All organisms require the pyridine nucleotides NAD(P)(H) to perform various cellular functions, but their role in methylotrophy is not well understood. Methylotrophic organisms are able to use organic substrates without carbon-carbon as a sole source of energy and carbon. In metabolism, pyridine nucleotides provide reducing power for the cell to perform redox reactions in catabolic and anabolic processes. In contrast to energy-limited modes of growth, methylotrophy is predicted to be reducing power-limited as the oxidation of methanol or methylamine is coupled to phosphorylation of ATP and energy production. Furthermore, internal concentrations of NAD(P)(H) are predicted to be saturating so the balance between catabolic and anabolic charge ratios may be of greater importance than the relative concentrations. Consistent with these predictions, fluctuations in NADPH levels are observed during as an immediate response when the facultative methylotroph Methylobacterium extorquens AM1 is transitioned from multi- to single-carbon substrates. Known mechanisms to recycle pyridine nucleotides include the decoupling of NAD(P)H using NAD(P) kinases/phosphatases or transhydrogenases and the use of enzymes with dual specificity or isoenzymes. We propose a transhydrogenation cycle between the EMC and PHB pathway to be an important mechanism to maintain redox homeostasis in M. extorquens AM1. To demonstrate this, I will use select OMIC approaches to analyze wild-type strains during succinate to methanol transitions and compare wild type to an mtdB mutant, a strain without a functional methylene-H₄MPT dehydrogenase B (MtdB). In this mutant, the assimilatory blocks on methanol and methylamine are presumably due to the disruption of a key pyridine-nucleotide producing step. These results will provide increased understanding of the mechanisms used to balance production with consumption of redox equivalents and re-distribute carbon flux in M. extorquens AM1. Insight from this work has implications for engineering methylotrophs for biotechnological applications including the production of biofuels and value-added chemicals.

This study investigated the effects of environmental pollution in Elliott Bay, central Puget Sound, by analyzing the microscopic biota collected from the sediment-water interface, in summer 1998 and 2007. The focus was on Foraminifera, single-cell microorganisms with shells made of calcium carbon or agglutinated sediment.  Many Foraminifera are sensitive to changes in the physical parameters of their environment and thus, they can be good indicators of pollution. Elliott Bay is surrounded by the city of Seattle with its large maritime industrial presence. City development and economic growth near Elliott Bay led to a focus on environmental concerns. On the southern shoreline of the Bay there are many heavy industries and Federal Superfund sites that had discharged large amounts of industrial pollution from the Duwamish River and Elliott Bay. In our study, sediment samples collected by the Washington State Department of Ecology were processed and the assemblage of Foraminifera species were obtained from  each of 16 samples. The species composition, species richness and density (number of individuals per sample) for each sample were analyzed.  Using this data, comparisons were made between those samples collected in 1998 and those collected from exactly the same sites in 2007. The results show that there are 12 different species found in the Bay, and the assemblages are dominated by the species Elphidiella hannai, Haplophragmoides planissimum, and the pollution-tolerant Eggerella advena. Large population of Eggerella advena in Elliott Bay, and the exclusion of other less tolerant species, indicates a physically stressed environment. By comparing foraminiferal assemblages from Elliott Bay with samples from less anthropogenically impacted locations within the Puget Sound, we can assess the degree of environmental damage to Elliott Bay.

Sleep apnea is a common syndrome where a person has an irregular breath rate only during sleep, making it difficult to be detected and diagnosed. Yet, in the U.S. about two percent of children are affected by this syndrome and thus at risk of some health issues. There are some prevalent breath detection products on the market but they remain inaccessible due to the undesired prices and limited range of detection. In the breath detection project I was participating in, we intended to detect the breath rate by wireless technology, e.g. home-based WiFi. Our design would ease the cost of production by zero assembly and installation of the hardware components and would achieve the detection for anywhere inside a house (whole-home detection) by wireless signal transmissions in line-of-sight, none-line-of-sight, and through-the-wall scenarios. The detection system broadcasts the wireless signals in a home environment, receives the reflected signals from the targeted person, and then computes the frequency of the target’s breathing over a period of detection. We expected that frequency plots would be uniform for a normal person but irregular for a patient with sleep apnea. One practical implication of performing such frequency analysis in the whole-home environment is that children’s health monitoring can still happen without parental interference.

This study aimed to analyze benthic foraminiferal assemblages to determine if environmental changes from pollution and other factors have affected the health of the Bremerton area marine ecosystems. Foraminifera are shelled, single-celled microorganisms that can act as good indicators of environmental change. Sinclair and Dyes Inlets border Bremerton and are connected by the Port Washington Waterway in Kitsap County, Washington. Bremerton is the home of Puget Sound Naval Shipyard and is listed as a federal Superfund site due to accumulations of industrial pollution. Additionally, agricultural and residential pollutants have been released into Dyes Inlet from local streams. Foraminifera were collected from 40 sediment samples provided by the Washington State Department of Ecology from 1998-2012. Samples were analyzed for foraminiferal species composition, diversity, and density (number of inidividuals/gram sediment). Results show 22 species were found, however most samples had low diversities and densities. Ten samples contained no foraminifera. In addition, numerous euhedral gypsum crystals were found in 13 samples in both inlets. Gypsum is a calcium sulfate mineral, and occurrences of subaquatic gypsum crystals in non-evaporitic settings are unusual. Samples with gypsum showed either lower foraminiferal densities or no foraminifera present. Sulfur isotope analysis of the crystals indicates they formed in a sulfate-reducing environment. The comparitively high Total Organic Carbon (TOC) values in gypsym-containing samples suggests that decomposition of organic material may lead to this sulfate-reduction. Additionally, significant dissolution observed in the calcium carbonate shells of many foraminifera indicates pH values of the sediment were low. This can also result from the degradation of organic matter. Thus, foramiferal assemblages within Sinclair and Dyes Inlets are responding markedly to conditions in sediments, establishing the efficacy of using foraminifera as a tool for monitoring benthic ecosystems in Puget Sound.

Patients with Alzheimer’s disease experience progressive loss of memory and thinking skills, gradual declines in the ability to carry out simple tasks and, in severe cases, full blown dementia. The brains of patients with Alzheimer’s accumulate extracellular plaques of a family of beta amyloid peptides (strings of amino acids cleaved from a larger protein called Amyloid Precursor Protein) in multiple regions of the cerebral cortex resulting in losses of synapses and neurons. Given the fact that beta amyloid peptides have been evolutionarily conserved, our group became interested in the potential normative roles of these peptides. Observations by a member of the Martin lab led to the hypothesis that small concentrations of beta amyloid may have a proliferative effect on cells. My project explores this idea with experiments that compare the proliferative effects of various members of the beta amyloid family and dose-response experiments that determine optimal concentrations for proliferative effects. In my experiments, various concentrations of beta amyloid 1-40 (and, as controls, comparable levels of a scrambled beta amyloid peptide 40-1) are added to a standard culture medium along with 10% embryonic serum (serum without detectible levels of beta amyloid peptides) and established required growth factors. The cells’ division rates within those two week periods are determined for individual clones though crystal violet staining. From these initial experiments, we have concluded that cells are indeed able to divide more rapidly in media that has beta amyloid 1-40 present and that further attempts to determine optimal concentrations should focus within the range of 1-10 nM and above 10 nM to the point at which we anticipate evidence of toxicity. Future experiments will probe changes in gene expression resulting from treatment with optimal concentrations of beta amyloid for the enhancement of cell proliferation.

The purpose of this project is to develop and refine a technique for taking photographs to be included in an atlas of the foraminifera of Puget Sound. Foraminifera are shelled single-celled organisms that live at the sediment/water interface in marine environments. Their sensitivity to environmental conditions makes them useful in monitoring ecosystems on the sea floor. The atlas will contain photographs and detailed descriptions of each foraminifera species. Despite their importance as an environmental monitoring tool, foraminifera have never been comprehensively studied in Puget Sound. This catalogue will become a useful tool for future workers in research and environmental monitoring. Key to this atlas is the inclusion of clear, accurate photographs of each species of foraminifera. Scanning Electron Micrographs yield excellent images, but these often do not portray the organism as it is viewed through a light microscope. Thus, the inclusion of photomicrographs is essential. Photography becomes quite technical when dealing with objects of a microscopic scale while still trying to produce a clear image with depth of field. With the Puget Sound Foram Project we work with specimens that can be as small as a tenth of a millimeter in size, so producing a high quality publishable picture takes time and a specialized technique. Using a light microscope, each specimen has to be photographed several times, focusing at a different depth each time. Once all of the layers have been captured, the pictures are then synchronized and edited in such a way that we get one final image that has both clarity and depth of field. In addition, each specimen must be photographed from two or more angles to illustrate critical features. Photography for research purposes can be a complicated and lengthy process but is essential for not only supplementing data but conveying vital information.

X-linked Myotubular myopathy (XLMTM) is a congenital myopathy caused by mutations in the myotubularin (MTM1) gene. Clinical symptoms of the disease including muscle weakness, respiratory failure, and hypotonia. XLMTM is caused by deficiency of the protein myotubularin necessary for normal muscle excitation-contraction. Dogs harboring a canine MTM1 gene model the disease and provide valuable insight due to phenotype similarities with patients. Affected dogs exhibit muscular atrophy, weakness, and stilted gait. A neuromuscular assessment score was developed to measure neurologic impairment in affected dogs. My project involved validation of this previously tested neurological assay by an independent observer. I was the independent observer who reviewed and scored neurological examination forms. I also reviewed and scored videotaped examinations and conducted statistical analyses looking at the correlation of the results within groups of dogs and between groups of dogs. Intra- and inter-reliability were performed by another observer and myself and wild type (n=6) and affected dogs (n=4) were scored. Prior to beginning the study, I received training on the assessment scoring system, including how to perform and analyze clinical tests. The intra-reliability test involved repeated examinations of wild type dogs and generated a consistent score in 88% (46/52) of exams based on interpretation of written neurological examinations and 100% (9/9) of exams based on scoring videotaped examinations at two time-points. The inter-reliability tests generated a consistent score in 54/71 examinations (76%) based on scoring of pre-recorded neurological exams and generated a consistent score in 12/15 cases (80%) based on scoring video-taped exams. The two observers scored dogs within 1 point in 68/71 (96%) of cases based on written notes of the neurological examination and within 1 point in 15/15 (100%) of videotaped examinations. Together, these data support the reliability of the neurological assessment score in XLMTM dogs.

Duchenne muscular dystrophy (DMD), is a degenerative disease caused by a recessive X-chromosome mutation, resulting in lack of the protein dystrophin. Affected males experience profound muscular weakness in childhood and by adolescence the heart and respiratory muscles are affected. The Golden Retriever muscular dystrophy (GRMD) dog model displays phenotypes analogous to patients and is considered the premier preclinical model. We evaluated changes in limb function by assessing gait in GRMD (n=3), wildtype (n=2), and in GRMD dogs injected with an adeno-associated virus carrying a dystrophin replacement gene in a single limb (n=3). Dogs were walked at a self-selected pace along an instrumented carpet to measure gait parameters. The average velocity over time of non-injected GRMD dogs was 60.39% of wildtype. Treated dogs displayed slightly increased velocities (67.17% of normal). By the final timepoint, velocity in all groups was comparable. The average paw pressure of the non-injected GRMD group increased 8.05%, while the average paw pressure for the wildtype decreased by 12.75%. The ratio of mean paw pressure for untreated GRMD: wildtype increases from 0.819 to 1.014 over time. We therefore determined that paw pressure might be a sensitive indicator of a treatment effect. To estimate the minimum sample size required for future studies, a power of analysis was performed. Results indicate that n=14 is required to detect differences between groups with a confidence level of 5% and power of 80%. Given that human patients suffer multiple symptoms of muscular weakness analogous to the dogs, including reduced limb function, changes in treated GRMD dogs as measured through gait analysis may evaluate the efficacy of the gene therapy as treatment for DMD. Human patients suffer multiple symptoms from muscular weakness (non-ambulatory, cardiac impairment, etc). Future studies hope to transition treatment to human patients to improve ambulation and increase quality of life.