Melatonin is a product of the pineal gland and is postulated to play an antigonadotropic role in the reproductive system of mammals. The reproductive system of non-seasonally breeding mammals is believed to be not as responsive to melatonin treatment as that of seasonally breeding mammals. Recently, there has been increasing support from in vivo and in vitro studies, for the hypothesis that melatonin has negative effects on sperm physiology, especially on sperm motility. High and/or low seminal concentrations of melatonin have been associated with abnormalities in human sperm motility and concentration. In this study, I examined the effects of melatonin on the testis, epididymis and sperm physiology, using in vivo and in vitro experiments, in a non-seasonally breeding mammal. Treatment, in vivo, with exogenous melatonin for six weeks did not inhibit testosterone production or spermatogenesis, nor did it affect the mass of the testes and epididymides at dissection, the concentration the morphology of speimatozoa. However, melatonin in vivo had a small, but significant negative effect on sperm motility and sperm motility index. In vitro incubation of spermatozoa Fith melatonin reduced the percentage (%) of forward progressive movement (fpm), increased the % reduction in fpm, reduced the vigor or quality of sperm motility, reduced the sperm motility index, and delayed and/or prolonged the transition of one pattern of sperm motility to the subsequent patterns. Melatonin increased the pH of the culture medium, and the increased pH, and the ethanol utilized as a solvent for melatonin, both negatively affected all the sperm motility parameters that were assessed in my study. The effects of ethanol increased with time, and the effects of pH increased with both time and increasing pH. Melatonin in vitro did not inhibit capacitation and the acrosome reaction, but it delayed the onset and the progression of capacitation and the acrosome reaction. These results suggest that while melatonin did not inhibit spermatogenesis in the Wistar rat, it may influence sperm motility. Therefore, the presence of high concentrations of melatonin in the reproductive fluids may inhibit sperm motility. With further detailed research, melatonin may have a potential use as a contraceptive drug.

The multiple functions of the pineal gland have been collectively interpreted as constituting a general anti-stressogenic role. The adrenal glands play a central role in maintaining homeostasis. The major neuroendocrine consequence of long-term stress is elevated circulating glucocorticoid levels. In this study, the effect of chronic, oral hydrocortisone treatment on pineal biochemistry was investigated in male Wi star rats of the albino strain. The results show that seven days of oral hydrocortisone treatment endows the pineal gland with the ability to increase melatonin synthesis in organ culture. The increase is accompanied by a rise in NAT activity, cyclic AMP levels and enhanced specific binding to the pineal B-adrenergic receptors. It appears that hydrocortisone sensitizes the pineal gland to stimulation by B-adrenergic agonists. thus rendering the pineal more responsive to B-adrenergic agonists. Further studies were directed at demonstrating an anti-stressogenic function for the pineal gland by investigating whether the principal pineal indole, melatonin. could protect against the deleterious effects of elevated. circulating drocortisone levels. The results show that chronic, oral hydrocortisone treatment significantly increases liver tryptophan pyrrolase activity. The catabolism of tryptophan by tryptophan pyrrolase is an important determinant of tryptophan availability to the brain, and therefore, brain serotonin levels. The findings show that melatonin inhibits basal and hydrocortisone-stimulated liver tryptophan pyrrolase apoenzyme activity in a dose-dependent manner. This inhibition suggests that melatonin may protect against excessive loss of tryptophan from circulation and against deficiencies in the cerebral serotinergic system which are associated with mood and behavioural disorders. It was shown that another deleterious effect of chronic hydrocortisone treatment is a significant increase in the number of glutamate receptors in the forebrain of male Wistar rats. The increase in receptor number observed in this study is probably due to an increase in the synthesis of glutamate receptors and is associated with a marked reduction in the affinity of the glutamate receptors for glutamate. possible to demonstrate an receptor number or the For practical reasons, it was not effect of melatonin on either glutamate affinity of glutamate receptors for glutamate in rat forebrain membranes. In view of the neurotoxic effect of glutamate in the eNS, the functional significance of recently described glutamate receptors in the pineal gland was investigated. The results show that 10-4 M glutamate significantly inhibits the isoprenaline-stimulated synthesis of N-acetylserotonin and melatonin in organ culture when the pineal glands were pre-incubated with glutamate for 4 hours prior to stimulation with isoprenalin and when glutamate and isoprenaline were administered together in vitro. GABA, a glutamate metabolite could not mimic the decrease in isoprenalinestimulated melatonin, and it is likely that the observed effects were directly attributed to glutamate. Incubation of the pineal gland with 10-4 M glutamate in organ culture did not affect HIOMT activity in pineal homogenates, but significantly elevated both basal and isoprenaline-stimulated NAT activity. It was concluded that glutamate only inhibits melatonin synthesis in intact pineal glands and not in pineal homogenates. The present study has provided further support for an interaction between the pineal and the adrenal glands. There is an ever increasing likelihood that melatonin is an anti-stressogenic hormone and that the pineal gland may have a protective role to play in the pathology of stress-related diseases.

The main goals in electroanalytical sensing are towards improved sensitivity and selectivity, or specificity, of an analyte. There are several approaches to achieving these goals with the main approach being modification of an electrode surface with synthetic or natural catalysts (enzymes), polymers and also utilisation of nanostructured materials. At present, there is a strong movement towards hybrid sensing which couple different properties of two or more surface modification approaches. In this thesis, a range of these surface modifications were explored for analysis and detection of two main analytes: the amino acid, tryptophan (Trp); and, the neurotransmitter, dopamine (DA). Specifically, this thesis aimed to utilise these methods to enhance the sensitivity and selectivity for Trp over an interferent, the indoleamine, melatonin (Mel); and, DA over the vitamin, ascorbic acid (AA). For Trp detection, immobilisation of an enzyme, Tryptophanase (Trpase) resulted in poor selectivity for the analyte. However, enhanced sensitivity and selectivity was achieved through pH manipulation of the electrolyte medium at a Nafion®-modified electrode surface for both Trp and Mel. At pH 3.0, the Mel and Trp anodic peak potentials were sufficiently resolved allowing for an LOD of 1.60 and 1.62 nM,respectively, and permitting the accurate analysis of Trp in a dietary supplement containing Mel. Multi-walled carbon nanotubes (MWCNTs) suspended in Nafion® exhibited further increases in the signal responses of these analytes at pH 3.0 and 7.4 with minimal change in the resolution of the anodic peaks. A lower sensitivity was, therefore, observed at the Nafion® and MWCNT modified electrode compared to the Nafion®-modified electrode at pH 3.0 with LODs of 0.59 and 0.80 nM exhibited for Trp and Mel, respectively. Enhanced selectivity for Trp in the presence of Mel can be achieved with MWCNTs in the presence of metallotetrasulphonated phthalocyanines (MTSPcs) particularly at pH 3.0, owing to cation exchange effects. However, the lack of sensitivity towards Trp, and even Mel, at this CoTSPc and MWCNT modified electrode remains a drawback. For DA, detection at the MWCNT and Nafion® surface resulted in improved sensitivity over that of both the bare electrode (613.0 nM) and the Nafion® modified electrode (1045.1 nM) with a calculated LOD of 133.9 nM at this layer. Furthermore, improvements in the selectivity of DA were achieved at the Nafion® and MWCNT modified electrode as exclusion of AA (150 μM) was achieved. At the MWCNT and CoTSPc surface, AA was excluded up to 130 μM with sensitivity for DA extending as low as 14.3 nM, far greater than observed for Trp and Mel. These concentrations are well within physiological concentration ranges and represent the most significant solution yet in terms of AA exclusion and enhanced sensitivity for DA. An examination of the surface layering by impedance spectroscopy and atomic force microscopy indicates that the success of the hybrid sensor utilising CoTSPc and MWCNTs lay in improved dispersion of MWCNTs and improved electron transfer kinetics, facilitated by the net charge of the materials present. This thesis, thus, showed the utility of a judicious selection of synthetic and biological catalysts, polymers and carbon nanomaterials towards a hybrid approach to the electrochemical sensing of Trp, Mel, DA and AA with focus on sensitivity and selectivity of these analytes.

Sleep health and sufficient sleep are particularly important during adolescence when important physical, cognitive, emotional, and social changes occur. Given the potential role of PAX6 in pineal development and circadian regulation, adolescents with PAX6 haploinsufficiency may be more likely to experience sleep-related problems compared to adolescents without these deletions or mutations. Haploinsufficiency of PAX6 can result from WAGR syndrome, a contiguous gene deletion syndrome in which multiple genes are involved, or point mutations and microdeletions affecting only PAX6, which result in isolated aniridia. The purpose of this dissertation is to examine pineal volume, melatonin concentrations, and sleep disturbance in individuals with PAX6 haploinsufficiency, as well describe validity of self-report measures of sleep problems in adolescents. Results are presented in three papers. Although PAX6 haploinsufficiency is rare and minimal research has focused on the role of PAX6 in circadian regulation, irregular patterns of sleep-wake rhythm have been studied in children and adolescents with neurodevelopmental disorders (e.g. autism spectrum disorders), another population with possible abnormalities in melatonin physiology. The first paper presents an integrative review to synthesize the literature regarding the sleep-related measures currently being used to assess sleep disturbance in adolescents with a neurodevelopmental disorder. The second paper reports significantly reduced pineal volume, reduced melatonin secretion, and greater parent-report of sleep disturbance in individuals with PAX6 haploinsufficiency versus controls. Paper 3 further characterizes the sleep-related-phenotype associated with an abnormality in the PAX6 gene using self-report questionnaires and actigraphy in adolescents with PAX6 haploinsufficiency, as well as performs preliminary validation studies on age-appropriate self-report tools to measure sleep in adolescents. Results demonstrate similar self-reported daytime sleepiness, sleep disturbance, and sleep-related impairment in adolescents with PAX6 haploinsufficiency compared to the healthy comparison group; however, actigraphy data documented increased time from lights off to sleep in the PAX6 haploinsufficiency group. Self-reported sleep questionnaire scores and objective actigraphy variables (e.g. total sleep time) were significantly correlated in the healthy comparison group only; however, a lack of correlation among sleep-related measures in adolescents with PAX6 haploinsufficiency suggests potential limitations in using self-reported sleep measures in this population. This study used a combination of physiological and patient-reported health measures, and although WAGR syndrome and isolated aniridia due to PAX6 insufficiency are rare disorders, describing the sleep-related phenotypes in this population contributes to knowledge of assessment and treatment of sleep disorders in general, facilitating research in additional adolescent populations.

Charles University in Prague Pharmaceutical Faculty in Hradec Králové Department of Pharmacology and Toxicology Student: Anastasiya Shkut Supervisors: Mgr. Jana Mandíková, Virginia Motilva Ph.D. Title of diploma thesis: The role of melatonin in SIRT1 and p-AMPK regulation in HT-29 cell line. Sirituin 1 (SIRT1) is NAD+ dependent deacetylase present in wide range of organisms including mammals. It was found to extend life span in yeasts during calorie restriction (CR) conditions. SIRT1 deacetylates many regulator proteins and thus control metabolic status of cell as well as AMP-activated kinase (AMPK), which is also energy regulator enzyme depending on NAD+ levels in cell. Both of them play roles in cancer and could influence autophagy, although the exact mechanism remains unclear. We focused our study on hormone melatonin, which has anti-inflammatory and anti-cancer effects, to study its influence on human colon cancer cell line HT-29. If it has impact on SIRT1 and AMPK and what is hierarchic relationship between SIRT1 and AMPK. Also we observed its possible influence on autophagy. We used Western blotting (WB) technique and from our results it seems that melatonin has significant effect on SIRT1, which might activate AMPK as well as autophagy. Nevertheless some of results did not have sufficient...

Melatonin is an indolamine hormone presumably synthesized by retinal photoreceptors, and may act as a paracrine signal of darkness within the retina. Previous studies have suggested that melatonin, acting through specific receptors, may be involved in cyclic retinal functions such as photoreceptor outer segment disc shedding and phagocytosis, and modulation of neurotransmitter release in the inner retina. The goal of this study was to determine if melatonin receptor mRNA is expressed in the neural retina and retinal pigment epithelium (RPE) of Xenopus laevis. Sheets of RPE, devoid of contaminating cells, were obtained from Xenopus eyes, and epithelial cultures were subsequently established on microporous membrane filters in a defined medium. Total RNA was isolated from whole brain, neural retina, fresh RPE sheets, and cultured RPE cells. RNA expression of the three known Xenopus melatonin receptor subtypes (MEL1A, 1B, and 1C) was determined by reverse- transcription/polymerase chain reaction (RT/PCR) amplification, followed by Southern hybridization with RNA probes. PCR-amplified cDNA encoding melatonin receptor subtypes 1B and 1C, but not 1A, were detected in reverse-transcribed RNA obtained from brain, neural retina and RPE. RPE cells grown in culture for two weeks also demonstrated 1B and 1C receptor RNA expression. This study suggests that RNA encoding the 1B and 1C melatonin receptor subtypes is expressed in the neural retina and RPE of Xenopus retina, and the expression persists in RPE cells when grown in culture. The expression of melatonin receptor RNA in the RPE may reflect a regulatory role for melatonin in some diurnal events that occur in this tissue, such as phagocytosis of photoreceptor outer segment membranes, and intracellular migration of pigment granules.

We investigated the transfer of three antioxidants - melatonin, S-adenosyl methionine (SAM) and various forms of vitamin E - across the term, normal human placenta. The transport technique involved the single, isolated placental cotyledon system in vitro. Melatonin crossed the placenta rapidly, equally to the freely diffusible marker, antipyrine. There was no biotransformation of the agent. SAM was transferred slowly, similarly to passively transported L-glucose as a marker. There was a breakdown of SAM to at least one other derivative; the process appeared to be nonenzymatic. Vitamin E was transferred slowly, at a rate only 10% of L-glucose. The natural RRR (nonracemic) form of vitamin E was transported best. Free vitamin, rather than the acetate seems to be transferred best, a finding that will require further study.

Biosensors monitor physiological activities for diagnosis and treatment of disease. Molecularly imprinted polymers (MIPs) are a viable synthetic approach for molecular recognition in biosensing. For biosensing purposes, the most important properties in MIP optimization are sensitivity and selectivity towards a desired analyte. This study aims to optimize MIP sensitivity and selectivity by varying the amount and type of cross-linker used in the synthesis of cortisol and melatonin. The four cross-linkers tested were trimethylpropane trimethacrylate (TRIM), ethyleneglycodimethacrylate (EGDMA), divinylbenzene (DVB), and pentaerythritol triacrylate (PETRA). Based on literature, the following ratios were used for the template molecule to functional monomer to cross-linker in MIP synthesis: for EGDMA cross-linked polymers, 1:6:30; for TRIM and PETRA cross-linked polymers, 1:8:8, 1:6:3, and 1:8:35; for DVB cross-linked polymers, 1:6:30, 1:4:16, and 4:1:60. The polymers were ground and washed, then suspended in a polyvinyl matrix which was spin-coated onto an organic electrochemical transducer (OECT). The device performance was evaluated using electrochemical impedance spectroscopy. For each device, the impedance was measured in electrolyte solutions containing target molecules in concentrations ranging from 1 pM to 100 uM. The impedance was plotted against the analyte concentration to give the sensing slope, which is a measurement for the binding affinity of the polymer. For a device to be considered sensitive, its sensing slope should be greater than its non-imprinted counterpart by a factor above the error margin (+/- 1.79). Of the devices tested, CM1835T (highly cross-linked with TRIM) showed sensitivity towards cortisol, but lacks selectivity towards cortisol over its structural analog, estradiol. Of the melatonin selective polymers, MM163T (low cross-linking with TRIM), MM1630D, and MM4160D (both highly cross-linked with DVB) all showed promising results in sensitivity to melatonin. Overall, the results indicate that high degrees of cross-linking in MIPs improve sensitivity for large, rigid, non-aromatic molecules such as cortisol; however there is no correlation between selectivity and the degree of cross-linking. Meanwhile, divinylbenzene as a cross-linker improves sensitivity and selectivity towards aromatic analytes such as melatonin and estradiol. This study could be improved upon by further characterization of imprinted and non-imprinted polymers, investigation of molecular dynamics, and optimization of devices.

Studies on rat islet preparations have shown melatonin inhibits MAO activity and thereby reduces glucose mediated insulin release. The objective of this work is to investigate the effects of melatonin on insulin release, and on glucose mediated insulin release in the intact and in the pinealectomized rat.

The objectives of this study were to determine: the effects of maternal melatonin (MEL) supplementation during late gestation on the histological and molecular regulation in the Longissimus dorsi (LM) muscle of fetal bovine offspring, composition and gene expression of fetal perirenal (PR) adipose tissue, and LM gene expression in postnatal offspring at birth and d 195 of age. Maternal supplementation of MEL during late gestation resulted in no difference in calf fetal body weight or birth weight. However, at d 195 of age, calves from MEL treated dams had an average body weight increase of 20 kg. Fetal LM weight and length tended to be increased in calves from MEL treated dams. Fetal gene expression of calves from MEL treated dams resulted in: increased LM adenosine monophosphate-activated protein kinase-α (AMPK) and decreased PR adiponectin (ADIPOQ), CCAAT enhancer binding protein alpha (CEBPA), proliferator activated receptor gamma (PPARg), and stearoyl-CoA desaturase (SCD). The improved metabolic status of LM coupled with the decrease in adipogenic gene expression, could result in calves from MEL treated dams having improved lean muscle accretion and reduced overall adiposity during postnatal development.