Mountain Scholar :: Browsing by Author "Tobet, Stuart, committee member"

Browsing by Author "Tobet, Stuart, committee member"

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Open Access
A biosensor system with an integrated CMOS microelectrode array for high spatio-temporal electrochemical imaging
(Colorado State University. Libraries, 2019) Tedjo, William, author; Chen, Thomas, advisor; Tobet, Stuart, committee member; Collins, George, committee member; Wilson, Jesse, committee member
The ability to view biological events in real time has contributed significantly to research in life sciences. While optical microscopy is important to observe anatomical and morphological changes, it is equally important to capture real-time two-dimensional (2D) chemical activities that drive the bio-sample behaviors. The existing chemical sensing methods (i.e. optical photoluminescence, magnetic resonance, and scanning electrochemical), are well-established and optimized for existing ex vivo or in vitro analyses. However, such methods also present various limitations in resolution, real-time performance, and costs. Electrochemical method has been advantageous to life sciences by supporting studies and discoveries in neurotransmitter signaling and metabolic activities in biological samples. In the meantime, the integration of Microelectrode Array (MEA) and Complementary-Metal-Oxide-Semiconductor (CMOS) technology to the electrochemical method provides biosensing capabilities with high spatial and temporal resolutions. This work discusses three related subtopics in this specific order: improvements to an electrochemical imaging system with 8,192 sensing points for neurotransmitter sensing; comprehensive design processes of an electrochemical imaging system with 16,064 sensing points based on the previous system; and the application of the system for imaging oxygen concentration gradients in metabolizing bovine oocytes. The first attempt of high spatial electrochemical imaging was based on an integrated CMOS microchip with 8,192 configurable Pt surface electrodes, on-chip potentiostat, on-chip control logic, and a microfluidic device designed to support ex vivo tissue experimentation. Using norepinephrine as a target analyte for proof of concept, the system is capable of differentiating concentrations of norepinephrine as low as 8µM and up to 1,024 µM with a linear response and a spatial resolution of 25.5×30.4μm. Electrochemical imaging was performed using murine adrenal tissue as a biological model and successfully showed caffeine-stimulated release of catecholamines from live slices of adrenal tissue with desired spatial and temporal resolutions. This system demonstrates the capability of an electrochemical imaging system capable of capturing changes in chemical gradients in live tissue slices. An enhanced system was designed and implemented in a CMOS microchip based on the previous generation. The enhanced CMOS microchip has an expanded sensing area of 3.6×3.6mm containing 16,064 Pt electrodes and the associated 16,064 integrated read channels. The novel three-electrode electrochemical sensor system designed at 27.5×27.5µm pitch enables spatially dense cellular level chemical gradient imaging. The noise level of the on-chip read channels allow amperometric linear detection of neurotransmitter (norepinephrine) concentrations from 4µM to 512µM with 4.7pA/µM sensitivity (R=0.98). Electrochemical response to dissolved oxygen concentration or oxygen partial pressure (pO2) was also characterized with deoxygenated deionized water containing 10µM to 165 µM pO2 with 8.21pA/µM sensitivity (R=0.89). The enhanced biosensor system also demonstrates selectivity to different target analytes using cyclic voltammetry to simultaneously detect NE and uric acid. In addition, a custom-designed indium tin oxide and Au glass electrode is integrated into the microfluidic support system to enable pH measurement, ensuring viability of bio-samples in ex vivo experiments. Electrochemical images confirm the spatiotemporal performance at four frames per second while maintaining the sensitivity to target analytes. The overall system is controlled and continuously monitored by a custom-designed user interface, which is optimized for real-time high spatiotemporal resolution chemical bioimaging. It is well known that physiological events related to oxygen concentration gradients provide valuable information to determine the state of metabolizing biological cells. Utilizing the CMOS microchip with 16,064 Pt MEA and an improved three-electrode system configuration, the system is capable of imaging low oxygen concentration with limit of detection of 18.3µM, 0.58mg/L, or 13.8mmHg. A modified microfluidic support system allows convenient bio-sample handling and delivery to the MEA surface for sensing. In vitro oxygen imaging experiments were performed using bovine cumulus-oocytes-complexes cells with custom software algorithms to analyze its flux density and oxygen consumption rate. The imaging results are processed and presented as 2D heatmaps, representing the dissolved oxygen concentration in the immediate proximity of the cell. The 2D images and analysis of oxygen consumption provide a unique insight into the spatial and temporal dynamics of cell metabolism.
Open Access
A metabolomics approach for examining synbiotic protection against infectious enteric pathogens
(Colorado State University. Libraries, 2019) Nealon, Nora Jean, author; Ryan, Elizabeth P., advisor; Dean, Gregg, committee member; Henry, Charles, committee member; Tobet, Stuart, committee member
Infectious gastrointestinal diseases contribute to billions of global cases of human illness annually. Salmonella enterica serovar Typhimurium and human rotavirus represent two human health challenges, where escalating multidrug resistance and poor vaccine efficacy warrant the development of alternative treatments. Health-promoting probiotic microorganisms are becoming increasingly studied for their production of bioactive small molecules that confer protective effects against enteric pathogens. Among probiotics, Lactobacilli, Bifidobacteria and E. coli Nissle form synbiotics with rice bran, the prebiotic-rich outer coating of brown rice, to enhance animal protection against S. Typhimurium infection and human rotavirus diarrhea compared to probiotics or rice bran alone. Despite these beneficial interactions of probiotics and rice bran, a knowledge gap exists in our understanding of the synbiotic small molecules driving these protective effects, especially across probiotic species differences in small molecule production. To test our overarching hypothesis that probiotic species would metabolize rice bran into distinct suites of small molecules that suppressed pathogen function, we first applied the cell-free supernatant from L. paracasei, L. fermentum, and L. rhamnosus cultured with rice bran to S. Typhimurium and observed magnitude-dependent growth suppression across synbiotics. Both L. paracasei and L. fermentum supernatants exhibited enhanced growth suppression compared to their probiotic-only treatments and contained differentially abundant antimicrobial lipids, amino acids, and nucleotides that have not been previously characterized for antimicrobial functions. The cell-free supernatant of the L. paracasei and L. fermentum synbiotics were fractionated and applied to S. Typhimurium to identify the small molecules driving their enhanced Salmonella growth suppression. Metabolite profiles were also compared across synbiotics. Each synbiotic produced several bioactive fractions that suppressed Salmonella growth. While both L. fermentum and L. paracasei bioactive fractions contained abundant lipids, L. fermentum fractions were selectively-enriched in the energy metabolite fumarate and L. paracasei fractions were uniquely-enriched with amino acids (imidazole lactate, ornithine) suggesting that Lactobacillus spp. probiotics could differentially metabolize rice bran to drive Salmonella growth suppression with different suites of small molecules. To examine probiotic metabolism of rice bran in mammalian systems, we compared the intestinal and blood metabolomes of healthy adult mice and gnotobiotic, neonatal pigs that were fed combinations of probiotics and rice bran to the metabolomes of animals consuming rice bran or probiotics alone. In mice, a notable difference following 15 weeks consumption of B. longum fermented was that the arginine metabolite N-delta-acetylornithine was significantly increased in B. longum fermented rice bran compared to rice bran alone and was elevated in both the colon tissue and blood of mice consuming fermented rice bran compared to rice bran alone. In gnotobiotic neonatal pigs, three weeks of prophylactic supplementation with E. coli Nissle and L. rhamnosus GG and rice bran were more effective at reducing human rotavirus diarrhea compared to pigs given these probiotics or rice bran alone. Approximately 300 colon and blood metabolites that were differentially-abundant between synbiotic-consuming pigs versus pigs consuming probiotics alone were identified, over 50% of which were lipids and amino acids. Similar modulations lipid and amino acid metabolites (sphingolipids, diacylglycerols, arginine metabolites) were identified in the colon tissue and blood of mice and pigs consuming the synbiotic treatments. Consequently, the association of these metabolite profiles with human rotavirus diarrhea protection, when combined with their presence in two mammalian models, provides strong rationale for these infectious enteric disease protective roles harbored by these metabolites. The results of these studies provide a role for synbiotics in the prevention of infectious gastrointestinal diseases. For the first time, high-throughput metabolomics analyses were applied to identify differential bioactive metabolite production by Lactobacillus spp. + rice bran synbiotics that suppressed S. Typhimurium growth, as well as to compare bioactive metabolites produced by B. longum, L. rhamnosus GG, and E. coli Nissle in mice and pigs that were protective against human rotavirus diarrhea. The contributions of amino acids and lipids to the enhanced capacities of these synbiotics compared to probiotics or rice bran alone can be studied further for their mechanisms of action on pathogens. Ultimately, these bioactive synbiotic metabolites can guide the optimization and development of broad-spectrum antimicrobials and other prophylactic agents that protect against infectious enteric diseases across the human and animal lifespan.
Open Access
Advancing impulsive Raman spectroscopy and microscopy for biological applications
(Colorado State University. Libraries, 2024) Smith, David R., author; Bartels, Randy, advisor; Wilson, Jesse, advisor; Tobet, Stuart, committee member; Jost, Dylan, committee member
Chemically sensitive, label-free spectroscopy and microscopy is a critical tool for the study of many complex and dynamic biological systems. The development of the impulsive stimulated Raman scattering (ISRS) techniques in this thesis represent important steps forward in addressing the ability to interrogate Raman vibrations in complex and scattering samples, particularly low frequency Raman modes.
Open Access
Analysis and modeling of cells, cell behavior, and helical biological molecules
(Colorado State University. Libraries, 2011) Benoit, Steven Richard, author; Putkaradze, Vakhtang, advisor; Shipman, Patrick, committee member; Estep, Don, committee member; Marconi, Mario, committee member; Tobet, Stuart, committee member
Mathematical models of biological systems have evolved over time and through the introduction and growth of computer simulation and analysis. Models have increased in sophistication and power through the combination of multi-scale approaches, molecular and granular dynamics simulations, and advances in parallelization and processing speed. However, current cell models cannot accurately predict behaviors at the whole-cell scale, nor can molecular models predict accurately the complex shape assumed by large biological molecules including proteins, although significant progress is being made toward this goal. The present work introduces new models in three domains within biological systems modeling. We first discuss a phenomenological model of observed cell motions in developing tissue that characterizes cells according to a best-fit generalized diffusion model and combines this data with Voronoi diagrams to effectively visualize patterns of cell behavior in tissue. Next, we present a series of component models for cells and cell structure that support simulations involving tens to hundreds of cells in a way that captures behaviors ignored by existing models, including pseudopod formation, membrane mechanics, cytoskeletal polymerization / depolymerization, and chemical signal transduction. The resulting models exhibit many of the behaviors of real-world cells including polarization and chemotaxis. Finally, we present a method for analysis of biological molecules that form helical conformations that includes long-range electrostatic interactions as well as short-range interactions to prevent self-intersections. We consider the stability of molecules with repeating monomers that include off-axis charge concentrations and derive energy landscapes to identify stable conformations, then analyze helical stability using geometric methods.
Open Access
Androgen signaling in the placenta
(Colorado State University. Libraries, 2014) Cleys, Ellane Rachael, author; Bouma, Gerrit, advisor; Clay, Colin, advisor; Tobet, Stuart, committee member; Di Pietro, Santiago, committee member
Placental estrogen signaling is known to regulate placental trophoblast function and differentiation. However, the role of placental androgen signaling has never been investigated, despite the rise of maternal serum androgens throughout gestation. Recent findings have shown increased maternal serum androgen in patients with the placental induced disorder preeclampsia. Preeclampsia, a maternal hypertension and proteinuria condition instigated by insufficient trophoblast differentiation and invasion into maternal spiral arteries, is also associated with increased placental expression of androgen receptor and an increased risk of incidence in patients with polymorphisms in androgen receptor that decrease androgen signaling. These findings suggest a crucial role for placental androgen signaling. Moreover, research investigating androgen's role in cancer progression has shown that many androgen responsive genes regulate cell proliferation, differentiation to invasive phenotypes, and tissue vascularization, all processes necessary for normal placental development. Androgen signaling in tumor tissues is further regulated by androgen receptor complexes with histone lysine demethylases. These complexes are recruited to androgen response elements in DNA and dynamically regulate histone tail modifications for transcription initiation. This led us to the overall hypothesis that (1) androgen signaling in trophoblast cells is important for placental development, and (2) androgen receptor complexes with histone lysine demethylases in the placenta to regulate vascularization, growth and invasion factors in trophoblast cells. To test this hypothesis, we utilized a prenatal androgenization ewe model as well as human first trimester placental samples and immortalized human trophoblast cell lines. Using the prenatal androgenized ewe model, we report for the first time expression of histone lysine demethylases in the placenta. Furthermore, we showed androgen receptor complexes with histone lysine demethylases and is recruited to an androgen response elements in the 5'untranslated flanking sequence of vascular endothelial growth factor in the sheep placenta. We also report that histone lysine demethylase are present in human first trimester syncytiotrophoblast and complex with androgen receptor in immortalized trophoblasts. Additionally, we demonstrated that androgen receptor complexes with histone lysine demethylases are also present in choriocarcinoma ACH-3P and BeWo cells. Dihydrotestosterone treatment in these cells led to down-regulation of androgen responsive genes, specifically KDM3A and MMP2. Inhibition of androgen receptor through flutamide treatment altered mRNA levels for genes regulating vascularization, including HIF1α, PPARα, and PPARy. Hypoxia also decreased CYP19 levels, however, further investigation is needed to confirm dihydrotestosterone and flutamide effect on protein expression in trophoblast cells. These data suggest that histone lysine demethylases complex with androgen receptor to regulate androgen responsive genes, including those directing placental vascularization and development. However, further experiments are needed to confirm the necessity of histone lysine demethylases for targeted androgen signaling in trophoblast cells and to determine if androgen directly regulates trophoblast differentiation and invasion. These findings suggest androgen signaling may play a critical role in placental development.
Open Access
Automated methods for quantifying the tortuosity of microvascular networks
(Colorado State University. Libraries, 2012) Dodd, Melody, author; Putkaradze, Vakhtang, advisor; Tobet, Stuart, committee member; Shipman, Patrick, committee member
Networks of microscopic blood vessels can be studied for changes in morphology that correlate with biological abnormalities. Tortuosity, or vessel twistiness, is one of these morphological properties, and it can be surprisingly difficult to quantify. The purpose of this thesis is to present the development, testing, and analysis of new automated methods to measure and quantify the tortuosity of microvascular networks. We will explain necessary automated image processing techniques and background information before presenting our new metrics for measuring network tortuosity. Experiments using the methods will be presented, including a full analysis of the results. We will use the results from these experiments to justify our final conclusions and recommendations regarding the performance of the methods.
Open Access
Behavioral effects of estrogen receptor beta acting locally to regulate the expression of tryptophan hydroxylase 2 (THP2) in serotonergic neurons of the dorsal raphe nuclei
(Colorado State University. Libraries, 2008) Donner, Nina Caroline, author; Handa, Robert J., advisor; Tjalkens, Ronald, committee member; Clay, Colin McKeown, committee member; Tobet, Stuart, committee member
Affective disorders often involve serotonin (5-HT)-related dysfunctions and are twice as common in women than men. Interactions between estrogen and the brain 5-HT system have long been proposed to contribute to sex differences in mood and anxiety disorders, but the mechanisms underlying this phenomenon have yet to be revealed. Estrogen signaling is mediated by two different receptors termed estrogen receptor alpha and estrogen receptor beta. While estrogen receptor alpha (ERalpha) has mainly reproductive responsibilities, in brain, estrogen receptor beta (ERbeta) has been shown to attenuate anxiety- and despair-like behaviors in rodent models. However, little is known about ERbeta regulation of function in the brainstem raphe nuclei. The raphe nuclei are the main 5-HT system of the brain, and projections from the dorsal raphe nuclei (DRN) innervate many important forebrain and limbic areas. The work presented in this thesis addressed the possibility that ERbeta may be involved in the regulation of 5-HT gene expression specifically in DRN neurons. My studies examined the effects of systemic versus local, intracerebral application of the selective ERbeta agonist diarylpropionitrile (DPN) and the nonselective ERligandestradiol (E) on tryptophan hydroxylase 2 (TPH2) mRNA expression within the DRN of female rats. TPH2 is the brain-specific, rate-limiting enzyme catalyzing 5-HT synthesis, and is expressed in every 5-HT neuron. Thus, it provides an excellent tool to assess the capacity for 5-HT production with the DRN. In these studies, TPH2 mRNA expression was assessed via in situ hybridization. In addition, relevant behavioral parameters were tested in all animals to evaluate each compound’s effect on two closely related, but yet different mental states, anxiety-like and despair-like behavior. Both, chronic systemic and chronic local DPN administration to ovariectomized (OVX) female rats significantly enhanced TPH2 mRNA expression in mid- and caudal subregions of the DRN after 8 days of treatment. Respective controls received systemic vehicle (27% hydroxypropyl-beta-cyclodextrin) or blank control pellets. Local application of DPN caused a stronger effect than systemic drug delivery. Chronic local delivery of E (0.5 μM) increased TPH2 mRNA expression in the same subregions of the DRN as did DPN, but its overall effect was weaker compared to the selective ERbeta agonist. Interestingly, while systemic DPN-administration confirmed the anxiolytic nature of ERbeta in two separate anxiety tests (elevated plus maze and open field test), the effect was lost when DPN was delivered locally. However, local DPN- as well as E-treatment both resulted in attenuated despair-like behavior, as measured in the forced-swim test. Chapter 3 describes the experimental design, results and interpretation of these studies in depth. Taken together, my data indicate that local actions of ERbeta agonist onto DRN neurons are sufficient to decrease despair-like behavior, whereas ERbeta stimulation of other brain regions is necessary to alter anxiety-like behaviors. Correspondingly, ERbeta acts locally to control TPH2 mRNA expression and presumably 5-HT synthesis in the certain subregions of the rat DRN. These results suggest an important role of ERbeta for regulating cellular events in the female DRN, and offer new opportunities for therapeutic treatments of depressive disorders.
Open Access
Comparison of EEG preprocessing methods to improve the performance of the P300 speller
(Colorado State University. Libraries, 2011) Cashero, Zachary, author; Anderson, Charles, advisor; Chen, Thomas, advisor; Tobet, Stuart, committee member; Ben-Hur, Asa, committee member
The classification of P300 trials in electroencephalographic (EEG) data is made difficult due the low signal-to-noise ratio (SNR) of the P300 response. To overcome the low SNR of individual trials, it is common practice to average together many consecutive trials, which effectively diminishes the random noise. Unfortunately, when more repeated trials are required for applications such as the P300 speller, the communication rate is greatly reduced. Since the noise results from background brain activity and is inherent to the EEG recording methods, signal analysis techniques like blind source separation (BSS) have the potential to isolate the true source signal from the noise when using multi-channel recordings. This thesis provides a comparison of three BSS algorithms: independent component analysis (ICA), maximum noise fraction (MNF), and principal component analysis (PCA). In addition to this, the effects of adding temporal information to the original data, thereby creating time-delay embedded data, will be analyzed. The BSS methods can utilize this time-delay embedded data to find more complex spatio-temporal filters rather than the purely spatial filters found using the original data. One problem that is intrinsically tied to the application of BSS methods is the selection of the most relevant source components that are returned from each BSS algorithm. In this work, the following feature selection algorithms are adapted to be used for component selection: forward selection, ANOVA-based ranking, Relief, and recursive feature elimination (RFE). The performance metric used for all comparisons is the classification accuracy of P300 trials using a support vector machine (SVM) with a Gaussian kernel. The results show that although both BSS and feature selection algorithms can each cause significant performance gains, there is no added benefit from using both together. Feature selection is most beneficial when applied to a large number of electrodes, and BSS is most beneficial when applied to a smaller set of electrodes. Also, the results show that time-delay embedding is not beneficial for P300 classification.
Open Access
Crexens™: an expandable general-purpose electrochemical analyzer
(Colorado State University. Libraries, 2019) Yang, Lang, author; Chen, Tom, advisor; Collins, George J., committee member; Wilson, Jesse, committee member; Tobet, Stuart, committee member
Electrochemical analysis has gained a great deal of attention of late due to its low-cost, easy-to-perform, and easy-to-miniaturize, especially in personal health care where accuracy and mobility are key factors to bring diagnostics to patients. According to data from Centers for Medicare & Medicaid Services (CMS) in the US, the share of health expenditure in the US has been kept growing in the past 3 decades and reached 17.9% of its overall Gross Domestic Product till 2016, which is equivalent to $10,348 for every person in the US per year. On the other hand, health care resources are often limited not only in rural area but also appeared in well-developed countries. The urgent need and the lack of health resource brings to front the research interest of Point-of-Care (PoC) diagnosis devices. Electrochemical methods have been largely adopted by chemist and biologist for their research purposes. However, several issues exist within current commercial benchtop instruments for electrochemical measurement. First of all, the current commercial instruments are usually bulky and do not have handheld feature for point-of-care applications and the cost are easily near $5,000 each or above. Secondly, most of the instruments do not have good integration level that can perform different types of electrochemical measurements for different applications. The last but not the least, the existing generic benchtops instruments for electrochemical measurements have complex operational procedures that require users to have a sufficient biochemistry and electrochemistry background to operate them correctly. The proposed Crexens™ analyzer platform is aimed to present an affordable electrochemical analyzerwhile achieving comparable performance to the existing commercial instruments, thus, making general electrochemical measurement applications accessible to general public. In this dissertation, the overall Crexens™ electrochemical analyzer architecture and its evolution are presented. The foundation of the Crexens™ architecture was derived from two separate but related research in electrochemical sensing. One of them is a microelectrode sensor array using CMOS for neurotransmitter sensing; the other one is a DNA affinity-based capacitive sensor for infectious disease, such as ZIKA. The CMOS microelectrode sensor array achieved a 320uM sensitivity for norepinephrine, whereas the capacitive sensor achieved a dynamic range of detection from 1 /uL to 105 /uL target molecules (20 to 2 million targets), which makes it be within the detection range in a typical clinical application environment. This dissertation also covers the design details of the CMOS microelectrode array sensor and the capacitive sensor design as a prelude to the development of the Crexens™ analyzer architecture. Finally, an expandable integrated electrochemical analyzer architecture (Crexens™) has been designed for mobile point-of-care (POC) applications. Electrochemical methods have been explored in detecting various bio-molecules such as glucose, lactate, protein, DNA, neurotransmitter, steroid hormone, which resulted in good sensitivity and selectivity. The proposed system is capable of running electrochemical experiments including cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), electrochemical capacitive spectroscopy (ECS), amperometry, potentiometry, and other derived electrochemical based tests. This system consist of a front-end interface to sensor electrodes, a back-end user interface on smart phone and PC, a base unit as master module, a low-noise add-on module, a high-speed add-on module, and a multi-channel add-on module. The architecture allows LEGO™-like capability to stack add-on modules on to the base-unit for performance enhancements in noise, speed or parallelism. The analyzer is capable of performing up to 1900 V/s CV with 10 mV step, up to 12 kHz EIS scan range and a limit of detection at 637 pA for amperometric applications with the base module. With high performance module, the EIS scan range can be extended upto 5 MHz. The limit of detection can be further improved to be at 333 fA using the low-noise module. The form factor of the electrochemical analyzer is designed for its mobile/point-of-care applications, integrating its entire functionality on to a 70 cm² area of surface space. A glutamine enzymatic sensor was used to valid the capability of the proposed electrochemical analyzer and turned out to give good linearity and reached a limit of detection at 50 uM.
Open Access
Design of a multi-sensor platform for integrating extracellular acidification rate with multi-metabolite flux measurement for small biological samples
(Colorado State University. Libraries, 2019) Obeidat, Yusra M., author; Chen, Tom, advisor; Pasricha, Sudeep, committee member; Collins, George, committee member; Tobet, Stuart, committee member
To view the abstract, please see the full text of the document.
Open Access
Design of integrated on-chip impedance sensors
(Colorado State University. Libraries, 2014) Kern, Tucker, author; Chen, Thomas W., advisor; Pezeshki, Ali, committee member; Tobet, Stuart, committee member
In this thesis two integrated sensor systems for measuring the impedance of a device under test (DUT) are presented. Both sensors have potential applications in label-free affinity biosensors for biological and bio-medical analysis. The first sensor is a purely capacitive sensor that operates on the theory of capacitive division. Test capacitance is placed within a capacitive divider and produces an output voltage proportional to its value. This voltage is then converted to a timedomain signal for easy readout. The prototype capacitive sensor shows a resolution of 5 fF on a base of 500 fF, which corresponds to a 1 % resolution. The second sensor, a general purpose impedance sensor calculates the ratio between a DUT and reference impedance when stimulated by a sinusoidal signal. Computation of DUT magnitude and phase is accomplished in silicon via mixed-signal division and a phase module. An automatic gain controller (AGC) allows the sensor to measure impedance from 30 Ω to 2.5 MΩ with no more than 10 % error and a resolution of at least .44 %. Prototypes of both sensing topologies were implemented in a .18 μm CMOS process and their operation in silicon was verified. The prototype capacitive sensor required a circuit area of .014 mm2 and successfully demonstrated a resolution of 5 fF in silicon. A prototype impedance sensor without the phase module or AGC was implemented with a circuit area of .17 mm2. Functional verification of the peak capture systems and mixed-signal divider was accomplished. The complete implementation of the impedance sensor, with phase module and AGC, requires an estimated .28 mm2 of circuit area.
Open Access
Development of a nitric oxide measurement method in tissue media
(Colorado State University. Libraries, 2012) Bishop, Cherelle M., author; Reynolds, Melissa, advisor; Henry, Charles, committee member; Tobet, Stuart, committee member
Nitric oxide (NO) is involved in many biological pathways such as vasodilatation and cellular migration. The biological roles of NO have been most heavily investigated using cell and tissue culture models. The limitations with current analytical measurement methods used most commonly with these studies, however, are that they often do not record in real-time or measure NO directly. This makes it difficult to understand the concentration dependent response activity of NO. To overcome these limitations, a measurement method has been developed that enables the real-time measurement of NO in buffered tissue media (pH 7.4, buffered with CO2 gas, 37 °C). The design of our system included multi-volume custom sample cells with a pH probe and multiple gas supply inputs, a flow regulated CO2 gas system and a chemiluminescence detector. Results demonstrated the expected first-order NO release kinetics using a model NO donor (MAHMA/NO) in phosphate buffered saline (PBS) over a specified volume range. The following half-lives were found: 63±2 s (2 mL), 65±2 s (6 mL), 63±4 s (8 mL) and 67±9 s (10 mL). Using this method at these buffer volumes, an experiment was conducted using 11 mM MAHMA/NO stock used to demonstrate that NO release was linearly proportional with respect to buffer volume with a linear fit of R2 =0.9936. The linearity of NO release allowed NO release measurements of 4.4 x 10-7 M MAHMA/NO concentration in 10 mL PBS achieving NO recovery of 117±2 and MAHMA/NO decomposition half-lives 66±2. The analysis of a 10-7 M MAHMA/NO was not measurable previously using other chemiluminescence methods. Subsequent results in tissue media buffered with 5% CO2 at a controlled rate of 20 mL/min showed statistically similar kinetic rates 68±5 s (2 mL) to that of the PBS, demonstrating the ability to measure NO in real time under tissue conditions. The simultaneous pH measurements confirmed that the pH was constant at 7.4 during the NO release portion of the experiment, an important aspect to maintain accurate kinetics. Using this method for NO release measurement in tissue media, another NO donor, DETA/NO, was used to look at steady-state release for 1.5 h. The total NO release was 0.12±0.02 (nmol) and the NO release rate was 22±3 (fmol/s). This is the first analytical measurement method that enables detection of NO release from NO donors in buffered tissue media method mimicking in vitro condition.
Open Access
Dihydrotestosterone attenuates endotoxin, cytokine, and hypoxia-induced vascular inflammation
(Colorado State University. Libraries, 2011) Osterlund, Kristen Leanne, author; Handa, Robert, advisor; Gonzales, Rayna, committee member; Amberg, Gregory, committee member; Garrity, Deborah, committee member; Tobet, Stuart, committee member
Vascular inflammation plays a key role in the etiology of cardiovascular disease, particularly stoke. Vascular inflammation is under the control of several transcription factors, including nuclear factor kappa B and hypoxia inducible factor-1 alpha (HIF-1α). Activation of these transcription factors can lead to the production of inflammatory mediators such as cyclooxygenase-2 (COX-2). COX-2 plays a role in vascular inflammation, cerebral ischemia-induced injury, and has been implicated as a source of reactive oxygen species (ROS). Inflammatory mediators, such as endotoxin or cellular breakdown products released following injury, are known to signal through the Toll-like receptor 4 (TLR4). TLR4 activation leads to NFκB activation and subsequent production of COX-2. Like COX-2, TLR4 has also been implicated in injury-induced oxidative stress and cerebral ischemia damage. Previous studies have demonstrated that gonadal steroid hormones can also modulate vascular inflammation. Both protective and detrimental effects of androgens on the cardiovascular system have been reported. Since the potent androgen receptor (AR) agonist dihydrotestosterone (DHT) can be converted to 3β-diol, an estrogen receptor (ER) β-selective agonist, I hypothesized that ERβ may mediate some of the protective effects of androgens, while the AR may mediate some of the detrimental effects. The overall goal of this dissertation was to determine the mechanisms by which androgens can influence the vascular inflammatory response under both physiological and pathophysiological conditions. The hypothesis to be tested was that DHT influences vascular inflammation under both physiological and pathophysiological conditions. In my first set of experiments, using Western blot, I found that DHT increases expression of the vascular inflammatory mediator COX-2 under physiological conditions in human coronary artery vascular smooth muscle (VSM) cells and human brain VSM cells. This effect of DHT was attenuated in the presence of the AR antagonist bicalutamide. This data indicates that the pro-inflammatory effect of DHT under normal physiological conditions is AR mediated. In my second set of experiments, I examined the effects of DHT on vascular inflammation under a variety of pathophysiological conditions. Surprisingly, I found that DHT decreased cytokine-induced COX-2 expression and oxidative stress, endotoxin-induced COX-2 and TLR4 expression in human VSM cells. Furthermore, DHT also decreased hypoxia induced HIF-1α and COX-2 expression in human brain VSM cells and rat pial arteries. Finally, I found that DHT decreased hypoxia with glucose deprivation (HGD)-induced HIF-1α, COX-2 and TLR4 expression in human brain VSM cells. DHT`s anti-inflammatory effects during cytokine or HGD-induced inflammation in human brain VSM cells were not blocked by the AR antagonist bicalutamide, indicating that they were not AR mediated. These results led me to my second hypothesis, that DHT's anti-inflammatory effects are ERβ-mediated. In my third set of experiments, I found that the DHT metabolite/ERβ selective agonist 3β-diol also decreased cytokine-induced COX-2 expression in human brain VSM cells. Furthermore, DHT's ability to reduce cytokine-induced COX-2 expression in human brain VSM cells was inhibited by the non-selective estrogen receptor antagonist ICI 182,780 and the selective ERβ antagonist PHTPP. The mRNAs for steroid metabolizing enzymes in the pathway necessary to convert DHT to 3β-diol were detected in human brain VSM cells, as were AR and ERβ mRNAs. Therefore, DHT appears to be protective against cerebrovascular inflammation via conversion to 3β-diol and subsequent activation of ERβ in human brain VSM cells. The results of these studies indicate that: 1) DHT increases COX-2 expression under unstimulated/physiological conditions via an AR-dependent mechanism. 2) DHT decreases cytokine-, endotoxin,-hypoxia, and HGD-induced COX-2 expression via an AR-independent mechanism. 3) DHT decreases cytokine-induced reactive oxygen species. 4) DHT decreases hypoxia-induced HIF-1α expression. 5) DHT decreases HIF-1α and TLR4 expression during HGD via an AR-independent mechanism. 6) DHT's effect to attenuate cytokine-induced COX-2 expression is ERβ-mediated.
Open Access
Functional organization of a cortical-medullary neural circuit mediating organismal adaptation to stress
(Colorado State University. Libraries, 2023) Pace, Sebastian A., author; Myers, Brent, advisor; Hentges, Shane, advisor; Tobet, Stuart, committee member; Foster, Michelle, committee member
Hindbrain regions responsible for epinephrine and norepinephrine production are critical for orchestrating stress responses, maintaining physiological equilibrium and integrating afferent information. The nuclei central to hindbrain epinephrine and norepinephrine production, create a neural network that interfaces with forebrain and spinal cord regions, facilitating the integration of neuroendocrine and autonomic functions. Despite significant strides in our comprehension of stress response systems, questions concerning the roles of sex, stress history, and circuit mechanisms endure. In this study, we unveil and characterize a prefrontal-medullary circuit crucial for the suppression of stress responses. First, anterograde and retrograde tract-tracing studies demonstrated a stress-reactive vmPFC-RVLM circuit. Activation of this vmPFC-RVLM circuit mitigates glucocorticoid stress reactivity in both males and females, by targeting non-catecholaminergic neurons. Therefore, vmPFC-RVLM circuit activation may utilize local inhibitory neurons to limit catecholaminergic activation. To better understand how chronic stress affects the medulla, we explored the impact of chronic stress on signaling machinery and revealed elevated tyrosine hydroxylase (TH) levels in both male and female rats following chronic variable stress (CVS). To understand how CVS interacts with the vmPFC-RVLM circuit, we used an intersectional TeLC (Tetanus toxin - light chain) approach to disrupt the circuit and evaluate multiple stress response systems. In males, circuit disruption and CVS largely left behavioral and cardiovascular stress reactivity unaltered, however, some neuroendocrine endpoints were affected. Conversely, females exposed to circuit disruption and chronic stress exhibited heightened stress reactivity in glycemic, corticosterone, and arterial pressure responses, coupled with avoidant-like behaviors. These findings underscore the sex-specific necessity of the vmPFC-RVLM circuit in countering chronic stress-related outcomes, emphasizing a greater protective role in females relative to males. To gain deeper insights into the role of vmPFC inputs to the RVLM in females, we once again utilized a circuit-based TeLC approach, employing in situ hybridization (ISH) coupled with immunohistochemistry (IHC) to assess TH and phenylethanolamine N-methyltransferase (PNMT) transcript density across various VLM subregions. Notably, the TeLC-induced elevation of PNMT expression in females suggests that disrupting this circuit could potentially enhance epinephrine production by RVLM neurons, potentially intensifying stress reactivity post-CVS. This comprehensive study demonstrated the critical role of the vmPFC-RVLM circuit in modulating stress responses and revealing female-specific effects in mitigating physiological, behavioral, and transcriptional outcomes after chronic stress. These findings emphasize the significance of the vmPFC-RVLM circuit in managing stress reactivity in the context of chronic stress and identify the circuit as a potential candidate for reducing stress responding.
Open Access
Inducible photoreceptor degeneration model in goldfish
(Colorado State University. Libraries, 2011) Varland, Dezaray D., author; Vigh, Jozsef, advisor; Gionfriddo, Juliet, committee member; Ishii, Douglas, committee member; Madl, James, committee member; Tobet, Stuart, committee member
Photoreceptor degenerative diseases are among the leading causes of vision loss and there is presently no known cure. The future success of biological and prosthetic vision rescue approaches following photoreceptor loss remains questionable, due to the morphological and functional changes occurring in the remaining retinal circuitry. In the current study we sought to establish a chemically-induced photoreceptor degenerative model in goldfish, based on the ability of teleost to regenerate their retina following damage. N-methyl-N-nitrosourea (MNU) was chosen to chemically induce the photoreceptor degeneration, because it has been found to be potent, and selective in mammalian studies. We hypothesized that MNU would induce selective and complete photoreceptor loss in the goldfish retina as well as the consequent morphological changes observed in mammalian retinas. Under anesthesia, fish received a direct, intraocular injection of MNU into the posterior chamber of one eye whereas the contralateral eye served as sham-injected control. The effects of MNU were determined by standard immunohistochemical methods using known, well-established molecular markers of retinal cells. The MNU induced unilateral, selective, and dose-dependent photoreceptor degeneration: up to ~60% of photoreceptors lost the injected eye of the goldfish within 7 days, followed by nearly complete regeneration by ~50 days post-injection. Repeated MNU treatments did not increase the magnitude of degeneration, but delayed the regeneration. Unlike in mammals, MNU did not destroy all of the photoreceptors in fish. The incomplete photoreceptor degeneration together with the quick regeneration may be responsible for preventing the development of chronic morphological and functional consequences. However, the regeneration observed after MNU treatment is promising. Inducing total photoreceptor degeneration in fish retina, possibly by combining MNU with other factors shown to destroy photoreceptors (i.e. strong light) could provide an all-encompassing natural model for studying the potential of stem cell-based vision rescue approaches after photoreceptor loss.
Open Access
Low power biosensor and decimator design
(Colorado State University. Libraries, 2013) Scholfield, Kristin, author; Chen, Tom, advisor; Collins, George, committee member; Tobet, Stuart, committee member
This paper examines the use of low power circuits applied to biosensors used to observe neurotransmission. The term "biosensors" in the broadest sense describes many devices which are used to measure a biological state e.g. neural signal acquisition. The methods for developing biosensors are just as diverse, but one common thread is that many biomedical devices are battery operated and require low power for mobility. As biosensors become more complex they also require more functions such as data storage, digital signal processing, RF transmission etc. The more functions a sensor needs, the tighter the constraint for power consumption on a battery operated device becomes. In order to solve this problem, biosensors are increasingly being designed for low power consumption while weighing tradeoffs for performance and noise. Designers accomplish this by lowering the supply voltage, which reduces the overall size, and thus the load, of the devices. The amount of individual components will also be reduced, allowing for a smaller, faster device. Biosensors are important because they grant the ability for scientists to better understand complex biological systems. While many other methods exist for observing biological systems, electrochemistry is a practical method for measuring redox reaction because it senses chemical reactions on the surface of an electrode. The reaction will create a current, which can be interpreted via electronics. With the use of electrochemistry, scientist can cheaply and practically observe changes occurring between cells. On the engineering side, modern silicon processes provide small, tightly packed microelectrodes for high spatial resolution. This allows scientists to detect minute changes over a small spatial range. With an array of electrodes on the scale of 1000s, electrochemistry can be used to record data from a sizable cellular sample. Such an array could be used to identify several biological functions such as communication between cells. By combining known electrochemistry methods with low power circuit designs, we can create a biosensor that can further advance the understanding of the operation of cells, such as neurotransmission. The goal of our project is to create a device that uses electrochemistry to detect a redox reaction between a chemical, such as nitric oxide, and an electrode. The device needs to be battery operated for mobility and it must contain all needed electronics on chip, including amplification, digital signal processing, data transmission etc. This requires a surface of electrodes on chip that can handle the environment needed for a living tissue such as: specific temperature, pH and humidity. In addition, it requires a chip that is low power and which produces little heat. This thesis describes two separate designs, both of which are part of a final biosensor design that will be used for the detection of nitric oxide. The first design is a biosensor microelectrode array. The array will be used along with electrochemistry to detect the release of nitric oxide from a living tissue sample. The electrodes are connected to a chain of electronics for on chip signal processing. The design runs at a voltage of 3V in a 0.6µm CMOS process. The final layout for the microelectrodes measured approximately 4.84mm2 with a total of 8,192 electrodes and consumed 0.310mW/channel. The second design is a low power decimator for a sigma-delta analog to digital converter designed for biomedical applications. The ADC will be used along with a chain of amplifying electronics to interpret the signals received from the microelectrode array. The design runs at a voltage of 0.9V in a 0.18µm CMOS process. Its final layout measured approximately 0.0158mm2 and consumed 3.3uW of power. The ADC and microelectrode array were designed and fabricated separately to ensure their validity as standalone designs.
Open Access
Low-power switched-capcitor amplifier and Sigma-Delta modulator design for integrated biosensor applications
(Colorado State University. Libraries, 2013) Selby, Ryan, author; Chen, Tom, advisor; Collins, George, committee member; Tobet, Stuart, committee member
Neurotransmitters are chemicals present in living tissue which regulate biological functions. Some neurotransmitters which are present in the brain, such as nitric oxide (NO), are believed to play a role in the process of cellular migration during development. Today there is no practical way to measure gradients of neurotransmitters across pieces of tissue in both the spatial and temporal domains. Single electrode systems can be used to determine neurotransmitter concentrations at specific locations, but do not provide spatial resolution. Dyes and marking compounds can be used to locate concentrations of neurotransmitters across a piece of tissue, but kill the tissue in the process, thus limiting temporal resolution. Integrated silicon biosensor arrays have been proposed as a method for detecting neurotransmitters in both the spatial and temporal domains. Using large arrays of microelectrodes placed at pitches comparable to the size of individual cells, a high resolution chemical image of neurotransmitters could be captured in real time. For such an array, a large number of electronic components are necessary. Two such components are high precision amplifiers and analog-to-digital converters which are necessary to amplify the extremely small chemical signals and then convert them to digital values such that they can be stored and analyzed. These components must be low power to avoid generating heat, and small in size in order to limit total silicon area. This thesis proposes the design of a low power switched-capacitor amplifier and Sigma-Delta modulator for use as an analog-to-digital converter. The switched-capacitor amplifier achieves a gain of 40dB with -63.7dB total harmonic distortion while using 6.82μW and occupying 0.076mm2 silicon area. The Sigma-Delta modulator achieves a signal-to-noise ratio of 86.8dB over 2kHz signal bandwidth and uses 9.1μW while occupying 0.043mm2 silicon area. Both of these designs were implemented in a 0.18μm CMOS process with a supply voltage of 900mV and their functionality verified was in silicon.
Open Access
Microbes in the mucosa: impacts of the mucosal immune system and oral vaccination with Lactobacillus acidophilus on the gut microbiome
(Colorado State University. Libraries, 2021) Fox, Bridget E., author; Dean, Gregg, advisor; Abdo, Zaid, advisor; Tobet, Stuart, committee member; Ryan, Elizabeth, committee member
The mucosal immune system is constantly balancing between the clearance of pathogens, tolerance of self-antigen and food, and maintenance of homeostasis within the microbiota. Vaccination via mucosal routes is advantageous because it provides protection at local mucosal sites and systemically. However, induction of efficacious responses are often difficult due to the inherent barriers of the mucosal tissues. We have developed a probiotic-based mucosal vaccination platform that utilizes recombinant Lactobacillus acidophilus (rLA) to overcome these obstacles presented in oral vaccination. Here, we sought to determine whether repeated administration of rLA alters the intestinal microbiome as a result of L. acidophilus probiotic activity (direct competition and selective exclusion) or from the host's mucosal immune response against the rLA vaccine. To address the latter, IgA-seq was employed to characterize shifts in IgA-bound bacterial populations. Additionally, we determined whether using rice bran as a prebiotic would influence the immunogenicity of the vaccine and/or IgA bound bacterial populations. Our results show that the prebiotic influenced the kinetics of rLA antibody induction, and that the rLA platform does not cause lasting disturbances to the microbiome. Nucleotide-binding oligomerization domain containing 2 (NOD2) has presented itself as an essential regulator of immune responses within the gastrointestinal tract. This innate immune receptor is expressed by several cell types, including both hematopoietic and nonhematopoietic cells within the gastrointestinal tract. Mice harboring knockouts of NOD2 only in CD11c+ cells were used to better characterize NOD2 signaling during mucosal vaccination with rLA. We show that NOD2 signaling in CD11c+ cells is critical for mounting a humoral immune response against rLA. Additionally, disruption of NOD2 signaling in CD11c+ cells results in an altered bacterial microbiome profile in both vaccinated and unvaccinated mice.
Open Access
Multifunctional nanowire scaffolds for neural tissue engineering applications
(Colorado State University. Libraries, 2012) Bechara, Samuel Leo, author; Popat, Ketul, advisor; Tobet, Stuart, committee member; Legare, Marie, committee member; Rollin, Bernard, committee member; Sladek, John, committee member
Unlike other regions of the body, the nervous system is extremely vulnerable to damage and injury because it has a limited ability to self-repair. Over 250,000 people in the United States have spinal cord injuries and due to the complicated pathophysiology of such injuries, there are few options available for functional regeneration of the spinal column. Furthermore, peripheral nerve damage is troublingly common in the United States, with an estimated 200,000 patients treated surgically each year. The current gold standard in treatment for peripheral nerve damage is a nerve autograft. This technique was pioneered over 45 years ago, but suffers from a major drawback. By transecting a nerve from another part of the body, function is regained at the expense of destroying a nerve connection elsewhere. Because of these issues, the investigation of different materials for regenerating nervous tissue is necessary. This work examines multi-functional nanowire scaffolds to provide physical and chemical guidance cues to neural stem cells to enhance cellular activity from a biomedical engineering perspective. These multi-functional scaffolds include a unique nanowire nano-topography to provide physical cues to guide cellular adhesion. The nanowires were then coated with an electrically conductive polymer to further enhance cellular activity. Finally, nerve growth factor was conjugated to the surface of the scaffolds to provide chemical cues for the neural stem cells. The results in this work suggest that these multifunctional nanowire scaffolds could be used in vivo to repair nervous system tissue.
Open Access
N-linked glycosylation is fundamentally linked to the surface expression of neuroligins
(Colorado State University. Libraries, 2023) Cast, Thomas, author; Chanda, Soham, advisor; DeLuca, Jennifer, committee member; Di Pietro, Santiago, committee member; Tobet, Stuart, committee member
N-linked glycosylation is one of the most prevalent forms of post-translational modification, decorating secreted and cell-surface transmembrane proteins as they are trafficked along the secretory pathway. While well-characterized in most tissues, non-canonical N-glycan diversification has been reported to occur in the central nervous system. Chapter 2 of this dissertation describes the importance of N-linked glycosylation for the neuroligin family of synaptic cell-adhesion molecules (NLGN1-4). NLGNs play a crucial role in regulating synaptic transmission strength by recruiting neurotransmitter receptors to synapses. Mutation of N-glycosylated residues increased retention of each NLGN isoform in the endoplasmic reticulum (ER), consequentially reducing their ability to interact with presynapses. Pharmacological inhibition of various stages of the N-glycan maturation pathway further revealed that only the initial transfer of the polysaccharide is essential for the surface expression of NLGN proteins. Chapter 3 characterizes a missense mutation identified in the NLGN4 gene of a patient with autism. This mutation, p.Arg101Gln (R101Q), is directly upstream of a conserved N-linked glycosylation site, which played a universal role for the surface localization of each NLGN isoform. Biochemical and cellular analysis revealed the NLGN4-R101Q variant to be immaturely glycosylated and mistrafficked, retained in the ER similarly to N-glycan site mutants. In neurons, the mistrafficked R101Q variant failed to reproduce the excitatory synaptogenic effects of NLGN4-WT, indicating an overall loss-of-function phenotype. Further, equivalent RQ mutations introduced in other NLGN isoforms mimicked the glycoprotein maturation and surface expression defects. Together, these findings reveal a profound overall significance of N-glycans for NLGNs and the conserved role of a specific N-linked glycosylation site for promoting the forward trafficking of NLGN protein.