Life is abundant on planet Earth but we have relatively few locations in which we can study environments similar to those on other celestial bodies. One possible terrestrial feature that is similar to those on some of the moons of Saturn and Jupiter is Lake Vostok, located under 4000 meters of ice in Antarctica. If we can prove that life can survive in this desolate environment that has been covered by ice for the last 35 million years, there is a heightened chance that extraterrestrial life exists within our own solar system. To study this area, we are designing a probe that will melt through the ice and take measurements from the lake to determine if bacteria or other life forms can survive in such a harsh environment. The main goals of my project were to create a mechanism that will allow the probe to move vertically in the lake via user control, determine the optimal cable and wire storage method for such a large endeavor, and eventually design a housing for the probe that can withstand immense pressure without leaking. I did this by using 3D computer aided design software and pressure and stress analysis tools. From these designs, I manufactured actual prototypes to test my concepts. Eventually, this work will lead to a functioning probe that will survey Lake Vostok and other sub-glacial lakes in an attempt to find evidence of life.

Salmonella enterica serovar Typhimurium is a Gram-negative pathogen that causes gastroenteritis. Components of the outer and inner membrane, characteristic of Gram-negative bacteria, contribute to Salmonella’s mechanisms to avoid detection and killing by the host immune system. The anionic bacterial cell surface is disrupted by cationic antimicrobial proteins (CAMP) that increase outer membrane (OM) permeability and access to the inner membrane. To resist CAMP killing Salmonella activates the PhoPQ two-component system, which is essential for pathogenesis. Because many PhoPQ-regulated enzymes are dispensable during infection, we conducted a forward genetic screen that identified new components of the PhoPQ regulon involved in maintaining the OM permeability barrier. Sequencing revealed mutations in genes encoding a variety of membrane proteins including transporters, phospholipid and lipopolysaccharide biosynthetic enzymes and efflux pumps, as well as regulatory proteins. Amongst the results, yejM mutants expressed the highest levels of membrane damage. YejM is an inner membrane protein with a periplasmic enzymatic domain that is critically involved in maintaining the OM permeability barrier. Structural alignments suggest that YejM is a member of the alkaline phosphatase superfamily. Attempts at full length deletions of yejM were unsuccessful suggesting the transmembrane domain is essential for viability, whereas periplasmic deletion mutants were viable but expressed varied levels of OM damage. Complementation with periplasmic deletion mutants and plasmids carrying the periplasmic domain rescued the OM permeability defect. Using structural models generated by a Protein Homolog/analogy Recognition Engine (PHYRE), we located putative metal binding sites involved in catalysis. To investigate the enzymatic mechanism, specific metal-binding residues were mutated via Polymerase Chain Reaction. We aim to determine if these residues contribute to the OM permeability defect of the yejM mutant Salmonella.

Conventional belief has held that the icy regions of our solar system and planet are too cold to support life. However, new research has suggested that below the surface of some moons, like Saturn's Enceladus, lay pockets or oceans of liquid water. In addition, lakes, rivers, and whole ecosystems have been discovered beneath glaciers, areas previously thought devoid of such features. Unfortunately, accessibility remains a primary challenge to further study of these areas. One Applied Physics Laboratory experiment aims to provide a simple and practical alternative to current ice bore-hole technology by developing a heated probe that simply melts through the ice collecting data as it descends. As part of this project, I designed and developed a thermal temperature string, to be deployed behind the probe as it descends, to monitor the conditions of the glacier at depth. The result is a sensor array capable of providing data on the internal glacial temperature for prolonged periods of time. The thermal string consists of a number of sensors evenly spaced along a 100-meter bus wire and controlled by a microcontroller. This microcontroller samples the temperature of the ice at each sensor and stores the resulting data in onboard memory. Currently, the prototype setup transmits the data to a computer, which employs software I have written to interpret this data and display the sensor temperatures. In the future, the software will apply a quadratic calibration correction to achieve expected accuracies of ±0.05°C or better, compared to ±0.5°C without it. We plan to use this sensor array as part of the available instrumentation onboard the probe in places like the Greenland ice sheet or Antarctica. The design can also be modified for other types of sensors or experiments.

Retrotransposons exist as genetic elements within chromosomal DNA that can replicate themselves and move to new locations within a genome, using a process involving an RNA intermediate. They are a widespread class of transposable elements, abundant in plants, fungi, mammals and yeast. One type of retrotransposon is a Long Interspersed Nuclear Element (LINE). Unlike most retrotransposons, LINEs have no long terminal repeats (LTR) and they possess a poly A tail which defines the 3’ terminus of the element. They are several kilobases long and contain two open reading frames (ORFs) encoding a gag protein (ORF1) and endonuclease and reverse transcriptase domains (ORF 2) conferring the element’s ability for retrotransposition. Due to incomplete insertion attempts, many 5' truncated copies of LINE elements are present in the Rhododendron genome. A family of LINE elements called RLINE and several related 5’ truncation fragments have been discovered in Rhododendron williamsianum. I used PCR primers to amplify the regions of the genome in R. williamsianum where truncated RLINE insertions were found. The amplified DNA was then sequenced by Sanger sequencing. I am analyzing ten 5’ truncation fragments ranging from 692- 2918 base pairs. By phylogenetic analysis of aligned sequences common to the various full-length and deleted RLINES, I will endeavor to determine the evolutionary relationships of the truncated fragments to the full length RLINE and to one another. So far, I have sequenced and compared 8 different RLINE fragments of Rhododendron williamsianum, and will continue characterizing RLINE insertions.