going forward, we will be able to perform site directed mutagenesis on DNA constructs for cgLDH and cathepsin-L. After this, we can then express the site mutated DNA in order to make new forms of the protein of interest that contain enhanced catalytic and structural properties. The main goal is the systematically change cathepsin-L, through the site directed mutagenesis, in order to mutate it to the point where it matches the chemical and physical properties of silicatein-ɑ.

This program provided me a wonderful opportunity to not only sharpen my pre-existing laboratory and research skills and knowledge, but also to gain new ones. Thanks to professor Chang, I have made antibiotic containing plates, performed chemical transformations with E. coli, performed plasmid mini-preps towards DNA analysis, as well as protein purification and isolation through affinity chromatography. I also had the pleasure of working on a scientific research paper that was presented at the 2018 Gordon Research Conference (GRC) on Biomineralization, which was held at Colby-Sawyer College in New London, New Hampshire from July 28th through August 3rd. One of the biggest advantages of this program has been how I was able to perform different molecular biochemistry protocols and reactions hands on, when most students only learn about them in class. Professor Chang guided both Hayley and myself throughout this entire summer, and provided us with invaluable knowledge and techniques that I will carry with me through the rest of my career as a student.

Undergraduate Research Grant Summer 2018, Roca

This research project was largely an anthropological ode to the contemporary histories of urban farming and seed saving in the Bronx. I fused my last undergraduate research project, carried out during the 2017-18 academic year, into this project that has taken a more creative, visual approach.  The inspiration for this was born from the positive feedback I received on our poster for the showcase in the spring.  On the poster, I tracked migration routes of communities affected by urbanization of the early 20th century with seeds and beans relative to their cultural foodways.  I told the story of the contemporary uprising of urban gardening in communities of color through visuals and captions containing my research points.  The food justice movement, having sprung up from burnt out lots in the 70s, mass displacement and terrible food policy, is resilient and powerful. This makes for a visually-rich as well as politically profound moment of history that I feel compelled to unearth and respectfully honor.  This project helped my academic experience go full-circle—taking the typical archetypes of methodology to the street—drawing inspiration from both academics and the everyday person putting the actual labor into what is shaping the movement on the ground.

Our research so far has taken an unexpected turn, incorporating more of a conversation of seeds and climate change into the mix.  A recent New York Times feature, titled Losing Earth: The Decade We Almost Stopped Climate Change, by Nathaniel Rich, inspired us to touch on the irreversibility of climate change and the role of seed saving in this moment of time.  In a seemingly hopeless political and environmental climate, how does seed saving/banking/engineering outside of the lab have a stake in it all, and what does that look like?  We are posing this additional question to those within the movement we are conducting interviews with.  This is adding some rich audio content behind the moving visuals we are capturing.  Our accomplishments thus far are gathering visuals and audio to piece together creatively.  The time and focus this project requires is a challenge, however this experience has shaped my time management in a transformative and self-disciplined way.  We plan to pursue this film project beyond this research grant, and hopefully enter it into small short film festivals.

My mentor has helped me grow exceptionally as a dedicated researcher.  Her support and encouragement has motivated me to develop my research beyond the start and end date of a grant.  Her questions challenge me and push the research to incorporate narratives that are so often neglected in the larger picture.  I am extremely grateful to study and research under someone as accomplished and brilliant as Dr. Denise Santiago.

End of Summer Final Report

I, fortunately, had the opportunity to conduct research with Dr. Aditi Paul this summer regarding employee empowerment in a corporate environment and how to implement effective management techniques. I began to research the different leadership styles and how they are typically incorporated to increase productivity levels and staff satisfaction on a wider scale. Dr. Paul and I have an immense interest in organizational communication and how it can be utilized as a strength to ensure organizations are being effective in their standard operating procedures. Dr. Paul and I met early in the summer to discuss exactly what the research process would look like and how it could analyze a significant concept. We first created a timeline for the research. Thankfully, I will have the opportunity to continue working with Dr. Aditi Paul throughout the year as I will be continuing forward with this research topic to serve as my Senior Honors Thesis. The timeline that we constructed listed all of the stages this research project would maintain. Dr. Paul and I identified a basis for what we wanted to achieve and what we were hoping to fully analyze. In efforts to specify, we decided to focus on leadership styles, primarily. I began to conduct a literature review that is meant to be used as a solid foundation for my research. This will allow us to maintain a form a measurement that will be used to properly and thoroughly analyzed the empowerment levels of employees through effective leadership.

I, primarily, searched through numerous scholarly articles regarding leadership styles, discovering that the main techniques fall under the scope of either Transformational Leadership, Transactional Leadership or Laissez-Faire Leadership; as well as a multitude of subcategories and sections that will also be analyzed and described in my research. I also took it upon myself to conduct my own organic analysis by reading literary works that would provide further insight on effective leadership. I have read through the book, Creating Magic, by Lee Cockerell and have discovered the importance of serving on a surface level within organizations. This was identified to fall under the Transformational form of leadership. I have begun to read Creative Confidenceto gain further insight on the subject matter.

Thereafter, Dr. Paul and I ensured that we stabilized our topic by meeting to discuss the quality of our topic and how we would measure it. We also discussed the importance of identifying our “X” and “Y”. By looking at the concepts through a quantitative lens, we are able to understand and orient ourselves regarding the details and potential results of the research. Overall, Dr. Paul has helped me immensely in identifying an effective research process that works with me and my specific schedule.  She has assisted me in identifying my goals for my research, but also for what I would like to achieve with my research in terms of identifying possible conferences and platforms to discuss my findings. This program allowed me to conduct research on a larger scale, which is extremely beneficial for me. I am incredibly grateful for the opportunity to garner the critical thinking and research skills here at Pace. As an aspiring law student, it is quintessential that I put into practice this type of hands-on, in-depth project. The structure and techniques that I have learned through this program have provided me with the scholarly insight I need to continue being successful both in conducting this research, but also in furthering my academic career.

Persuade to Date: A meta-analyses of advertising appeals of online dating applications from American, European, and Asian countries – Final Report

The tile of the research I have been working on for the past 2018 summer time  vacation is “Persuade to Date: A meta-analyses of advertising appeals of online dating applications from American, European, and Asian countries.” The investigation is still in progress, therefore, I will describe the goals, methodology, and what I have learnt from this experience.

We—My  Professor, Aditi Paul and me—were working together on discovering persuasiveness of the core messages used by the most popular dating websites within the European, American (Northern and Southern) countries. It will be done by doing a cross-national and cross-continental analysis in order to explore the differences of the advertising appeals. If the differences will be discovered, we will consider among others, cultural, economic, and social factors.

The first thing I had to take care of, was collecting a data set consisting of all the most popular services in a certain country from previously given continents. To find it, I used the Google search where I was typing the following: “online dating application” or “online dating website” and the name of the country I was researching. I assumed that the search results that showed up, were one of the most popular for a certain nation. To extend my data set, I was also using the ratings of the most successful services. It was even more helpful in determining which online dating services are popular in a certain society. Subsequently, I collected their advertising appeals and next, I put all of them into the table. The table was created for each country separately. It consisted of name of the dating service, core message, and the references. On the next step, I had to create an Excel file which will be used as a material for coding. The following information was provided in a given order to each of the excel column: continent, country, advertising message, added features, the original language of the provided advertising message, determination whether the dating service automatically translated the website into English, determination whether the service is global or local, determination whether the service is an application or the website.

After collecting the data (the marketing messages/advertising appeals) from all of the dating websites I found, my Professor prepared a document with seven different motivations which make the potential customers join a certain dating service in order to do coding manual. The motivations are the following:

M1 (Motivation1)- Relationship; fall in love, build a serious relationship etc.

M2-Sex; find a sexual partner, seek someone with the same sexual interests etc.

M3-Peer pressure; because everyone is doing it, to be trendy etc.

M4-Socialize; make new friends, flirt, broaden social network etc.

M5-Entertainment; for fun, combat boredom etc.

M6-Design; ease of use, visually appealing, free of cost, geolocality etc.

M7-Identity; Try new identities, escape from who you are, live out a non-sexual fantasy etc.

The process of coding is time consuming and it requires us to be very careful, objective, and patient. All of the cases from the sample might be different, therefore it is very important to consider them separately. On our last summer meeting we have completed coding motivations for the European nations inter-coder reliability is still pending), we have also been trained on coding the primary visual element of the websites –video/photo, gender, race, and age of the subjects depicted in the primary visual element.

Throughout the research work, my Professor was always supportive, full of patience and open for any questions I had. Before taking any next steps of our study, she was always explaining to me not only the methodology, but also the goals and purposes. As a result, I could understand everything what I was doing, and moreover, this awareness let me ask the questions which were even more helpful in preventing us from being biased.

This experience showed me the importance of having an unbiased and objective approach in order to discover and understand the unknown. This little change in the way we perceive the world can extend the human abilities to get to know more.

The Perceived Impact of Therapy Dogs on College Students – Final Report

Over the summer, Dr. Maxam and I had an amazing time conducting qualitative research on the perceived impact that therapy dogs have on college students. Through interviewing multiple Pace faculty members who are connected with therapy dogs, it is made clear that interacting with therapy dogs on campus has a positive impact on students.

Beginning this research, I needed to do some background research on this topic. As a dog lover, I know a lot about dogs, but there were things I needed to learn before conducting this research. For instance, I learned the distinction between therapy dogs and service dogs, and stressed that throughout my research. Therapy dogs are not service dogs. Service dogs are specifically trained to perform tasks to help a person who is differently abled. Therapy dogs are trained to provide comfort, affection, and stress relief on college campuses, in hospitals, and other very stressful environments.

Though the research conducted showed that students are positively impacted, there are some negative aspects to having therapy dogs on campus. Some students simply may not like dogs, and other students may be allergic. However, these events with therapy dogs give students the option to participate. Another concerned raised by this research is students who have phobias of dogs. Some of these students have spent time with these therapy dogs on campus, and the dogs have helped them conquer their fear.

Ultimately, Dr. Maxam and I are conducting this research to implement a formal therapy dog program at Pace. Throughout our research, we learned that therapy dog programs have many components, and we had to see how this components could be implemented on the Pleasantville and New York City campuses. Things we need to consider include the cost, training, who would be the dog’s owner, if we would need multiple dogs, and where they would stay on campus. Once we have decided how the program should be modeled, we will present it, along with our research that shows the positive impact of therapy dogs on students, to President Krislov.

Pace University currently works with the Good Dog Foundation and has therapy dogs on campus on alternating Wednesdays, and during final exam week. These therapy dog events are very popular on campus. These events particularly seem to help college students with stress reduction. After spending time with a therapy dog, students are visibly in a better mood. For many students, seeing a therapy dog, or any dog on campus, immediately brightens their mood, as it reminds them of their own dog at home. For these students, the therapy dogs act as a springboard for conversation, and they talk about how much they miss their dog and how interacting with a therapy dog helps them feel less homesick and adjust to college life easier.

Working with Dr. Maxam has been such an amazing experience. She and I both love dogs more than anything, so it was a great experience being able to research something that she and I are both super passionate. Whenever I needed help during my research, Dr. Maxam was one call away. I am so appreciative of Dr. Maxam for trusting me to conduct this research, and for the unconditional support she provided me. Together we pieced together ideas and components of what a therapy dog program would look like at Pace, and we were lucky enough to have our research be chosen to be presented at the International Journal of Arts & Sciences Multidisciplinary Conference at the University of London. I cannot thank Dr. Maxam enough for this opportunity. I am so grateful for where this research project has taken me, and I cannot wait to see what happens with our research in the future.

Final Report on “A Comprehensive Guide to Volunteerism in Westchester”

Working on this report has been one of the most challenging things I’ve done thus far in my educational career. The work I’ve had to do has pushed me outside of my comfort zone and for that, I am honored to have had the opportunity to work on this project. A study showed that New York State ranked 49th out of 51 in volunteerism. So the goal of this project was to determine approximately how many volunteers there are in Westchester County, how many hours they are volunteering, provide recommendations of what volunteer managers in Westchester County need to improve their services, and use the data collected to provide recommendations on ways Westchester County can improve and promote volunteerism. While this report is still a work in progress, the chosen research methodology was mixed-method, with a quantitative survey distributed to various volunteer managers across Westchester County and qualitative follow-up interviews to be conducted. The data received from this survey would be compared to the aforementioned Bureau of Labor Statistics NYS volunteering data. Our study was modeled after one done on Ulster County by The Benjamin Center for Public Policy Initiatives at SUNY New Paltz.

With over 1000 nonprofit organizations in New York State, our goal was to have between 100 and 200 responses for at least a 10% response rate. In order to achieve this, I worked in conjunction with Nonprofit Westchester and Volunteer NY! to reach out to volunteer managers from different organizations and encourage them to take the survey. To date, we have received 149 submitted surveys. Contacting these volunteer managers has definitely been the most difficult part of this entire project because it forced me to leave my comfort zone. I usually revert to just emailing people so that I don’t have to deal with awkward phone interactions but, in this case, the most effective method of communication was a phone call. Having to make all of these phone calls taught me how to be more confident in cold-calling people for a cause and also taught me how to be clear and succinct. These skills help me two-fold. First and foremost, they help me in my career. I’m a journalism major so cold-calling sources is an everyday part of my job. Having to do this for the research project has made me more confident in doing so for work and has actually helped me land some very important sources for stories that I’m working on at my current internship in a nonprofit newsroom. The second area in which it’s helped me is in my position as an advocate for change. Being an advocate means stepping up and calling on others to make change, like our elected officials. I am now more confident and feel more capable of my abilities to successfully do so.

With this project, I was lucky enough to have not just one, but two mentors guiding me. Whether it was through our weekly email updates or weekly phone calls, they were always there to answer any questions I had about absolutely anything. They’ve helped me become a better researcher because they’ve pushed me to think outside the box. When the time came for me to begin looking at the data, they provided me with guiding questions so as not to become overwhelmed by the mass amounts of information the survey left us with. Both of them have been extremely supportive of my endeavors related to the project and otherwise. They both have provided me with affirmations that what I’m doing is valuable to this project, which has helped me push forward.

I plan on working with both Heather and Tyler to see this report to completion. I surround myself with a community, especially at Pace, where volunteerism is made a priority. To see that report about New York State was shocking and is part of the reason why I feel so honored in participating in this research project. Working on this project has altered how I see and understand the community around me. In one of my prior blog posts, I noticed the shocking differences in funding for these organizations. It seemed almost unreal to me that nonprofit organizations were operating with millions of dollars as their budget. Nonprofits are often synonymous with not having much to work with, but these survey results thus far have shown me otherwise. Another thing that was, for some reason, shocking to me, was that most of the nonprofits that participated in our survey are completely volunteer run. This aligns more with the “nonprofits have no money” train of thought but seem strange when the data is saying that nonprofits are operating with millions of dollars in budget. This brings about an entirely different set of questions. Nonetheless, I am excited to see what final results the survey will produce and how it all comes together in our report.

Iterative Site-Directed Mutagenesis Towards the Directed Evolution of Enzymes, Final Report

This summer, Arianna, Dr. Chang and I researched the topic of Iterative Site-Directed Mutagenesis Towards the Directed Evolution of Enzymes. Our hopes were to learn about the self-assembling properties of silicateins and use this information to develop new forms of cathepsin-L to adopt these properties. This would involve determining which amino acid residues are responsible for silicateins self-assembling properties and  then making changes to amino acids within cathepsin-L.

The beginning of our summer was spent getting Arianna and I comfortable with using various lab equipment. We prepared materials such as antibiotic-containing plates, chemical transformations with e. coli, and mini-preps towards DNA analysis. These steps aided in learning how to follow procedures accurately. After these preliminary procedures we took some time to do computational work and create our poster which was presented at the 2018 Gordon Research Conference on Biomineralization, held at Colby-Sawyer College in New London, New Hampshire from July 28 – August 3. My section of the poster involved searching for possible future uses of biosilica prepared with silicatein. I used Web of Knowledge to search through literature and found that biosilica has potential to be used in regenerative medicine as well as 3D printing. After some research of my own I came to the conclusion that there is very limited information focusing on future uses of silicatein, and I am curious as to why this is.

Initially, some techniques we were supposed to learn included: protein mutagenesis and characterizing hydrophobic and electrostatic protein-protein interactions. Tentative methods were: purifying, manipulating and characterizing proteins; forming and refining silica mineralization assays in the absence and presence of proteins; completing scanning and transmission electron microscopy, atomic force microscopy, focus-ion beammilling, and finally, analyzing complex datasets and interpreting the results. Unfortunately, we did not get to complete as much as we wanted due to unsuccessful protein-expression systems.

We started with trying to express the protein lactate dehydrogenase from the Mackerel ice fish (c. gunnari). The first system used a pUC57 vector purchased from GeneWiz containing the cgLDH gene. The goal was to express the cgLDH gene in the DH5-ɑ e. coli strain purchased from New England Biolabs. This attempt was unsuccessful despite the vector’s compatibility with the DH5-ɑ e. coli cells. The system was not able to produce and isolate our protein. For our second attempt, we used a pET11a vector with the cgLDH gene inserted purchased from GeneScript and T7 Express e. coli from New England Biolabs. Using a NanoDrop 1000 spectrophotometer we once again measured no protein present. After performing a more sensitive assay, Dr. Chang concluded we produced the desired protein, but in very small quantities. I would like to find out why we are not seeing the amount of protein one might expect, and which step of the system is hindering production.

I believe our next step is to modify our procedure to optimize growth conditions and produce the protein in large quantities. The expression of cgLDH is important for a research project that is in collaboration with research groups at Albert Einstein College of Medicine. This means that even though cgLDH is not directly related to our current summer research project, we hope cathepsin-L can be expressed using T7 Express e. coli and the pET11a vector as well.

This is my first time working in a research lab, so each step along the way has been a new opportunity for me to learn. From the first day learning how to use the autoclave, to our last meeting when we went through an entire protein-expression system, I have learned a wealth of new lab skills. Exposing myself to new academic experiences and growing as a student as well as a team member are the key things I would like to achieve from this project. Through our research, I hope to expand my knowledge of proteins, enzymes, and the amino acids which are the building blocks behind them.

I have certainly learned some important lessons from this project. I have learned that in research there is a lot of trial and error that goes on, and sometimes you can work for hours or even days on a single project and have it be unsuccessful. I’ve learned from Dr. Chang’s example that you can’t let these setbacks throw you off or deter you from trying a new procedure. I’ve also learned that it is important to use your resources and have those who are experts with certain materials help you if you are unsure where the process is failing. Our experience this summer leaves me determined to perfect our protein-producing system in the future so we can continue with our project.

 

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Evaluating The Function of Genes Implicating Glioblastoma Multiforme (GBM) Formation Using C. Elegans – Final Report

Evaluating The Function of Genes Implicating Glioblastoma Multiforme (GBM) Formation Using C. Elegans

The purpose of this project was to determine if genes implicating Glioblastoma Multiforme (GBM) formation play a role in cell division in Caenorhabditis elegans (C.elegans). To be able to detect genes predisposing to GBM, it is important to understand the role of cell division. Mutations in cell division cause the proliferation of cancerous cells. In order for cell division to produce malignant cell growth, genes responsible for proper cell division have to be altered. Gene regulation is usually affected making some cells do things that they should not be doing, like overproducing proteins or growth factors that may be useful for the destructive cancerous cells. To detect GBM genes, it is important to know what genes have increased variance in GBM affected patients. Genetic variation is important in cancer, because it causes evolutionary genetic susceptibility to cellular malfunctions, such as damage in DNA repair and replication. Using the Backes et al. (2012) study, we used the data published on GBM patients, which yielded multiple genes that had increased or abnormal genetic variance. These genes were not declared as markers of GBM, although with their suspicious presence in the studied GBM patients implicates that those genes may have a role in brain cancer development. There were some genes in the publication’s dataset that had high variance, but those genes were ignored because they have been already declared as cancerous genes and predisposition markers from other research studies.

To understand whether these genes have a role in GBM, we used C.elegans as a model organism to understand the morphological and cellular significance of the suspected genes. C.elegans is a nematode that possesses a nervous system. Its nervous system comprises of 302 neurons and 56 glial cells. (Oikonomou and Shaham, 2011) These cells have an influence on sensory and motor functions. We implicated in this study that if we knockout a suspected GBM gene from C.elegans, and observed changes in glial cell development using behavioral assay and advanced microscopy, then will be able to confirm the suspected genes association with GBM.

Using several online databases, including Wormbase, Ensembl and Ortholist, we were able to find 10 genes that had orthologous genes with C.elegans. We had to research the background of the 10 genes to pinpoint which ortholog had any association with glial cells or the nervous or neuromuscular system in C.elegans. One gene, Lev-9, a levamisole resistant gene, was one of the few genes from the bunch that had a connection to the nervous system in the nematode. Utilizing the services of a company that specializes in knocking out selected genes in organisms, we were able to order two strains of Lev-9: RB1717 and ZZ16. The two strains with similar genotypes allowed us to investigate the effects of the Lev-9 gene knockout.

Figure 1: Above graphs the median progeny count for two strains of Lev-9 C.elegans: RB1717 and ZZ16; and control, N2 (wild-type) strain. Median progeny count is taken from several tests performed using the Lev-9 strains and control. Blue indicated the number of unhatched eggs found on the plate. Orange indicates the amount of early larval stage (L1-L3) organisms inhabit a plate. Purple indicated the total amount of progeny present on the plate when counted after 48 hours of incubation in 20 C.

 

According to the data, it is clear that there is some statistical significance in the results we have collected. To determine significance, we conducted four tests on the N2 control and three tests each for both RB1717 and ZZ16 Lev-9 strain C.elegans. In Figure 1, the total progeny count is substantially lower in ZZ16 than in RB1717 in comparison to the control. We expected ZZ16 and RB1717 to have less progeny, since this would indicate that there are problems with cell division. In each test, ZZ16 still remained in its L4 stage, and unlike the other strains, it appeared that it needed a longer period of incubation time in order to develop into an adult. This suggests that ZZ16 may have developmental issues and a harder time laying eggs at the same pace as a wild-type. On the contrary, RB1717 appeared to develop at an increased rate and yielded an even higher progeny count than N2. This result was surprising, because we expected RB1717 to behave more closely as ZZ16 since they are both mutants from the same gene.

From the data we have collected, we can start discussing how the progeny count is affecting the nematode and whether this data is relevant to glial cells. We predicted that the organisms would have a deteriorated nervous system and that the neural tube would also be affected by the KO of the Lev-9 gene. The organisms did exhibit abnormal behavior and uncoordinated movement when observed under a 10X microscope. Body curling and slowed movements were two of the most obvious observations, when compared to a normal organism (control). This raised question whether glial cells have been affected. To determine whether glia are directly affected, we will need to follow up on our behavioral results with a much more advanced experiment.

Keeping in mind that there is no research adequate enough to understand to what extent GBM is hereditary, and the details of it being linked to cell division, this study has the potential to help provide significant insight for the gap of knowledge in genetic inheritance and variability in GBM. GBM has significant molecular characterization for its category as a tumor, and not all therapies like chemotherapy or radiotherapy have not been found to be completely effective. This study may contribute to biomedical and bioengineering professionals that are working on solutions for cancer development prevention and those who are finding simpler ways for disease prognosis. Additionally, this study may add information to new studies concerning C.elegans and cell reproductive health. It is also greatly anticipated that the contributions from this research can provide more direction for the development of both better drugs and therapies for life-threatening mutations in cell division. it is anticipated that the present and future studies may help reshape the current predisposition testing for cancer. For a bigger picture, it is hoped that the small, but significant information from this research and other studies can redesign treatment options, and revolutionize education on how cancer and may other diseases work. With the help of my mentor, this project has definitely enriched and enhanced my education in the fundamental knowledge of the reproductive biology and cellular biology. I hope that my research experience can be a motivation to others interested in the scientific field and that determination and commitment has the potential to create great things.

 

 

References:

Backes C, Harz C, Fischer U, et al. New insights into the genetics of glioblastoma multiforme by familial exome sequencing. Oncotarget. 2015;6(8):5918-5931.

Oikonomou G, Shaham S. The glia of Caenorhabditis elegans. Glia. 2011 Sep; 59(9):1253-63. Doi:10.1002/glia.21084.