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Material Information
- Title:
- Chemistry Sessions 1 and 2
- Creator:
- SUNY Oswego
Koeppe, Julia ( Speaker )
Baker, Matthew ( Speaker )
Bushen, Slater ( Speaker )
Farrell, Kevin ( Speaker )
Gomez, Chelsea ( Speaker )
Root, Lauren ( Speaker )
Hidoyatov, Muhammadzohir ( Speaker )
Jandev, Vikrant ( Speaker )
Brand, Katherine ( Speaker )
Roque, Johann ( Speaker )
- Publisher:
- SUNY Oswego
- Publication Date:
- 04/01/2022
- Copyright Date:
- 2022
Notes
- Abstract:
- Presenter: Slater Bushen. Title: Determining the enzyme function of structure 3CBW. Abstract: The purpose of this project was to use computational and experimental methods to determine the function of an enzyme for which the structure is known but the function is unknown. The selected structure is available in the Protein Data Bank (PDB) with ID 3CBW. Initial analyses were performed by using computational sites and databases, including BLAST, Dali, and Pfam for sequence and structure alignments. Next, Motimate was used for active site alignment and comparison to known enzymes. Finally SwissDock was used with suitable ligands to identify possible substrates for testing experimentally. These computational tools allowed us to hypothesize that 3CBW is a hydrolase which specifically cleaves mannan substrates. We next expressed 3CBW in a culture of E. coli cells and purified it. We then used chromogenic substrates to study the ability of 3CBW to catalyze hydrolysis reactions. We confirmed that 3CBW behaves as a generic hydrolase with substrates such as pnitrophenyl acetate and p-nitrophenyl butyrate. We further determined that 3CBW behaves specifically as a mannanase to hydrolyze azcl-galactomannan. We are currently using site-directed mutagenesisto change the proposed active site of 3CBW to determine the effect of the mutation on enzyme activity and confirm the location of the active site in the structure. ( ,,,,,, )
- Abstract:
- Presenter: Kevin Farrell and Chelsea Gomez. Title: CFH Expression and Purification. Abstract: The complement system is part of the innate immune system found in mammals. It is responsible for providing defense against pathogens such as bacteria and funguses, and it plays a role in the removal of damaged cells. Complement can be activated via three different pathways to start a cascade that results in various effector functions. Our lab is interested in eluding a molecular level understanding of the protein interactions that regulate complement activation. In particular, we study complement factor H (CFH) which is a 150 kDa glycoprotein made up of 20 globular complement control protein units. CFH is a known regulator of C3 and also interacts with thrombomodulin. From recent studies, there are certain domains in CFH that have shown more contributions in the interactions than other parts of the protein. For our research, domains 1-4 are to be investigated to observe the potential interactions these 4 domains have with other proteins. We know we have successfully transformed 2 different strains of P. pastoris containing the first 4 domains of CFH by screening. Currently we are experimenting with the expression and purification of CFH. Recent problems have arisen due to the low amount of protein being expressed by the yeast cells. Work will be focused on adjusting the growth conditions of the yeast cells to potentially increase the amount of protein expressed. Work will also be done to come up with an efficient purification scheme to successfully purify the CFH fragment.
- Abstract:
- Presenter: Lauren Root. Title: Determining The Function of 3DS8. Abstract: The main goal of this project is to determine the function of the enzyme 3DS8. It is already known that 3DS8 is mainly found in bacteria and is predicted to be an alpha-beta hydrolase based on computational analysis including both sequence and structure alignment. This means that it is likely that 3DS8 is an enzyme that catalyzes the hydrolysis of bonds in its substrate. The computational analysis also showed that 3DS8 was mainly aligned with other protein domains of unknown function, meaning that we do not know the specific substrates that the enzyme hydrolyzes or whether it may even be a different type of enzyme. The next step was to go into the lab and test 3DS8 for hydrolysis activity with different substrates. 3DS8 was expressed in E. coli cells and then purified. Its activity was tested by combining purified 3DS8 with PBS buffer and a chromogenic substrate. We then measured the absorbance of the reaction mixture over a 40 minute time period as the hydrolysis product was yellow. The results of this kinetic analysis were then analyzed to determine whether or not 3DS8 was able to catalyze the hydrolysis of the substrate.
- Abstract:
- Presenter: Muhammadzohir Hidoyatov. Title: Copper-catalyzed Alkenylation of Ketones Using Primary Alcohols and Primary Aldehydes. Abstract: Unsaturated ketones have been used in many life saving drugs, food preservatives, and pesticides. They can be synthesized using metal (I) catalyzed α-alkenylation of ketones using primary alcohols and primary aldehydes. Use of alcohols and aldehydes which are abundant and cheap makes these reactions budget friendly. A new methodology using copper catalyzed reactions with the use of N-phenylpicolinamide (NPPA) as ligands was developed to provide higher yields at low temperatures. The results with various metal catalysts, solvents at various temperatures as well as various electron rich and poor substrates will be discussed.
- Abstract:
- Presenter: Vikrant Jandev. Title: Detecting Cortisol Using LC-MS/MS. Abstract: Cortisol plays a vital role in the body's response to stress. Cortisol can be detected through enzymelinked immunosorbent assay (ELISA). The project's specific aim is to compare the liquid chromatography-tandem mass spectroscopy method (LC-MS/MS, with four transitions) to two different ELISA's we currently use in our laboratory to analyze cortisol in hair and nails. In collaboration with SUNY-Upstate-Medical, we used TSQ Quantis Plus MS to analyze the samples. For analysis, five quadruplicate samples were prepared: commercial milled hair; D-cortisol; D-cortisol after acetone chase; D-cortisol that went through the entire procedure; commercial hair with D-cortisol added before acetone chase; and commercial hair with D-cortisol that went through the entire procedure. This "spike experiment" aimed to see how effective the extractions were while using the Dcortisol as an internal standard. We analyzed them using ELISA (which cannot distinguish between Dcortisol and cortisol; Salimetrics and Arbor Assay kits were used) and LC-MS/MS (which can distinguish between D- cortisol and cortisol). Next, we will investigate how to improve LC conditions and check the linearity of hair and nail cortisol concentrations in different amounts of hair and nails.
- Abstract:
- Presenter: Katherine Brand. Title: Validation Experiments for Cortisol Extraction in Nails. Abstract: The focus of my effort is the validation of the process to extract and measure cortisol from human nail samples. Cortisol is at the end of the negative feedback loop of the HPA axis, comprised of the hypothalamus, pituitary, and adrenal glands, which regulates responses to environmental stressors, so cortisol is an ideal target molecule for studies concerning the stress levels of the participants. Determining the ideal conditions and proper procedures for quantifying cortisol concentrations in nail samples is crucial before any extramural samples can be processed and verifiable results can be produced. In our presentation, we will provide answers on how much cortisol is lost during the wash, how many washes are needed, how much mass is lost while drying the samples, how many methanol and acetone extractions are necessary to effectively collect cortisol, and how much weight of nails is optimal for analysis.
- Abstract:
- Presenter: Johann Roque. Title: Analysis of a Photo-Degradable Polyester. Abstract: Polymer chemistry is a rapidly growing subfield of chemistry revolving around the development of plastics. Within this field, the ability to create polymers (i.e., plastics) with controlled degradation capabilities has garnered much attention from the community. This focus derives from the fact that millions of tons of plastic are produced and disposed of each year. Due to the robust nature of plastics, they do not naturally degrade and instead build up in the environment. This presentation outlines research towards the development of a new degradable polymer that may contribute to the mitigation of the plastic waste issue. Previous students working on this project have developed a plastic capable of controllably degrading in the presence of ultraviolet light; however, the particular way in which this material degrades on the molecular level has yet to be explored. To better understand how our polymer degrades, two control molecules were synthesized, representing different degradation pathways inherent to the polymer. These molecules were analyzed to verify that the desired product had been synthesized. Subsequently, experiments using these molecules were carried out to determine the degradation path of our polymer. This investigation has broadened our knowledge of how our polymer degrades in the presence of UV light.
- Summary:
- Session 1 Chair: Julia Koeppe. Session 2 Chair: Mathew Baker.
- Acquisition:
- Collected for SUNY Oswego Institutional Repository by the online self-submittal tool. Submitted by Zach Vickery.
Record Information
- Source Institution:
- SUNY Oswego Institutional Repository
- Holding Location:
- SUNY Oswego
- Rights Management:
- All applicable rights reserved by the source institution and holding location.
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