The primary focus of this thesis is to establish the clinical utility of corticosteroid injections mixed with local anesthetics, when compared to only local anesthetics, for relief of pain in patients with chronic non-cancer pain. Chronic non-cancer pain is common and causes significant pain and suffering to patients, and economic burden to health care system. Injection of steroids is an option, either by targeting the painful structure or the associated neural elements. Steroids are commonly mixed with local anesthetics in the hope of prolonging the pain relief. Since there is no evident inflammation in most chronic pain conditions, and because existing clinical studies do not consistently support its effectiveness in various clinical conditions, the potential value of adding steroids is questionable. This clinical question has been addressed through a comprehensive systematic review and meta-analysis of randomized control trials comparing injections of steroid with local anesthetics against only local anesthetics for chronic non-cancer pain conditions. Our review found that there is paucity of good quality randomized controlled studies specifically addressing this comparison. Although a large numbers of studies were identified, there was a small effect favoring steroids in studies measuring pain relief by dichotomous outcomes. Overall confidence in the effect estimates were limited due to serious concerns of bias, significant heterogeneity and variability in studies, leading to low quality. A majority of the included studies did not aim to capture the full spectrum of adverse effects. Future studies addressing this clinical question should aim to be of optimum size, must aim to limit the threat of bias, and capture all patient important outcomes including pain relief. / Thesis / Master of Science (MSc)

<p> Bisphenol-A (BPA), used in the production of polycarbonate plastics and epoxy resins, has established estrogenic properties. Early pregnancy in mice is highly sensitive to exogenous estrogens, particularly during the period of blastocyst implantation. Accordingly, I assessed pregnancy outcome, implantation, urinary hormone levels and uterine morphology following BPA exposure. Subcutaneous injections of BPA administered on days 1 through 4 of gestation reduced litter size at a dose of 3 mg/animal/day and decreased the proportion of parturient females at 10 mg/animal/day. Hysterectomies performed on day 6 of pregnancy confirmed a significant disruption of implantation occurring at doses as low as 6 mg/animal/day. Urinary progesterone levels were also reduced by 10 mg/animal/day. Uterine luminal area expanded substantially in response to increasing doses of BPA. Luminal epithelial cell height increased following exposure to 10.125 mg/animal, whereas there were no differences in the number of corpora lutea among conditions. The proportion of cells staining positively for estrogen receptors was affected non-monotonically, showing highest levels at 3.375 mg/animal and lowest levels at 10.125 mg/animal. Similarly progesterone receptor expression measured through western blots related non-monotonically to dose, being highest at 3.375 mg/animal and diminishing with increasing dose. Effects of a single administration of BPA on days 0, 1, or 2 of gestation were also investigated. A single dose of 10 mg reduced the number of implantation sites when given on day 0 or 1, and 6 mg did so on day 1, but there was no such effect of any dose administered on day 2. Exposure to low, environmentally-relevant doses of BPA did not result in any clear reproductive or hormonal effects. These studies highlight the detrimental effects BPA exposure induces during early pregnancy and provides further evidence of its weak estrogenic properties in vivo.</p> / Thesis / Doctor of Philosophy (PhD)

(1) Background: From a young age, boys are more often affected by tooth wear than girls. This suggests an influence of the male sex hormone (testosterone) on the aetiology of tooth wear. The aim of the present study was to investigate the incidence of tooth wear in relation to steroid hormone levels in children. (2) Methods: 1022 test persons aged between 10 and 18 (491 male, 531 female) from the LIFE Child study underwent medical and dental examination. Tooth wear was measured through clinical inspection. Blood samples were taken to determine hormone levels (testosterone, SHBG). The level of free testosterone was calculated from the ratio of testosterone to SHBG. Using multivariable methods, the incidence of tooth wear was analyzed as a function of hormone levels, while controlling for confounders such as age, sex, social status, and orthodontic treatment. (3) Results: The incidence of tooth wear increased with age in both sexes. Boys showed significantly more often attrition facets than girls (17.5% vs. 13.2%, p < 0.001). Subjects with tooth wear showed significantly higher free testosterone levels than those without (males: p < 0.001, females: p < 0.05). After controlling for confounding variables, the risk of tooth wear increased by approximately 30.0% with each year of life (odds ratio [OR]boys = 1.29, 95% confidence interval [CI] = 1.04–1.56; [OR]girls = 1.32, 95% CI = 1.08–1.61). In addition, the risk of tooth wear increased by 6.0% per free testosterone scale score only in boys (OR = 1.06, 95% CI = 1.01–1.12). (4) Conclusions: Tooth wear is common in children and in adolescents, and it increases steadily with age in both sexes. The stronger increase and the higher prevalence among male adolescents can be explained by the additional effect of free testosterone.

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<p>Sex steroids are critical for the post-natal development of the female reproductive system, and are involved in ovulatory cycling and pregnancy. In mice, <em>Mus musculus</em>, female development, cycling, and pregnancy can be affected by the urine of conspecifics, which is known to contain active steroids. Specifically, puberty can be accelerated (the Vandenbergh effect), estrous cycling can be prolonged (the Lee-Boot effect) or synchronized (the Whitten effect), and blastocyst implantation can be disrupted (the Bruce effect). Since steroids alone can affect females in ways that are indistinguishable from these social reproductive effects, I hypothesized that urinary steroids of conspecifics may be absorbed by females, arrive in the reproductive system, and thereby affect females through known mechanisms. First I showed that tritium-labelled 17β-estradiol (³H-E₂) injected into males is excreted in their urine, and that application of urine from these males to the nose of an inseminated female results in detectable levels in her uterus. When I paired inseminated females with non-sire males injected with ³H-E₂, radioactivity was detected in the brain and reproductive tissues of the females. This was the first demonstration of steroids from one animal directly entering the body of another. Similar results were found when I exposed juvenile females to adult males injected with ³H-E₂, and when I exposed nulliparous adult females to same-strain ³H‑E₂- or ³H-progesterone- (³H-P₄) treated adult males or females. Taken with the existing literature, these results suggest that steroid transfer may underlie various social reproductive phenomena in mice, with potential implications for many other species.</p> / Doctor of Philosophy (PhD)

Introduction: A subgroup of severe asthmatics (SA) remain uncontrolled with persistent airway eosinophilia despite high dose glucocorticosteroids (GCS) termed “steroid insensitivity.” A significant proportion of this subgroup get recurrent airway infections. Phosphoinositide-3 kinase (PI3K) pathway may contribute to both GCS insensitivity and further susceptibility to recurrent infections. Objectives: The effect of GCS and PI3K antagonism was evaluated on GCS induced eosinophil apoptosis as a mechanism of GCS insensitivity in healthy, mild to moderate and severe asthmatics. Furthermore, we investigated the relationship between PI3K activation, histone deacetylase (HDAC) and macrophage receptor with collagenous structure (MARCO) expression in SA±recurrent bacterial bronchitis. Methods: Blood eosinophils, isolated from healthy subjects (HS) and asthmatics, were incubated with increasing concentrations of dexamethasone (0.1, 1 and 10 uM) and a pan-PI3K antagonist (LY294002 at 1 and 2 uM). Cell viability was assessed using PrestoBlue cell viability assay, Trypan Blue exclusion test and PE-Annexin-V/7-AAD apoptosis detection (flow cytometry). Furthermore, PI3K activity, MARCO and HDAC levels were measured in macrophages isolated from healthy subjects and SA (±history of recurrent bacterial bronchitis). In a subgroup, sputa were examined for in situ PI3K activity and gene expression of PI3K isoforms by digital PCR. Results: In HS, a time-dependent increase in eosinophil apoptotic% was observed (1.9% at baseline, 25.7% at 16h and 31% at 24h) (p=0.06, p=0.005 respectively). Dexamethasone 10uM increased it to 40.1% at 16h (p=0.03). Dexamethasone induced eosinophil apoptosis was less in severe asthmatics (34.2%) vs. to mild-to-moderate asthmatics (46.7%) (p=0.05) suggesting steroid insensitivity. This was not reversed by co-incubation with LY294002 2uM (39.9% vs. 53.3%) (p=0.04) (Fig. 1B). Asthmatics with recurrent lung infections had higher blood PI3K activity (demonstrated as inverse of biotinylated-PIP3, p=0.02), MARCO expression (p=0.01), and trend for lower HDAC expression (p=0.067) vs. healthy donors. PI3KCD (encoding catalytic 𝛿 isoform) gene expression relative to HPRT1 (housekeeping gene) was increased in SA-infection group (p=0.03). A higher number of asthmatics with recurrent lung infections were on oral corticosteroids (p=0.015) and replacement immunoglobulins (p=0.016). Conclusions: Evaluating dexamethasone-induced apoptosis in blood eosinophil can assess GCS sensitivity ex vivo. Severe asthmatics demonstrate GCS insensitivity, which was not reversed by a pan-PI3K antagonist. PI3K activity is increased in SA with a previous history of recurrent lung infections, which is associated with a decrease in HDAC and MARCO expression. Targeting PI3K pathway, specifically the 𝛿 isoform, may be a potential therapeutic target in SA with mixed-granulocytic bronchitis. / Thesis / Master of Science in Medical Sciences (MSMS) / Eosinophils are a type of blood cell that is responsible for causing asthma symptoms and flare-ups. Steroids (in both inhaled and oral forms) decrease eosinophils and so are the main treatment for asthma. But a group of asthmatics continues to have symptoms and eosinophils in their lung secretions even with maximum dose of steroids. Some of these asthmatics also get repeated bacterial lung infections. In blood samples, we studied one of the proteins (phosphoinositide-3 kinase, PI3K), which may be responsible for poor response to steroids and repeated infections. We looked at the effect of steroids on eosinophils extracted from blood samples of healthy people, asthmatics with mild disease and asthmatics with severe disease. We also measured PI3K levels and related proteins in blood and sputum samples from healthy people, asthmatics who get repeated bacterial lung infections (more than two in the previous two years) and asthmatics without recurrent lung infections. We found that eosinophils obtained from asthmatics with severe disease were harder to kill with steroids. But, this was not reversed with a PI3K blocking agent. We also found that asthmatics with a previous history of repeated bacterial lung infections had a higher PI3K level. High PI3K activity was associated with a decrease in two other proteins – histone deacetylase (HDAC) and macrophage receptor with collagenous structure (MARCO). Decrease in HDAC can result in a lower response to steroids, which may result in an increase in eosinophils in the lungs. Lower levels of MARCO can cause a patient to have higher sensitivity to recurrent bacterial infections. Overall, this may lead to a dangerous cycle where repeated lung infection will lead to high PI3K activity, which will cause worse asthma control and more infections. More studies are needed to evaluate drugs that block PI3K, which may be helpful in asthmatics who are less responsive to steroids and also get repeated infections.

Tesis por compendio / [EN] During the last 25 years, eel populations have declined considerably, this species is considered outside safe biological limits. The life cycle of the eel is quite complex including transoceanic migration whose conditions are still so unknown that even the natural spawning process has never been observed. Eels cease feeding during migration and that is why their energy reserves such as lipids play a crucial role and thus their in deep study is essential to obtain gametes (eggs and sperm) quality as the first step for their reproduction in captivity. In the last years, many efforts have been directed at the study of the influence of fatty acids on the reproductive performance in females, but not in male eels. The present manuscript describes the results obtained in two experiments. The first experiment (which led to the three first articles included in the memory) focused on the effect of fatty acids along maturation, their relationship with steroid hormones and their effect on sperm quality parameters. The second experiment involved the comparison of different diets designed, considering the previously acquired insights into the most influential fatty acids on male eel reproduction, and the assessment of their influence on the reproductive performance of males. The first experiment was used to assess the changes in the fat content, as well as fatty acids, in different tissues of male eels hormonally induced to sexual maturation under different thermal regimes (two variables and one constant). Males finished spermatogenesis earlier with a constant temperature of 20 ºC, suggesting that eel spermatogenesis is tightly regulated by temperature. The fat content did not change significantly in the muscle, but was increased in the liver and decreased in testes during testicular development. With regard to fatty acids, during sperm maturation, the liver was highlighted as the main site of synthesis. Finally, EPA, ARA and DHA remained constant in testes, while the level of the rest of fatty acids decreased significantly. Using samples from the same experiment, the correlation between the main steroid hormones and fatty acids at different stages of testicular development were investigated. Similarly as occurs in mammals, EPA and DHA were highlighted as possible modulators of androgen synthesis. The set of the results obtained suggests new perspectives concerning the functions and interactions between fatty acids and steroids in fish spermatogenesis. Also, with samples from the first experiment, a study was carried out seeking to establish the possible correlations between different fatty acids and several sperm quality parameters. Correlations between several highly unsaturated fatty acids with parameters such as volume, the percentage of motile sperm and sperm velocity were found. Finally, with the acquired knowledge so far, a second experiment was carried out designing feeds with different fatty acid percentages in order to evaluate the influence of the diets on sperm quality. The results showed that high levels of DHA and EPA in the diets induce higher volumes and high sperm motility. This study allowed obtaining results applicable to the design of optimum broodstock diets for this species. / [ES] Durante los últimos 25 años las poblaciones de anguila han disminuido considerablemente, de modo que la especie se considera fuera de los límites biológicos de seguridad. El ciclo vital de la anguila es bastante complejo incluyendo una migración transoceánica cuyas condiciones son todavía tan desconocidas que incluso el proceso natural de desove no se ha observado nunca. Las anguilas dejan de alimentarse durante la migración y es por ello que sus reservas energéticas en forma de lípidos tienen un papel crucial y su estudio en profundidad es importante para obtener gametos (huevos y esperma) de calidad como primer paso para su reproducción en cautividad. En los últimos años se han dirigido muchos esfuerzos al estudio de la influencia de los ácidos grasos en el rendimiento reproductivo de las hembras, pero no en el de los machos de anguila. El presente documento describe los resultados obtenidos en dos experimentos. El primer experimento (que dio lugar a los tres primeros artículos incluidos en la memoria) se centró en el efecto de los ácidos grasos durante la maduración, su relación con las hormonas esteroideas y su efecto sobre los parámetros de calidad espermática. El segundo experimento consistió en la comparación de diferentes dietas, diseñadas considerando los conocimientos adquiridos previamente sobre los ácidos grasos más influyentes en la reproducción de machos de anguila, y en la evaluación de su influencia en el rendimiento reproductivo de los machos. El primer experimento sirvió para evaluar los cambios en el contenido graso, así como en los ácidos grasos, en diferentes tejidos de machos de anguila hormonalmente inducidos a la maduración sexual bajo diferentes regímenes térmicos (dos variables y uno constante). Los machos concluyeron la espermatogénesis más temprano a una temperatura contante de 20 ºC, sugiriendo que la espermatogénesis de anguila está estrechamente regulada por la temperatura. El contenido en grasa no varió significativamente en el músculo, se incrementó en el hígado y bajó en el testículo a lo largo del desarrollo testicular. Con respecto a los ácidos grasos, durante la maduración del esperma, el hígado destacó como el principal sitio de síntesis. Finalmente, EPA, ARA y DHA permanecieron constantes en el testículo, mientras que el nivel del resto de ácidos grasos bajó significativamente. Usando muestras del mismo experimento, se investigaron las correlaciones entre las principales hormonas esteroideas y los ácidos grasos en los diferentes estadios del desarrollo testicular. De forma similar a lo que ocurre en mamíferos, EPA y DHA destacaron como posibles moduladores de la síntesis de andrógenos. El conjunto de los resultados obtenidos sugiere nuevas perspectivas sobre las funciones y las interacciones entre los ácidos grasos y los esteroides en la espermatogénesis de peces. También con muestras del primer experimento, se llevó a cabo un estudio que buscó las posibles correlaciones de los diferentes ácidos grasos con varios parámetros de calidad espermática. Se encontraron correlaciones entre varios ácidos grasos altamente insaturados con parámetros como el volumen, el porcentaje de espermatozoides móviles y la velocidad del esperma. Finalmente, con los conocimientos adquiridos hasta el momento, se realizó un segundo experimento para el que se diseñaron piensos con diferentes porcentajes de ácidos grasos con el fin de evaluar la influencia de las dietas sobre la calidad del esperma. Los resultados mostraron que niveles altos de DHA y EPA en las dietas inducen mayores volúmenes y una alta motilidad del esperma. Este estudio permitió obtener resultados aplicables al diseño de dietas óptimas para reproductores de esta especie. / [CA] Durant els últims 25 anys les poblacions d'anguila han disminuït considerablement, de manera que l'espècie es considera fora dels límits biològics de seguretat. El cicle vital de l'anguila és bastant complex incloint una migració transoceànica les condicions de la qual són encara tan desconegudes que fins i tot el procés natural de fresa mai ha estat observat. Les anguiles deixen d'alimentar-se durant la migració i és per això que les seves reserves energètiques en forma de lípids tenen un paper crucial i el seu estudi en profunditat és important per a obtenir gamets (ous i esperma) de qualitat com a primer pas per a la reproducció en captivitat. En els últims anys s'han dirigit molts esforços a l'estudi de la influència dels àcids grassos en el rendiment reproductiu de les femelles, però no en el dels mascles d'anguila. El present document descriu els resultats obtinguts en dos experiments. El primer (que dona lloc als tres primers articles inclosos en la memòria) es va centrar en l'efecte dels àcids grassos durant la maduració, la seva relació amb les hormones esteroides i el seu efecte sobre els paràmetres de qualitat espermàtica. El segon experiment va consistir en la comparació de diferents dietes, dissenyades considerant els coneixements adquirits prèviament sobre els àcids grasos més influents en la reproducció dels mascles d'anguila, i en l'avaluació de la seva influència en el rendiment reproductiu dels mascles. El primer experiment va servir per avaluar els canvis en el contingut de gras, així com en els àcids grassos, en els diferents teixits de mascles d'anguila hormonalment induïts a la maduració sexual baix diferents règims tèrmics (dos variables i un constant). Els mascles van concloure la espermatogènesis més prompte a una temperatura de 20 ºC, suggerint que l'espermatogènesis d'anguila està estretament regulada per la temperatura. El contingut en greix, no va variar significativament en el múscul, es va incrementar en el fetge i va baixar en els testicles al llarg del desenvolupament testicular. Pel que fa als àcids grassos, durant la maduració de l'esperma, el fetge va destacar com al principal lloc de síntesi. Finalment, EPA, ARA y DHA van romandre constants en el testicle, mentre que el nivell de la resta d'àcids grassos va baixar significativament. Emprant mostres del mateix experiment, es van investigar les correlacions entre les principals hormones esteroides i els àcids grassos en els diferents estadis de desenvolupament testicular. De manera semblant al que ocorre en mamífers, EPA i DHA van destacar com a possibles moduladors de la síntesi d'andrògens. El conjunt dels resultats obtinguts suggerixen noves prespectives sobre les funcions i les interaccions entre els àcids grassos i els esteroides en l'espermatogènesi dels peixos. També amb mostres del primer experiment, es va dur a terme un estudi que va buscar les possibles correlacions dels diferents àcids grassos amb diversos paràmetres de qualitat espermàtica. Es van trobar correlacions entre diversos àcids grassos altament insaturats amb paràmetres com el volum, el percentatge d'espermatozoides mòbils i la velocitat de l'esperma. Finalment, amb els coneixements adquirits fins al moment, es va realitzar un experiment per al qual es van dissenyar pinsos mb diferents percentatges d'àcids grassos per tal d'avaluar la influència de les dietes sobre la qualitat de l'esperma. Els resultats van mostrar que nivells alts de DHA i EPA en les dietes indueixen a majors volums i una alta motilitat de l'esperma. Aquest estudi va permetre obtenir resultats aplicables al disseny de dietes òptimes per a reproductors d'aquesta espècie. / Baeza Ariño, R. (2015). Roles of lipids and fatty acids through the spermatogenesis of European eel (Anguilla anguilla) [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/49988 / Compendio