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Browsing by Author "Tobet, Stuart A., advisor"

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    Anatomic plasticity and functional impacts of neural – immune and neural – epithelial signaling in the intestine
    (Colorado State University. Libraries, 2021) Schwerdtfeger, Luke A., author; Tobet, Stuart A., advisor; Chicco, Adam, committee member; Myers, Brent, committee member; Ryan, Elizabeth, committee member
    The intestinal wall is a multicompartmental barrier tissue composed of over 25 distinct cell types with integrated and complex signaling both within and between compartments. The gut wall is also a large endocrine organ comprised of cells capable of producing dozens of peptides used for hormonal and other signaling functions. However, the mechanistic roles that neural secretions play in regulating the gut epithelial barrier in health and disease are not well known. Additionally, frequently used models available for studying intestinal function outside of the body lack the complexity to investigate neural – epithelial and neural – immune signaling interactions. Using a bifurcated approach to method development, we created two culture systems for maintaining the full thickness of the intestinal wall ex vivo. One method allows for culture of mouse or human organotypic intestinal slices that maintain the gut wall for 6 or 4 days, respectively. This system does not however, maintain a true luminal – epithelial barrier as seen in the in vivo gut. The second method, a microfluidic organotypic device (MOD) enables maintenance of explanted mouse or pig intestinal tissue for up to 3 days ex vivo, with an intestinal barrier intact. These two methods allow for investigating and cross-validating of numerous biological questions now previously possible using traditional culture models. Neuronal fiber proximity to gut epithelia has been shown, with goblet, tuft and enteroendocrine cells being closely opposed by fibers. Goblet cells secrete mucopolysaccharides, a first line of defense separating luminal microbiota from host tissue. I have recently shown that vasoactive intestinal peptide (VIP) can regulate goblet cell production in organotypic slices of mouse ileum. This peptide is also in close proximity to Paneth cells in the base of the crypt, and enteric mast cells. There were sex differences in baseline mast cell neuronal proximity, quantities, and cell size in mouse ileum. Further, mast cells showed a sex difference in responses to lipopolysaccharide challenge. Further investigation of neurosecretory factor regulation of immune and epithelial function is needed, both in goblet cells and other secretory epithelia like anti-microbial producing Paneth cells, and in immune components like mast cells. Graphical illustration of the dissertation project is included below.
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    Development of fluidic devices to facilitate more accessible monitoring of human health
    (Colorado State University. Libraries, 2024) Cherwin, Amanda E., author; Henry, Charles S., advisor; Tobet, Stuart A., advisor; Snow, Christopher, committee member; Abdo, Zaid, committee member
    In December of 2023, the World Health Organization (WHO) Director-General Tedros Adhanom Ghebreyesus outlined the 'Five P's' of global health priorities: Promoting health, Providing health, Protecting health, Powering health, and Performing for health. Despite the mantra of 'prevention is better than cure,' many countries still prioritize treating the sick over proactive health promotion, leading to inadequate prevention of non-communicable diseases (NCDs). Access to healthcare services poses a significant barrier to early recognition and treatment of health issues, particularly in low-income communities. To address these challenges, harnessing the power of science and technology becomes imperative. Powering health involves leveraging scientific research and collaboration to understand disease mechanisms better. Physiologically relevant models, such as microfluidic systems, offer insights into disease progression. Microfluidics, especially when combined with 2D and 3D culture systems, enhances functionality by mimicking physiological conditions. These devices provide cost-effective solutions for diagnostic challenges, bridging the gap between in vitro and in vivo studies. Protecting health requires a deeper understanding of organ systems. Chapter 2 examines a microfluidic model of the gut, an organ that plays a critical role in maintaining overall health. Two devices are discussed, an organotypic device for maintaining ex vivo gut tissue explants, and an electrochemical sensor module for monitoring relevant molecules such as oxygen or hydrogen peroxide within the tissue media. Dysbiosis in the gut microbiome has been linked to various pathologies, emphasizing the need for accurate models for studying gut barrier integrity. Ex vivo models using microfluidic devices offer promising avenues for studying disease mechanisms. The devices described in Chapter 2 serve as an effective model of the intestinal barrier that can be closely monitored in real-time. Providing health involves making effective healthcare solutions universally accessible. Point-of-care (POC) diagnostics, facilitated by microfluidics, enable rapid and cost-effective disease detection. Capillary-driven flow microfluidic devices enhance accessibility by eliminating the need for bulky external pumps, making POC testing feasible even in resource-limited settings. Combining the concepts of Powering and Providing health leads to the development of innovative diagnostic devices. Capillary-driven flow microfluidics enables the development of portable devices for diagnosing conditions from viscous sample matrices like blood and saliva. These devices offer less invasive and more accessible alternatives to traditional diagnostic methods, potentially revolutionizing healthcare delivery. Chapter 3 describes a capillary flow device used to quantify levels of two salivary biomarkers (Galectin-3 and S100A7) correlated to Heart Failure (HF) outcomes. This rapid, noninvasive, accessible POC test can drastically improve the quality of life for HF patients, particularly in rural and resource-limited areas. Using an electrochemical detection method, we demonstrate successful multiplexed detection of both biomarkers in spiked buffer solutions. Chapter 4 focuses on microfluidic devices probing rheological properties of whole blood related to Sickle Cell Disease (SCD) and clotting using capillary flow. For the SCD device, our goal was to develop a low-cost Point-of-Care (POC) multiplexed device for rapid and accurate identification of SCD phenotypes using three key reagents tied to altered sickle cell blood rheology: calcium chloride, sodium metabisulfite, and adenosine diphosphate. We developed an integrated device where whole blood reacts with reagent pads, enabling rapid assessment of a patient's SCD phenotype to inform appropriate treatment. We also introduced the Paper-based Clotting Analysis Test (PCAT) for efficient, low-cost analysis of primary hemostasis. Current methods for monitoring hemostasis are expensive and slow. Our capillary flow device uses whole blood moving at high flow rates for sustained durations to induce thrombus formation. This dissertation bridges the gap between effective health monitoring and accessibility through fluidic devices using either pump-driven or capillary-driven flow. Chapters detail the development of microfluidic systems for monitoring intestinal barrier function, detecting biomarkers in saliva for Heart Failure prognosis, and processing blood samples for Sickle Cell Disease phenotyping and clotting analysis. Ultimately, these devices hold the potential to transform healthcare management, particularly in underserved communities.
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    Role of gamma-aminobutyric acid (GABA) in hypothalamic nuclear development
    (Colorado State University. Libraries, 2008) McClellan, Kristy Michelle, author; Tobet, Stuart A., advisor
    The hypothalamus is involved in energy balance, appetite regulation, stress and anxiety, reproduction including sex behavior, and aggression. It is divided into cell groups based on cell staining, functional similarities and projections. This dissertation looks at the development of three of these cell groups, the ventromedial nucleus (VMN), the paraventricular nucleus (PVN), and the arcuate nucleus (ARC). The VMN and PVN have a unique pattern of GABA expression in elements surrounding the nuclei, with a void of GABA within the region of the developing nuclei. The ARC, on the other hand, has dense GABAergic immunoreactive elements throughout the nucleus. Because of the expression pattern of GABA immunoreactivity surrounding the VMN and PVN, it is likely that GABA is acting as a boundary cue to influence migrating neurons. Chapter 2 reviews the development of the VMN, including a discussion of the heterogeneity of the nucleus, a description of what cues are involved in cell migration, and descriptive information on the directions of cell movement during development. Chapter 3 examines the role of GABA on cell migration within the VMN and ARC. A live slice culture system allowed visual tracking of cell movement in the VMN and ARC. There was a difference between the average movement speeds of cells in the VMN vs. cells in the ARC, and upon addition of GABA receptor antagonists to the slices, VMN cells increased in speed while ARC cells did not change. In mice lacking functional GABAB receptors there was a change in cell positions of neurons containing immunoreactive estrogen receptor (ER)α, which may be due to changes in cell movements and migration. There was no change in amount of cell positions of immunoreactive ERα cells in the ARC. Chapter 4 looks at the role of GABA and brain derived neurotrophic factor (BDNF) in PVN development. There was a sex-selective effect in GABAB R1 subunit knockout mice of positions of cells containing immunoreactive ERÎα, indicating that females may be particularly more susceptible to changes in GABA signaling as it may be influencing the final positions of cells. There was also a decrease in BDNF expression in GABABR1 subunit knockout mice, suggesting that GABA may play a role in cell differentiation. In conclusion, several lines of evidence indicate roles for GABA in the development of the hypothalamus, in particular, within the VMN and PVN.
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    The vasculature of the paraventricular nucleus of the hypothalamus: influence of development, gamma-aminobutyric acid (GABA) receptors, and prenatal glucocorticoids
    (Colorado State University. Libraries, 2014) Frahm, Krystle A., author; Tobet, Stuart A., advisor; Hentges, Shane, committee member; Tamkun, Michael, committee member; Garrity, Deborah, committee member
    The paraventricular nucleus of the hypothalamus (PVN) is a critical brain region that regulates many homeostatic and stress responses. In addition to its dense cytoarchitecture, it also contains a vast network of blood vessels. These blood vessels within the mouse PVN have a higher density than other brain regions, which develops postnatally. Loss of gamma aminobutyric acid (GABA) signaling or prenatal dexamethasone (dex) treatment decreased the blood vessel density. Dex also decreased blood brain barrier (BBB) competency while increasing desmin-immunoreactive pericytes at postnatal day (P)20. Long-term consequences included a decrease in GFAP contact with blood vessels selectively in dex-treated females, and an increase in depression-like behaviors in dex-treated males. Chapter 2 examines the blood vessel density within the PVN. Initially the blood vessel density is similar than surrounding brain regions, then after P8 there was an increase that resulted in a highly vascularized network around P20. The highest densities were restricted to the rostral and mid regions of the PVN, where the neuroendocrine neurons are housed. In addition, mice lacking a functional GABAB receptor had a significant decrease in blood vessel density in the mid region at P20. The protein endocan has been proposed to be a "tip cell" marker, indicating angiogenesis. To further characterize the postnatal angiogenic period within the PVN, recently developed antibodies against endocan were used. Chapter 3 provides evidence that endocan is normally expressed in the mouse brain but not restricted to tip cells. In addition, prior perfusion with fluorescein isothiocyanate (FITC) prevents endocan-immunoreactivity (ir) and provides a novel method for identifying non-functional blood vessels. Chapters 4 and 5 show that excess fetal glucocorticoids alters the BBB within the PVN at two time points. At P20, there was a loss of BBB integrity accompanied by an increase in desmin-ir pericytes on a reduced blood vessel network due to dex-treatment for both prepubertal males and females. In contrast at P50, the blood vessel density and BBB were no longer disrupted following fetal dex-treatment. However, there was a decrease in glial fibrillary acidic protein (GFAP)-ir astrocytes in dex-treated females and an increase in desmin-ir pericytes in dextreated males. In conclusion, the work set forth in this dissertation indicates that the dense vascular network within the PVN develops postnatally and is susceptible to regulation by both exogenous and endogenous factors.