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Complete resection is correlated to better patient outcome in aggressive cancers such as glioblastoma. Optical biopsy refers to a family of techniques utilizing optical properties of living targets to make diagnoses where a biopsy would conventionally be used. Such a technology can potentially guide neurosurgeons in removing glioblastomas. Diffuse reflectance (DR) and Time-resolved fluorescence (TRF) have previously been investigated for their ability to measure biomarkers indicative of cancer. One of the difficulties faced in using TRF as a diagnostic tool is that multiple endogenous fluorophores will simultaneously contribute to the signal. This makes it difficult to attribute fluorescence lifetimes or spectral changes to one type of molecule in the tissue. This thesis focuses on the challenge of separating the components in a TRF measurement and their fractional contributions. A DR-TRF instrument was designed and built and characterized using fluorescent dyes. An orthonormal basis deconvolution method combined with a Fourier-domain method were tested for their ability to unmix fluorescent components in a hyperspectral TRF measurement. This method was tested on dye mixtures and retrieved fluorescence lifetimes of 4.6±0.4 ns and 2.7±0.2 ns in a mixture of Fluorescein and Coumarin-6 at concentrations of 5 μM each. It was also tested on an ex-vivo brain tissue where the fluorescence was approximated as a sum of 2 components. / Thesis / Master of Applied Science (MASc)

Protein-protein interactions are important for biological processes. Therefore, many small molecules target a specific protein or interaction in the cell to have biological consequence. While we can measure some protein-protein interactions in a test tube, many proteins cannot be purified making it difficult to properly test that a drug is “on target”. An alternative is to measure these interactions in live cells. We express the proteins of interest fused to fluorophores allowing the use of fluorescence techniques. Förster Resonance Energy Transfer (FRET) provides a molecular level ruler to measure the distance, within a few nanometers, between two proteins. FRET indicates binding. The gold standard for measuring FRET in live cells is by quantifying changes in fluorescence lifetime using Fluorescence lifetime imaging microscopy (FLIM). The change in fluorescence lifetime is inversely proportional to the ratio of bound to non-bound proteins. Tradition FLIM-FRET microscopy is too slow for screening applications. Our aim was to develop a highly multiplexed confocal system for rapid FLIM-FRET acquisition. We present the development of multiple prototypes for confocal multiplexing. In this work, our final design includes 32×32 multiplexed excitation points which scan the sample using refractive window scanners. We coupled this excitation scheme to a 64×32 time-gated single-photon avalanche photodiode (SPAD) sparse array detector. This multiplexed setup allows the use of the sparse array with high frame rate and sub-nanosecond time-gating to achieve high throughput FLIM acquisition. Using our multiplexed FLIM prototype we measured Bcl-2 family protein-protein interactions in live cells (310×310 μm FOV) with two-channel confocal FLIM in 1.5 s. Protein binding affinities were estimated by measuring the changes in FRET as a function of acceptor to donor ratio. The resulting speed of this system meets requirements for implementation in screening applications. / Thesis / Candidate in Philosophy / Inside a cell, proteins are the “workers” and they interact with each other, doing that work. Many of these interactions are important for the cell to live. Pharmaceutical companies may design drugs that can interfere with a specific interaction in order to cause an effect in the cell. Scientists are interested in measuring these interactions and we can do this by “taking a picture” of the interaction using a specialized microscope. One of the major issues with these microscopes is that it takes scientists a long time to collect pictures of these interactions. This means only a few drugs can be tested in a day. To speed up the drug discovery and testing we want to design faster microscopes that can test hundreds of drugs in a day. In my thesis I contributed to building a state-of-the-art super fast microscope. We made progress in steps, and by the third attempt we successfully measured interactions in cells in seconds! Our new microscope is ~400x faster than current technologies. We hope that this research will be useful to speed up drug discovery in the future.

Companies routinely perform life tests for their products. Typically, these tests involve running a set of products until the units fail. Most often, the data are censored according to different censoring schemes, depending on the particulars of the test. On occasion, tests are stopped at a predetermined time and the units that are yet to fail are suspended. In other instances, the data are collected through periodic inspection and only upper and lower bounds on the lifetimes are recorded. Reliability professionals use a number of non-normal distributions to model the resulting lifetime data with the Weibull distribution being the most frequently used. If one is interested in monitoring the quality and reliability characteristics of such processes, one needs to account for the challenges imposed by the nature of the data. We propose likelihood ratio based cumulative sum (CUSUM) control charts for censored lifetime data with non-normal distributions. We illustrate the development and implementation of the charts, and we evaluate their properties through simulation studies. We address the problem of interval censoring, and we construct a CUSUM chart for censored ordered categorical data, which we illustrate by a case study at Becton Dickinson (BD). We also address the problem of monitoring both of the parameters of the Weibull distribution for processes with right-censored data. / Ph. D.

Data for this study were age-adjusted linear scores on all cows scored between 1980 and 1993. Lifetime production information on these cows and their herdmates was used to calculate Relative Net Income adjusted for opportunity cost (RNIOC) for the 2,249,835 cows with an 84 month herdlife opportunity. The effect of inbreeding was analyzed using both a fixed and animal model, with little difference in results. Inbreeding depressed RNIOC by $12.69 in a fluid market and $11.53 in a manufacturing market per 1% increase in inbreeding. Addition of somatic cell information in the profit function had little effect. Heritabilities of profit functions were .16 and .14 for a fluid and manufacturing market, respectively. Animal model estimates of inbreeding depression were +.16 days, -6.7 days and -5.1 days for age at first freshening (AFF), days of productive life (DPL) and days in milk (TDIM), respectively. Inbreeding decreased first lactation mature equivalent milk, fat, and protein by 23.7 kg, .85 kg, and .76 kg, respectively and lifetime milk, fat, and protein production by 176.9 kg, 6.4 kg, 5.6 kg, respectively per 1% increase in inbreeding. Inbreeding had little effect on conformation traits. Effects of inbreeding were cumulative, exacting a larger effect on lifetime profit functions than on individual traits, when expressed as a percent of additive standard deviation. This study gives evidence that though not alarming, inbreeding has a deleterious effect on the lifetime performance of dairy cattle. / Master of Science

As environmental change continues and increases, understanding how species will respond to change and how these responses may affect populations will be important for conserving and managing species. Red-cockaded Woodpeckers (Picoides borealis) are well-studied and provide an ideal system in which to examine ecological and evolutionary questions related to life histories because monitoring them accurately is relatively easy, their behavior is well-described and structured. In this study, I examined the following questions using long-term data (1980-2013) from two study sites in North Carolina: 1) what traits contribute most to lifetime fitness, 2) how have traits changed over time and how is climate change influencing life history, and 3) to what extent are traits that contribute to fitness and that are changing over time heritable in this species. I found that a multitude of factors contribute to different aspects of fitness, including: parental age and hatch date affecting survival to year one and probability of attaining breeding status; and lay date, clutch size, age at first reproduction, and variance in clutch size affecting lifetime fitness. I also found that many traits were changing over time including lay date, clutch size, partial brood loss, and survival to year one. These traits were strongly influenced by local climate variables at each study site, but it is not clear that climate has changed over time at the study sites to account for all the observed changes in life history traits. Habitat improvement has also played a role as evidenced by increased fledgling production in terms of raw numbers (without accounting for covariates). I also found that lay date, clutch size, and partial brood loss had low heritabilities after accounting for other random and fixed effects. These results indicate that Red-cockaded Woodpeckers at these two study sites are shifting traits successfully in response to changing conditions, and that these changes are in the direction that increases aspects of fitness. These shifts indicate that individuals are plastic with respect to these traits, but most of the variance in traits was related to external habitat-associated factors rather than additive genetic variance or environmental × genotype interactions. / Ph. D.