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The Cytosolic Iron-Sulfur Assembly (CIA) pathway is essential for metabolic catalysis, DNA replication, transcription and translation, and iron homeostasis in eukaryotic cells. We studied this pathway in yeast yet there is a human homolog. The CIA pathway synthesizes and transports iron-sulfur clusters to apoproteins. The targeting complex is composed of three proteins- Cia1, Cia2, and Met18. This complex is responsible for transporting iron-sulfur clusters to proteins that lack it by interacting with apoproteins. Little is known on how this complex interacts and recognizes apoproteins that require the iron-sulfur clusters. Cia1 and Cia2 form a sub-complex. By having the structure of this sub-complex, we will be able to understand the function of this complex. We were able to obtain a sub-complex through various purification methods. We grew Cia1 and Duf59 (A truncation of Cia2) by heterologously expressing both in bacteria. Cia1, containing both a his-tag and strep-tag, was purified on a nickel column by its his-tag through affinity chromatography. Duf59, and its his-tag, is expressed as inclusion bodies, so it was purified through denaturing and refolding the inclusion bodies. The two proteins were mixed to form the sub-complex. The Cia1-Duf59 complex was purified through two tandem columns. With the purified complex, we set a crystal tray to understand the optimal conditions in which protein crystals grow. Since no crystals have grown, different purification methods or different conditions might be required to obtain the optimal buffer conditions.
Previous work in the Elliott lab utilized bioinformatics to identify a unique di-heme peroxidase from Burkholderia thailandensis, BthA, conserved upstream an unreported phosphatase, referred to as PhosA. The conservation of these two genes is present in all strains of Burkholderia. The biophysical characterization of PhosA is one of our approaches to understanding the physiological role of this protein and its relationship to BthA with respect to biological function. By use of traditional biochemical and biophysical techniques, we expressed and purified PhosA, a putative purple acid phosphatase (PAP) found in B. thailandensis, a gram negative soil bacteria. PAP proteins are commonly found in mammals, plants and bacteria, though the role of mammalian PAPs are most understand.1 Our goal is to express and purify the recombinant PhosA. By use of traditional biophysical and biochemical techniques, we purified PhosA and analyzed the protein by UV-Visible spectroscopy. PhosA has been previously found to contain a di-metal center, identified as Fe-Zn, which gives rise to the purple color due to a charge transition between the FeIII center and a key Tyr residue found in the metal binding motif. The characterization of PhosA is important to further understand the role of BthA as well as the role of this phosphatase in B. thailandensis.
Fungi and bacteria are the primary decomposers of dead organic material (e.g., plant litter) and facilitate the turnover from carbon stored in biomass to the atmosphere. Although fungi and bacteria are naturally found to co-exist in the environment, the nature of fungal-bacterial interactions is poorly understood, limiting our ability to determine the specific mechanisms behind biogeochemical cycling processes. To investigate this, we developed a computational technique to quantify the growth rate of decomposer fungi in competition with bacteria on plant litter during a one month incubation. We observed that initial fungal growth rate was not significantly affected by the presence of other fungi or bacteria, but was observed to significantly decrease upon bacterial enclosure. Our data also show that fungal growth rate did not predict fungal growth response to bacteria, and is presumed to be regulated by an unidentified fungal characteristic. We also observed that Cladosporium herbarum in particular, facilitates the formation of an inhibition zone immediately surrounding the fungal colony, perhaps as part of a self-defense mechanism, while additionally accompanied by a dramatic shift in colony color. The findings of this study emphasize that elucidating the nature of microbial interactions is necessary in order to better understand their impact on the carbon cycling process through global ecosystems.
Sea urchin larval skeletal development provides a simple model for understanding developmental patterning. Ectodermal signals direct the migration of primary mesenchyme cells (PMCs), which pattern the skeleton according to their arrangement. Chlorate inhibits the creation of Sulfated Proteoglycans (SPGs), a ventral PMC patterning cue, by blocking sulfate from binding to proteoglycans. This lack of patterning cue inhibits PMCs from being positioned ventrally, thus eliminating ventral components of the larval skeleton. The timing of each stage of skeletal development is, as of yet, incompletely described in both control and treated embryos. To document when skeletal elements develop, we photographed developing embryos in two different environments. We observed the timing of skeletal element development in control and chlorate-treated embryos. We found that control embryos develop heterosynchronously, with each element initiating and growing over a range of times. Chlorate treatment arrests growth of the skeleton at the triradiate stage.
OLEDs are light emitting diodes that are comprised entirely of organic materials rather than inorganic semiconductors. OLEDs have the same function as the more commonly known LEDs, yet the organic materials used provide many advantages economically and environmentally. Currently, the creation of stable blue light emitting diodes is holding back the transition to OLED technologies. The properties of OLEDs allows them to overcome the restrictions presented by everyday LEDs. OLEDs do not require a backlight which allows them to be lighter, thinner, and flexible so they can conform to any surface and have increased energy efficiency. OLEDs are capable of being inkjet printed reducing production costs significantly. Small molecules based off of benzobisoxazole cores are synthesized through condensation and cross coupling reactions. The process of creating small molecules consists of experimenting with different combinations of cores and electron donors of which the efficiencies are then measured. We synthesized a new electron deficient core that is benzobisoxazole based. The core has has a 472 emission which is sky blue. Since this core is electron deficient, theoretically it is will be it easier for charge transfer to occur. This property is important because it increases the efficiency and reduces the amount of energy lost to heat.
Coral reefs are some of the most diverse and valuable ecosystems on Earth, whose benefits range from advances in medicine to economic productivity. When coral are stressed by changes in environmental conditions such as temperature, light, or nutrients, they expel the symbiotic algae living in their tissues, causing them to turn completely white. This phenomenon is known as “bleaching”. When coral are bleached, they lose their essential nutrients and are therefore more susceptible to disease and mortality. NF-kB transcription factors are involved in immunity and stress responses in higher animals. We hypothesize that the bleaching is an immune disease and that NF-kB plays a role in the coral death. In this project we are studying NF-kB in the endangered coral Orbicella faveolata. The goal of this project is to create a mutation in the Of-NF-kB to determine if the regulation of NF-kB is similar in corals and humans. We have used PCR, gel electrophoresis, and DNA purification to create a Serine to Alanine triple mutation in a series of predicted regulatory phosphorylation sites. These studies will improve our understanding of the NF-KB transcription factor in corals and how it may be related to coral bleaching and death. In the long term, these studies may help in decreasing environmental damage to the coral reef ecosystem.
Currently Lithium-ion batteries play a large role in technology, but moving to larger industries increases safety risks due to high temperature applications. Although the carbonate based organic solvent electrolytes used in current Lithium-ion batteries have ideal low viscosities and high conductivities, their low boiling points are hazardous. To improve the thermal stability of the batteries, Phosphonium based ionic liquid electrolytes are studied as replacement electrolytes. Four Phosphonium ionic liquids with two counter-ions were synthesized and tested using Differential Scanning Calorimetry (DSC) and conductivity testing. The results of these tests found that the melting points of the ionic liquids decrease as the alkyl chain length increases from 2 to 6 carbons (e.g. from no melting point to 51.56 ⁰C). Moreover, exchanging a halide counter ion for bis(triflouromethane)sulfonamide (TFSI) further lowered the melting point and increased conductivity.
When a neuron is activated, it fires, transmitting information from one neuron to the next. The information is passed in the form of neurotransmitters (chemical signals) or action potentials (electronic signals) at the synapse. The synapses we were interested in studying were located in the piriform cortex, the part of the brain that perceives smell. Neurons in the piriform cortex form specific ensembles in response to sensory stimuli to form a behavioral reaction 3. The goal of this experiment was to learn and understand more about synaptic connectivity. We hypothesized that through learning an odor discrimination task, the synaptic connectivity within the ensemble of neurons associated with the odor would be strengthened. Behavior can be conditioned by providing a specific stimulus paired with a reward or punishment in order to elicit a particular response. In this study, Go/NoGo odor discrimination task was developed to teach the mice to associate an odor to the command stay or go. The mice received a shock if they reacted improperly to the exposed odor. In order to make this an efficient behavioral task, the number of trials per block and the concentration of the odor were tested. It was determined that 100 trials per block and higher odor concentrations led to the mice learning the task quicker. Once the mice successfully learn the behavioral task, a virus expressing channelrhodopsin will be injected into the piriform cortex of mice genetically modified to express Cre (neuronal activity marker) and tdtomato (fluorescent protein) in the next step of the experiment. The synaptic connectivity of the labeled neurons will then be compared to unlabeled neurons through the use of optogenetics to stimulate the neurons and electrophysiology to record the synaptic responses. The recording will allow us to test the hypothesis and determine if synaptic connectivity was strengthened due to the behavior task.
The receptor tyrosine kinase, RET is critical to the development of cells of the nervous system, spermatogenesis, and kidney maturation. Inducing RET activation has the potential to promote neuronal survival, but the delivery of the natural ligands to the brain remains a challenge due to their inability to cross the blood brain barrier. Small molecules, however, can overcome this challenge, and are therefore safer drug candidates. Our goal is to conjugate a bivalent scaffold to a small molecule capable of mimicking the native protein, inducing RET activation. To test the feasibility of our approach we are using artemin peptide fragments conjugated to a bivalent cyclobutane molecule. This presentation explains the methodology of peptide synthesis using flow chemistry, as well as the use of Click Chemistry to link the peptide fragments to the cyclobutane scaffold. Future testing of the hybrids will hopefully demonstrate the potential of using scaffolds in small molecule drug design to replace large, bivalent ligands.
Larval skeletons of the sea urchin Lytechinus variegatus are formed by the pattern of the primary mesenchyme cells (PMCs), which are positioned by signals from the ectoderm. Lipoxygenase (LOX) is a gene that is expressed in the ectoderm and necessary for the formation of midline skeletal elements. We tracked the skeletal patterning of DMSO-treated embryos to determine when different skeletal elements emerged in a control setting. We then treated cultures of embryos with MK886, a drug that inhibits LOX activity. Following treatment, we imaged the embryos hourly between 18 hours post fertilization (hpf), and 36hpf. We were able to analyze the development of the embryos and define time points in which the skeletal elements developed. Analyzing the MK886 data, we also found that the embryos treated with MK886 develop at a slower rate than the DMSO-treated embryos.
Despite the fact that signaling pathways are usually studied in isolated environments free of proteins that are not involved in those specific pathways, signaling pathways do not take place in such sterile condition in real life. Therefore, the project focuses on the effect that multiple receptors can have on one single pathway, which is also known as crosstalk. The receptors of interest are Rearranged during transfection (RET), Insulin receptor (IR), and Insulin like growth factor 1 receptor (IGF1R); all are receptor tyrosine kinase. While RET is activated by Artemin (ART), IR, and IGF1R are activated by Insulin like growth factor 2 (IGF2). These growth factors are used to treat NB41A3 cells. Some cells were treated only with different concentrations of ART while others were treated with both constant concentration of IGF2 and varying concentrations of ART. Western blot was performed with lysates from these cells. The targeted proteins were pAKT, pPDK-1, pIRS-1, and PI3K, which are phosphorylated along the AKT pathway. Only pPDK-1 showed a clear signal. In the future, the goal is to successfully detect all four proteins and identify where in the pathway does the crosstalk take place.
There are major three forms of cell death: necrosis, autophagic cell death, and apoptosis; however, other forms exist. One of the less well known forms of cell death is phagoptosis, which occurs in the Drosophila ovary. At stage 10 a subset of the follicle cells undergo a radical shift in purpose, these new stretch follicle cells invade the nurse cell cyst and strangle the nurse cells. Unknown cytoskeletal machinery is clearly at work in the ovary. To unravel that mystery αPS3, WASp and Rac1 RNAi flies were crossed with GR1-mCD8-GFP flies to explore which of these known cytoskeletal and cytoskeletal associated proteins were involved in the movement of the follicle cells. Through our crosses we found that only the GR1 driving WASp RNAi impacted the development of follicle cells and have a strange morphology. In addition to that, we also found that the cytoskeletal protein Rac2 is upregulated in late stages of development. This is an exciting find and our lab will further investigate the role of WASp and Rac2 in cytoskeletal events.