The development of simple and affordable diagnostics is critical to the diagnosis, monitoring, and management of disease. Nucleic acid amplification has become the gold standard for many diagnostic applications due to its high sensitivity, specificity, and low limit of detection. Fluorescent dyes typically detect the success of amplification, but require expensive optical equipment including lasers and filters. A cheap and visually distinctive method for detecting nucleic acid amplification will simplify and lower the cost of molecular diagnostics. This research project aims to develop a simple colorimetric reaction for detection of loop-mediated isothermal amplification (LAMP) reactions. LAMP produces large amounts of pyrophosphate by-product during amplification. Pyrophosphate complexes many metals and can displace dyes bound to metals in solution to create color changes. Our goal is to identify a set of chemicals consisting of colored dye and metal ions that will present an unambiguous color change denoting success (or lack thereof) of amplification. Using knowledge of metal-ligand formation constants, we have determined a group of metal-dye complexes that may be viable candidates for detection and are currently testing them in the laboratory. One example is indigo carmine dye. In the presence of pyrophosphate, the dye stays blue (a positive test), while the lack of pyrophosphate will turn the dye clear (a negative test). We are in the process of fully understanding the chemistry of this reaction and further optimizing the process to improve its viability. Exploration of other metal-dye complexes, as well as continuing investigation of the literature, will be conducted to improve the present results. This colorimetric reaction has the potential to simplify and lower the cost of DNA detection.

Mitochondria are essential organelles in eukaryotic cells and are most commonly known for their prominent role in energy production. In addition, mitochondria are involved in other important cellular processes such as lipid biosynthesis and apoptotic cell death. Mitochondria also have dynamic properties including fusion, division, and movement, which are all essential to maintain mitochondrial function. Given the plethora of essential cellular activities that require mitochondrial function, it is not surprising that defects in mitochondrial function are implicated in many different human diseases and disorders including diabetes and myopathies. Mitochondrial movement is important for the distribution of mitochondria, which is important in all cells, but especially important for asymmetric cells such as neurons. Transport is also required for mitochondrial fusion – the two organelles must move close enough to each other to fuse. Mitochondrial fusion in particular is required to maintain mitochondrial DNA and therefore, mitochondrial function. We are interested in the mechanism of mitochondrial movement and the connection between mitochondrial fusion and transport. Mitochondria move on microtubules using a complex of proteins. Miro 1 and Miro 2 are anchored to the mitochondrial outer membrane and interact with two adaptor proteins, TRAK1 and TRAK2. The TRAK proteins also interact with Kinesin, which binds to the microtubules and mediates microtubule-directed movement. Understanding the assembly and regulation of this complex and the stoichiometry of its components will provide insights into the mechanism of microtubule-directed mitochondrial movement. Our goal is to clone TRAK1 and TRAK2 complementary DNA (cDNA) into expression vectors for protein production in Escherichia coli cells and then to develop a purification protocol for TRAK1 and TRAK2. This will facilitate biochemical analysis of the mitochondrial motility complex.

Acute viral respiratory infections are responsible for much of the infectious disease in the world. Of the known respiratory viruses, influenza is probably the most dangerous. In addition to the relative lack of knowledge on how influenza produces systemic symptoms, little is known about how it affects the central nervous system (CNS) and subsequent recovery outcomes. Following an acute viral infection, the body undergoes a state of acute stress response (APR). APR is a complex array of physiological responses that leads to changes in behavior and body temperature, anorexia, immobility, sleepiness, and malaise. Anecdotal and personal experience suggests that sleep becomes more prolonged while one is battling influenza infection. Following an infection, cytokines and other inflammatory regulators affect sleep and body temperature caused by influenza. Cytokines are signaling molecules that are generally involved in the immune response following trauma or infection. IL1β, a focus of this study, is a particular cytokine that has been extensively studied for its involvement in physiological sleep regulation in normal healthy organisms and in those subjected to an immune challenge. IL1β alters the sleep profile post-infection by enhancing non-eye rapid movement sleep (NREMS). Such increase in NREM sleep has been demonstrated to increase the probability of survival due to microbial challenge in rabbits. I will focus on the involvement of IL1β and its receptor (ILR1) in influenza-induced NREMS APR responses. A transgenic mouse strain that selectively expresses IL1β receptors on neuronal cells in the brain will be used for this study. I hypothesize that ILR1 on neuronal cells in the brain is necessary for the virus-induced APR following an infection. Whether more sleep (specifically NREMS) promotes, as enhanced by IL1β, and less sleep impairs, recovery from infection remains to be determined.

Solar power is an increasingly significant resource as we look to move from fossil fuels to a cheap and sustainable source of energy. Solar cells can be manufactured using organic semiconductors by using cheap roll-to-roll printing techniques, however, the efficiency of such organic photovoltaic (OPV) devices is currently too low for cost-effective implementation. These devices utilize a built-in electric field, resulting from the difference in potential energy between the device’s electrodes, to collect photogenerated charges at the electrodes as current. Fine-tuning of the electrode energy levels also dictates the amount of voltage that can be generated by the device. As a result, the ability to chemically alter the electrode energy levels can greatly influence the overall power output of the solar cell. Phosphonic acid self-assembled monolayers (SAMs) are molecules that can be chemically attached to a variety of electrode interfaces and have been shown to alter the energy levels of certain electrodes. I am investigating the effect that attaching SAMs to the zinc oxide surface of an OPV electrode will have on both the built-in voltage and power conversion efficiency of an OPV cell. In order to test the hypothesis that the SAMs can affect OPV performance, I have fabricated solar cells with SAMs in order to compare them with solar cells without SAMs. Experiments that measure the built-in voltage of the device and investigate the lifetime of photogenerated charges will help to clarify the specific role that the SAMs play in affecting OPV device performance. If this hypothesis is correct, we have the potential to achieve higher power conversion efficiency than what is currently reported in these devices and this may lead to a greater emphasis towards understanding the role of interfaces in developing sustainable solar technology.

Childhood Apraxia of Speech (CAS) is a rare, severe, persistent pediatric motor speech disorder of largely unknown genetic cause that affects the ability to acquire intelligible speech and coordinate the volitional production of sounds by sequencing muscle movements for the production of words. Developmental Dyslexia is a common and specific childhood learning disorder defined as a significant impairment in reading ability that cannot be explained by deficits in intelligence, learning opportunity, motivation, or sensory acuity. Various dyslexia studies suggest that slowed global processing speed, especially when sequential modes are involved, may be a brain-based endophenotype of dyslexia under genetic control. Impaired sequential processing, hence, may be a biomarker shared by CAS and Developmental Dyslexia. Our group recently identified a de novo heterozygous deletion of a single gene in a proband with severe CAS, raising the question whether similar deletions are causal in other cases as well. Here we report a genome-wide Single-Nucleotide Polymorphism (SNP) and CNV findings in a cohort of 20 affected individuals in nine multigenerational families with well-characterized CAS and six unrelated individuals with Developmental Dyslexia. All participants met clinical research criteria for CAS as part of a larger study on familial motor speech sound disorder or Developmental Dyslexia. The genome-wide copy-number variation study of the selected individuals was completed using the Illumina HumanOmniExpress-24 BeadChips and Human Exome BeadChips. Analysis of the SNP content and CNV for overlapping variations across the genomes of these affected individuals give us insight into genomic regions associated with the clinical phenotype of sequential processing deficit in multiple domains.

The toll-like receptor (TLR) proteins are expressed on cells of the innate immune system and recognize structural motifs present on microbial pathogens. Recognition of pathogens by TLRs activates an intracellular signaling cascade and activation of NF-kB, a transcription factor that is critical for expression of inflammatory mediators. Previous studies from our laboratory have shown that single nucleotide variations present in human TLR1 lead to altered expression of TLR1 and are associated with increased risk of death in patients with sepsis. We tested the hypothesis that variations of mouse Tlr1 which correspond to common human variants would result in comparable changes in TLR1 activity. We used site-directed mutagenesis to introduce coding variations in the Tlr1 cDNA. After transfecting the variants into cultured human cells, we stimulated them with Pam3CSK4, a triacylated lipoprotein known to be recognized by the TLR1/TLR2 complex. We then used a bioluminescent luciferase reporter system to measure the levels of NF-kB transcriptional activity. Tlr1 with an Ile allele at position 605 showed increased activity relative to a Leu allele, and the Tlr1 with a Ser allele at position 605 showed increased activity compared to Leu, but to a lesser degree than the Ile allele. Tlr1 with a Ser allele at position 251 showed increased activity compared to Asn, but elucidating its specific effect will require further investigation. These results indicate that variations in the transmembrane domain of mouse TLR1 result in alterations in function comparable to that of human variants in the same region. These findings suggest that mouse models may be used to study TLR1 variants’ effect on the immune response in humans. This could lead to a better understanding of the mechanisms leading to poor clinical outcomes for patients with sepsis and other inflammatory diseases.

Traumatic Brain Injury (TBI) is a leading cause of mortality and permanent neurological disability. Microglial activation and inflammation contribute to the onset and progression of TBI-induced pathology. In this study we aimed to identify the factors that activate brain inflammatory pathways in the early responses to TBI, and the mechanisms by which inflammation is sustained during the late stages of pathology. We have previously demonstrated that the microglial expressed receptor, CX3CR1, regulates microglia activity. We have also shown that lack of CX3CR1 leads to deficits in cognitive function and synaptic plasticity. Here we hypothesized that the loss of the CX3CR1 receptor - and consequently, the loss of normal microglia function - would increase pathologic conditions in the brain after TBI and cause measurable defects in behavioral and cognitive abilities. To this end, three month old male CX3CR1 wild type and CX3CR1 knockout mice were subjected to a mild TBI (0.5mm depth) using a controlled cortical impact (CCI) animal model. Control mice (sham operations) received the same anesthesia and craniotomy, but were not subjected to CCI. All animals were humanely euthanized and transcardially perfused at fifteen and thirty days following TBI. I specifically participated in assisting TBI surgeries, behavioral testing, transcardial perfusions, brain sectioning, and immunohistochemistry. Our results indicate that lack of CX3CR1 at early time points protects neuromotor function, reduces cell death, and is associated with expression of microglia that have an anti-inflammatory phenotype. However, the long term effect of CX3CR1 receptor loss following TBI induces neuronal cell death, as well as neurobehavioral and cognitive deficits, which are associated with an increase in expression of microglia that have a proinflammatory phenotype. These results point to the presence of CX3CR1 in the early stages following a TBI as essential in slowing the progression of the neuropathology. In conclusion, CX3CR1, the fractalkine signaling receptor, plays a central role in regulating neuroinflammation after a TBI.