Osteoarthritis (OA) is a prevalent joint disease without a cure and leading cause of disability worldwide. Knee OA is the most common and expected to increase with a growing, aging population. OA is clinically diagnosed by the measure of joint space narrowing on a radiograph and pain scores, though OA is now known to be a disease of the whole joint and characterized by cartilage degradation, subchondral bone sclerosis, synovitis, meniscal erosion and inflammation. There are currently no disease-modifying therapies because the pathogenesis and progression of these events are unknown. Recent studies have demonstrated the importance of subchondral bone in OA initiation. Using an advanced imaging technique called individual trabecula segmentation (ITS) to decompose the trabecular network into individual plates and rods, subtle microstructural changes were identified beneath both intact and damaged cartilage in human OA. These findings were also observed during OA initiation before cartilage changes in a model of guinea pig OA. The aims of this study were to further investigate these bone microstructural changes and investigate how they affect the mechanical properties of trabecular bone and overlying articular cartilage. In the first aim, the Dunkin-Hartley guinea pig model of spontaneous OA was used to quantify ITS-based trabecular microstructural changes in the knee joint during OA initiation in both a longitudinal and cross-sectional analysis. In the longitudinal study, microstructural changes in the subchondral bone were quantified from μCT images and voxel-based modeling and remodeling. In the cross-sectional study, structural, biomechanical and biochemical properties of articular cartilage, subchondral bone plate and trabecular bone were analyzed. In both the longitudinal and cross-sectional analyses, there was a trend towards increased thickness, a significant decrease in porosity, and increased mineralization in the subchondral bone plate. There was an increase in the plate-to-rod (PR) ratio due to a loss of trabecular rods, an increase of trabecular plates, and thickening of trabecular plates before any visible histological changes in the cartilage. Voxel-based bone modeling and remodeling analysis confirmed that there was a loss of rods and not merely that the rods were turning into plates. This confirmed our hypothesis that trabecular rod loss precedes cartilage damage and could be a potential therapeutic target. There was a trend towards an increase in the apparent elastic modulus of bone followed by a reduction in the modulus, with the stiffest and most drastic reduction in the medial tibial plateau, which coincided with cartilage fibrillations and a trend towards reduction in the cartilage aggregate modulus. The tissue-level mechanical properties of the trabecular bone are due to both microstructural changes in the trabecular network and material changes in the tissue modulus. Micro-indentation of the trabecular bone revealed a trend towards an increase in the tissue modulus in the medial tibial plateau, followed by a reduction in the tissue modulus. This suggested that during OA progression, there is rapid formation of lower-quality bone with a reduced capacity to mechanically support the joint. In summary, there were structural and mechanical bone changes observed before histological, mechanical or biochemical changes in the articular cartilage. In the second aim, a mechanically-driven subchondral bone computational model was developed. Under equilibrium conditions to simulate aging, there was no change in bone volume fraction and there was a shift from plate-like to rod-like trabecula. There was a slight decrease in the apparent elastic modulus of the bone. Under increased applied strain to simulate the effects of obesity, there was an increase in bone volume fraction, due to both a rod loss and plate thickening. This change came from rod loss, rods thickening and becoming plates, as well as rods and plates merging together. These microstructural changes caused an increase in the apparent elastic modulus of bone. This study demonstrated that the microstructural changes observed in OA can be simulated by increasing the applied strain. Taken together, these studies demonstrate how bone microstructural changes in OA initiation affect the mechanical integrity of the subchondral bone and could cause abnormal stress distributions in the overlying cartilage and promote cartilage degradation. Therapies that prevent bone loss, like bisphosphonates, could be investigated to prevent this initial rod loss as a means of potentially slowing or reversing OA progression.

The shadow economy is a topic that has been around for many years now. The increasing regulations on cash and the endeavours public authorities made to enlarge the tax base show a clear intention from the public sector to pursue any activity that is carried out outside the borders of what is taxable. This paper uses the Currency Demand Approach (CDA) to estimate the monetary base M0 for Portugal, Italy, Greece, and Spain and for each year from 2002 to 2021 and, subsequently, calculate the size of the shadow economy as a percentage of GDP. To estimate the CDA equation, we employed fixed effects panel data regression. The results show an average shadow economy value of 9,33% for Greece; 13,43% for Italy; 10,78% for Portugal; and 11,11% for Spain. The results have also been compared with those of previous studies that have estimated the shadow economy of other countries using the MIMIC approach, showing that the CDA tends to give lower estimates. Additionally, a common trend was found for the studied countries since after the financial crisis of 2008 and 2014, all of them reached peak values in their shadow economies.

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The nutritional value of an amino acid (AA) is determined by its bioavailability, however concept of digestibility is mostly used in practical situations. Four studies were conducted to test 2 exogenous factors that were hypothesized to affect the AA digestibility in non-ruminant animals. In study 1, broiler chicks were randomly allotted to 4 dietary treatments of control and distillers dried grains with solubles (DDGS, 20%) diets supplemented or not with a novel mixture of carbohydrases. Results indicated the ability of carbohydrase mixture to increase energy utilization of the DDGS diet, with significant improvements in AA digestibility, consequently improving growth performance of broilers. Study 2 examined effect of the carbohydrase mixture in pigs fed a high DDGS diet. Ileal cannulated growing pigs (n = 8, 64.3 ± 0.5 kg) were allotted to 4 dietary treatments in a replicated 4 Ã 4 Latin Square design. Control and DDGS (40%) diets were supplemented or not with a mixture of carbohydrases. Numeric increases for AA digestibility, along with a decreased tendency of urinary energy output suggested a possibility for improved nutrient utilization in pigs when carbohydrases were supplemented to 40% corn DDGS diet. Collectively, carbohydrase seems less effective for swine applications due to greater water content and consequently a lower viscosity in pig digesta. Next, study 3 showed changes in AA digestibility and endogenous AA losses (EAAL) when pigs were challenged orally with Salmonella enterica serovar Typhimurium. Nursery pigs (n = 48, 17.9 ± 0.5 kg) were randomly assigned to a 2 Ã 2 factorial arrangement consisting of two diets (control or N-free) and inoculation (sterile broth or 9.8 Ã 10^9 CFU of Salmonella). Measurements at 24 and 72 h post-inoculation indicated that AA digestibility of pigs is impaired through the initial phase of Salmonella infection and gradually restored, but not fully by 72 h. Finally, study 4 determined the dynamic fluctuations of EAAL and subsequent AA digestibility in response to Salmonella Typhimurium measured at multiple time points. Ileal cannulated pigs (n = 8, 76.0 ± 1.4 kg) were randomly assigned to either a control or a N-free diet and challenged orally with 1.3 Ã 10^10 CFU of Salmonella. Inflammatory diarrhea was associated with reduced AA digestibility and increased EAAL showing respective peak values at 8-16 h post-inoculation. Alterations in AA digestibility and EAAL were gradually recovered to near pre-inoculation values by 56-64 h post-inoculation, but showed impaired digestibility at 72-80 h post-inoculation. / Ph. D.

Five experiments were conducted, utilizing 512 crossbred weanling pigs to determine the P (Exp. 1) and Ca (Exp. 2 and 3) equivalency values of microbial phytase based on performance, rib mineralization and P and Ca digestibility estimates, and to investigate the possible interactions of phytase and citric acid (Exp. 4 and 5). In Exp. 1, adding phytase to low P diets linearly increased ADG (P < .001), rib shear force (P < .01), shear energy (P < .02), ash weight (P < .001) and ash percent (P < .001), Ca (P < .001) and P (P < .001) digestibility and digestible Ca (P < .001) and P (P <.001). Added P linearly increased ADG (P < .003), rib shear force (P < .003) shear energy (P < .001), ash weight (P < .001) and ash percent (P < .01), Ca (P < .02) and P (P < .001) digestibility and digestible Ca (P < .02) and P (P <.001). Based on phytase and P linear or nonlinear response equations for ADG, rib shear force, shear energy, and ash weight, P digestibility, and digestible P, the average equivalency of 500 U/kg of phytase was .78 g of P per kg of diet. In Exp. 2, dietary addition of phytase linearly increased rib ash % (P < .03), Ca (P < .001) and P (P < .001) digestibilities, and digested Ca (P < .001) and P (P < .001), but had no effect (P > .10) on ADG and rib shear force and ash weight. Added Ca linearly increased ADG (wk 3-4, P < .04), and rib shear force (P < .001), ash percentage (P < .001) and ash weight (P < .01), and digested Ca (P < .001), but P digestibility (P = .07) and digested P (P = .08) were numerically decreased. In Exp. 3, added phytase linearly increased ADG (wk 3-4, P < .002), feed efficiency (wk 3-4, P < .02), rib ash weight (P < .001), Ca total tract digestibility (P < .001), and Ca (P < .001) and P (P < .001) ileal digestibilities. Added Ca linearly increased ADG (wk 3-4, P < .02), feed efficiency (wk 3-4, P < .01), rib ash percentage (P < .001) and ash weight ( P < .001), shear force (P < .03) and energy (P < .008), and total tract (P < .001) and ileal (P < .001) digestible Ca. Based on phytase and Ca linear or nonlinear response equations for ADG in wk 3-4, measurements of rib mineralization, and digestible Ca, 500 U of microbial phytase was estimated to be equivalent to 1.08 g and .78 g of Ca in Exp. 2 and 3, respectively. In Exp. 4 and 5, dietary phytase addition linearly increased rib shear force (P < .004 and P < .02), shear energy (P < .001), dry bone weight (P < .001), ash weight (P < .001) and ash percent (P < .001). Calcium (P < .001) and P (P < .001) digestibilities were also improved in both experiments when phytase was added. Addition of citric acid in both experiments, reduced dietary pH and stomach digesta pH (P < .05). The addition of citric acid improved ADG (P < .05), feed efficiency (P < .04) and Ca digestibility (P < .05) in Exp. 4, but decreased Ca digestibility in Exp. 5 and had no effect on performance. In Exp. 5, the addition of 2.0% citric acid to the diet supplemented with 500 U/kg of phytase caused a decrease (P < .04) in the phytase activity recovered in the stomach digesta resulting in a phytase by citric acid interaction (P < .02). In summary, the addition of 500 U/kg microbial phytase to weanling pig diets, causes the release of approximately .78 g of P and .93 g of Ca, thus decreasing the need for supplemental P and Ca. The addition of citric acid to phytase supplemented diets does not appear to enhance the efficacy of microbial phytase based on the results of these studies. / Master of Science

My dissertation research focused on studying mechanisms of immunomodulation by probiotic lactobacilli on innate and T cell immune responses induced by rotavirus infection and vaccines in a gnotobiotic pig model of human rotavirus (HRV) infection and vaccination. We first studied the effects of probiotics on antigen-presenting cells (APCs) through TLR activation. We found that a mixture of Lactobacilli acidophilus strain NCFM (LA) and L. reuteri (ATCC# 23272) induced strong TLR2-expressing APC responses and virulent HRV induced a TLR3 response. Probiotics and HRV had an additive effect on TLR2- and TLR9-expressing APC responses, consistent with the adjuvant effect of lactobacilli. Dose effects of LA on T cell immune responses were investigated. We found that low dose LA significantly enhanced frequencies of HRV-specific IFN-γ producing CD4⁺ and CD8+ T cells whereas high dose LA reduced frequencies of HRV-specific IFN-γ producing CD4+ T cells. Low dose LA reduced frequencies of induced regulatory (iTreg) cells and TGF-β expression in the iTreg cells whereas high dose LA increased frequencies of iTreg cells and IL-10 expression in the iTreg cells. The dose effects of LA were independent of HRV infection/vaccination. In addition, we demonstrated that TCR-γδ T cells play an important role in modulating immune responses to rotavirus infections. All three γδ T cell subsets showed evidence of activation after HRV infection by increasing TLR2, TLR3, TLR9 expression and IFN-γ production during the acute phase of infection. There was an additive effect between lactobacilli and HRV in inducing total γδ T cell expansion in ileum and in recruiting the cells from blood. HRV infection induced a significant expansion of the CD2+CD8+ γδ T cell subset in the ileum. This subset mainly exerts regulatory functions as evident by expressing FoxP3, secreting TGF-β and IL-10 or increasing production of the anti-inflammatory cytokines by CD4+ and/or CD8+ αβ T cells in the co-cultures. CD2+CD8- and CD2-CD8- γδ T cell subsets have mainly pro-inflammatory and anti-viral functions as evident by secreting IFN-γ or promoting CD4+ αβ T cell proliferation and IFN-γ production. This knowledge will facilitate the development of more effective vaccination and therapeutic strategies to protect children and young animals against rotavirus gastroenteritis. / Ph. D.