Mecanismos de regulación de la síntesis de la melatonina para el control de la presión intraocular

Tesis de la Universidad Complutense de Madrid, Facultad de Óptica y Optometría, Departamento de Bioquímica y Biología Molecular IV, leída el 10/11/2017Ocular hypertension, although not considered a disease itself, it leads to glaucoma which eventually cause irreversible vision loss. Consequently, glaucoma therapeutic approach is to lower intraocular pressure (IOP). Nonetheless, anti-glaucoma drugs come often with several side effects that can lead to treatment withdrawal in some cases. In this context, melatonin and its analogs emerges as potential complimentary/alternative treatment, offering the advantage of hypotensive and antioxidant properties. The present PhD thesis aim to investigate endogenous melatonin content in the aqueous humor and describes mechanisms involved in detecting increased IOP, along with a focus on the possibility of melatonin receptors to interact with different receptors with the intention to find a more effective combined therapeutical approach. Finally, to study the attribution of the crystalline lens to the aqueous humor melatonin content and to discover the regulation of its synthesis in this ocular structure. Results showed a correlation between melatonin levels in the aqueous humor and IOP. Melatonin levels were found higher in patients with elevated IOP. Using a glaucomatous animal model (DBA/2J) it was possible to corroborate our findings. In vitro assays pointed to the participation of the TRPV4 channel, which is sensitive to pressure, among other stimuli. Activation of the mentioned channel in human immortalised nonpigmented ciliary body epithelial cells resulted in an increment of melatonin secretion through the increase of aralkylamine N-acetyltransferase (AANAT) expression, the first enzyme in melatonin synthesis. In addition, short term assays showed that the TRPV4 activation leaded to AANAT phosphorylation through a cascade of intracellular events that involves the participation of calmodulin and calcium-calmodulin dependent protein kinase II. This phosphorylation activates AANAT and therefore melatonin synthesis is stimulated...La hipertensión ocular, aunque no se considera una enfermedad en sí, es un factor de riesgo para desarrollar glaucoma, que eventualmente causa pérdida irreversible de la visión. En consecuencia, el enfoque terapéutico más habitual en el glaucoma es reducir la presión intraocular (PIO). No obstante, los fármacos anti-glaucomatosos presentan varios efectos secundarios que pueden conducir a la retirada del tratamiento en algunos casos. En este contexto, la melatonina y sus análogos emergen como un tratamiento complementario/ alternativo, ofreciendo la ventaja de poseer propiedades hipotensoras y antioxidantes. La presente tesis doctoral tiene como objetivo investigar el contenido de la melatonina endógena en el humor acuoso y describir los mecanismos implicados en la detección de los cambios en la IOP. Igualmente se estudia la capacidad de los receptores de melatonina para interactuar con otros receptores con la intención de encontrar un enfoque terapéutico combinado más eficaz. Finalmente, esta tesis también estudia la contribución del cristalinoen su aporte de melatonina al humor acuoso. Los resultados han mostrado una correlación entre los niveles de melatonina en el humor acuoso y la PIO. Los niveles de melatonina se encontraron más altos en pacientes con PIO elevada. El uso de modelo animal glaucomatoso (DBA/2J) corroboró nuestros hallazgos de una manera consistente. Por otro lado, los ensayos in vitro señalaron la participación del canal TRPV4, proteína que es sensible a la presión,como principal activador de la sintesis de la melatonina. De este modo, la estimulación de este canal en células epiteliales del cuerpo ciliar no pigmentadas inmortalizadas humanas dio como resultado un incremento de la secreción de melatonina a través de un aumento de la expresion de la Aralquilamina N-acetiltransferasa(AANAT), la primera enzima en la síntesis de melatonina. Además, los ensayos a corto plazo mostraron que la activación de TRPV4 induce a la fosforilación de AANAT, y su activación, a través de una cascada de eventos intracelulares implica la participación de la calmodulina y la calmodulina quinasa II dependiente de calcio-calmodulina...Unidad Docente de Bioquímica y Biología MolecularFac. de Óptica y OptometríaTRUEunpu

POST-MORTEM INTERVAL ESTIMATION THROUGH METABOLOMICS OF PINEAL GLAND

This study investigates metabolomic profiling of post-mortem pineal glands using 1H-NMR spectroscopy. Significant diurnal variations in metabolites correlated with the post-mortem interval, offering a novel approach for time-of-death estimation

Photoperiod, adrenal corticosterone and the development of avian glaucoma

Three hundred and fifty Rhode Island Red cockerels were maintained under three lighting conditions from day one after hatching to 10 weeks of age to investigate the possible relationships between photoperiod, avian glaucoma and adrenal function. Other parameters studied included body, comb, testicular and thyroid weights. The lighting treatments included (1) diurnal light/dark or 12L:12D, (2) continuous light or 24L:0D, and (3) continuous dark or 0L:24D. Both 24L:0D and 0L:24D conditions significantly increased avian eyeball weight, adrenal weight and plasma content of corticosterone compared with the 12L:12D regimen (P \u3e 0.05). There was a highly significant reduction in eye depth (P \u3c 0.01) when birds were raised under continuous light or continuous darkness. A non-significant reduction in thyroid weight was produced by keeping chicks under total illumination and also under 24 hours of darkness. Constant light greatly enhanced testicular growth while the continuous absence of light drastically reduced testes weight in comparison with testicular growth under alternating light and dark. Body weight, comb weight, and intraocular pressure were not significantly affected by any of the treatments. At least two peaks of plasma corticosterone were seen under each treatment, but the corticosterone diurnal rhythm was not disturbed even though the levels of the hormone were increased significantly (P \u3c 0.05) by the absence of an alternating light and dark treatment

Aerospace Medicine and Biology: A continuing bibliography, supplement 216

One hundred twenty reports, articles, and other documents introduced into the NASA scientific and technical information system in January 1981 are listed. Topics include: sanitary problems; pharmacology; toxicology; safety and survival; life support systems; exobiology; and personnel factors

The role of 24-hour intraocular pressure fluctuation on glaucoma progression in primary open angle claucoma

Introduction: The accurate assessment of patient’s intraocular pressure profile is critical in the management of primary open angle glaucoma. Although it is a multifactorial disease, IOP remain the only treatable risk factor for the condition. Currently, wide diurnal IOP fluctuation has been identified as an independent risk factor of glaucoma progression. Therefore a single IOP reading taken in our clinic will failed to detect diurnal IOP fluctuation in glaucoma patients. The recognition of diurnal IOP may also explain the progression of visual field in patients who appear to be controlled. Objectives: To study the pattern of 24-hour intraocular pressure fluctuation in primary open angle glaucoma patients and compare the 24 hours mean intraocular pressure, peak trough and IOP fluctuation between POAG patients with and without visual field progression. Patients and Methods: A comparative cross sectional study was conducted involving POAG patients. They divided into non-progressed and progressed group based on AGIS score. Patients were admitted in the ward and IOP measured by GAT at 4-hourly interval for 24-hour. Mean intraocular pressure, peak trough and IOP fluctuation IOP were compared between non-progressed and progressed groups. Analysis was conducted using repeated measure ANOVA and independent t-test A total of 68 patients (36 non-progressed and 32 progressed) were recruited. Mean age for all recruited patients were 68.3±8.6 years old. There was significant more Male among patient with progressed group than in non-progressed group (P=0.022). Mean follow-up was 6.7±3.8 years in progressed group and 5.0±2.6 years in non-progressed group. Patient in progressed group has significantly more moderate to severe glaucoma (P=0.001) and 71.9% of them were on more than two topical pressure lowering agents (P=0.001). There was significantly higher 24-hour mean peak IOP (P=0.001) and wider 24-hour mean IOP fluctuation (P=0.003) in progressed group compared to non-progressed group. There was significant higher mean IOP at 1200 hour in progressed group (P=0.003). Both group showed afternoon peak pattern. Conclusion: 24-hour IOP profile showed that there was significant higher 24-hour mean peak IOP and wider 24-hour mean IOP fluctuation found in progressed group. Both group showed pattern of afternoon peak

Standardization of sample collection, isolation and analysis methods in extracellular vesicle research

The emergence of publications on extracellular RNA (exRNA) and extracellular vesicles (EV) has highlighted the potential of these molecules and vehicles as biomarkers of disease and therapeutic targets. These findings have created a paradigm shift, most prominently in the field of oncology, prompting expanded interest in the field and dedication of funds for EV research. At the same time, understanding of EV subtypes, biogenesis, cargo and mechanisms of shuttling remains incomplete. The techniques that can be harnessed to address the many gaps in our current knowledge were the subject of a special workshop of the International Society for Extracellular Vesicles (ISEV) in New York City in October 2012. As part of the “ISEV Research Seminar: Analysis and Function of RNA in Extracellular Vesicles (evRNA)”, 6 round-table discussions were held to provide an evidence-based framework for isolation and analysis of EV, purification and analysis of associated RNA molecules, and molecular engineering of EV for therapeutic intervention. This article arises from the discussion of EV isolation and analysis at that meeting. The conclusions of the round table are supplemented with a review of published materials and our experience. Controversies and outstanding questions are identified that may inform future research and funding priorities. While we emphasize the need for standardization of specimen handling, appropriate normative controls, and isolation and analysis techniques to facilitate comparison of results, we also recognize that continual development and evaluation of techniques will be necessary as new knowledge is amassed. On many points, consensus has not yet been achieved and must be built through the reporting of well-controlled experiments

Configuration of a multi-layered multi-disk tablet for specialized drug delivery

M. Pharm., Dept. of Pharmacy and Pharmacology, Faculty of Health Sciences, University of the Witwatersrand, 2011Chronotherapy is a form of therapy where treatment is administered according to a schedule that corresponds to an individual’s biological clock. Research demonstrates that the body’s natural processes follow a 24-hour pattern, or circadian rhythm. In addition, symptoms of disease fluctuate according to this 24-hour pattern. These diseases, termed chronotherapeutic disorders may include amongst other disorders, hypertension, cardiovascular disease and asthma. Common therapy for these disorders involves the use of controlled zero-order release formulations. Here, the same quantity of drug is released over a period of time. Although beneficial, these formulations are not ideal in the treatment of chronotherapeutic disorders. Treatment of these disorders aims to release drug at specific periods, only when it is required, such that therapy coincides with the body’s natural rhythm. Ideally, drug should be released in pulses with two or more pulses released from the dosage form. In this manner, the patient is exposed to drug only when required, reducing the number of dosages, reducing side-effects and ultimately increasing patient compliance. Therefore, the aim of this research was to develop a Multi-Layered Multi-Disk Tablet (MLMDT) that incorporates two drug-loaded disks enveloped by three polymeric layers. The proposed system, to be used in the treatment of chronotherapeutic disorders, is designed to provide a lag phase and then two pulses of drug release separated by a ‖switch-off‖ phase. During the ―switch-off‖ phase no drug is released from the system. Initially, preliminary screening studies were performed on various polymeric materials to assess their effectiveness to generate the desired drug release profile. Of the numerous polymer combination and ratios, only a few were relevant and were subsequently tested further. From the preliminary studies it was ascertained that the composition of disk 2 was critical in generating the ―switch-off‖ phase separating the two pulses. Artificial Neural Networks (ANN); a computational technique that simulates the thinking process of the human brain was employed for optimization. Results from this technique outlined the polymer combination suitable for the optimized MLMDT. The optimized formulations were subjected to friability, hardness and uniformity of mass analysis as well as swelling, erosion and magnetic resonance imaging techniques to observe and confirm the performance of the MLMDT during dissolution. In addition, textural analysis, computational modeling and temperature modulated differential scanning calorimetry techniques were used to elucidate any incompatibilities or complexes formed. In vitro drug release analysis revealed that the MLMDT generated a lag phase followed by two pulses of drug release over the 24 hour period. The two pulses were separated by a ―switch-off‖ phase. To confirm data obtained during preclinical in vitro testing, animal studies were undertaken using the Large White Pig model. Pigs were dosed with conventional products and the optimized MLMDT. Blood samples collected over a 24 hour period were analyzed using Ultra Performance Liquid Chromatography to determine the drug concentration in blood. Drug concentration analysis from conventional products revealed increasing plasma concentrations up to 2 hours followed by a steady decline in concentration while the developed MLMDT displayed two pulse drug release separated by a ―switch-off‖ phase