Aquaporin proteins significantly increase water permeability across tissues and cell membranes. Ocular tissues, including the trabecular meshwork (TM) and retinal pigment epithelium (RPE), are especially reliant on aquaporin mediated water movement for ocular homeostasis. Even though bulk fluid movement is paracellular through the TM and transcellular through the RPE, both express aquaporin-1 (AQP1). The role and regulation of AQP1 as it relates to homeostasis in different ocular tissues is not well understood. I hypothesized that ocular tissues respond to external mechanical and molecular cues by altering AQP1 expression and function in order to regulate ocular fluid movement and maintain homeostasis.To test how AQP1 function is altered in response to external cues in order to maintain tissue-specific homeostasis, I addressed the following two aims. The first aim was directed at determining how mechanical strain, an external stimulus that routinely affects TM function, influences AQP1 expression and TM homeostasis. Primary cultures of human TM were subjected to static and cyclic stretch and then analyzed for changes in AQP1 expression by western blot and cell damage by activity of lactate dehydrogense (LDH) in conditioned media. The results show AQP1 expression and LDH release significantly increased with static stretch. Analysis of LDH release with respect to AQP1 expression revealed an inverse linear relationship (r² = 0.7780).The second aim was directed at characterizing signaling mechanisms responsible for regulating fluid transport in RPE, previously shown to be dependent upon AQP1. I treated primary cultures of human RPE with either atrial natriuretic peptide (ANP) or 8-bromo-cyclic guanosine monophosphate (8-Br-cGMP) in the presence or absence of Anantin (ANP-receptor inhibitor) or H-8 (Protein Kinase G inhibitor). The results show that ANP and 8-Br-cGMP significantly increased apical to basal net fluid movement (p < 0.05, n = 3). Inhibition of these effects was successful with Anantin treatment but not with application of H-8.The data presented demonstrate a novel role of protection for AQP1 in TM, and also expand upon cGMP dependent regulation of RPE fluid transport. The combined studies indicate tissue specific AQP1 regulation may offer new avenues to target water movement in treatment of ocular pathologies.

In vivo, biomechanical stress plays an important role in tissue physiology and pathology, affecting cell and tissue behavior. Even though conventional outflow tissues in the eye are constantly exposed to dynamic changes in intraocular pressure (IOP), the effects of such biomechanical stressors on outflow tissue function have not been analyzed. In particular, changes in IOP with each heartbeat have been measured in human eyes approximating 2.7 mmHg/sec. The purpose of this dissertation is to determine the effect(s) of ocular pulse on conventional outflow tissue regulation and the effect that contractility plays in this mechanical stress-mediated response. The central hypothesis directing this research is that cyclic intraocular pulsations (i.e. ocular pulse) play a significant role in conventional outflow facility.In order to address our hypothesis we studied the effect of biomechanical stressors on conventional outflow physiology using three different strategies: (1) by comparing conventional outflow endothelial cells to blood and lymphatic capillary endothelia, we gained a better understanding of the effects of biomechanical stress on conventional outflow tissue physiology, (2) by modifying the anterior segment perfusion model, we were able to measure the effect of ocular pulse on conventional outflow facility, and (3) by exposing trabecular meshwork cell monolayers to cyclic biomechanical pressure oscillations in the presence of compounds known to affect trabecular meshwork contractility, we were able to analyze the effect of rho-kinase-mediated contractility on the ocular pulse-associated response.Perfused human and porcine anterior segments showed a significant ocular pulse-mediated decrease in outflow facility; in addition, perfused trabecular meshwork monolayers showed an increase in intra-chamber pressure when exposed to cyclic pressure oscillations. This effect was blocked by Y27632 inhibition of rho-kinase-mediated contraction.In conclusion, the work shown in this dissertation demonstrates for the first time that trabecular outflow tissues are capable of responding to a physiologically-relevant cyclic biomechanical stress. This response can be observed as an increase in outflow resistance that translates to lower baselines in outflow facility of anterior segments and lower hydraulic conductivity of trabecular meshwork monolayers. In addition, we concluded that the observed ocular pulse-mediated response of trabecular meshwork cells is regulated by rho-kinase-induced contractility.

Primary open angle glaucoma (POAG) is a leading cause of blindness worldwide. Elevated intraocular pressure (IOP), resulting from increased aqueous humor outflow resistance, is a major risk factor for the development and progression of POAG. Outflow resistance in the trabecular outflow pathway is mainly (50-75%) generated in the juxtacanalicular connective tissue (JCT), and partially (25-50%) in the portion distal to the inner wall of Schlemm’s canal. The details of how aqueous humor flows through these tissues and how resistance in these tissues is regulated are not fully understood in normal and POAG eyes. Aqueous humor outflow was shown to be “segmental”, with discontinuous active regions of aqueous humor filtration along the trabecular outflow pathway that can be labeled with perfused fluorescent tracers and measured as effective filtration area (EFA). In this study, we investigated the relationship between changes in EFA along the trabecular outflow pathway and outflow facility/IOP under two experimental conditions. The first experiment was designed to increase outflow facility by using netarsudil, a recently approved Rho kinase inhibitor class glaucoma medication, in normal human donor eyes. The second experiment was designed to increase IOP with topical steroid treatment for 5 weeks in mice. The purpose of this study is to verify whether EFA can be modulated by netarsudil or steroid treatment and to demonstrate the morphological changes that may be responsible for the changes of EFA. We analyzed EFA along the trabecular outflow pathway and found that elevated/reduced EFA correlated with increased outflow facility/IOP. Guided by EFA, we performed detailed morphological comparison between the active and inactive portions of aqueous humor filtration tissue to evaluate possible structural changes involved in EFA regulation. We found that increased EFA was associated with a loosened JCT structure and dilated episclearal veins, while decreased EFA was associated with a compacted JCT structure, increased deposition of curly collagen and/or fibrillary structure in the trabecular meshwork, and increased basement membrane continuity. Our data suggest that the netarsudil/steroid-induced morphological changes in the trabecular outflow pathway can result in either an increase or decrease in EFA, which in turn contributes to the regulation of outflow facility/IOP. / 2020-10-24T00:00:00Z

Orientadores: Mônica Barbosa de Melo, José Paulo Cabral de Vasconcellos / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-25T15:53:03Z (GMT). No. of bitstreams: 1 Oliveira_MarianaBorges_M.pdf: 6901699 bytes, checksum: b84b4ac92949fdb082625b01ad6c66a1 (MD5) Previous issue date: 2014 / Resumo: O glaucoma engloba um grupo de doenças que têm como característica comum a atrofia progressiva do disco óptico com alteração correspondente de campo visual, decorrente da perda de células ganglionares da retina. O aumento da pressão intraocular (PIO) é o principal fator de risco para o desenvolvimento do glaucoma, mas tem sido sugerida a existência de outros aspectos relevantes, tais como alterações no metabolismo do óxido nítrico, na regulação do fluxo vascular, sinais de estresse oxidativo e alterações no sistema imunológico. O glaucoma primário de ângulo aberto (GPAA), a forma mais prevalente entre os glaucomas, está associado a uma série de fatores de risco para sua instalação e desenvolvimento, além da PIO. Os demais fatores de risco são: idade (relação direta), raça (mais frequente e grave em indivíduos da raça negra), miopia e história familiar de glaucoma. A maioria dos casos de GPAA apresenta um padrão complexo de hereditariedade, em que variantes de suscetibilidade contribuem para a gravidade da doença. O objetivo deste estudo do tipo caso-controle foi avaliar o papel de polimorfismos nos genes da interleucina alfa (IL1A), interleucina beta (IL1B) e fator de necrose tumoral alfa (TNFA) em relação à suscetibilidade ao GPAA. Após avaliação oftalmológica, foram recrutados 214 indivíduos não relacionados portadores de GPAA. A análise molecular foi realizada por meio de técnicas de PCR e sequenciamento direto. Neste estudo observou-se que o alelo C do polimorfismo -31 C/T, presente na região promotora do gene IL1B, confere risco para o glaucoma (p=0,002). Este alelo está contido em um "TATA" box, influenciando na transcrição deste gene. Existe um desequilíbrio de ligação entre os SNPs -31 C/T e -511 T/C do gene IL1B e este último também está associado a um risco aumentado para o glaucoma (p=0,006). Com relação aos demais SNPs estudados (+3954 C/T do gene IL1B, -889 C/T e +4845 G/T do gene IL1A e -238 G/A e -308 G/A do gene TNFA), não houve associação com o glaucoma. Este estudo sugere que há relação das variantes "C" do SNP -31 C/T e "T" do SNP -511 T/C do gene IL1B com a etiologia do GPAA em uma amostra da população brasileira / Abstract: Glaucoma comprises a group of diseases characterized by progressive atrophy of the optic disc and corresponding visual field loss, as consequence of retinal ganglion cells death. Augmented intraocular pressure (IOP) is the main risk factor for glaucoma development, but it has been suggested that there are other relevant aspects that may lead to neuronal and trabecular meshwork (TM) damage and contribute to the development of glaucoma, including alterations in nitric oxide metabolism, oxidative stress and vascular and immune system dysregulations. Primary open angle glaucoma (POAG), the most prevalent form of glaucoma, is associated with several risk factors to its initiation and progression besides IOP. The additional risk factors are: age (direct correlation), race (more frequent in black subjects), myopia and family history of glaucoma. The majority of POAG cases presents a complex pattern of heritability, in which susceptibility variants contribute to disease severity. The aim of this case-control study was to evaluate the role of single nucleotide polymorphisms (SNPs) in the interleukin alpha (IL1A), interleukin beta (IL1B) and tumor necrosis factor alpha (TNFA) genes in relation to POAG susceptibility. After ophthalmologic evaluation, 214 unrelated subjects with POAG were recruited. The molecular analysis was performed by PCR and direct sequencing techniques. In this study it was observed that the C allele of the -31 C/T promoter polymorphism of the IL1B gene confers risk for glaucoma (p=0.002). This allele disrupts a "TATA" box, influencing the transcription of the IL1B gene. There is linkage disequilibrium between the -31 C/T e -511 T/C SNPs, and the latter is also associated with an increased risk for glaucoma (p=0.006). Regarding the others studied SNPs (+3954 C/T of the IL1B gene, -889 C/T and +4845 G/T of the IL1A gene and -238 G/A and -308 G/A of the TNFA gene), there is no association with glaucoma. This study suggests the relationship of the "C" variant of -31C/T and "T" variant of -511T/C of IL1B gene with POAG etiology in a sample of the Brazilian population / Mestrado / Oftalmologia / Mestra em Ciências Médicas

Primary open-angle glaucoma (POAG) is a leading cause of blindness in the world. A primary risk factor for POAG is elevated intraocular pressure (IOP), caused by increased aqueous humor outflow resistance. Currently, lowering the IOP is the only effective way of treating glaucoma; however, the cause of increased outflow resistance remains unclear. This thesis will present a series of studies which investigated structures of the trabecular outflow pathway, including Schlemm’s canal endothelium, juxtacanalicular tissue, and trabecular beams, and their roles in regulating aqueous outflow resistance. The studies were conducted in both human and animal models using ex vivo ocular perfusion as well as in vitro microfluidic systems. In the first study, we investigated the effects of Y27632, a derivative of Rho-kinase inhibitor that is being developed as next generation glaucoma drug with unclear IOP lowering mechanism, on aqueous humor outflow dynamics and associated morphological changes in normal human eyes and laser-induced ocular hypertensive monkey eyes. In the second study, we developed and validated a novel three-dimensional microfluidic system using lymphatic microvascular endothelial cells. The microfluidic system can be used to study Schlemm’s canal endothelial cell dynamics and aqueous humor transport mechanism in the future. In the last study, we characterized the morphological structure, distribution, and thickness of the endothelial glycocalyx in the aqueous humor outflow pathway of human and bovine eyes. Together these studies will help define new directions for therapy that will help control IOP and preserve vision throughout a normal life span.

Primary open-angle glaucoma (POAG) is one of the leading causes of blindness worldwide. The only modifiable risk factor for POAG is elevated intraocular pressure, resulting from increased aqueous humor production or decreased drainage. Resistance to drainage in the aqueous outflow pathway is believed to reside in the juxtacanalicular connective tissue (JCT) and to be modulated by the inner wall (IW) endothelium of Schlemm’s canal (SC); however, the mechanisms that increase resistance in POAG remain unclear. To cross the IW, aqueous humor passes through I-pores on giant vacuoles (GVs) or B-pores between adjacent endothelial cells. Additionally, outflow around the circumference of the eye is segmental, or non-uniform, and fluorescent tracers can be used to label areas of high-flow and non-flow. The morphological differences in the endothelial cells of SC and their GVs in high- vs. non-flow areas have not been fully elucidated. In this project, we investigated the role of GVs and pores in the IW endothelial cells of SC in regulating segmental outflow in human eyes. We used serial block-face scanning electron microscopy to generate thousands of serial images and visualize these structures in 3D at the ultrastructural level. First, we 3D-reconstructed 45 individual IW cells and their GVs and quantified the number of connections each cell makes with the underlying JCT matrix/cells. We found that cells in high-flow areas made significantly fewer connections to JCT matrix/cells compared to cells in non-flow areas. Secondly, we analyzed 3,302 GVs for I-pores and basal openings and found a significantly greater percentage of GVs with both basal openings and I-pores in high-flow area compared to non-flow area, suggesting this type of GVs form a channel through which aqueous humor passes from JCT to SC. We also found that GVs with I-pores were significantly larger than those without I-pores. Our results suggest that decreasing number of cellular connections and increasing number of GVs with pores may be potential strategies to increase the amount of high-flow area and aqueous outflow for glaucoma treatment. Together, these studies add to our understanding of the role of GVs and pores in regulating segmental flow around the eye.

No description available.

This thesis is focused on the material known as the Mesolithic portable art. Earlier research have interpreted the material as representative art relating to ideology, mythology, prestige, ritual practices,and tribalism. Such interpretations are based on theoretical frameworks that build on hylomorphism and Cartesian metaphysics. By a change of theoretical framework, to a new animistic perspective based on a combination of Gilles Deleuze and Félix Guattari’s rhizome theory, Tim Ingold’s meshwork and Giordano Bruno’s theory of bonds in general, followed by the use of ChantalConneller’s method rhizomatic chaîne opératoire, I conclude that the motifs on the tools and pendants are communications to the animated subjects that make up and inhabit the environment. Furthermore, I conclude that the binary positions of function and ritual cannot be applied when studying the formgenerating process of this material, as the tools and pendants along with their applied motifs are a result of what is in between these binary positions.

Durch Ausübung physiologischer Grundfunktionen spielen Ionenkanäle eine entscheidende Rolle für die reguläre Funktion von Zellen. Zum besseren Verständnis ihrer Struktur und Funktion sind Untersuchungen natürlicher und künstlicher Ionenkanäle wichtige Werkzeuge. Großes analytisches und therapeutisches Potential ist in der Untersuchung künstlicher Kanäle in lebenden Zellen vorhanden, was bisher wenig Beachtung fand. In der vorliegenden Arbeit wurde die Wirkung der künstlichen Ionenkanäle THF-gram, THF-gram-TBDPS sowie linked-gram-TBDPS auf elektrophysiologische Eigenschaften boviner Trabekelwerkszellen des Auges anhand von Patch-Clamp-Untersuchungen im Whole-Cell-Modus analysiert. Die Untersuchung brachte folgende Erkenntnisse: 1. Die Inkorporation aller drei Verbindungen war erfolgreich, was sich durch Anstieg der Stromdichte und Verschiebung des Umkehrpotentials zeigte. 2. Einbau von THF-gram und THF-gram-TBDPS war mit dem Überleben der Zellen vereinbar, während linked-gram-TBDPS aufgrund einer sehr potenten Antwort bereits bei sehr geringen Konzentrationen zum raschen Zelltod führte. 3. Eine Asymmetrie der Stromantwort zugunsten stärkerer Auswärtsströme wurde bei THF-gram und in schwächerer Ausprägung bei THF-gram-TBDPS festgestellt. Linked-gram-TBDPS zeigte keine derartige Asymmetrie. 4. Unter Verwendung von Cs+ als Ladungsträger war der beobachtete Anstieg der Stromdichte bei allen drei Verbindungen eindeutig stärker als unter physiologischen Bedingungen (Na+/K+). 5. Die dargestellten Erkenntnisse sind ein erster Schritt zur therapeutischen Anwendung von künstlichen Ionenkanälen. Eine Weiterentwicklung in Richtung höherer Selektivität und besserer Kontrolle ist jedoch genauso erforderlich wie die Klärung der klinischen Umsetzbarkeit. / Ion channels play a pivotal role for regular cell function. To better understand their structure and function, investigation of both natural and artificial ion channels is being performed to date. Investigation of artificial channels in living cells hides a big potential, however, little attention has been paid to this field so far. In this work, the effect of the artificial ion channels THF-gram, THF-gram-TBDPS and linked-gram-TBDPS on electrophysiological properties of bovine trabecular meshwork cells was investigated with the patch-clamp-technique. Following results were obtained: 1. Incorporation of all three compounds was successful, which was proven by increase of current density and cell depolarisation. 2. The cells survived after incorporation of THF-gram and THF-gram-TBDPS but not after linked-gram-TBDPS, which resulted in cell death at very low concentrations. 3. Larger outward currents were observed with THF-gram and, at a lower extent, with THF-gram-TBDPS. Linked-gram-TBDPS did not show such an asymmetry. 4. With Cs+ as charge carrier all three compunds showed a stronger increase of current density than under physiological conditions (Na+/K+). 5. The decribed results are a first step towards therapeutic application of artificial ion channels, however, further development towards higher selectivity and better control is as necessary as clarification of clinical feasibility.

Starting from the overall Revitalizing Small Millets in South Asia (RESMISA) project objective and question, how to increase millet production and consumption, I will use the same to enter Chintaguda and understand how that can be accomplished in the village. As millets do not occupy a significant aspect of the lives of people in Chintaguda, I essentially sought to understand the general decision-making logics that operate therein. This objective will help me understand which factors and their interactions influence activities around socio-ecological engagements. I aim to devise a framework to comprehend these logics and the complexities found in Chintaguda by using social theories. These will help me stitch together a narrative for Chintaguda that will contextualize the people’s relationship to millets. This study will show the various ways people can and do relate to millets. / February 2016