Pigmentation disorders occur in multiple conditions (Hakozaki et al., 2006:105). Although many modalities of treatments are available, none are completely satisfactory (Briganti et al., 2003:101). Two cytokines normally present in the skin, transforming growth factor–beta1 (TGF–81) and tumour necrosis factor–alpha (TNF–9), have been shown to inhibit melanin synthesis (Martinez–Esparza, 2001:972). The stratum corneum has been commonly accepted as the main barrier to percutaneous absorption. Many techniques have been applied to overcome this barrier properties and to enhance penetration with varying success (Pellet et al., 1997:92). The objective of this study was to investigate the topical delivery of the above mentioned peptide drugs with aid of the Pheroid drug delivery system. Pheroid technology is a delivery system that promotes the absorption and increases the efficacy of dermatological, biological and oral medicines in various pharmacological groups (Grobler et al., 2008:4). Pheroid entraps drugs with high efficiency and delivers them with remarkable speed to target sites (Grobler, 2004:4). In order to avoid degradation of these peptides, bestatin hydrochloride (an aminopeptidase inhibitor), was used (Lkhagvaa et al., 2008:386). Topical drug delivery was achieved by means of vertical Franz cell diffusion studies performed over a 6 and 12 h period. ELISA (enzyme linked immunosorbent assay) detection was used to detect cytokine concentrations. Entrapped cytokine solutions were monitored by confocal laser scanning microscopy (CLSM). Upon removal of donor and receptor compartments, skin discs were subjected to tape stripping in order to establish the amount of active present within the stratum corneum and epidermis as well as the remaining dermis (Pellet et al., 1997:92). When comparing the two studies with each other, it is evident that the diffused concentration values obtained with PBS (phosphate buffer solution, pH 7.4) was lower than that obtained with the Pheroid drug delivery system. Both cytokine concentrations were successfully delivered topically as a minimum of concentrations for both actives were detected. This positive result was confirmed as well by the amount of active detected in stratum corneum–epidermis and epidermis–dermis solutions. / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2011.

Pigmentation disorders occur in multiple conditions (Hakozaki et al., 2006:105). Although many modalities of treatments are available, none are completely satisfactory (Briganti et al., 2003:101). Two cytokines normally present in the skin, transforming growth factor–beta1 (TGF–81) and tumour necrosis factor–alpha (TNF–9), have been shown to inhibit melanin synthesis (Martinez–Esparza, 2001:972). The stratum corneum has been commonly accepted as the main barrier to percutaneous absorption. Many techniques have been applied to overcome this barrier properties and to enhance penetration with varying success (Pellet et al., 1997:92). The objective of this study was to investigate the topical delivery of the above mentioned peptide drugs with aid of the Pheroid drug delivery system. Pheroid technology is a delivery system that promotes the absorption and increases the efficacy of dermatological, biological and oral medicines in various pharmacological groups (Grobler et al., 2008:4). Pheroid entraps drugs with high efficiency and delivers them with remarkable speed to target sites (Grobler, 2004:4). In order to avoid degradation of these peptides, bestatin hydrochloride (an aminopeptidase inhibitor), was used (Lkhagvaa et al., 2008:386). Topical drug delivery was achieved by means of vertical Franz cell diffusion studies performed over a 6 and 12 h period. ELISA (enzyme linked immunosorbent assay) detection was used to detect cytokine concentrations. Entrapped cytokine solutions were monitored by confocal laser scanning microscopy (CLSM). Upon removal of donor and receptor compartments, skin discs were subjected to tape stripping in order to establish the amount of active present within the stratum corneum and epidermis as well as the remaining dermis (Pellet et al., 1997:92). When comparing the two studies with each other, it is evident that the diffused concentration values obtained with PBS (phosphate buffer solution, pH 7.4) was lower than that obtained with the Pheroid drug delivery system. Both cytokine concentrations were successfully delivered topically as a minimum of concentrations for both actives were detected. This positive result was confirmed as well by the amount of active detected in stratum corneum–epidermis and epidermis–dermis solutions. / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2011.

Introdução: A alteração dos níveis normais de marcadores séricos e urinários ocorre na presença de dano renal associado à uropatia obstrutiva. Valores séricos e e urinários de TGF beta1 e CA 19-9 ainda não foram avaliados em modelo experimental de uropatia obstrutiva. Material e Métodos: Ratos foram divididos em sete grupos: referência, sham operation, nefrectomia unilateral, ligadura completa de ureter unilateral, obstrução parcial de ureter unilateral, obstrução parcial de ambos ureteres, nefrectomia unilateral associada à obstrução parcial do ureter contralateral. Morfometria renal e ureteral, concentrações séricas e urinárias de TGF beta1 e CA 19-9 e expressão tecidual renal de CA 19-9 foram analisadas. A correlação destes marcadores com os grupos submetidos a obstrução completa, obstrução parcial ou sem obstrução foi realizada. Resultados: Achados anatomopatológicos correlacionaram-se positivamente à intensidade da obstrução ureteral e negativamente aos níveis urinários de CA 19-9. Subexpressão acentuada do CA 19-9 foi observada em unidades renais com obstrução completa. Não foram encontradas diferenças estatisticamente significativas para os marcadores TGF beta1 urinário, TGF beta1 sérico e para o CA 19-9 sérico Conclusões: O CA 19-9 urinário correlacionou-se negativamente com o grau de obstrução ureteral. A análise imuno-histoquímica demonstrou a expressão do CA 19-9 no citoplasma das células epiteliais tubulares, sugerindo produção renal do marcador. O TGF beta1 sérico e urinário não apresentaram modificações de acordo com o grau de severidade e tempo de obstrução, o que pode estar relacionado a remodelamento renal menos intenso em resposta à uropatia obstrutiva nestes ratos / Introduction: Abnormal levels of serum and urinary markers occur in the presence of renal damage associated to obstructive uropathy. Urinary and serum TGFbeta1 and CA 19- 9 have not yet been evaluated in an experimental model of obstructive uropathy. Material and Methods: Rats were divided into seven groups: reference, sham operation, unilateral nephrectomy, complete unilateral ureteral obstruction, partial unilateral ureteral obstruction, partial bilateral ureteral obstruction, and unilateral nephrectomy with contralateral partial ureteral obstruction. Kidney and ureter morphometry, TGFbeta1 and CA 19-9 serum and urinary concentrations and CA 19-9 renal tissue expression were analysed. Correlation of these markers to complete, partial obstruction or unobstructed groups was performed. Results: Pathological findings correlated positively with the degree of ureteral obstruction, but negatively with urinary CA 19-9 levels. Marked underexpression of CA 19-9 was observed in kidneys with complete ureteral obstruction. No statistically significant differences were found for urinary and serum TGFbeta1 and also for serum CA 19-9. Conclusions: Urinary CA 19-9 correlated negatively with ureteral obstruction grade. Immunohistochemistry depicted CA 19-9 expression on epithelial tubular cells cytoplasm, suggesting renal origin. Serum and urinary TGFbeta1 did not show alterations in response to severity and length of urinary obstruction, which might be associated with less intense renal remodeling

Orientador: Cláudio Chrysostomo Werneck / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-23T04:57:03Z (GMT). No. of bitstreams: 1 Nery-Diez_AnaClaudiaCoelho_D.pdf: 21192177 bytes, checksum: 572bd54be1e7496c1fd325e94812ecc1 (MD5) Previous issue date: 2013 / Resumo: As mutações no gene da fibrilina-1, presente na fibra elástica, estão relacionadas à síndrome de Marfan, doença genética autossômica dominante. Acredita-se que a maioria dos seus sintomas esteja relacionada a uma hiper-ativação do fator TGF-?. Quando camundongos modelo para síndrome são tratados com losartan apresentam uma melhora significativa nos sinais clínicos. Assim, neste estudo, investigamos o papel de fibrilina-1 na análise de trombose arterial em modelos de camundongos para a síndrome de Marfan (Fbn1mg?/+). Foram analisados a formação de trombos, os níveis de plaquetas, APTT, PT e TT, agregação e adesão plaquetária, parâmetros hemodinâmicos, níveis de TGF-?, atividade das MMPs, bem como a morfologia das plaquetas, das fibras elásticas e dos trombos. Foi observado que os animais Fbn1mg?/+ necessitam de cerca de 120±21,07 minutos para formarem o trombo, enquanto os animais selvagens precisam de 58±7,16 minutos. Mas, quando os animais Fbn1mg?/+ foram tratados com anti-hipertensivos losartan e captopril, ocorreu uma recuperação no tempo de formação do trombo, com redução de 57,5% e 67,5% no tempo, respectivamente. Além disso, constatou-se que os animais Fbn1mg?/+ apresentam um aumento na atividade das MMPs e TGF-? ativos, quando tratados com os anti-hipertensivos, houve uma diminuição apenas na atividade das MMPs. Ademais, não foi verificada diferença significativa entre todos os outros parâmetros analisados. Este estudo sugere que, de alguma forma, as drogas interferem na remodelação da matriz elástica, devido a uma diminuição da atividade das metaloproteinases de matriz e, consequentemente, leva á recuperação na formação do trombo / Abstract: Recent works show that an increased activation of the TGF-? is associated with most of the symptoms of the Marfan syndrome. Studies using mouse models of Marfan treated with losartan have been shown to prevent the degradation of the elastic matrix. Other investigators suggest that the metalloproteinases and the noncanonical ERK signaling are involved in the breakdown of the elastic fiber, which results in the aneurysm. In this study we investigated the role of fibrillin-1 in the arterial thrombosis model using mouse models of Marfan. We analyzed thrombus formation, platelet levels, APTT, PT and TT time, hemodynamic parameter, TGF-? levels, MMP activity as well as platelets, elastic fibers morphology and thrombus. This study demonstrated that Fbn1mg?/+ mice take about 120±21,07 minutes for the thrombus to be formed when compared with wild type, 58±7,16. When these Fbn1mg?/+ mice were treated with antihypertensive losartan and captopril the time taken for the thrombus formation was reduced in 57,5% and 67,5%, respectively. The activity of metalloproteinases and activity TGF-? was increased in the Fbn1mg?/+, however no significant difference between the hemodynamic parameters, levels of totals TGF-?, morphological platelets, elastic fiber and thrombus were observed. Finally, the results suggested that fibrillin-1 interferes with this process and antihypertensive affect the physiology of Fbn1mg?/+ mice. This study suggested that somehow drugs interfere with elastic matrix remodeling due to a decrease in the activity of matrix metalloproteinases which results in the recovery time of thrombus formation / Doutorado / Bioquimica / Doutora em Biologia Funcional e Molecular

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À la fin du 19e siècle, Dr. Ramón y Cajal, un pionnier scientifique, a découvert les éléments cellulaires individuels, appelés neurones, composant le système nerveux. Il a également remarqué la complexité de ce système et a mentionné l’impossibilité de ces nouveaux neurones à être intégrés dans le système nerveux adulte. Une de ses citations reconnues : “Dans les centres adultes, les chemins nerveux sont fixes, terminés, immuables. Tout doit mourir, rien ne peut être régénérer” est représentative du dogme de l’époque (Ramón y Cajal 1928). D’importantes études effectuées dans les années 1960-1970 suggèrent un point de vue différent. Il a été démontré que les nouveaux neurones peuvent être générés à l’âge adulte, mais cette découverte a créé un scepticisme omniprésent au sein de la communauté scientifique. Il a fallu 30 ans pour que le concept de neurogenèse adulte soit largement accepté. Cette découverte, en plus de nombreuses avancées techniques, a ouvert la porte à de nouvelles cibles thérapeutiques potentielles pour les maladies neurodégénératives. Les cellules souches neurales (CSNs) adultes résident principalement dans deux niches du cerveau : la zone sous-ventriculaire des ventricules latéraux et le gyrus dentelé de l’hippocampe. En condition physiologique, le niveau de neurogenèse est relativement élevé dans la zone sous-ventriculaire contrairement à l’hippocampe où certaines étapes sont limitantes. En revanche, la moelle épinière est plutôt définie comme un environnement en quiescence. Une des principales questions qui a été soulevée suite à ces découvertes est : comment peut-on activer les CSNs adultes afin d’augmenter les niveaux de neurogenèse ? Dans l’hippocampe, la capacité de l’environnement enrichi (incluant la stimulation cognitive, l’exercice et les interactions sociales) à promouvoir la neurogenèse hippocampale a déjà été démontrée. La plasticité de cette région est importante, car elle peut jouer un rôle clé dans la récupération de déficits au niveau de la mémoire et l’apprentissage. Dans la moelle épinière, des études effectuées in vitro ont démontré que les cellules épendymaires situées autour du canal central ont des capacités d’auto-renouvellement et de multipotence (neurones, astrocytes, oligodendrocytes). Il est intéressant de noter qu’in vivo, suite à une lésion de la moelle épinière, les cellules épendymaires sont activées, peuvent s’auto-renouveller, mais peuvent seulement ii donner naissance à des cellules de type gliale (astrocytes et oligodendrocytes). Cette nouvelle fonction post-lésion démontre que la plasticité est encore possible dans un environnement en quiescence et peut être exploité afin de développer des stratégies de réparation endogènes dans la moelle épinière. Les CSNs adultes jouent un rôle important dans le maintien des fonctions physiologiques du cerveau sain et dans la réparation neuronale suite à une lésion. Cependant, il y a peu de données sur les mécanismes qui permettent l'activation des CSNs en quiescence permettant de maintenir ces fonctions. L'objectif général est d'élucider les mécanismes sous-jacents à l'activation des CSNs dans le système nerveux central adulte. Pour répondre à cet objectif, nous avons mis en place deux approches complémentaires chez les souris adultes : 1) L'activation des CSNs hippocampales par l'environnement enrichi (EE) et 2) l'activation des CSNs de la moelle épinière par la neuroinflammation suite à une lésion. De plus, 3) afin d’obtenir plus d’information sur les mécanismes moléculaires de ces modèles, nous utiliserons des approches transcriptomiques afin d’ouvrir de nouvelles perspectives. Le premier projet consiste à établir de nouveaux mécanismes cellulaires et moléculaires à travers lesquels l’environnement enrichi module la plasticité du cerveau adulte. Nous avons tout d’abord évalué la contribution de chacune des composantes de l’environnement enrichi à la neurogenèse hippocampale (Chapitre II). L’exercice volontaire promeut la neurogenèse, tandis que le contexte social augmente l’activation neuronale. Par la suite, nous avons déterminé l’effet de ces composantes sur les performances comportementales et sur le transcriptome à l’aide d’un labyrinthe radial à huit bras afin d’évaluer la mémoire spatiale et un test de reconnaissante d’objets nouveaux ainsi qu’un RNA-Seq, respectivement (Chapitre III). Les coureurs ont démontré une mémoire spatiale de rappel à court-terme plus forte, tandis que les souris exposées aux interactions sociales ont eu une plus grande flexibilité cognitive à abandonner leurs anciens souvenirs. Étonnamment, l’analyse du RNA-Seq a permis d’identifier des différences claires dans l’expression des transcripts entre les coureurs de courte et longue distance, en plus des souris sociales (dans l’environnement complexe). iii Le second projet consiste à découvrir comment les cellules épendymaires acquièrent les propriétés des CSNs in vitro ou la multipotence suite aux lésions in vivo (Chapitre IV). Une analyse du RNA-Seq a révélé que le transforming growth factor-β1 (TGF-β1) agit comme un régulateur, en amont des changements significatifs suite à une lésion de la moelle épinière. Nous avons alors confirmé la présence de cette cytokine suite à la lésion et caractérisé son rôle sur la prolifération, différentiation, et survie des cellules initiatrices de neurosphères de la moelle épinière. Nos résultats suggèrent que TGF-β1 régule l’acquisition et l’expression des propriétés de cellules souches sur les cellules épendymaires provenant de la moelle épinière. / At the end of the 19th century, Dr. Ramón y Cajal, a scientific pioneer, discovered that the nervous system was composed of individual cellular elements, later called neurons. He also noticed the complexity of this system and mentioned the impossibility of new neurons to be integrated into the adult nervous system. One of his famous quotes: “In adult centers the nerve paths are something fixed, ended, immutable. Everything may die, nothing may be regenerated” is representative of the prevalent dogma at the time (Ramón y Cajal 1928). Key studies conducted in the 1960-1970s suggested a different point of view. It was demonstrated that new neurons could be born during adulthood, but this discovery created an omnipresent skepticism in the scientific community. It took 30 years for the concept of adult neurogenesis to become widely accepted. This discovery, along with more advanced techniques, opened doors to potential therapeutic avenues for neurodegenerative diseases. Adult neural stem cells (NSCs) reside mainly in two niches in the brain: the subventricular zone of the lateral ventricles and the dentate gyrus of the hippocampus. Under normal conditions, neurogenesis level is relatively high in the SVZ whereas some steps are rate-limiting in the hippocampus. In contrast, the spinal cord is rather defined as a quiescent environment. One of the main questions that arose from these discoveries is: how do you activate adult NSCs in order to increase neurogenesis levels? In the hippocampus, environmental enrichment (including cognitive stimulation, exercise and social interactions) has been shown to promote hippocampal neurogenesis. The plasticity potential of this region is important as it could play a crucial role in rescuing learning and memory deficits. In the spinal cord, studies conducted in vitro demonstrated that ependymal cells found around the central canal have self-renewal and multipotency capacities (neurons, astrocytes, oligodendrocytes). Interestingly, it turns out that in vivo, following a spinal cord lesion, ependymal cells become activated, can self-replicate, but can only give rise to glia cell fate (astrocytes and oligodendrocytes). This new post-injury function shows that plasticity can still occur in a quiescent environment and could be exploited to develop endogenous spinal cord repair strategies. v As mentioned above, NSCs play important roles in normal brain function and neural repair following injury. However, little information is known about how a quiescent NSC becomes activated in order to perform these functions. The general objective of this project was to investigate the mechanisms underlying activation of neural stem cells in the adult central nervous system. My specific aims were to address this question using adult mice in two complementary models: 1) activation of hippocampal NSCs by environmental enrichment, and 2) activation of spinal cord NSCs by injury-induced neuroinflammation. Moreover, 3) to gain new insights into the molecular mechanisms of these models, we will perform transcriptomics studies to open new lines of investigation. The first project is expected to provide us with new insights into the basic cellular and molecular mechanisms through which environmental enrichment modulates adult brain plasticity. We first evaluated the contribution of individual environmental enrichment components to hippocampal neurogenesis (Chapter II). Voluntary exercise promotes neurogenesis, whereas a social context increases neuronal activation. We then determined the effect of these components on behavioural performances and transcriptome using an eight-arm radial maze to assess spatial memory, novel object recognition, and RNA-Seq, respectively (Chapter III). Runners show stronger spatial short-term memory recall, whereas mice exposed to social interactions had a better cognitive flexibility to abandon old memory. Surprisingly, RNA-Seq analysis indicated clear differences in the expression of modified transcripts between low runners and high runners, as well as for social interacting mice (within the complex environment). The second project consists of discovering how ependymal cells acquire NSC properties in vitro or multipotentiality following lesions in vivo. A RNA-Seq analysis revealed that the transforming growth factor-β1 (TGF-β1) acts as an upstream regulator of significant changes following spinal cord injury (Chapter IV). We therefore confirmed the presence of this cytokine after lesion and investigated its role on proliferation, differentiation, and survival of neurosphere-initiating cells from the spinal cord. Our results suggest that TGF-β1 regulates the acquisition and expression of stem cell properties of spinal cord-derived ependymal cells.

Liver disease is now a leading cause of non-AIDS related morbidity and mortality in people living with HIV (PLWH). The present study investigated the interplay between adverse lifestyle factors that are prevalent in PLWH, biological mediators of liver pathogenesis, and a non-invasive measure of liver fibrosis (FIB-4 index) in HIV mono- and HIV/HCV co-infected individuals. The results of this investigation in the Miami Adult Studies of HIV (MASH) cohort show that the odds of liver fibrosis progression significantly increased over two years for HIV mono-infected participants who drank alcohol hazardously (OR 3.038, P=0.048), and had BMI ≥ 28kg/m2 (OR 2.934, P=0.027). Cocaine use reduced the odds of advancing one stage of liver fibrosis (OR 0.228, P=0.038), but an interaction between high BMI and cocaine use slightly raised the odds by 4.8% of liver fibrosis progression (P=0.072). HIV/HCV co-infected participants showed interactions between cocaine use and high BMI with increased FIB-4 stage (OR 4.985, P= 0.034), however no lifestyle factors could independently predict FIB-4 stage in this group. Biological mediators previously associated with liver pathogenesis were associated with higher FIB-4 index over 2 years in a subset of (n=65) HIV mono-infected participants. Plasma measures of oxidative stress (% oxidized glutathione: OR 4.342, P= 0.046), hepatocyte-specific apoptosis (Cytokeratin-18 (CK-18): OR 1.008, P=0.021), and microbial endotoxin (lipopolysaccharide (LPS): OR 1.098, P= 0.097) were associated with having higher odds of progressing at least one stage of FIB-4 over 2 years. The same biological mediators were also associated with liver fibrosis within HIV infected people who also had a harmful lifestyle characteristic. FIB-4 index was significantly associated with % oxidized glutathione in obese subjects (β=0.563, P=0.018), TGF-β1 in cocaine users (β=0.858, P=0.027), and CK-18 in HIV infected individuals without any adverse lifestyle factors (β=0.435, P=0.015). Taken together, the findings of these studies describe interrelationships between HIV disease status, lifestyle, and biological mediators of liver fibrosis. The results show interactions between lifestyle conditions and the mediators of liver fibrosis may account for higher rates of liver disease in HIV infection. Research is warranted to develop personalized therapeutics for PLWH to curb the burden of liver disease.

Indiana University-Purdue University Indianapolis (IUPUI) / Skeletal manifestations including short stature, osteoporosis, kyphoscoliosis, and tibial dysplasia cumulatively affect approximately 70% of patients with neurofibromatosis type 1 (NF1). Tibial pseudarthrosis, the chronic non-union of a spontaneous fracture, is a debilitating skeletal malady affecting young children with NF1. These non-healing fractures respond poorly to treatment and often require amputation of the affected limb due to limited understanding of the causative mechanisms. To better understand the cellular and molecular pathogenesis of these osseous defects, we have established a new mouse model which recapitulates a spectrum of skeletal pathologies frequently observed in patients with NF1. Nf1flox/-;Col2.3Cre mice, harboring Nf1 nullizygous osteoblasts on a Nf1+/- background, exhibit multiple osseous defects which are closely reminiscent of those found in NF1 patients, including runting (short stature), bone mass deficits, spinal deformities, and tibial fracture non-union. Through adoptive bone marrow transfer studies, we have demonstrated that the Nf1 haploinsufficient hematopoietic system pivotally mediates the pathogenesis of bone loss and fracture non-union in Nf1flox/-;Col2.3Cre mice. By genetic ablation of a single Nf1 allele in early myeloid development, under the control of LysMCre, we have further delineated that Nf1 haploinsufficient myeloid progenitors and osteoclasts are the culprit lineages mediating accelerated bone loss. Interestingly, conditional Nf1 haploinsufficiency in mature osteoclasts, induced by CtskCre, was insufficient to trigger enhanced lytic activity. These data provide direct genetic evidence for Nf1’s temporal significance as a gatekeeper of the osteoclast progenitor pool in primitive myelopoiesis. On the molecular level, we found that transforming growth factor-beta1 (TGF-β1), a primary mediator in the spatiotemporal coupling of bone remodeling, is pathologically overexpressed by five- to six- fold in both NF1 patients and in mice. Nf1 deficient osteoblasts, the principal source of TGF-β1 in the bone matrix, overexpress TGF-β1 in a gene dosage dependent fashion. Moreover, p21Ras dependent hyperactivation of the Smad pathway accentuates responses to pathological TGF-β1 signals in Nf1 deficient bone cells. As a proof of concept, we demonstrate that pharmacologic TβRI kinase inhibition can rescue bone mass defects and prevent tibial fracture non-union in Nf1flox/-;Col2.3Cre mice, suggesting that targeting TGF-β1 signaling in myeloid lineages may provide therapeutic benefit for treating NF1 skeletal defects.