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Vitamin D, folate, and cobalamin (vitamin B12) are crucial micronutrients in human physiology that are necessary for healthy calcium, phosphorus, and single-carbon metabolism. Recent studies have indicated that these vitamins also affect the inflammatory response in ways unrelated to their well-characterized deficiencies. Accordingly, analysis of their effect on chronic inflammatory diseases like Crohn's disease (CD) is warranted. This investigation examines the effects of vitamin deficiency on macrophages and intestinal epithelial cells upon exposure to Mycobacterium avium paratuberculosis (MAP,) a pathogen capable of triggering CD, to model the inflammatory response in clinical CD patients. ELISA analysis of CD patient plasma established that MAP-positive patients have lower folate, vitamin B12, and active vitamin D (calcitriol) than MAP-negative patients. Next, we investigated the effects of folate and vitamin B12 deprivation on macrophages to assess inflammatory cytokine expression, oxidative stress, and macrophage apoptosis. We determined that folate and B12 deprivation exacerbates inflammation while preventing infected macrophages from successfully undergoing apoptosis, whereas supplementation reversed these effects. Then, we examined the role of vitamin D in regulating cathelicidin expression during MAP infection. MAP infection blocked the conversion of inactive vitamin D (calcifediol) to calcitriol, thereby interrupting the expression of the antimicrobial peptide cathelicidin. Calcitriol treatment restored cathelicidin production, reduced inflammation and bacterial viability, and reduced oxidative stress in co-cultured macrophages, Furthermore, cathelicidin knockdown abolished calcitriol's beneficent effects. These studies detail the importance of vitamin availability for healthy immune functionality. The attenuation of inflammation during MAP infection further indicates that CD patients, who are at elevated risk of vitamin deficiency, may benefit from supplementation or clinical screening for low vitamin levels.

Migraines affect an estimated 1.04 billion people worldwide annually. To address the global prevalence of migraines, new migraine treatments should be of utmost importance. To date, magnesium hydroxide [Mg(OH)2] and magnesium sulfate are two common treatments for delivering magnesium to patients with low intracellular levels, which are believed to be linked to migraines. One major downside to using Mg(OH)2 as an over-the-counter treatment for magnesium delivery is the possibility of side reactions in the acidic environment of the stomach, further affecting the pH stability necessary during uptake in the small intestine. To address these downfalls, Mg(OH)2 was synthesized with capping agents betaine and citrate [Mg(OH)2 B/C] to protect the Mg(OH)2 particles from side reactions in the acidic environment of the stomach. The addition of betaine is expected to improve particle absorption, distribution, and potential to cross the blood-brain barrier via the betaine transporter while citrate is expected to decrease particle size and reduce particle aggregation. Fourier transform infrared and ultraviolet-visible spectroscopy validated the presence of key Mg(OH)2 B/C functional groups present in Mg(OH)2 citrate and Mg(OH)2 betaine controls. Dynamic light scattering confirmed the average hydrodynamic diameter of Mg(OH)2 decreased with capping agents betaine and citrate from 960 nm to 660 nm. Likewise, the average pH of the Mg(OH)2 B/C particle solution in gastric juice over 48 hours was 7.6, slightly above neutral pH (pH ˜ 7.0-7.4) and within the pH range of the small intestine (pH ˜ 6-8). Scanning electron microscopy energy dispersive x-ray spectroscopy analyzed the atomic composition of the Mg(OH)2 B/C particle core, revealing a high density of magnesium and oxygen in the Mg(OH)2 particle core. In gastric juice, the high density of magnesium and oxygen were less affected in Mg(OH)2 B/C particles compared to uncoated Mg(OH)2, confirming the hypothesis capping agents betaine and citrate help protect the Mg(OH)2 core structure from side reactions in acidic environments. Lastly, cell viability studies on J774 human macrophage cells with Mg(OH)2 B/C particles, Mg(OH)2, milk of magnesia, Mg(OH)2 citrate, and Mg(OH)2 betaine confirmed normalized cell viability percentages for all treatments within a magnesium concentration range of 159 to 0.2 mM were above 100%. Thus, this study introduces a pH-stable magnesium delivery system that is safe to use while protecting the Mg(OH)2 core against the acidic environment of the stomach for use in migraine patients.

After the completion and failure of 44 phase III clinical trials, Traumatic Brain Injury (TBI) continues to be a leading cause of disability, morbidity, and mortality amongst military personnel and civilians of all age groups. TBI is characterized by primary and secondary injury processes of which the secondary injury is defined by oxidative stress, increased energy demands, mitochondrial dysfunction, neuroinflammation, and more; characteristics which are also shared with neurodegenerative diseases. Creatine (Cr) is one of the most abundantly used and studied supplements in the fitness industry which, when in the form of Phosphoryl-Creatine (PCr), directly aids in the conversion of ADP to ATP, particularly in metabolically stressed conditions where oxygen is unavailable, and hypothesized to be a JAK2 inhibitor. Similarly, Tannic Acid (TA) is a polyphenol naturally available in teas and nuts that provides neuroprotective effects against TBI through the PGC-1?/Nrf2/HO-1 pathway. However, bioavailability of these compounds in the brain is limited through oral supplementation. Therefore, increasing the local concentration of these compounds in the brain parenchyma may provide therapeutic benefits after cerebral injury. In this study, efficiently loaded, TA-based Creatine nanoparticles (NPs) were synthesized as a potential therapeutic for secondary TBI and related neuroinflammatory conditions. This nanosystem demonstrates surface chemistry augmentation, high loading efficiency, and biodegradation with 24 hours. Purified NPs had an average hydrodynamic diameter of 200 nm, an average surface charge of -44mV, and a polydispersity index (PDI) of 0.171. Purified particles also demonstrate long shelf life and stability over many months, suggesting this inexpensive formulation could be utilized as a cheap therapeutic in underserved, low-income areas.

The increasing incidence of chronic diseases worldwide has encouraged the discovery of treatment alternatives for chronically ill patients. To increase patient compliance and diminish secondary effects, delayed drug release systems have been developed. However, current pharmaceutical coatings still face limitations in targeting, loading efficiency, and pH tunability when administered orally. In this thesis, we demonstrate the potential of using atomic layer deposition (ALD) as a technique to coat 5-Aminosalicylic acid (5-ASA)—a drug to treat inflammatory bowel disease—to control 5-ASA's release throughout the gastrointestinal tract. 5-ASA was coated with 300 cycles of Al2O3 and 200 cycles ZnO ALD and was characterized by Fourier transform infrared spectroscopy (FTIR), scanning transmission electron microscopy (SEM), high resolution transmission electron microscopy (TEM), energy dispersive x-ray spectroscopy (EDX), and UV-visible spectroscopy, to confirm 5-ASA as a viable ALD substrate and its ability to be studied at a wavelength of 298 nm for release rate characterization. Three different form factors of 5-ASA—pellets, films, and powders— which were coated with 300 cycles of Al2O3 ALD were studied via UV-Vis in acidic HCl pH 4 media. To further understand the etching rate of Al2O3 films, quartz crystal microbalance (QCM) crystals were coated following the same ALD protocols used in our UV-Vis studies. Based on the results gathered from 5-ASA coated with Al2O3 ALD, equivalent studies were made with ZnO inorganic film coatings via ALD. This thesis demonstrates and encourages ALD's potential in coating 5-ASA as a proof-of concept to achieve delayed and controlled drug release that is tunable based on the ALD coating thickness and chemistry.

Candida albicans is a fungal microbe that is often present inside of humans in the mouth and gastrointestinal tract. It shares a mostly commensal relationship with its hosts but can develop into an opportunistic pathogenic infection under conditions of immune suppression. Oral thrush or candidiasis is an uncomfortable condition resulting from excessive growth of Candida albicans in the oral cavity. Candidiasis is prone to progressing into more threatening symptoms without proper treatment. There are few effective antifungal medicines used for treatment and the problem of antimicrobial resistance is growing. Alcaligenes faecalis is a bacterial microbe that does not pose a significant threat to human health in many cases. It is also present in the human gastrointestinal tract and shares an inhibitory relationship with Candida albicans. Streptococcus mutans is also a bacterial microorganism present in the oral cavity and GI tract of humans. It is one of the primary factors related to dental decay, one of the most common modern health issues humans face. In cases of dental caries, Candida albicans and Streptococcus mutans have been found to have positive correlation in the biofilm coating teeth. This study examines the effects on microbial growth under the presence of all three organisms in a tri-culture. The results of this experiment could help better understand how to inhibit growth of Candida albicans and Streptococcus mutans and promote oral health.

Tumor Necrosis Factor alpha antagonists (anti-TNFα) have been extensively used for Crohn's disease (CD) treatment. Even though they may control CD symptoms initially, treatment response varies among patients, which seems to depend on single nucleotide polymorphisms (SNPs) in TNFα receptors superfamily 1A and 1B (TNFRSF1A/B). Most importantly, M. tuberculosis infection has been strongly associated with these medications, but no studies have elucidated the effects of anti-TNFα on CD associated with MAP (Mycobacterium avium subspecies paratuberculosis; a possible causative agent of CD, and closely related to M. tuberculosis). Here, we investigated the effects of recombinant inflammatory cytokines and anti-TNFα therapeutics on macrophages infected with MAP isolated from CD patient. We also tested the prevalence of MAP and the significance of nine SNPs in TNFα, TNFRSF1A and TNFRSF1B from the blood of 54 CD and 50 healthy subjects by IS900 nPCR. Both PEGylated and non-PEGylated forms of anti-TNFα increased MAP viability by nearly 1.5 Log CFU/mL, while rIL-6 and rIL-12 induced MAP viability at 5.42 ± 0.25 and 4.79 ± 0.14 Log CFU/mL, respectively. In contrast, rTNFα reduced MAP survival in infected macrophages by 2.63 Log CFU/mL. Expression of TNFα, IL-6, and IL-12 was upregulated by three folds following MAP or M. tuberculosis infection compared to other bacterial strains (P < 0.05). Four SNPs (TNFα:rs1800629, TNFRSF1A:rs767455, TNFRSF1B:rs1061624 and TNFRSF1B:rs3397) were overrepresented significantly (P < 0.05) among CD patients compared to healthy controls. The TNFRSF1A:rs767455 GG genotype was found in 15/54 CD patients (28%), while it was only found in 2/50 healthy controls (4%) [OR = 9.2, 95% CI: 1.98-42.83]. The TNFRSF1B:rs3397 TT genotype was found in 15/54 CD patients (28%) compared to (4/50) healthy controls (8%) [OR = 4.4, 95% CI: 1.36-14.14]. Furthermore, the SNPs TNFRSF1A:rs767455 and TNFRSF1B:rs3397 were associated with downregulating their corresponding genes significantly (P < 0.05). MAP infection was predominantly found among CD patients in comparison to healthy controls (57% vs 8%, respectively), which was also dependent on the SNPs TNFRSF1A:rs767455 and TNFRSF1B:rs3397. Our SNP haplotype analysis of TNFRSF1A:rs767455 and TNFRSF1B:rs3397 indicates that the G – T haplotype is significantly distributed among CD patients (46%) and MAP infection susceptibility is also associated with this specific haplotype (31%). The data indicate MAP positive CD patients receiving anti-TNFα could result in favorable conditions for MAP infection, which explains the poor response of many CD patients to this treatment, leading to adverse outcomes ultimately.

Metabolic dysregulation accompanying SARS-CoV-2 infection is a key determinant of disease severity. In this study, we performed extensive data mining of multiple existing single-cell RNA seq datasets of COVID-19 BALFs, in combination with high-dimensional immune cell profiling of PBMCs from COVID-19-infected patients, to get a comprehensive, systemic profile of the immunometabolic regulation of adaptive immunity during severe COVID-19. Hypoxia, a hallmark of COVID-19 ARDS, was found to elicit a global metabolic reprogramming in effector lymphocytes. In response to oxygen and nutrient-deprived microenvironments, these cells shift from aerobic respiration to increase their dependence on anaerobic processes including glycolysis, mitophagy, and glutaminolysis to fulfill their bioenergetic demands. We find that these metabolically reprogrammed CD8 and NK cells, under persistent antigen stimulation, become exhausted, displaying impaired cytotoxic function and anti-viral efficacy. We demonstrate that dysregulated metabolism significantly impairs memory lymphocyte differentiation, including the formation of memory NK and tissue resident CD8 memory cells. Unsupervised clustering techniques revealed multiple distinct, differentially abundant CD8 and NK memory cell states that are marked by high glycolytic flux, mitochondrial dysfunction, and cellular exhaustion, further highlighting the connection between disrupted metabolism and impaired memory cell function in COVID-19. Overall, our findings provide novel insight on how SARS-CoV-2 infection affects host immunometabolism and anti-viral response during COVID-19.

<p>Localization and subtype distribution of the receptors for neurotensin and opiods in canine small intestine was studied by the radioligand binding technique.</p> <p>Extensive dissection procedure was developed to separate a) longitudinal muscle (LM) layer containing myenteric plexus (MP), b) the circular muscle (CM) layer containing the deep muscular plexus (DMP) and c) the submucosa containing the submucous plexus (SMP).</p> <p>Purified membranes were prepared from the LM, MP, CM, DMP and SMP by differential and the density gradient centrifugation and using the markers 5'-nucleotidase (for smooth muscle plasma membranes), NADPH cytochome C reductase (for endoplasmic reticulum), cytochrome C oxidase (for mitochondrial membranes), specific binding of [^3H]saxitoxin for the neuronal membranes and the content of the vasoactiev intestinal polypeptide immunoreactive material as a measure of intact synaptosomes.</p> <p>The fractions enriched in the membranes from LM, CM, DMP, MP and SMP were used to study the distribution and properties of neurotensin receptors using [^125]Tyr^3-neurotensin and of opioid receptors using [^3H]diprenorphine, [^3H]etorphine and [^3H]ethylketocyclazocine.</p> <p>Neurotensin receptors were confined to the CM, DMP and SMP. These receptors has the similar affinity and recognition properties at their high affinity sites (Kd 0.1 - 0.2 nM). The low affinity receptors on the DMP were of lower affinity than their CM counterparts (Kd 40 nM vs 3nM). The receptors on the CM differed from those on the DMP in their radiation target size (mw - 190, 000 da on the CM and - 120, 000 da. on the DMP). Reduced disulfide bridges were required for the binding to both the CM and DMP neurotensin receptors. However, in vitro, the excitation, but not the inhibition, of the circular muscle strips to added neurotensin was abolished on reduction of the disulfide bonds.</p> <p>Opioid receptors were present on the DMP, MP and SMP but not on any smooth muscle. The receptors on all the three plexuses had similar affinity for the non-selective opioid ligand ([3H]diprenorphine (Kd - 0.1 - 0.2 nM). Both the DMP and MP contained -40-45% 5-subtypes of opioid receptors as assessed in competition studies. All three plexuses contained similar porportion of k-subtype (10-15%), assessed by competition studies with dynorphin [1-13] and U-50488H, and confirmed by saturation experiments with [^3H]ehtylketocyclazocine with or without sheilded 5-receptors. Ionic regulation of these receptors was similar to those observed for the opioid receptors in other systems.</p> <p>Therefore the action of neurotensin on the motility of the canine small intestine may be the combined result of the its action on the smooth muscle and on the modulation of the release of other mediators presynaptically at the DMP level. The action of opioids on the motility is probably through the modulation of the release of other mediatorset the DMP and MP level: direct action of opioids on the smooth muscle is not supported by the present studies.</p> / Thesis / Doctor of Philosophy (PhD)

The intent of this thesis is to determine whether the deubiquitinating enzyme ubiquitin specific protease 30 (USP30) is cleaved by Omi/HtrA2 (hereafter referred to as Omi) protease during mitochondrial stress. USP30 is a mitochondrial protein that is anchored in the outer mitochondrial membrane and has components in the intermembrane space (IMS) as well as in the cytoplasm. USP30's IMS component has a six-amino-acid sequence that is very similar to Omi's consensus cleavage sites. Under normal conditions, Omi resides exclusively within the IMS; therefore, if Omi were to cleave USP30, it would target the part of the protein located in the IMS component. Omi is known to play a crucial role in a variety of diseases including cancers, neurodegenerative, and metabolic disorders. Since Omi is a serine protease, it is assumed to carry its normal function through the direct cleavage and degradation of specific substrates. If USP30 deubiquitinase is a bona fide substrate of Omi, this will provide new and important information on the mechanism by which Omi regulates the polyubiquitination process during mitochondrial stress.