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Iterative Size Reduction of Bead Placement in Nanosphere Lithography

Description

Nanosphere lithography is a high throughput procedure that has important implications
for facile, low cost scaling of nanostructures. However, current benchtop experiments have
limitations based on the placement of molecular species that exhibit greater than singlemolecular binding. In addition, reliance

Nanosphere lithography is a high throughput procedure that has important implications
for facile, low cost scaling of nanostructures. However, current benchtop experiments have
limitations based on the placement of molecular species that exhibit greater than singlemolecular binding. In addition, reliance upon bottom-up self-assembly of close-packed
nanospheres makes it problematic to resolve images using low-cost light microscopes due to the
spacing limitations smaller in magnitude than light wavelength. One method that is created to
resolve this issue is iterative size reduction (ISR), where repetitive ‘iterative’ processes are
employed in order to increase the precision at which single molecules bind to a given substrate.
ISR enables inherent separation of nanospheres and therefore any subsequent single molecule
binding platforms. In addition, ISR targets and encourages single-molecule binding by
systematically reducing binding site size. Results obtained pursuing iteratively reduced
nanostructures showed that many factors are needed to be taken into consideration, including
functionalization of nanosphere particles, zeta potential, and protonation-buffer reactions.
Modalities used for observation of nanoscale patterning and single-molecule binding included
atomic force microscopy (AFM) and ONI super-resolution and fluorescence microscopy. ISR
was also used in conjunction with zero mode waveguides, which are nanoapertures enabling realtime single molecule observation at zeptoliter volumes. Although current limitations and
obstacles still exist with reproducibility and scalability of ISR, it nonetheless exhibits limitless
potential and flexibility in nanotechnology applications.

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2020-05

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Does chronic unpredictable restraint produce dendritic retraction in long-shaft CA3 hippocampal neurons?

Description

Major Depressive Disorder (MDD) is a widespread mood disorder that affects more than 300 million people worldwide and yet, high relapse rates persist. This current study aimed to use an animal model for depression, unpredictable intermittent restraint (UIR), to investigate

Major Depressive Disorder (MDD) is a widespread mood disorder that affects more than 300 million people worldwide and yet, high relapse rates persist. This current study aimed to use an animal model for depression, unpredictable intermittent restraint (UIR), to investigate changes in a subset of neurons within the hippocampus, a region of high susceptibility in MDD. Adult male and female Sprague-Dawley rats were randomly assigned to four treatment groups based on sex (n = 48, n = 12/group). Half of the rats underwent UIR that involved restraint with orbital shaking (30 min or 1 h) for 2-6 consecutive days, followed by one or two days of no stressors; the other half of the rats were undisturbed (CON). UIR rats were stressed for 28 days (21 days of actual stressors) before behavioral testing began with UIR continuing between testing days for nearly 70 days. Rats were then euthanized between 9 and 11 days after the last UIR session. Brains were processed for Golgi stain and long-shaft (LS) neurons within the hippocampal CA3a and CA3b regions were quantified for dendritic complexity using a Camera Lucida attachment. Our findings failed to support our hypothesis that UIR would produce apical dendritic retraction in CA3 hippocampal LS neurons in both males and females. Given that UIR failed to produce CA3 apical dendritic retraction in males, which is commonly observed in the literature, we discuss several reasons for these findings including, time from the end of UIR to when brains were sampled, and the effects of repeated cognitive testing. Given our published findings that UIR impaired spatial ability in males, but not females, we believe that UIR holds validity as a chronic stress paradigm, as UIR attenuated body weight gain in both males and females and produced reductions in thymus gland weight in UIR males. These findings corroborate UIR as an effective stressor in males and warrant further research into the timing of UIR-induced changes in hippocampal CA3 apical dendritic morphology.

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2020-12

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Towards Purification of human TRPV1 Pore Domain

Description

The transient receptor potential channel subfamily V member 1 (TRPV1) functions as the heat and capsaicin receptor. It can be activated by heat, protons, pungent chemicals, and a variety of other endogenous mediators of nociception. TRPV1 is a non-selective cation

The transient receptor potential channel subfamily V member 1 (TRPV1) functions as the heat and capsaicin receptor. It can be activated by heat, protons, pungent chemicals, and a variety of other endogenous mediators of nociception. TRPV1 is a non-selective cation channel consisting of 6 transmembrane domains (S1-S6), with helices S1-S4 forming the sensing domain and the S5-S6 helices forming the pore domain. Understanding the TRPV1 channel is imperative due to its relation to a variety of human diseases, including cancer, type II diabetes, hyper and hypothermia, and inflammatory disorders of the airways and bladder. Although TRPV1 is the best-studied thermosensitive-TRP channels of all the 28 family members, the molecular underpinning and the contributions of the human TRPV1 pore domain in thermo-sensing remains elusive. Recently, the human TRPV1 sensing domain was found to contribute to heat activation. It was found to undergo a non-denaturing temperature-dependent conformational change. This finding triggered interest in studying the function and the role of the human TRPV1 pore domain in the heat activation process. Specifically, to identify whether heat activation is intrinsic to the pore domain. This thesis paper explores and optimizes the purification protocol of the human TRPV1 pore domain through three different methods. The first method was using a denaturant, the second method was increasing the length of the histidine tags through Q5 insertion, and the third method was incorporating the protein construct into nanodiscs. In addition to the above three methods, size exclusion chromatography and ion-exchange chromatography were utilized after thrombin cleavage to separate the human TRPV1 pore domain from the cleaved MBP deca-histidine tags as well as the impurities.

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2020-12

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Integrated Breast Biopsy Bioimpedance Sensor

Description

Breast microcalcifications are a potential indicator of cancerous tumors. Current visualization methods are either uncomfortable or impractical. Impedance measurement studies have been performed, but not in a clinical setting due to a low sensitivity and specificity. We are hoping to

Breast microcalcifications are a potential indicator of cancerous tumors. Current visualization methods are either uncomfortable or impractical. Impedance measurement studies have been performed, but not in a clinical setting due to a low sensitivity and specificity. We are hoping to overcome this challenge with the development of a highly accurate impedance probe on a biopsy needle. With this technique, microcalcifications and the surrounding tissue could be differentiated in an efficient and comfortable manner than current techniques for biopsy procedures. We have developed and tested a functioning prototype for a biopsy needle using bioimpedance sensors to detect microcalcifications in the human body. In the final prototype a waveform generator sends a sin wave at a relatively low frequency(<1KHz) into the pre-amplifier, which both stabilizes and amplifies the signal. A modified howland bridge is then used to achieve a steady AC current through the electrodes. The voltage difference across the electrodes is then used to calculate the impedance being experienced between the electrodes. In our testing, the microcalcifications we are looking for have a noticeably higher impedance than the surrounding breast tissue, this spike in impedance is used to signal the presence of the calcifications, which are then sampled for examination by radiology.

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2018-05

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Assessing the Navigational Abilities of an Acoustically Active Cannula

Description

The purpose of this thesis experiment was to design and create an Acoustically Active Cannula (AAC), which is furnished by a piezoelectric crystal placed at its tip that produces an acoustic navigation signal. I tested the functionality of the cannula

The purpose of this thesis experiment was to design and create an Acoustically Active Cannula (AAC), which is furnished by a piezoelectric crystal placed at its tip that produces an acoustic navigation signal. I tested the functionality of the cannula in vitro and demonstrated its navigational abilities in vivo in anesthetized pigs. This experiment was based upon ultrasound science and technology, and thus some practical experience with conventional (B-mode) and Doppler ultrasound was achieved as well. The results of bench and experimental animal studies indicated proper functionality of the AAC for identification and spatial navigation of its tip with color Doppler ultrasound imaging.

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2018-05

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Ambassador Communities, Hybrid Spectatorship, and Other Indicators of Success in eSports

Description

The academic study of eSports, or professional competition through the medium of video games, has tended to focus on players' motivations to play and watch eSports as well as marketing concerns of huge eSports corporations. Instead of utilizing marketing or

The academic study of eSports, or professional competition through the medium of video games, has tended to focus on players' motivations to play and watch eSports as well as marketing concerns of huge eSports corporations. Instead of utilizing marketing or psychology to analyze this phenomenon, I investigate three areas of focus in accordance with available literature: the fans and their characteristics, the design of the game itself, and the relationship between fans and the game's developer. This investigation was conducted by first examining existing literature surrounding eSports fans, then collecting public domain data such as Reddit posts, forum posts, and YouTube videos, and last by studying interviews with developers and players. With this thesis, I apply a fan studies approach to eSports by creating a series of indicators based in each of the three focus areas which can be utilized as a systematic method of evaluating an eSport's popularity and growth.

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2018-05

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Characterization of Food Intake and Weight Gain Responses in Rats on a High Fat Diet

Description

With the influence of the Western Diet, obesity has become a rising problem in the country today. Western Diet is characterized by the overconsumption of processed food that is low in nutritional values and high in saturated fats. Study showed

With the influence of the Western Diet, obesity has become a rising problem in the country today. Western Diet is characterized by the overconsumption of processed food that is low in nutritional values and high in saturated fats. Study showed that every two out of three adults in the United States are either overweight or obese. Being obese increase the risk of many other disease such as diabetes, cardiovascular disease and insulin resistance. Besides being a great health concern, obesity is also cause a great financial burden. Many efforts have been made to understand the defense against obesity and weight loss. The goal of this study was to understand the characterization of food intake and weight gain responses when imposed on a high-fat diet (HFD) using rats. It was predicted that weight gain would be dependent on energy intake and it would have a significant effect on adiposity compared to energy intake. Data showed that energy intake had high significance with adiposity whereas weight gain showed no significance. Also for the rats that were on HFD, the obesity-prone (OP) rats exhibited a great amount of weight gain and energy intake while the obesity-resistance (OR) rats showed a similar weight gain to the controlled group on low-fat diet (LFD) despite being hyperphagic. This suggests that OR is characterized by equal weight gain despite hyperphagia but this alone cannot explain the boy defense against obesity. More research is needed with a larger sample size to understand weight gain responses in order to fight against the epidemic of obesity.

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2018-05

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Analysis of Yeast and Fungi in Children with ASD vs. Neurotypical Controls

Description

The gastrointestinal (GI) tract is home to a complex and diverse microbial ecosystem that contributes to health or disease in many aspects. While bacterial species are the majority in the GI tract, their cohabitants, fungal species, should not be forgotten.

The gastrointestinal (GI) tract is home to a complex and diverse microbial ecosystem that contributes to health or disease in many aspects. While bacterial species are the majority in the GI tract, their cohabitants, fungal species, should not be forgotten. Children with autism spectrum disorder (ASD) often suffer from GI disorders and associated symptoms, implying a role the bacterial and fungal gut microbiota play in maintaining human health. The irregularities in GI symptoms can negatively affect the overall quality of life or even worsen behavioral symptoms the children present. Even with the increase in the availability of next-generation sequencing technologies, the composition and diversities of fungal microbiotas are understudied, especially in the context of ASD. We therefore aimed to investigate the gut mycobiota of 36 neurotypical children and 38 children with ASD. We obtained stool samples from all participants, as well as autism severity and GI symptom scores to help us understand the effect the mycobiome has on these symptoms. By targeting the fungal internal transcribed spacer (ITS) and bacterial 16S rRNA V4 regions, we obtained fungal and bacterial amplicon sequences, from which we investigated the diversities, composition, and potential link between two different ecological clades. From fungal amplicon sequencing results, we observed a significant decrease in the observed fungal OTUs in children with ASD, implying a lack of potentially beneficial fungi in ASD subjects. We performed Bray-Curtis principal coordinates analysis and observed significant differences in fungal microbiota composition between the two groups. Taxonomic analysis showed higher relative abundances of Candida , Pichia, Penicillium , and Exophiala in ASD subjects, yet due to a large dispersion of data, the differences were not statistically significant. Interestingly, we observed a bimodal distribution of Candida abundances within children with ASD. Candida's relative abundance was not significantly correlated with GI scores, but children with high Candida relative abundances presented significantly higher Autism Treatment Evaluation Checklist (ATEC) scores, suggesting a role of Candida on ASD behavioral symptoms. Regarding the bacterial gut microbiota, we found marginally lower observed OTUs and significantly lower relative abundance of Prevotella in the ASD group, which was consistent with previous studies. Taken together, we demonstrated that autism is closely linked with a distinct gut mycobiota, characterized by a loss of fungal and bacterial diversity and an altered fungal and bacterial composition.

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2018-05

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Assessing the Influence of Extracellular Mitochondria on Neuroinflammation

Description

A prominent aspect of Alzheimer’s disease (AD) is the presence of neuroinflammation is mediated by the activation of microglial cells, which are the immune cells in the central nervous system (CNS) that express an array of cytokines that may promote

A prominent aspect of Alzheimer’s disease (AD) is the presence of neuroinflammation is mediated by the activation of microglial cells, which are the immune cells in the central nervous system (CNS) that express an array of cytokines that may promote an inflammatory response. The main cytokines produced are: tumor necrosis factor-alpha (TNF-), interleukin-1β (IL-1β), and interleukin-6 (IL-6). The presence of these cytokines in the CNS may lead to neuronal death, to the production of toxic chemicals (such as nitric oxide), and to the generation of amyloid beta (a major pathological feature of AD). Previous studies have shown that modulation of the inflammatory response in the nervous system can potentially prevent and/or delay the onset of neurodegenerative diseases such as AD. Therefore, it is important to identify the process that induces CNS inflammation. For example, mitochondrial lysates have been found to produce an inflammatory response due to their ability to stimulate TNF-, Aβ, and APP mRNA [10]. Interestingly, extracellular mitochondria have been detected in the brain due to neurons degrading old mitochondria extracellularly. Therefore, we set out to study the effect of whole mitochondria isolated by differential centrifugation from human neuroblastoma cells (BE(2)-M17 cells) on the neuroinflammatory response in a human microglia model (THP-1 cells). Despite our best efforts, in the end it was unclear whether the mitochondrial fraction or other cellular components induced the inflammatory response we observed. Thus, further work with an improved mitochondrial isolation method should be carried out to address this issue.

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2018-05

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Over-expression of a putative multi-heme cytochrome c from Heliobacterium modesticaldum

Description

Heliobacterium modesticaldum (H. modesticaldum) is an anaerobic photoheterotroph that can fix nitrogen (N2) and produce molecular hydrogen (H2). Recently, the Redding and Jones labs created a microbial photoelectrosynthesis cell that utilized these properties to produce molecular hydrogen using electrons provided

Heliobacterium modesticaldum (H. modesticaldum) is an anaerobic photoheterotroph that can fix nitrogen (N2) and produce molecular hydrogen (H2). Recently, the Redding and Jones labs created a microbial photoelectrosynthesis cell that utilized these properties to produce molecular hydrogen using electrons provided by a cathode via a chemical mediator. Although this light-driven creation of fuel within a microbial electrochemical cell was the first of its kind, its production rate of hydrogen was low. It was hypothesized that the injection of electrons into H. modesticaldum was a rate-limiting step in H2 production. Within the H. modesticaldum genome, there is a gene (HM1_0653) that encodes a multi-heme cytochrome c that may be directly involved in this step. From past transcriptomic experiments, this gene is known to be very poorly expressed in H. modesticaldum. Our hypothesis was that increasing its expression with a strong promoter could result in faster electron transfer, and thus, increased H2 production in the photoelectrosynthesis cell. In order to test this hypothesis, different promoters that could lead to high expression in H. modesticaldum were included with a copy of HM1_0653 in various plasmid constructs that were first cloned into E. coli before being conjugated with H. modesticaldum. Cloning in E. coli was possible with the newly derived transformation system and by reducing the copy-number of the vector system. When overexpressed in E. coli, the protein appeared to be expressed, but its purification proved to be difficult. Moreover, conjugation with H. modesticaldum was not achieved. Our results are consistent with the idea that high level overexpression in H. modesticaldum was toxic. An inducible promoter may circumvent these issues and prove more successful in future experiments.

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2018-05