Blog 4: End of Year UGR Report

Research involves critical thinking, problem solving and patience. Researching about infertility has helped me realize how this issue has impacted so many people. The process of researching has enhanced my critical thinking skills. Any questions that came along the path of research required me to consult with the latest literature published in various scientific journals. I was able to practice problem solving and most of all I was able to learn from my mistakes. From the start of researching to now I have come a long way and I have greatly enhanced my research skills. I was also able to present my findings at the Society of Fellows event at Pace University and won an award for my research. I am truly grateful for the Undergraduate Research Grant which helped fund my project.

Mammalian fertilization begins with the fusion of two gametes, sperm, and egg. The spermatozoa enter the female reproductive tract and are required to migrate to the oviduct. The oviduct is where the spermatozoa meet the ovulated eggs. But the question is, how do these specialized cells know when and how to interact. At the cellular level, fertilization is a complex molecular interaction that may require multiprotein complexes on both gametes for sperm-egg fusion to occur. However, this process often is unsuccessful because of genetic mutations in the sperm. We have previously identified a sperm membrane protein interactome in Caenorhabditis elegans (C. elegans) that provided evidence that sperm membrane proteins interact extensively. We hypothesized that these interactions are dynamic during spermiogenesis and sperm maturation and are necessary for fertilization. In the interactome, FER-1 interacts directly with four other membrane proteins, including SPE-10. This project leverages the plethora of genetic resources available for C. elegans research to analyze how double mutant animals with mutations in  fer-1 & spe-10 (VC40852), fer-1 &spe-9 (VC20575) , and fer-1 & spe-12 (VC20650) impact fertility.

One hypothesis to explain the decrease of hermaphrodite self progeny of the double mutants (VC20650 and VC40852) is that the fer-1, spe-12 and spe-10 are required for successful fertilization similar to spe-36. It is possible that VC20650 and VC40852 double mutants are also embryonic lethal. RNA interference experimentation is necessary to determine the location of lethality during fertilization. Due to unforeseen circumstances the semi-quantitative assays were not completed at 16°C and 20°C. By determining the rate of fertility of the double mutants are these two remaining temperatures we will have a better understanding of why the mutants are infertile. Furthermore, we did not expect to see an increase in fertility for the VC20575 strain. This is interesting because the double mutant had a significant increase in fertility not only compared to the other mutants but the control (N2) C. elegans.

Further experimentation at 16°C and 20°C is needed to determine if there is any decrease in fertilization. As hypothesized before, since single mutations significantly decrease the rate of fertility, it is reasonable to assume that double mutations will result in a more significant decrease in the rate of fertility. However, if a significant increase in the rate of fertility is shown, as in the VC20575 strain, it can be concluded that either the mutations do not impact the encoded protein or one of these mutations changes the molecular complex formation and could compensate for the loss of one of these functions. Various genetic approaches have provided insight into genes necessary for sperm viability — understanding how one gene influences another will offer a comprehensive understanding of the network design underpinning infertility.

 

 

 

 

 

 

Blog Post 3

Over the past few months, I have made progress in the lab and gained positive results. To evaluate the functions of specific genes required for male fertilization, I looked for articles in the literature. I came across an assay that is used by many scientists to determine the rate of fertility in the model organism C. elegans. I used the Brood Sizing Assay to determine the rate of three different strains at 25 degrees Celsius. This experiment requires me to monitor the rate of fertility of the C. elegans daily and to accurately count the number of progenies produced.

During this process, I came across multiple problems. The first problem that occurred was that one of the strains became contaminated. To resolve this issue, I kept transferring the worms to new plates until the contamination reduced. I then began the assay, and this required me to use 90 plates per week. To do this, I had to manage my time well and make plates quickly and efficiently. The first batch of plates I made was not the best since the bacteria overgrew. I made the plates again, and they came out perfect. I will be using the new plates this week to re-do the assay at 20 Degrees Celsius. So far, I have seen that at 25 degrees Celsius, the mutants had reduced fertility compared to the control group meaning these mutations result in fertility defects.

UGR Blog Post 2

Fertilization is a complex molecular interaction that may require multiprotein complexes on both gametes for sperm-egg fusion to occur. My project aims to identify how potential multiprotein protein complexes are functioning and what their impact is on fertility and sperm-egg fusion. We have previously identified a sperm membrane protein interactome in Caenorhabditis elegans (C. elegans) that provided evidence that sperm membrane proteins interact extensively. My lab team has weekly meetings where we discuss our projects for the following week. I have been maintaining my worms and making sure they are healthy for future experiments. One of the difficulties I have faced is that one of my plates got infected, and this required me to follow better sterilization procedures.

We are analyzing the spe-36 gene, which was originally identified as a sterile strain. Further analysis revealed that spe-36 mutants are unable to fertilize eggs normally, but the cause is unknown. To better understand why spe-36 mutants are sterile, we are analyzing the mutants at different developmental stages. We are investigating three different strains N2 (wild-type), spe-36(as1), and spe-36(as1)asEx96. The spe-36(as1) mutant is the C. elegans without the gene of interest. And the spe-36(as1)asEx96 is the C. elegans with the gene of interest replaced and tagged with green fluorescent protein (GFP). We have conducted a brood size analysis at 16°C to assess fertility rate. We discovered that C.elegans with spe-36 mutants are unable to fertilize. When the gene is knocked in, the fertilization reaches close to the wild-type but not entirely. We are interested in seeing what will happen at 20°C and 25°C. We expect to see a slower fertilization rate at these temperatures since they are not the preferred temperature.

We have also identified two C. elegans strains with mutations in genes known to be necessary for fertilization to analyze the effect that missense mutations in multiple sperm membrane proteins have on sperm-egg fusion. Since I will be examining more than one mutation, we ordered different strains of C. elegans from the Caenorhabditis Genetics Center.The VC40852 strain has missense mutations in fer-1 and spe-10 and the VC20575 has missense mutations in fer-1 and spe-9. We are measuring the effect of these mutations on brood size and sperm-egg fusion. The analysis of both strains will provide insight into the genetic interactions between fer-1, spe-9, and spe-10 and allow us to generate a model of how these genes could be functioning together to mediate fertilization.

Blog Post 1: Using the Model Organism, Caenorhabditis elegans (C. elegans), to Evaluate the Function of Homologous Genes Hypothesized to Impact Male Infertility

Male infertility can result from low sperm production, abnormal sperm function, or defects in sperm delivery. The mammalian fertilization process begins with the fusion of two germ cells. More specifically, the spermatozoa enter the female reproductive tract and are required to migrate to the oviduct. The oviduct is where the spermatozoa meet the ovulated eggs. However, this process often is not successful because of genetic mutations in the sperm. I am interested in determining how different genes mutated in sperm impact the rate of infertility.

More specifically, I am interested in analyzing how double mutations influence infertility. As a model to study sperm development, I am using the nematode Caenorhabditis elegans (C. elegans). C. elegans is a well-established genetic system that can be used to determine how my genes of interests control spermiogenesis and how their misregulation could lead to infertility. Using Celegans as a model system, we are using a genome sequenced multi-mutation library, from the Million Mutation Project, to identify genes associated with defects in sperm. We will than analyze the rate of fertility of single mutants versus double mutants. We are going to use those orthologs and perform structure-function analysis using single mutants to understand better which domains of the gene the mutation impacts. In the end, this analysis will help us better understand infertility and will help us identify how specific genes interact with one another.

End of Year Report

Final End of Year Report

Throughout the 2018 – 2019 school year, I worked collaboratively with my faculty mentor, Dr. Elmer Mojica, on studying the chromatographic characterization of bee pollen from different countries around the world. The original overall objective was to determine the composition of different solvent extracts of bee products, specifically from the Philippines. This was successfully completed using the five solvents of methanol, ethanol, dichloromethane, hexane, and chloroform. This led to the natural curiosity to determine the antioxidant activity and phenolic content of said solvent extracts. Both objectives were met using the HP 6890 Gas Chromatography Mass Spectroscopy and a microplate reader where the DPPH and Folin-Ciolcalteu assays were performed. Being given the opportunity by the Undergraduate Research Initiative to brainstorm, conduct, and discuss my research via presentations and blog-posts has been a critical and valuable asset to my analytical research.

Before beginning the physical research in the lab, I established three major learning outcomes that I wanted to work diligently towards throughout the academic school year. The first goal I wanted to reach was the articulation of a proper research question with an effective objective. I wanted my research to have meaning and a purpose. For this reason, I chose to work with bee pollen in relation to the health and nutrition benefits that it could possibly have on humans. For example, working with the assay gave me an insight to the amount of antioxidant levels. Antioxidants prevent the formation of free radicals, which are often cancer-causing, in the human body. Establishing an understanding of what bee pollen consists of was imperative before moving onto further research. Next, I wanted to utilize an appropriate and efficient methodology to find an answer to my research question. I did so using instrumental methods and mass spectroscopy analysis. Lastly, I aimed to present the research in a conference setting. I applied with my research abstract and was gratefully accepted to present in an Undergraduate Poster Session at the American Chemistry Society Annual Conference in Orlando, Florida.  I can successfully say that I accomplished all three of these outcomes.

An imperative part of my research was the collaboration between my faculty mentor and myself. Dr. Mojica first helped introduce me to the GC-MS instrument and the sample preparation methods involved such as extraction and filtration methods using solvents and bee pollen. Once I was able to acquire the compounds that make up the pollen, he challenged the research to go even further by teaching me how to determine the phenol content and antioxidant levels. Two assays were used with the Biotek Cytation instrument, DPPH and Folin-Ciocalteu. The DPPH assay involved dissolving the DPPH solution and pollen extracts in methanol on a microtiter plate. The Folin-Ciocalteu assay involved diluted methanol mixed with Na2CO3. The absorbance was then measured at 745 nm post-incubation. We gathered that the phenol content was highest when methanol was used for extraction and lowest when hexane was used. The antioxidant amount was highest when extracted with chloroform and lowest when extracted with methanol. Without Dr. Mojica’s guidance and assistance in the assay processes, the procedure would not have reached the scientific caliber that it did. By meeting with him on a regular basis to discuss all aspects of the research, I heightened my ability to work with others in terms of problem-solving and effective communication.

The main initiative for my research project was being able to find an answer to the question: what components make up bee pollen? I wanted the answer to this to be used in future studies for human health benefits. While my study did not focus on how bee pollen benefits humans, it focused rather on what bee pollen consists of. For this reason, further analysis can be used to interpret the benefits of these components, specifically in terms of antioxidants. Although problems were encountered during the school year, most were fixed through collaborative problem solving. For example, during a reading of bee pollen in hexane, no peaks were found. Through doing several trials, we realized that a greater amount of hexane, in microliters, must be injected into the GC-MS for a proper reading to be established. We therefore injected 10 microliters as opposed to 5 microliters. For precise readings, it was necessary that trials be taken multiple times. It was then found that the non-polar solvents contained mainly non-polar compounds, specifically long-chain hydrocarbons.

The Undergraduate Research Initiative provided me with the motivation to ask, learn, and challenge my previous bee exploration. By collaborating with Dr. Mojica, I established a solidified understanding of the GC-MS instrumentation, filtering process, and two different assay methods. Through constant communication, we were able to establish a proper methodology and ability to solve rising issues in the lab. The conducting of chromatographic research on bee pollen has served as an educational and eye-opening experience with results that can be implemented by future researchers.

Blog 3: A Conclusive Review of my Research on the Characterization of Bee Products

March is a busy month in the world of Chemical research! I am currently preparing to attend the American Chemistry Society conference in Orlando, Florida, which will take place at the end of the month, along with the Dyson School Society of Fellows conference on March 9th. Although the plane tickets are booked and the conferences are registered for, the most important part remains in the works – the display poster. I aim to have the poster completed by Spring Break, as I am currently working on analyzing the major peaks on the gas chromatography-mass spectrometry chromatogram to understand which compounds can be found in the pollen.

Presenting at the Dyson Society of Fellow’s conference.

The extraction process for the solvents being used is now complete. I decided upon using four main solvents: ethanol, methanol, methylene chloride, and chloroform. Using a precision balance, I weighed 5 grams of pollen, specifically from Los Baños in the Philippines. Allowing the pollen to sit in a test tube with the solvent for about 24 hours results in a solution ready to be filtered. I used syringe filtration to filter each of the solutions. The filtered products were then put into mini centrifuge tubes and refrigerated for another 24 hours. Post-refrigeration, the filtered solutions were ready to be injected into the GC-MS. One sample was injected per day, using different injection needles as to avoid any cross-contaminations between the solvents.

Samples of bee pollen in five various solvents.

Experiments do not always go as planned. Through this experiment, I have learned to not only accept this, but embrace it as a learning opportunity. Whenever something does not go as planned, I work collaboratively with my faculty mentor to ask the questions “why did this go wrong?” and “how can we fix it?” One recent example of this was the possibility of contamination. Rather than rinsing out the injection needle and re-using them, the possibility of cross-contamination was eradicated by using multiple injection needles.

As the end of my research project comes near, I am focusing on an in-depth exploration and analysis of the results at hand. I am also currently working on a paper about my research in hopes of submitting it for publication. Overall, this research opportunity has provided me with investigative skills, critical thinking skills, while enhancing my wet lab skills as well.

Blog Post #2: Continuation of Research on the Methods of Extracting Bee Pollen

During the entirety of the Fall semester, I worked collaboratively with Dr. Mojica for 1-2 lab sessions per week. We met before, during, and after my time in the lab to establish the goals for the lab session, answer any relevant questions, and discuss a brief overview of the results attained. Using my lab notebook, I kept track of each step taken in this process, as well as by taking photo documentation. Dr. Mojica and I also worked to establish an abstract to submit to the American Chemical Society for the 257th National Meeting, taking place in the Spring of 2019 in Orlando, Florida. I was happily accepted, alongside some of my colleagues, to be given the opportunity to present my research at the conference with some of my colleagues. Since my study focuses on various extraction methods, it has been very beneficial to work with Dr. Mojica who, through a collaborative effort, has taught me several methods of extraction for various bee-deriving substances. With a passion for the topic of analysis, the initiative to continue my research this semester was simply to find an answer to the question at hand in hopes of using the results to help better understand how bee substances can help our society.

Throughout this semester, my UGR research has taken a turn from focusing primarily on propolis to being inclusive of pollen, as well. A recurring obstacle was noticed despite of the change in focus from propolis to pollen. It appeared that cross-contamination in the GC-MS instrument remained a prevalent issue. For this reason, we came up with a few problem-solving techniques, such as running a trial solely with the solvent before running it with the pollen. This aided in cleaning out the machine of any by-products that may have been lingering from prior research. Additionally, our addition of a new machine, described below, has helped us in the extraction process in an effort to avoid cross-contamination. Each week, we have worked to make continuous progress on this project, despite of any obstacles encountered along the way.

Fortunately, we ended the semester with a gift from NSF, the National Science Foundation. The National Science Foundation, an American government agency, provides financial aid to support research and education in the field of science. Dr. Mojica was given a grant for the Accelerated Solvent Extraction (ASE) machine, which I have had the benefit to start using. The ASE machine is used for the extraction of chemicals from a solid. I first grind the bee pollen into smaller pieces and let these pieces sit in 5 mL of the given solvent overnight. Then, I manually filter this solution using syringe filters. This is followed by an extraction in the ASE machine. Below is a photo of me with our new ASE machine and my first extraction using the machine! We are continuing to answer the question of why there are outside chemicals showing up in our results from the GC-MS and hope to have this issue solved by the end of the semester.

 

My first extract of pollen and methanol using our new ASE.

Chromatographic Characterization of Bee Propolis and Pollen from Around the World

The title of my research, as stated above, is the Chromatographic Characterization of Bee Propolis and Pollen From Around the World. I have chosen to analyze and compare the components that make up bee pollen and propolis from several species with varying origins, developing an understanding for how the type of extraction methods will affect the gathered results. The purpose of this research is to distinguish between the results gathered from using different extraction methods, while understanding the importance of the extracted components. Moving forward, we would like to understand the different health benefits that propolis can provide for humans and which benefits relate to which chromatographically discovered components.

Before stepping into the wet lab and performing physical research, Dr. Elmer Mojica and I worked collaboratively to perform literary research in order to finalize my research question and methodology being used. We studied papers from several scientific journals, such as the Food Research International and the Journal of the Science of Food and Agriculture. In today’s society, the focus of what is being put inside the foods and products consumed by humans is so vast and significant. For this reason, I expect to achieve a fully developed understanding of which extraction method is best, along with which components are found in which species of bees. I can then use this to understand which components humans are consuming when they go to health food stores to purchase propolis and pollen.

The methodology being used to answer my research question varies depending on the extraction method being used. In chemistry, extraction methods are used to separate substances that they are mixed with. I used my literary research to fully understand the Soxhlet, microwave, sonication, accelerated solvent extraction, and simple extraction. I aim to compare these methods with one another. I will first be using dichloromethane and methanol to make solutions with the propolis and pollen. I can then inject these solutions into the machine known as GC-MS, or gas chromatography-mass spectrometry. This provides us with a visual representation of the spectrum. We can then use this, alongside a library, to analyze which molecules make up the propolis and pollen. Refer to Image 1.0 for footage of the first bee pollen that we have begun analyzing.

Image 1.0 – Stingless Bee Pollen from Los Baños, Leguna, Phillippines

This semester has so far been successful with literary research and we have begun injecting samples into the GC-MS. We are looking forward to analyzing the results and successfully reaching our desired achievements to answer my research question.

Final Report – Kuno van der Horst’s Family

Victoria Noriega

08/17/2018

UGR Final Report

Kuno van der Horst’s family reached out to Prof. Hearst earlier this year telling him that they had found our previous paper, “The Helpers of the Secret Annex – A Study Guide,” from last summer while working through the Undergraduate Research Program. They were excited to find this information seeing as they had never seen anything about their father like this. They began to exchange emails and soon agreed to let us do another paper focused on Kuno. This is how we gathered most of our information. The van der Horst’s kindly allowed us to use the documents they had regarding their father and his family. This opened up the doors to Henny, his wife, and Catharina, his mother. We began to learn more about the family and the connection they had to Miep Gies, one of Anne Frank’s helpers.

From the help provided by the van der Horst family our research was practically complete. Since the family hardly had any information about them online the majority of our information came from the family themselves or previous research we had done last summer. It was a majority of personal memories from Kuno, Catharina, or Henny that we used and some interviews. These memories gave us their stories during World War II and the experiences they went through while living in Holland. The memories held details of Kuno hiding in Amsterdam with Miep and Jan Gies, Catharina helping to hide Jews before they personally lost their home from Nazis and of the things they all did to survive during the war.

This paper Prof. Hearst and I did this summer is similar to our paper from last summer and the experience has been just as great. It’s been very eye opening to read their personal stories during this time period and gain a little bit more knowledge on the environment of World War II. Having the opportunity to read their memories and to hear from their personal family has left me feeling in awe of how little time has passed from then to now. This has truly just left with me with a stronger sense of what to do during times like these and to read about people who set the example of not backing down during hard times has been a fulfilling experience. World War II has always been one of my favorite topics to learn about due to the intricacy and historical prominence it has inflicted for years to come and to get to discover more details that not many know about has only left me feeling proud of the work we’ve done.

 As this is now my second year participating in the program I’ve learned so much and grown since when I first began. My research skills have expanded and my ability to write has improved tremendously throughout the entire experience. It has helped me learn how to communicate with professionals and also my time management in getting things done to respond for the benefit of the project. At times I would get stressed feeling as if I had no idea what I was doing, but Professor Hearst would help me and I would find that it wasn’t that stressful and I did know what I was doing but I needed to push myself.

Ever since I first met Professor Hearst back in the 2017 spring semester he has helped me grow a writer and given me opportunities to expand on my writing credits. He has diligently worked with me to improve my technical skills and critical thinking when it comes to writing and researching. Through him I have also gotten to learn more about people I would have probably never gotten the chance to learn about. The last two papers have been a wonderful learning experience and it is all thanks to Professor Hearst.

Blog #2 “More Than a Footnote- Kuno van der Horst’s Anne Frank Connection.”

At first, for me, this project seemed difficult and daunting. I was no longer merely helping with the research, but I had to write for myself. It was nerve-wracking at first because in my head I didn’t have enough information to go off of only. To me, this project had first come to fruition to share the story of Kuno and his connection to Miep Gies and the Frank’s, but I still had questions about every detail of Kuno van der Horst’s life because I thought it’d make it easier for me to write and understand. Though, as I began I realized that wasn’t the case at all. As I got more comfortable with writing it and the story of the van der Horst family it all began to piece together. It no longer felt as daunting; it was still a lot of information to read through and decide if it should be included or not, but I didn’t feel as nervous. The main challenge I began to have was after all of the information was written down. The editing process made me realize how much more I need to work on my writing skills and my attention to detail. It showed me that it’s been quite some time since I’ve really written and looked at my writing in a critical manner. Although at times I felt like I caught on to mistakes before I’d send it back to Prof. Hearst there was always something I’d forget to correct. This has helped me immensely though to realize these mistakes and even though there are always new ones I can continue to learn and grow as a writer.

As Prof. Hearst and I are in the final stages of our project it’s hard to imagine the beginning of the summer when I was still trying to scramble enough ideas on how to piece together all of the information. The information that was provided to us by Kuno van der Horst’s family and their generous help gave us the opportunity to share a story that needed to be shared. The story of Kuno and his family is one that is probably shared with many families during this time period. A will to help and fight against an oppressive force and to help those more defenseless than you. Although there may be many stories like the van der Horst’s it doesn’t mean that not every story should be shared. From what I’ve learned through last summer and now is that to stand in the face of such trying times and to cower away only adds to you helping the people in the wrong. From Anne Frank’s helpers to Kuno van der Horst’s family these people risked their lives to do what was right. Just like the project from last summer it has only opened up my eyes more to the need to stand for what is right and to not be selfish in times of peril, but to risk your life knowing that you’re helping someone else. Leading into the future these historical stories should be a guiding light for everyone and give people the courage to keep fighting even if they want to give up, just keep fighting because the end result will not leave you with regret or sadness. It’ll leave you with pride knowing that you did everything in your power to go against something that was blatantly morally wrong.