2011 Research Symposium

COMPLETED RESEARCH



Study of pdr-1 Knockout using RNAi in Caenorhabditis elegans Al Hasan, Naila; Lucky, Nikki; Nisnisan, Marie
Professor: Dr. Rachel Hopp

Parkinson’s disease (PD) is second to Alzheimer’s as the most common neurodegenerative disease among older persons. PD affects dopaminergic neurons of the substantia nigra in the midbrain; it is characterized by movement related symptoms such as tremors, difficulty with walking, and uncoordinated gait. A gene implicated in PD is park2. It encodes for an E3 ubiquitin ligase enzyme called PARKIN, which is involved in elimination of unwanted, damaged, or excess proteins by targeting them for degradation through the Ubiquitin Proteosome System (UPS). By using animal models, such as nematode Caenorhabditis elegans, underlying mechanisms specific to PD can be studied. PARK2’s homolog in C elegans is pdr-1. RNA interference (RNAi) is a method employed to study the function of desired genes where a dsRNA is used to prevent an mRNA from producing a protein thereby inhibiting a gene of interest. RNAi knockdown of pdr-1 is known to express phenotypes such as mitochondrial stress, decreased lifespan, and truncated PDR-1, similar to PARKIN mutations observed in PD patients. To test this, experiments were carried out with wild type N2 strains. Specific gene sequence of pdr-1 was identified using OMIM and Wormbase; primers were attained from an E-RNAi web application. Fragments of the pdr-1 gene from an N2 worm lysate were then amplified by PCR, whose products were cloned into the established RNAi feeding vector, pPR244 (pDONRdT7). This cloned vector was transformed first into DH5alpha and then HT115 strains of E. coli. The latter strain is more difficult to transform, but lacks RNAse 3. As a result, pPR244-pdr-1 was transformed into HT115 (DE3) E. coli feeding strains and fed to the worms. Preliminary findings are expected to be severely uncoordinated movement and decreased lifespan phenotypes. Experimental results will be reported at the symposium.



Construction of Supramolecular Assemblies using Diruthenium (II) Complexes Al Hasan, Naila; Avi, Gimenez; Garcia, Nancy; Lee, Kevin; Tran, Tue
Professor: Dr. Robert Towery

The synthesis of supramolecular assemblies using metal-metal multiple bonded diruthenium complexes as building blocks is an interesting research area with great potential for design of molecular materials. Reaction of Ru2(CCH3O2-)4Cl with carboxylic acid groups of various amino acids was preformed. Two amino acids used in this study were alanine and glutamic acid. It is proposed that less crowded groups present on the amino acid would be easier to bind to the di-metal complex at the equatorial positions than bulkier side chains. Resulting complexes were characterized by ultraviolet-visible (UV-Vis) spectroscopy and gel electrophoresis. Spectral changes for these compounds were taken between 380 -700 nm, where the effect of amino acid substitution for the acetate fragments on diruthenium was indicated. Reactivity of diruthenium with that of a phosphate buffer was also investigated which yielded a peak at 348 nm due to the formation of a new phosphate complex; another peak was present at 424 nm as expected for the diruthenium complex. Upon dilution of the dimetal complex with HPLC grade water, the peak at 348 nm was reduced suggesting its presence to be due to addition of phosphate groups. Additionally, experiments with LiCl to encourage axial rather than equatorial substitutions and solubility tests as a means to characterize the above complexes were carried out. The diruthenium complexes were provided by the laboratory of Dr. Karl Kadish at University of Houston.



Determination of the extent to which ATP synthase F0 subunit 8 protein sequence is conversed in mammals Ali, Noman
Professor: Dr. Brenda Whaley

ATP synthase F0 subunit 8 protein is a single-pass mitochondrion membrane protein. Mitochondrial membrane ATP synthase produces ATP from ADP in the presence of a proton gradient across the membrane which is generated by electron transport complexes of the respiratory chain. F-type ATPases consist of two structural domains, which are F1 and F0. F1 contains the extramembraneous catalytic core and F0 contains the membrane proton channel, linked together by a central stalk and a peripheral stalk. Subunit 8 is part of this F0 domain. The purpose of this research was to determine the extent to which the 68 amino acids of ATP synthase F0 subunit 8 with the gene ID of 4509 and locus name of MT-ATP8 at position 8366-8572 on the mitochondrion DNA are conserved across species lines by comparing the protein sequence in the human to that of other mammals and identifying conserved regions by using the NCBI website. The research showed that 99% of the Neanderthal, 96% of the chimpanzee, and 47% of the mouse ATP synthase F0 subunit 8 protein sequence were identical to the human sequence. While, the sequence for ATP synthase F0 subunit 8 in chicken, zebra fish, and fruit fly shows little homology to protein sequence in humans. More specifically, it was found that animals belonging to the primate order shared more of this protein sequence than did non-primates.



Eukaryote vs. Prokaryote Bana, Anum; Varughese, Blessy; Bechtel, Robert
Professor: Dr. Jackie Horn

The purpose of this research was to perform bioremediation of oil using bacteria and fungus. Two different experiments were performed to observe the prokaryotic and eukaryotic aspect of bioremediation. In the bacteria experiment, three samples of soil were obtained from mechanic shops. Bacteria were isolated from these soils by a series of dilutions, reducing the amount of broth and increasing the amount of oil. Streak plate and gram-staining techniques were used to confirm the presence and identify the bacteria. Two methods were used to test for bioremediation. The last dilution from each soil was placed in 25 drops of oil to perform oil bioremediation. Mayco and Mobile Doctors were identified as the best oil degrading bacteria and degraded .1 cm of oil. Spread plate was also performed with a drop of oil to see if bacteria would grow on the drop or around it. Mayco and Mobile Doctors showed the best results. In the fungus experiment, Mushroom Spawn Spores, Pleurotus ostreatus, were grown under two conditions, inside and outside. Probes were used to monitor and observe the effect of oil on the fungus. The fungus was watered weekly and after two weeks oil was introduced. There was a decrease in oxygen content and an increase in carbon dioxide production, which confirmed fungus, was performing aerobic respiration and consuming the oil. The experimentation proved that bacteria and fungus both play a role in bioremediation of oil. Eukaryotic and Prokaryotic bioremediation can possibly be used for oil degradation.



Examining Water with Bacteria, Filters and Chlorine Bana, Anum
Professor: Dr. Betty Thomspon

The purpose of this experiment was to study bacteria in different samples of water, compare water from clean and dirty filters, and to see the effect of chloride on the number of bacteria. Water samples were obtained from natural waterways such as Willowridge Park and Lakehouse water from HBU, and also from filtered water sources such as Science Building water fountain, water from a clean refrigerator filter and water from a contaminated refrigerator filter. First a series of spread plates were performed to count the number of bacteria in each water sample. Willowridge Park had the most bacteria count with 4000 bacteria/mL, Lakehouse water had 1600 bacteria/mL, contaminated filter water had 2800 bacteria/mL, clean filter had 1080 bacteria /mL, and water fountain had 920 bacteria /mL. In addition gram-staining and methylene-blue staining was performed to identify microorganisms in the water. Gram positive bacillus was found in Willowridge park water and gram positive staphylococcus was found in Lakehouse water. This suggests that the filters removed the larger protists but allowed bacteria through the filter. When new filters were introduced, the number of bacteria significantly reduced. The two water samples, Willowridge park lake and contaminated filter water, had the highest bacteria count and were used for chloride testing. Chloride levels of 60 ppm and 100 pm were used to test the effect on bacteria. The bacterial counts reduced for contaminated filter water from 2800 microbes/mL to 27 microbes/mL for 60 ppm , and from 2800 microbes/mL to 13 microbes/mL for 100 ppm. For Willowridge Park the bacterial counts for 60 ppm were reduced from 2000 microbes/mL to 47 microbes/mL, whereas for 100 ppm concentration, all the bacteria died compare to the initial amount of 2000 microbes/mL. This data indicates that a higher concentration of chloride kills more bacteria.



Composting: Ingredients for Life Barrilleaux, Brandy
Professor: Dr. Betty Thompson

The purpose of this study is to determine the best combination of organic materials to produce nutrient rich compost. Different materials were combined and analytical chemical analysis was conducted to compare the best compost. Some examples of the different materials analyzed were: 1) various types of soil 2) paper (carbon source) 3) grass clippings (nitrogen source) 4) water 5) urine. Before composting, the soil was analyzed for nitrate, phosphate, pH, temperature, CO2, and O2 production for possible microbial action. Another preliminary test was performed on the soil to determine soil porosity and permeability. The potting soil had the highest porosity at 78%. The soil from a natural undisturbed habitat had a porosity of 74% and the HBU soil had a porosity of 56%. The potting soil also had the highest permeability with .200s-1. In at second was the farm soil with a permeability of .143s-1. Lastly, the HBU soil had very poor permeability with .063s-1. The materials were added in dissimilar combinations and set in the incubator. Readings were taken each week for three weeks. The final results show that the urine trial had the highest temperature at39◦ C, CO2 levels at 6934ppm demonstrating the highest rate of decomposition. The urine trial also portrayed the highest nitrate and phosphate levels which would give plants more nutrients. On the other end of the spectrum the HBU compost trial had the highest Oxygen levels at 20.85% representing low decomposition rates. This compost also demonstrated very low nitrate and phosphate levels making it not very efficient as compost.



Conservation of NADH Dehydrogenase Subunit 3 Bechtel, Robert
Professor: Dr. Brenda Whaley

The protein NADH dehydrogenase subunit 3 is a part of the NADH dehydrogenase complex that is commonly expressed among all animals. The NADH dehydrogenase complex is located on the inner mitochondrial membrane and works to transfer the electrons from the carrier NADH to the enzyme ubiquinone during the electron transport chain, oxidizing NADH to form NAD+. This process function is essential to aerobic respiration. Mutations in this subunit cause Leigh syndrome, which is marked by necrotic lesions in subcortical brain regions. The NADH dehydrogenase subunit 3 for homo sapiens is 115 amino acids in length, coded for on the mitochondrial DNA (GeneID = 4537) from the nucleotide positions 10059 to 10404 on the positive DNA strand. The DNA of the mitochondria is separate than that of the DNA of the cell itself and is instead located on a single circular chromosome. Since the NADH dehydrogenase complex serves such a vital role, one would expect the genetic sequence to be conserved among the animal kingdom. Initial research shows that the protein sequences of NADH dehydrogenase subunit 3 are somewhat conserved across phyla. For example, when compared to Homo sapiens, the chicken, Gallus gallus is seen to have a 55% amino acid sequence match, while the zebra fish, Danio rerio has a 58% amino acid sequence match with Homo sapiens. The extent to which this conservation occurs will be looked at more in-depth in this research. Specific domains of the protein will be analyzed for homology. The protein sequences will be analyzed to gain a better understanding of where mutations occurred from the common mitochondrial ancestor.



Investigating gene mutations in NADH dehydrogenase subunit 5, regarding conservation of protein function in mammals Dickerson, Johana
Professor: Dr. Brenda Whaley

NADH dehydrogenase subunit 5 (Accession number: YP_003024036, GeneID: 4540) is a multi-pass membrane protein found in the mitochondrial inner membrane of Homo sapiens. This 603 amino acid long protein is a fundamental subunit of the mitochondrial membrane respiratory chain, NADH dehydrogenase (Complex I), that helps transfer electrons from NADH to the respiratory chain via ubiquinone, resulting in NAD+ and ubiquinol. NADH dehydrogenase subunit 5 is also referred to by its gene locus abbreviation ND5. The gene that codes for this protein is located between nucleotides 12337 and 14148 of the mitochondrial genome. Genetic similarities can be observed between humans and other mammals by comparing the sequence of NADH dehydrogenase subunit 5 with that of orthologous proteins of said mammals. Neanderthals have one less amino acid in their protein, but the nucleotide sequences match up with a 99% identity, indicating that there is little difference between the proteins. A chimpanzee has nine fewer amino acids in its orthologous protein, but otherwise has a similar nucleotide sequence, presenting with a 94 % identity in which 1080 nucleotides are shared. However, when compared with a mouse, the NADH dehydrogenase subunit 5 not only has twelve more amino acids, but also only 753 nucleotides match, resulting in a 66% identity. Modifications in the genetic code of mammals are examined to determine where differentiation occurred. These mutations not only show how Homo sapiens evolved, but also reveal how the functions of mammals are similar on a cellular level.



Investigations of Diruthenium Complexes with Amino Acids using UV-Visible Spectroscopy — Do, Linh; Patel, Sarin
Professor: Dr. Robert Towery

Metal-metal multiple-bound complexes such as Ru2(Ac)4Cl can be used as a starting material for construction of supramolecular assemblies. A substitution of the acetate ligand by the carboxylic acid functional group of amino acids led to the formation of new complexes such as Ru2(C2H5NO2)4Cl. This demonstrates an increase in the solubility complex of these samples under physiological conditions. Experiments were performed by reacting the metal-metal complex with the amino acids glycine and aspartic acid. These reactions were carried out using 6x10-4 M and 1x10-3 M diruthenium solution with 1x10-3M glycine and 1x10-3 M aspartic acid dissolved in HPLC grade water at room temperature. Results were analyzed using a Shimadzu 1501 multiplex diode array spectrometer. Pure diruthenium absorbs at a wavelength of about 427nm. Mixtures of diruthenium with glycine and aspartic acid show a hyperchromic shift to 433nm and 430nm respectively from 427nm. The absorbance around 430nm shows the preservation of the metal-metal bond and the substitution of the axial acetate groups with amino acids. In addition to the synthesis of the new diruthenium compounds, it was also found that diruthenium reacts with excess phosphate in 0.14M pH6.7 phosphate buffer. A distinct peak was found at 350nm in phosphate buffer along with the 427 nm peak. It can be concluded that new compounds of diruthenium with phosphate, diruthenium with glycine, and diruthenium with aspartic acid were successfully synthesized.



Fractal dimension of Proteus mirabilis and Bacillus subtilis chemotaxis in semi-defined minimal nutrient media — Do, Linh; Patel, Sarin; Ali, Noman; Medi, Sai; Kumar, Ricki; Cook, Rebecca; Leal, Franklin; Gardner, Brittney; Dickerson, Johanna; Mandal, Tanaya; Amir, Syed
Professor: Dr. James Claycomb

A fractal is a geometric concept observed in an object that exhibits self-similarity over an extended finite scale range. An example of these phenomena exists in microbial growth. The morphology of microorganisms can be analyzed by fractal analysis. To illustrate fractalization, physicists created a variety of software that utilizes mathematical algorithms such as the box counting method to quantify the nature of fractals in objects. In our research, we are studying the fractal properties of bacterial growth to better understand microbial chemotaxis. By altering chemical components in certain media, bacteria and other microorganism’s growth patterns resemble fractals of branching tree limbs and blood vessels. For this experiment, medias’ pH conditions were modified to 9 and 7 using tris buffer with HCl, and to 5 using acetic acid buffer with sodium hydroxide. The three different pH conditions were applied to 1.5% bacto-agar with 0.1% peptone and 0.9% bacto-agar with 0.1% peptone to facilitate fractal growth of Proteus mirabilis and Bacillus subtilis at 37˚ C respectively. Images of the fractal growth will be processed and analyzed using computer programs called FracLac and Gimp. By doing this, we hope to quantify chemotaxis of P. mirabilis and B. subtilis.



Cytochrome c Oxidase Subunit I: Homology Comparison in the Animal Kingdom — Fonseca, Sharon
Professor: Dr. Brenda Whaley

Cytochrome c oxidase subunit I (GenBank accession number YP_003024028.1, Gene ID 4512) is a mitochondrial protein in Homo sapiens that is 513 amino acids long. The 1541 base pairs coding for this protein start at position 5904 and end at position 7445 on the positive strand of the mitochondrial chromosome. The official abbreviation for its gene locus is COX1. This enzyme is a multi-pass membrane protein found in the mitochondrial inner membrane and is part of electron transport chain. It is involved in catalysis of the reduction of oxygen to water. Subunit 1, part of the functional core of this enzyme complex, is the catalytic subunit. The copper A center of subunit 2 and heme A of subunit 1 of this enzyme transfers electrons from cytochrome c to the heme A3 and copper B bimetallic center associated with subunit 1 (the largest subunit). This subunit is directly associated with creating a proton gradient while simultaneously reducing oxygen to water. If homology of cycochrome c oxidase subunit I is compared across various species, it is seen that chimpanzees, which is closely related to Homo sapiens, exhibited 99% similarity in homology. Comparatively, the fruit fly, which is more distantly related to Homo sapiens, had only a 77% similarity in homology. Thus it can be inferred that orthologues from the animal kingdom that are more closely related and had a similar evolutionary paths generally reveal greater similarity of this Homo sapien’s mitochondrial protein.



The Mitochondrial Protein NADH Dehydrogenase subunit 6 — Fouad, Marie
Professor: Dr. Brenda Whaley

NADH dehydrogenase subunit 6 is a component of complex 1 in the respiratory chain found in the inner membrane of the mitochondria. Its basic function is to facilitate the transfer of electrons from NADH to the respiratory chain that carries out oxidative phosphorylation in the mitochondrial membrane. NADH dehydrogenase subunit 6 (GenBank accession #:YP_003024037.1) is a protein consisting of 174 amino acids that is coded by a gene (GeneID:4541) on the negative strand of mitochondrial DNA from position 14149 to 14673. The official abbreviation for this gene locus is ND6. There are protein products that are similar to human NADH dehydrogenase subunit 6 in other species, but the homology between these orthologues decreases as the species becomes less similar to humans. When compared to the human sequence of the protein the Neanderthal sequence is very similar with 99% identity, the chimpanzee comes in second with 96% identity and then the similarity drops by nearly half with the mouse sequence showing only 54% identity with the human sequence. Even less similarity is shown with other animals such as the zebra fish, which shows only 33% identity with the human version of the sequence. The sequences from the chicken and the fruit fly both show 36% identity with the human sequence, and the ranges covered by the fruit fly sequence are not very large. The homology of the orthologous protein clearly decreases with increasing taxonomic variation.



Construction and Cloning of a Recombinant DNA — Gastineau, Tracy; Luna, Paloma; Sparkman-Royo, Alexander
Professor: Dr. Nicole Pinaire

The objective of this experiment was to use various recombinant DNA technology procedures to clone a DNA fragment, extract, and map the resulting fragment. The recombinant DNA technologies procedures included ligation of a vector to a kanamycin resistance gene, transformation of the recombinant plasmid, and selection of successfully transformed cells although the latter was not conducted due to time constraints. The process of ligation involves the recombination of DNA from different sources into a useful DNA molecule. Successful ligation in the experiment would result in the insertion of the Kanr gene fragment into circular vectors producing recombinant plasmids. After subjecting the ligation reaction mixture to electrophoresis and observing its progression under a UV transilluminator, its recombinant plasmid band was measured at 0.7cm. The remaining ligation reaction mixture that was not used for electrophoresis was used for transformation, a process that separates the complex mixture of ligation products into individual bacterial colonies to purify it and indicate successful ligation. The Kanr gene served the purpose of a selective measure. Those vectors that were successfully transformed with the recombinant plasmid with the Kanr gene would grow on the LB plates containing kanamycin. The experiment yielded 1,200 transformants per μg of plasmid DNA, indicating that the experiment was successful.



Molecular Biology of DNA Amplification by PCR — Gastineau, Tracy; Luna, Paloma; Sparkman-Royo, Alexander
Professor: Dr. Nicole Pinaire

The objective of this experiment was to learn the general procedures of the Polymerase Chain Reaction and electrophoresis, observe the increase of product with increased number of cycles, and to quantify the length of the DNA fragment using a ladder of standard DNA fragments. The procedures of Polymerase Chain Reaction include a denaturation stage where the double stranded DNA is cleaved into two separate single stranded DNA strands, an annealing stage where primers and Taq polymerase attach to the strands of DNA, and an extension stage where oligonucleotides copy the DNA fragment in question. After PCR, the DNA is inserted into a gel along with a kilobase ladder that will undergo electrophoresis. Successful PCR and electrophoresis will show a dark band after staining. Measuring the distance that the band has traveled and comparing it to the ladder will show how many kilobases the DNA strand is. Once PCR and electrophoresis were undergone, the bands were measured to be at 2.7 cm from the well. From a log graph, the band were indicative of 760 bases for the DNA strand. The overall experiment was successful in that we were able to see the bands indicating that the Polymerase Chain Reaction was successful in isolating and amplifying the DNA fragment of interest. Overall results will be shown in person.



Comparing NADH Dehydrogenase Subunit 4 in Various Orthologues — Hassan, Nida
Professor: Dr. Brenda Whaley

NADH dehydrogenase subunit 4, also known as NADH-ubiquinone oxidoreductase chain 4 (Genbank accession number: YP_003024035.1, gene ID: 4538), is a mitochondrial protein in Homo sapiens (humans) that has 459 amino acids. The official abbreviation for the gene locus on the mitochondrial chromosome is ND4. NADH dehydrogenase subunit 4 is a multi-pass membrane protein found in the mitochondrion. The base pairs that make up the gene for this protein start on position 10760 and ends at position 12137 on the mitochondrial chromosome, including 1377 base pairs on the positive strand. This core subunit of the mitochondrial membrane respiratory chain NADH dehydrogenase (complex 1) belongs to the assembly needed for catalysis. It is involved in the transfer of electrons to the respiratory chain from NADH where the electron acceptor is ubiquinone. In this complex, two electrons are taken from NADH and transferred to ubiquinone that acts as a lipid-soluble carrier. Complex 1 moves protons across the inner mitochondrial membrane. This makes a proton gradient that facilitates the production of ATP by oxidative phosphorylation. A comparison of this protein’s sequence amongst orthologous proteins in other species, concluded that increasing taxonomic variation from Homo sapiens, leads to decreasing similarity in sequence. Orthologues that are from species with a close taxonomic relationship to Homo sapiens will have greater homology, quantitated by larger percentages of sequence identities. For example, when comparing NADH dehydrogenase subunit 4 chimpanzees to the Homo sapiens, the percent similarity in homology is 95% whereas when the Homo sapiens is compared to the fruit fly, the percent similarity in homology is 43%. Therefore, when comparing homology, results show orthologues in the animal kingdom that are closely related to Homo sapiens have similar homology.



Mitochondrial Protein: NADH Dehydrogenase Subunit 1 —  Ho Mei Ki
Professor:
Dr. Brenda Whaley

Mitochondria are organelles found in the cytoplasm of the cells of almost all eukaryotic organisms. NADH dehydrogenase subunit 1, otherwise known as NADH-ubiquinone oxidoreductase chain 1, is a mitochondrial encoded protein with the sequence length of 318 amino acids that is located in the inner mitochondrial membrane. It is the core subunit of the mitochondrial membrane respiratory chain NADH dehydrogenase (Complex I) and is believed to belong to the minimal assembly required for catalysis. Complex I functions in the transfer of electrons from NADH to the respiratory chain. The immediate electron acceptor for the enzyme is believed to be ubiquinone. The mitochondrial gene encoding NADH dehydrogenase subunit with the gene ID of 4535 is named as MT-ND1, also known as ND1 which is the official abbreviation for the gene locus. It is located from 3307 to 4262 on the mitochondrial genome. The sequence of NADH dehydrogenase subunit 1 which is the gene product of human mitochondrial DNA was compared with the similar protein found in other members of the animal kingdom, and the result showed that the similarity of sequences was highly conserved for the orthologous proteins in primates, but homology fell dramatically for sequences from species outside of the primate order.



Excitation Probabilities of a Time-Dependent Quantum System Interacting with an External Electric Field — Holmes, Joseph; Gardner, Brittnay; Do, Linh
Professor:
Dr. Gardo Blado

We conducted a computational study of a quantum system interacting with an external electric field using the time-dependent Schrödinger equation. In order to accomplish this, we solved time-dependent Schrödinger equation by applying the coupled-channel method. Once the Schrödinger equation was solved, we then applied our solution to a particle in a box interacting with a pulsed laser. This produced a graph depicting the excitation probabilities of the particle.



Spectrophotometric Titration of Iron(II) with o-Phenanthroline —  Leal, Franklin; Nguyen, Christine
Professor:
Dr. Eric Van Caemelbecke

The purpose of our experiment was to determine the stoichiometry of the reaction of Fe(II) with o-phenanthroline (o-phen). We tried to verify and replicate the literature stoichiometry of 3 moles of o-phen to 1 mole of Fe(II) using a spectrophotometric titration and mole ratio plot. Constructed mole ratio plot with linear regressions y = 0.4295x + 0.0082 and y = 1.31 yielded a stoichiometric ratio of 3.03, which compares favorably with the literature value of 3. We obtained a very good correlation factor for the very strongly binding linear part of our mole ratio plot on all of our trials with a value of at least 0.99 which is nearly linear. Through several trials and with careful preparation, delivery, and analysis of our solutions and instruments we were able to confirm the literature value with only 1% to 4% error for our trials.



Conservation of NADH subunit 4L Protein — Khan Lucero, Sadaf
Professor:
Dr. Brenda Whaley

NADH dehydrogenase subunit 4L is part of a large enzyme complex called Complex 1, and this subunit is believed to belong to the minimal assembly required for catalysis. Complex I is one of several enzyme complexes necessary for oxidative phosphorylation which are embedded in the inner mitochondrial membrane. Complex I is responsible for the first step in the electron transport process, the transfer of electrons from NADH to ubiquinone. Electrons are then passed from ubiquinone through several other enzyme complexes to provide energy for the generation of ATP. The accession number for NADH subunit 4L is YP_003024034 (GenBank), and the length of the protein is 98 amino acids long. This gene is located from 10470 to 10766 on the mitochondrial genome, 297 in length, and the locus name for the gene is ND4L. Defects in NADH dehydrogenase subunit 4L are a cause of Leber hereditary optic neuropathy (LHON). LHON is a maternally inherited disease that results in acute or sub-acute loss of central vision due to optic nerve dysfunction. Cardiac conduction defects and neurological defects have also been seen in some patients. The protein sequence of human mitochondrial-coded NADH dehydrogenase subunit 4L was compared to orthologues from other species in the animal kingdom. The similarities between the human sequence and that of other species fell significantly once the comparison was done with non-mammalian species. In particular, the similarities dropped down to less than about 50 percent as the comparison moved from mouse to chicken, zebrafish, and the fruit fly. This is similar to the homology seen for other Homo sapiens mitochondria proteins when their sequences are compared to orthologues in the animal kingdom.



Conservation of ATP synthase 6 subunit Fo Across Species Lines — Marachlian, Adriana
Professor: Dr. Brenda Whaley

ATP synthase is an enzyme that converts adenosine diphosphate (ADP) to adenosine triphosphate (ATP), thus providing organisms with a form of utilizable energy for different cellular functions, in or outside the cell. In eukaryotic organisms, ATP synthases are often found in the inner membrane of the mitochondrion, where much of the respiratory processes of the cell occur. ATP synthase 6 (ATP6) is one of these enzymes, which uses the proton gradient and electron transport machinery in the mitochondria to make ATP as well as to transport hydrogen ions across the membrane. ATP6 has two structural domains –F1, the catalytic domain, and Fo, the membrane proton channel domain. The Fo domain includes a subunit of 226 amino acids (YP_002124307.1) that is coded by the MT-ATP6 gene, which is found at positions 8527-9201 in the L-strand of the mitochondrial genome (Gene ID: 4508). The Fo subunit is of particular interest, because it has been found to be highly conserved in different species, such as Pan troglodytes and Mus musculus, when compared to humans. Also, the Fo subunit of ATP6 synthase is relatively conserved in lower species like Gallus gallus, Danio rerio, and Drosophila melanogaster. Furthermore, knowing the conservation level of ATP6 synthase Fo subunit is of significance in research because it dictates what organisms can be used to effectively study mutations in the MT-ATP6 gene, which is usually deficient in some neurological disorders, as well as ataxia and mitochondrial infantile bilateral striatal necrosis (MIBSN).



The Influence of Smoking on Heart Rate and Cardiac Functions Measured Using Electrocardiography Nisnisan, Marie; Medi, Sai Preteek
Professor: Dr. Rachel Hopp

An electrocardiograph exhibits how the heart is functioning by measuring the electrical impulses that are used to coordinate the heart’s characteristic pumping patterns. Electrocardiography is commonly used to assess cardiac function in clinical and research environments. This study explores the cardiac function of twelve college students between the ages of 18-25 years, half of the subjects were smokers and the other half were non-smokers. Subjects were asked to perform a series of physical activity conditions to influence their cardiac activity. An ECG was conducted after each participant performed the five following condition:lying down in a supine position, immediately after sitting up, hyperventilating for 30 seconds, performing twenty jumping jacks, and performing the Valsalva maneuver for 20 seconds. The Valsalva maneuver is a forced expiration attempt against blocked airways which increases intrathoracic pressure and has a characteristic effect on pulse and systolic Blood Pressure which could be monitored with an ECG. Results indicated that smokers were more responsive to the physical activity conditions in comparison with non-smokers, based on each individual’s ECG baseline. Smokers showed a larger differential in how their Q-T time intervals decreased compared to non-smokers when comparing responses from each condition. Smokers also exhibited a greater response to the effect of the Valsalva maneuver.



RNAi Regulation of Vab-10 in Caenorhabditis elegans — Olokode, Michael; Ajaroh, Egwono; Dickerson, Johana; Nguyen, Linh
Professor:
Dr. Rachel Hopp

The gene vab-10 is expressed in the nematode Caenorhabditis elegans. Vab-10, variable abnormal morphology, contains two spectraplakins, vab-10a and vab-10b. These spectraplakins are required for epidermal resistance under strain due to contraction of actin microfilaments linked to fibrous organelles. The human homolog of vab-10 is DST, dystonin, which when mutated in humans expresses bullous pemphigoid blistering disease. Previous experiments using RNAi regulation of vab-10 in C. elegans have resulted in slow growth, late embryonic arrest, protruding vulva and other phenotypes. PCR was performed to amplify vab-10 in constructing the feeding vector. To observe phenotypes, a feeding vector was prepared and inserted into RNase deficient E. coli. The C. elegans consume the E. coli thus the vector is successfully inserted into the worms. Knock down of vab-10 is expected to be observed in the C. elegans. When compared with bli-1 worms, vab-10 deficient worms are expected to express a similar phenotype. Results will be presented in the symposium.



Measuring Protein Solubility as a Function of pH — Olokode, Michael; Gastineau, Tracy
Professor:
Dr. Saul Trevino

Protein solubility is an important issue in the pharmaceutical industry and many biochemical studies such as x-ray crystallography or NMR. Protein solubility is expected to depend dramatically on pH. In this study, ammonium sulfate precipitation followed by UV-VIS spectroscopy was used to measure the solubility of the proteins Bovine Serum Albumin (BSA) and Lysozyme as a function of pH. The graphs for each protein showed that they do not follow the assumption that protein solubility follows a parabolic relationship. This suggests that the relationship between protein solubility and pH is more complex than previous studies suggest. Further testing is needed to obtain all solubility values over the entire pH range, and to further determine the relationship of protein solubility and pH. Hopefully this research will eventually help solve problems related to poor protein solubility in pharmaceutical and biochemical studies.



Examination of lipid homeostasis in Caenorhadhditis elegans by RNAi silencing of sbp-1BI — Patel, Sarin Mahesh; Medi, Sai Preteek; Thomasy, Dustin James
Professor:
Dr. Rachel Hopp

Sufficient management of energy supplies is a necessity for all living things. The prevalence of diseases such as diabetes, steatohepatitis, and obesity support the concerns and investigations regarding defects in energy storage, consumption, and more. Studies in model organisms, such as Caenorhabditis elegans have greatly improved our understanding of the development and process of diseases today. The C. elegans protein SBP-1, a homolog of human SREBP, has been identified as one of the transcriptional regulators of the entire fat and sterol pathway. Previous studies in RNAi silencing of sbp-1 show a negative effect in regards to C. elegans’ lipid homeostasis. In this study, the effects of RNAi silencing of sbp-1 in C. elegans were reexamined. Silencing of sbp-1 was carried out by first amplifying the sbp-1 gene sequence using PCR. The sbp-1 fragment was then inserted into pPR244 vector. Transformation was initially carried out in DH5 alpha cells for selection. Subsequently, the plasmid was isolated and transformed into HT115 cells for feeding. Observations of the preliminary results show that sbp-1 (RNAi) mutant C. elegans tend to be thinner when compared to wild-type worms of similar lengths. Additional experiments and trials will be performed to confirm these results and possibly observe the restoring effects of oleic acid.



Ants, Exposed! — Sakhuja, Shruti; Shahid, Urooj; Fonseca, Sharon; Hassan, Nida
Professor:
Dr. James Claycomb and Dr. Betty Thompson

The purpose of this research study was to demonstrate how tunneling and carbon dioxide production vary amongst ants that have been exposed to an electric field over an extended period of time. In order to conduct the tunneling experiment, 6 ant farms, with 15 ants in each farm, were placed between a parallel plate capacitor. At the end of the observation period, the lengths, widths, and number of tunnels in each farm were measured. The results obtained demonstrated that ants placed within the parallel plate capacitor tended to have fewer and shorter tunnels than ants that were not placed in the capacitor. On average, the total lengths of tunnels in ant farms that were under electrical stimulation were 38.68 inches. The tunnels that were not under electrical stimulation had an average total length of 44.30 inches. The average widths of each of the tunnels were almost identical. The average number of tunnels in the farms exposed to the electric field was about 8, whereas the average number in the farms not exposed to the electric field was 11. In order to perform the carbon dioxide experiment, 10 ants each were placed in 6 different jars for a 48 hour period, and a CO2 probe was used to measure CO2 levels. The results of this experiment indicate that ants exposed to an electric field produced much less CO2 than ants not exposed to the field. Ants placed between the parallel plate capacitors produced 586 ppm CO2, whereas ants in the control group produced 1432 ppm. The mean of CO2 production of the exposed ants was also lower than that in the control. Additional experimentation can be conducted to further corroborate these findings.



An Evolutionary Comparison of Cytochrome c Oxidase Subunit II in Animal Orthologues — Sakhuja, Shruti
Professor:
Dr. Brenda Whaley

Cytochrome c oxidase subunit II (GenBank accession number: YP_003024029.1, Gene ID: 4513) is a multi-pass inner mitochondrial membrane protein, containing 227 amino acids, that is found in Homo sapiens. The 683 base pairs that code for this protein are found at start position 7586 and end position 8269 on the positive strand of the mitochondrial DNA. The official abbreviation for the gene locus is COX2. Cytochrome c oxidase is a component of the electron transport chain in the cellular respiration process, and is involved in catalysis of the reduction of oxygen to water. Subunit II, one of the three cytochrome c oxidase subunits, specifically provides the substrate-binding site and is most likely the first electron acceptor in the electron transport chain. Subunit II of the enzymatic protein transfers the electrons originating in cytochrome c to the heme A and copper B bimetallic center of subunit I, using subunit II’s cofactor, copper A. If the cytochrome c oxidase subunit II protein is compared across different species lines, evolutionary similarities can be revealed. For instance, the chimpanzee, which is known to be closely related to the Homo sapiens species, demonstrated a 98% homology to the mitochondrial protein in Homo sapiens. Comparatively, the fruit fly, which is more distantly related to Homo sapiens revealed only a 59% homology. Therefore, orthologues from the animal kingdom that are more closely related to Homo sapiens exhibit a greater similarity in the cytochrome c oxidase subunit II protein of Homo sapiens.



Exposure to low levels of 17β-estradiol (E2) in vitro induces proliferation and enhances viability in MCF-7 and ZR-75-1 breast carcinoma cells — Slepicka, Bryan
Professor:
Dr. Betty Thompson

A variety of pharmaceutical estrogens and xenoestrogens (estrogen mimics) have been detected in varying concentrations in environmental surface waters. Many of these environmental estrogens have been introduced into the environment through the effluents of municipal waste water treatment plants (5). Once in the surface waters, the environmental estrogens are free to bind to and activate the estrogen receptor (ER) to induce its estrogenic effects in human and wildlife populations (1). Thus, the aim of this study was to determine whether environmental concentrations of 17β-estradiol (E2) in vitro, induces proliferation and enhances cell viability in the ER positive cell lines, MCF-7 and ZR-75-1. Based upon the data that was collected, low concentrations of 17β-estradiol were found to induce cell proliferation (concentration) and enhance % cell viability in ER positive MCF-7 and ZR-75-1 breast carcinoma cells in a dose-dependent fashion. The MCF-7 mean viable cell concentration for the 40 ng L-1 treatment wells on day 15 of a growth cure was 3.13 x 105. This represents a 1.75 x 105 difference in cell number for the 40 ng L-1 treatment wells as compared to the control, indicating that an environmental concentration of E2 induces proliferation in MCF-7 cells. In addition, the mean % cell viability for MCF-7 cells on day 15 ranged from 97.3% to 98.8% for the 40, 400, and 4,000 ng L-1 treatment wells, respectively. Experimental data collected for the ZR-75-1 cells also mimic these findings. Unexpectedly, the % cell viability in the ER negative MDA∙MB∙157 breast carcinoma cells also increased in response to higher concentrations of 17β-estradiol.



Human Mitochondrion Protein Cytochrome b and its Homology to Orthologous Proteins in the Animal Kingdom — Tadros, Mary
Professor:
Dr. Brenda Whaley

Cytochrome b is one of the 13 proteins encoded by the mitochondrion genome of Homo sapiens. The gene that codes for this protein is located on positions 14747-15887 of the mitochondrion genome and its GeneID is 4519. It is located on the positive coding strand and the gene locus can be abbreviated as CYTB. The length of the protein is 380 amino acids and its accession number is YP_003024038.1. Cytochrome b is as multi-pass membrane protein located in the inner membrane of the mitochondrion. It is a component of the ubiquinol-cytochrome c reductase complex (complex III or cytochrome b-c1 complex). This complex is a respiratory chain that generates an electrochemical potential coupled to ATP synthesis. It non-covalently binds two heme groups. Defects in the protein can cause mitochondrial dysfunction leading to different myopathies. Homology to orthologous proteins in the animal kingdom was studied to observe the conservation of cytochrome b. Percent identities observed revealed that cytochrome b is highly conserved in primates such as the Chimpanzee to a level of 94%. The percent identities of the sequences began to decrease as orthologous proteins in other animals outside of the primate order were analyzed, with the least similar being Drosophila melanogaster, the fruit fly with a percent identity of 62%.



Using RNA interference to silence ubc-20 in Caenorhabditis elegans for studies on Huntington’s disease — Tolbert, Jessica; Hlavaty, Victoria; Tavalaian, Venous
Professor: Dr. Rachel Hopp

Huntington’s disease is a genetic, neurodegenerative disease characterized by abnormal behavior changes, uncontrolled muscle movements, and dementia. This disease is caused by a (CAG)n, trinucleotide repeat, encoding glutamine, on the huntingtin (HTT) gene. RNA interference (RNAi) was the research technique used in this study to silence a huntingtin-associated gene in both mutant (with severely uncoordinated movements) and normal N2 Caenorhabditis elegans. RNAi of ubiquitin conjugating enzyme-20 (ubc-20) was performed in order to determine its relation to muscle coordination (a coordination similarly affected by Huntington’s disease) in C.elegans. Phenotypes characterized by Huntington’s disease are a result of the polyglutamine aggregation caused by the expanded, (CAG)n repeat. Huntingtin-interaction protein-2 (HIP2/UBE2K; homolog to ubc-20) interacts with HTT and plays a role in this polyglutamine aggregation. Based on previous studies, it is hypothesized that RNAi of ubc-20 will lead to a decrease in polyglutamine aggregation, and thus lead to more coordinated muscle movement in mutant worms and little change in normal worms. RNAi of ubc-20 for this experiment was conducted as follows: genetic sequences of ubc-20 were identified by OMIM/Wormbase and primer sequences were identified by E-RNAi web application. DNA of N2 worms were isolated and then PCR amplification of ubc-20 in C.elegans was conducted. BP clonase was used to insert cloned ubc-20 fragments into pPR244 vectors. This product was then transformed into E.Coli DH5alpha cells. Afterwards, a plasmid miniprep was performed, cleaning and isolating the DNA plasmid (containing the dsRNA constructs of ubc-20) from the DH5alpha cells. The purified plasmid DNA was then transformed into E.Coli HT115 cells. This RNAi feeding strain was then allowed to grow onto NGM-IPTG/Kan plates where N2 worms with normal phenotype and severely uncoordinated phenotype (unc-5) were introduced and observed for resulting phenotype after 48 hours. Results are pending and will be presented at the symposium.



The Homology of Cytochrome c oxidase subunit III within the Animal Kingdom, — Tran, Anh
Professor: Dr. Brenda Whaley

Cytochrome c oxidase subunit III is one of 13 protein coded by genes from the mitochondrial genome. It consists of 261 amino acids and the accession number is YP_003024032. The GeneID is 4514 and the locus is COX3. The COX3 gene begins at 9207 and ends at 9990 of the “+” strand of the mitochondrial genome. Cytochrome oxidase subunit III is one of three subunits that forms the functional core of Complex IV of the electron transport chain. Complex IV found on the inner mitochondrial membrane and cytochrome oxidase subunit III is part of the transmembrane domain. Complex IV mediates the transfer of electrons to oxygen and moves hydrogen ions across the inner mitochondrial membrane to ultimately facilitate the process of oxidative phosphorylation. The homology to the cytochrome oxidase subunit III human sequence gradually decreases to 66% as you compare it to Homo sapiens neanderthalensis (Neanderthal), Pan troglodytes (Chimpanzee), Mus musculus (Mouse), Gallus gallus (Chicken), Danio rerio (Zebrafish), Drosophila melanogaster (Fruit fly) in the animal kingdom.



RNA Interference of Mrp-1 on Caenohabditis elegans — Tran,Anh; Gastineau, Tracy; Marachlian El Yammouni, Adriana
Professor:
Dr. Rachel Hopp

The nematode Caenohabditis elegans expresses the gene Mrp-1, multidrug resistance-associated protein. Mrp-1 encodes an ATP-binding cassette (ABC) transporter most closely related to Mrp-1. RNAi regulation of Mrp-1 has been shown to cause phenotypes of worms being slimmer in size, abnormal vibrations, and hyperactivity. RNAi is the introduction of homologous dsRNA to target a gene’s product which results specific phenotypes. Wormbase and OMIM were used to identify the specific gene sequence of Mrp-1. The primers were determined by using the E-RNAi web application. Polymerase chain reaction (PCR) was used to amplify gene fragments of Mrp-1 from N2 worm DNA lysates. The PCR products were cloned into the pPR244 feeding vectors, an RNAi feeding vector was obtained from this. The cloned vector was transferred into a DH5alpha strain of E.coli, but contained RNAse that could degrade the RNA. Instead, HT115 was utilized since it lacked the RNAse. Mrp-1 gene was transformed into an HT115 (DE3) E.coli feeding strain and exposed to the worms. The results obtained showed that a majority of the worms expressed hyperactivity, were slender in size, and had irregular locomotive moments. Experiments shall be repeated to confirm results. Final results will be reported at the symposium.



Solution Hybridization Determination of Binding Ratios for Nucleic Acid Homopolymers — Tran,Anh; Ogega, Duke; Olokode, Michael; Mirza, Asma
Professor:
Dr. Robert Towery

Homopolymers of DNA and RNA have been useful as model systems in the elucidation of structure and properties of nucleic acids. Due to their commercial availability and well-known behaviors, polyadenylic acid (poly(A)) and polyuridylic acid (poly(U)) were used as complementary base pairs in this study. A Shimadzu 1501 Multiplex Diode-Array Spectrophotometer was used to determine an ultraviolet mixing curve which allowed detection of a duplex or a triplex in the solution mixtures. Two standard solutions, one poly((A)) 1.04 X 10^-4 g/L and the other poly((U)) 1.06 X10^-4 g/L were prepared in 0.14 M and pH 6.8 Phosphate Buffer. These two solutions were mixed in varying ratios according to the table below (as shown in the chart). After vortexing for 5 to 10 minutes, an absorbance reading at 260 nm was obtained for each mixture.The mol fraction of poly((A)) and poly((U)) were varied while the total nucleotide concentration was maintained at a constant amount. Triplex-forming DNA and nucleotides are known to downregulate and inhibit proliferation of cancer cells, and induce apoptosis.



Contractile Strength and Endurance as Related to Forearm Size in Females —  Delk, Stephanie; Kichline, Justin; Thomasy, Dustin; Valencia, Catherine
Professor:
Dr. Rachel Hopp

Muscle fibers are not individually controlled; they are directed in groups called motor units, each of which is activated by its own motor neuron. As the desired application of strength increases, more of these motor units are activated, increasing the force exerted. This activation process is referred to as motor unit recruitment (Phlanzer, 2004). At any time during a sustained contraction, some motor units will be contracting while others will be relaxing and recovering. This asynchronous recruitment allows the muscle tissue to maintain its strength over a greater interval of time than when undergoing a maximal contraction, in which all motor units are, activated simultaneously (Sherwood, 2007). This study explored the correlation between forearm size and motor unit recruitment therein. The purpose was to demonstrate the differences in muscle fatigue between physically active females and non-physically active females based on forearm size. An electromyogram (EMG) which recorded the changes in skin voltage due to skeletal muscle contraction was used to measure the magnitude of the contractions. Subjects repeated cycles of “clench- release- wait” with the hand dynamometer tool. The subject increased clench strength in equal increments so that the 4th clench was the maximum clench force. As the subject tried to maintain the maximum clench force, forearm muscles fatigued and the clench force decreased. Investigation measured the time of fatigue to 50% of the particular subject’s maximal clench force. Analysis of results concluded no correlation of arm fatigue based upon physical activity. Significant results based upon forearm size showed broad forearm size obtained a greater maximum clench force. Petite arm size showed greater fatigue while moderate forearm size showed the least arm fatigue. After statistical analysis of all data, we found significant evidence that forearm circumference correlated to the maximum clench force obtained along with rate of fatigue.



Influence of Peers and Behavioral Traits on Social Behavior and Underlying Neural Circuitry Valencia, Catherine
Professor:
BJ Casey, PhD

Risk taking behavior increases substantially in the presence of peers for many teens, but not all. It has been hypothesized that peers are secondary reinforcers that alter behavior, but this hypothesis has not been tested directly. In the current study we tested the role of peer interactions as reinforcers by using a task that parallels human and nonhuman primate studies of reinforcement learning. Specifically we tested whether peer interaction alters behavior and neural circuitry in a similar way as primary reinforcers (juice) and other secondary reinforcers (money). In addition, we examined whether personality traits (e.g., introversion/extraversion) influenced these changes. Differences in reward neural circuitry to the different amounts of peer interaction were measured using functional Magnetic Resonance Imaging (fMRI). Results show that participants who are extroverted show greater effects of interaction while introvert participants do not show as big an effect. We hypothesize that greater enhanced change in activity for reward neural circuitry will be present for outcomes with greater peer interaction. This data will give us a better perspective on why certain adolescents associated with key personality traits are more likely to engage in risky behavior in the presence of others. Our preliminary findings suggest that heightened risk taking in teenagers is likely to increase in those with extroverted personalities and be affected by those peers with whom they positively interact the most. A better understanding of how personality traits factor into such behavior can give way to prevention of health-risk behavior consequences among adolescents by helping identify those individuals at greater risk.



Construction, Calibration, and Deployment of an On-campus Weather Station — Winters, William
Professor:
Dr. Robert Towery

This research project deals with the construction, calibration and deployment of an on campus weather station. The main goal of this project is to better prepare faculty and students of Houston Baptist University for developing weather issues. In constructing the station, from Davis, preliminary thoughts centered around strictly weather related research. In collecting the data of temperature, wind speed and direction, humidity, wind chill, heat index, dew point, barometric pressure, and rain fall the University could monitor weather unique and absolutely applicable to its position in terms of relation to an incoming front and/or storm system. Creating a database with the weather data gathered, was used to determine short term and long term accuracy and reliability of our weather station. However as the research project proceeded, it took on a more personal stance. As bad weather and/or high winds approached the University we were concerned and anxious as to how durable the station is and how it would respond. Hopefully the installation of our station has laid the ground work and opened the door for students and for future classroom activities involving and relating to weather and climate.



PROPOSED RESEARCH



Characterizing the effects of proline to threonine point mutations in RNase Sa to investigate the mechanism of cataract formation — Cook, Rebecca; Lu, Vinh; Kumar, Ricki; Alonso, Nicolas; Dickerson, Johana; John, Julie; Mirza, Asma
Professor:
Dr. Saul Trevino

The aggregation of the P23T mutant Human-γ-D-Crystalline (HGD) plays a role in various forms of childhood cataracts. Studies suggest that while there is no change in HGD structure, the proline to threonine mutation results in significantly decreased solubility, which leads to cataract formation. The mechanism by which this protein aggregates is not well understood. In order to further characterize the effects of proline to threonine mutations in proteins, we propose to use RNase Sa as a model system. RNase Sa is a small protein with 96 amino acids that is well-suited for characterizing the effects of mutations. We plan to use site-directed mutagenesis to mutate the six proline residues in RNase Sa to threonine residues. We then plan to characterize the six proline to threonine point mutations of RNase Sa in terms of changes in solubility, structure, stability, and other information related to residue location. We intend to apply our findings to elucidate the mechanism of aggregation in the P23T HGD mutant and cataract formation.



Small Scale Application of the Curlometer Technique Lao, Jordan; Al Hassan, Naila; Kabiru, David
Professor:
Dr. Gardo Blado

Physicists implement the curlometer technique to indirectly measure the current density J, by calculating the differential form of the magnetic field, B. This technique’s primary application is to measure the current density produced in space by a planet’s external magnetic field. By establishing non-planar geometry, that is to say a three dimensional configuration of magnetic sensors, Stokes’ Theorem can be utilized to calculate the magnetic flux through the normal surface. Stokes’ Theorem states that the flux through a boundary is equal to the loop integral; as such, the loop integral of the magnetic field can be calculated around each face to determine the flux through that surface. In the simplest configuration, four magnetic sensors are used to create a tetrahedron. Since accurate calculation of the loop integral depends on the distance between the magnetic sensors at any given time, configuration of the geometry is of utmost importance. Therefore, on a small scale, this experiment works best with a regular tetrahedron in which each sensor is placed equidistantly. A current carrying wire is run through the middle of the configuration to simulate the magnetic flux. Using LoggerPro, the magnetic field can be measured at each point in the tetrahedron. Then, using the curl of the magnetic field, the current density can be determined using Ampere’s Law (µ0J= ∇xB). The divergence of the magnetic field, calculated by analyzing the three dimensional components of the magnetic field, can be used in conjunction with the curl to provide the percentage deviance from zero to ensure accuracy. Therefore, it is the goal of this experiment to measure the magnetic field to find the curl and indirectly measure the current density. In such a scenario, the curlometer technique would be applied in a small scale laboratory setting to elucidate the differential form of Ampere’s Law.