Os linfócitos T sofrem rígida regulação homeostática desde seu desenvolvimento até sua maturação, expansão clonal e morte celular. Compostos endógenos ou exógenos que alterem as funções fisiológicas dos linfócitos T podem modular passos importantes da resposta imune. Triptofano tem um papel importante na manutenção da homeostasia dos linfócitos T e é o precursor da síntese de melatonina. Neste estudo, avaliamos os efeitos de triptofano, melatonina e seus produtos de oxidação sobre os principais processos de ativação e desativação dos linfócitos T. Inicialmente, investigamos os efeitos biológicos de L-quinurenina (KYN, composto formado na via de metabolização do triptofano), melatonina e seus produtos de oxidação N1-acetil-N2-formil-5-metoxiquinuramina (AFMK) e N1-acetil-5- metoxiquinuramina (AMK) sobre a proliferação dos linfócitos T humanos e na produção de interferon-gamma (IFN-γ) e das interleucinas IL-2, IL-10 e IL-12. Observamos que KYN, melatonina, AFMK e AMK inibem a linfoproliferação e a liberação de IL-2 e de IFN-γ . O efeito inibitório destes compostos na síntese de IFN-γ não está associado às variações na produção das duas citocinas mais importantes na regulação de IFN-γ , ou seja, IL-10 e IL-12. Na seqüência, descrevemos a ação da melatonina e seus produtos de oxidação, sobre a morte celular induzida por ativação (AICD) de hibridomas de linfócitos T e os possíveis mecanismos de ação. Os resultados deste estudo mostram que tanto melatonina quanto AFMK e AMK são potentes inibidores da apoptose dos linfócitos T. Estes compostos inibem a ativação do Fator de Transcrição Nuclear de Células T ativadas (NFAT) e suprimem a expressão da molécula Fas Ligante (FasL), que é o evento imprescindível para a indução da AICD. Como conclusões, verificamos que os produtos formados tanto a partir da oxidação de triptofano quanto de melatonina são potenciais reguladores da resposta imune e podem participar dos efeitos imunossupressores, nos quais a depleção de triptofano tem sido sugerida como principal mecanismo de regulação dos linfócitos T. Adicionalmente, nossos achados podem ter utilidade na avaliação de possíveis efeitos indesejáveis durante a utilização terapêutica de melatonina. / T Lymphocytes are exposed to severe homeostatic regulation from development stage up to their maturation, clonal expansion and cell death. Endogenous or exogenous compounds altering the physiological functions of T lymphocytes can modulate important steps of the immune response. Tryptophan plays an important role for maintaining the homeostasis of T lymphocytes and is the precursor of the melatonin synthesis. In this study we evaluated the effects of tryptophan, melatonin and their oxidation products concerning the main activation and deactivation processes of T lymphocytes. Firstly, we analyzed the biological effects of L-kynurenine (KYN, a compound formed at the tryptophan metabolization pathway), melatonin and its oxidation products, N-acetyl-N-formyl-5-methoxykynuramine (AFMK) and N-acetyl-5- methoxykynuramine (AMK), concerning the proliferation of human T lymphocytes and the production of interferon-gamma (IFN-γ) as well as of interleukins IL-2, IL-10 and IL-12. It was observed that KYN, melatonin, AFMK and AMK inhibit the lymphocytes proliferation and the release of IL-2 and IFN-γ. The inhibitory effect of these compounds in the IFN-γ synthesis is not related to the variations on the production of the two most important cytokines for the IFN-γ regulation, that is, IL-10 and IL-12. After that, we reported the melatonin action as well as of its oxidation products, in what concerns activation-induced cell death (AICD) of T lymphocytes hybridomas and probable activation mechanisms. The results of this study have shown that melatonin as well as AFMK and AMK are powerful inhibitors of the T lymphocytes apoptosis. These compounds inhibit the activation of the nuclear transcription factor of activated T cells (NFAT) and suppress the expression of the Fas-Ligand molecule (FasL), which is the essential event to the AICD induction. We concluded that products formed by the oxidation of either tryptophan or melatonin are potential regulators of the immune response and may participate on immunosuppression effects, in which the tryptophan depletion is believed to be the main mechanism for T lymphocytes regulation. Added to that, our results may be helpful for evaluating possible unwanted effects during the therapeutical use of melatonin.

Copper is an extremely toxic metal in biological systems and thus, its availability to the system, must be effectively and efficiently controlled. Copper is vital for life, as it is essential for critical enzymes in biological systems. It is free copper in the biological systems that is toxic, as free copper induces free radical generation, which disrupts lipid membranes, interacts with DNA causing mutations, and eventually leads to cell death. Wilson’s disease is a inherited copper disease, which results in hepatolenticular disease. Copper is unable to be excreted, and thus accumulates, eventually spilling over into the bloodstream from the liver, and “poisons” the patient. The Wilson’s disease patient leads a short life, due to neurological and hepatological problems. There is no cure for Wilson’s disease, only chelation therapy using potent chelators such as penicillamine and EDTA. Zinc, in high doses, can be used to compete with copper absorption. This has proved to be the only successful therapy at present. This study investigates the possible use of melatonin as a copper binder/chelator. Melatonin has been shown to interact with copper in vitro. By binding/chelating to copper, melatonin may inhibit copper-induced free radical generation, and thus prevent copper from interacting with DNA to cause mutations and act as a cytotoxin. In vivo studies on copper (2mg/kg) administered for 2-weeks and 6-weeks were carried out on Wistar rats. The potential of melatonin (12mg/kg) to prevent copper-induced cellular damage was investigated. The results indicate that melatonin does not protect the lipid membranes from copper-induced lipid peroxidation. In vitro investigations using 1mM, 5mM and 10mM copper and 5mM melatonin, show that melatonin prevents copper-induced lipid peroxidation at a copper concentration of 1mM (p<0.001). The 5mM and 10mM copper induces less lipid peroxidation, compared to the 1mM copper. It has been reported that metal ions, antioxidants and chelating agents can influence peroxide decomposition during the assay. Melatonin (5mM) administration does not significantly prevent copper-induced lipid peroxidation at 5mM and 10mM copper. It is possible that due to melatonin’s relatively low concentration, it is unable to inhibit lipid peroxidation induced by the copper. The chemical nature of the interaction between melatonin and copper was also investigated, using NMR, IR and electrochemistry techniques. The NMR and IR techniques show that melatonin coordinates with Cu²⁺ and not Cu¹⁺, at the carbonyl group of melatonin. The electrochemistry experiments using cyclic voltammetry and adsorptive stripping voltammetry, show that melatonin forms a strong bond with Cu¹⁺. Cu²⁺ prefers binding to oxygen, and that is clearly seen in the NMR and IR. Cu¹⁺ prefers binding to nitrogen and then oxygen, and this is seen in the electrochemistry, as Cu¹⁺ is forced to bind through one of the nitrogens on the melatonin. Previously, it has been shown that melatonin binds/chelates with Cu²⁺. Histochemical investigations show that copper administration for 2-weeks and 6-weeks, causes extensive mitochondrial damage in liver and kidney’s proximal convoluted tubule epithelium cells. Melatonin (12mg/kg) co-administration with copper for 2-weeks and 6-weeks did not significantly protect the mitochondria from copper-induced damage. Copper-specific stains (rhodanine, silver sulphide and rubeanic acid) were used to stain liver, brain and kidney tissue samples. Rhodanine and silver sulphide were equally sensitive in staining copper in the 2-week samples, but not at all in the 6-week samples. This could not be explained. Rubeanic acid was ineffective in all samples tested. Thus, it appears that specific copper stains cannot be used in making a definitive diagnosis in cases of copper overload, and that specific copper stains do not always correlate with a high concentration of copper present in tissues. Pineal organ culture was used to determine the effect of copper administration on pineal indole synthesis. Exogenous (³H) tryptophan was administered to the pineal organ cultures, and the level of (³H) pineal indoles synthesised, were measured. Pineals from 2-week and 6-week copper/melatonin treated animals exhibited paradoxical 5- methoxytryptophol (ML) levels, as compared to the 2-week and 6-week copper treated animals. The 2-week copper/melatonin administered animals, showed a decrease in the ML level (p<0.01), and the copper/melatonin administered for 6-weeks, showed an increase in the ML levels (p<0.01). This indicates that melatonin interacts with the HIOMT enzyme. Pineals from 6-week copper/melatonin treated animals, as compared to the 6-week copper treated animals, showed an increase in N-acetylserotonin levels. This indicates that melatonin prevents the inhibition of the NAT enzyme. The final experiment was to determine in vitro, the effect of Cu²⁺ and Cu¹⁺ administration, on mitochondrial electron transport chain. Rat liver homogenate was incubated with and solutions of Cu²⁺ (10mM) and Cu¹⁺ (10mM) and melatonin (10mM). Cu²⁺ administration caused an inhibition of the electron transport at t=0 and t=60, whereas Cu¹⁺ administration at t=0 caused an inhibition of electron transport, but at t=60, Cu¹⁺ administration stimulated electron transport. Melatonin administered with Cu²⁺, resulted in an inhibition of the electron transport chain at t=0 and t=60. The findings of this study indicate that melatonin might have a potentially beneficial effect in copper overloading, by binding/chelating copper.

No description available.

Parkinson’s disease, one of the most common neurodegenerative disorders associated with ageing, is characterised by abnormal and profound loss of nigrostriatal dopaminergic neurons. The cause of Parkinson’s disease is unknown, but epidemiological studies suggest an association with pesticides and other environmental toxins, and biochemical studies implicate oxidative damage and mitochondrial impairment, particularly at the level of complex I enzyme. Recently, rotenone, a commonly used organic pesticide and a classical inhibitor of mitochondrial complex I has been reported to reproduce the specific features of Parkinson’s disease in rodents. The mitochondrial respiratory chain is one of the most important sites of reactive oxygen species production under physiological conditions. Toxic free radicals have been implicated in a variety of neurodegenerative diseases as well as ageing itself. Melatonin, a secretory product of the pineal gland is a multifaceted free radical scavenger and natural antioxidant. In the present study, the neuroprotective effects of melatonin against the environmental neurotoxin, rotenone was investigated. Initial studies showed that inhibition of mitochondrial complex I enzyme by rotenone induced superoxide radical generation. Melatonin, administered to the rat in vivo and in vitro was able to offer neuroprotection by curtailing the production of superoxide radicals induced by rotenone. Mitochondria, being the major target of rotenone, the effects of melatonin were investigated at the mitochondrial level. Melatonin was able to increase the electron transport chain activity thus preventing the respiratory inhibition by rotenone. The pineal hormone also counteracted the action of rotenone on complex I enzyme. These results suggest melatonin’s ability to potentially limit the free radical generation and thereby modulate the mitochondrial functions. The detection and measurement of lipid peroxidation is the evidence most frequently cited to support the involvement of free radical reactions in toxicology and in human disease. Melatonin also offered significant protection in vivo and in vitro against rotenone induced lipid peroxidation. Since iron plays a major role in oxidative damage and in the progression of Parkinson’s disease, the effect of melatonin on both rotenone and iron induced lipid peroxidation was investigated, the results of which show that melatonin affords protection and this was suggested to be due to its interaction with the rotenone-iron complex that might have formed. Electrochemical studies were further used to characterise the interactions between melatonin, rotenone and iron (III). Melatonin was shown to bind with iron and thus reducing their toxicity. Histological studies were undertaken to assess the effects of melatonin on rotenone induced toxicity on the dopaminergic neurons in the rat brain. Rotenone treated brains showed extensive neuronal damage whereas with melatonin less damage was observed. Rotenone induces apoptosis via reactive oxygen species production and apoptotic cell death has been identified in PD brains. Furthermore, the apoptotic cell death was detected and quantified by the TUNEL staining. Rotenone treated sections showed signs of apoptosis whereas with melatonin, less apoptotic damage was observed. The findings of this study indicate that the neurohormone, melatonin may protect against rotenone-induced neurodegeneration. Since melatonin production falls substantially during ageing, the loss of this antioxidant is theorized to be instrumental in the degenerative processes associated with advanced age. Considering how devastating diseases such as Parkinson’s disease, are to a patient and the patient’s families, the discovery of protective agents are a matter of urgency. Further investigations using the pesticide model will help to determine the involvement of environmental exposure in the pathogenesis of human diseases as well as to test therapeutic strategies for the treatment of such diseases.

Melatonin is a strong antioxidant that has previously been observed to increase uteroplacental blood flow and increase postnatal calf growth when supplemented during gestation. The objective of the current study was to examine the effects of melatonin implants on uterine blood flow and subsequent offspring growth. Commercial beef heifers and cows were artificially inseminated and assigned to one of two treatment groups supplemented with (MEL) or without (CON) melatonin from days 180 to 240 of gestation. Total uterine artery blood flow was increased in MEL- versus CON-treated cattle. Fetal and birth weight were not different between treatments. However, at castration, body weight was increased in calves from MEL-treated dams compared with CON-treated dams. Further research on placental vascularization and the mechanism in which melatonin impacts angiogenic factors is necessary to understand the relationship between melatonin and compensatory growth that occurs in postnatal offspring.

Prior studies have observed a link between night shift work and increased risk of breast cancer. Melatonin, a hormone related to circadian rhythm, has been proposed to lower breast cancer risk by inhibiting cell proliferation. The disruption of peak melatonin that occurs during night shift work could explain the increase in risk observed. Several studies have assessed whether higher melatonin levels are associated with decreased breast cancer risk, but results have been conflicting. We examined the relationship between urinary melatonin levels and breast cancer risk in a nested case-control study conducted within the Women’s Health Initiative Observational Study. First morning urine samples collected at baseline were assayed for melatonin levels in 258 women diagnosed with invasive breast cancer and 515 matched controls from three enrollment sites. Using conditional logistic regression to adjust for matching factors and established risk factors, results indicate no association between urinary melatonin levels and breast cancer risk. The mean creatinine adjusted melatonin levels for cases and controls were 16.30 ng/mg and 16.05 ng/mg, respectively. Compared to the lowest quartile of creatinine adjusted melatonin, the odds of breast cancer did not vary by quartile of creatinine adjusted melatonin, adjusted for known breast cancer risk factors: second quartile 0.84 (95% CI 0.52-1.38), third quartile 1.05 (95% CI 0.65-1.72) and fourth quartile 1.09 (95% CI 0.66-1.81). This study does not suggest that melatonin is protective against breast cancer and suggests that reasons other than melatonin suppression may explain the increased risk of breast cancer seen in night shift workers.

In this work we address two problems in computational chemistry relevant to biomolecular modeling. In the first project, we consider the conformer space of melatonin as a a representative example of “real-life” flexible biomolecules. Geometries for all 52 unique conformers are optimized using spin-component scaled MP2, and then relative energies are obtained at the CCSD (T) level near the complete basis set limit. These are then used to validate a variety of DFT methods with and without empirical dispersion corrections, as well as some lower-level ab initio methods. Basis set convergence is found to be relatively slow due to internal C-H…O and C-H…N contacts. Absent dispersion corrections, many DFT functionals will transpose the two lowest conformers. Dispersion corrections resolve the problem for most functionals. Double hybrids yield particularly good performance, as does MP2.5. In the second project, we propose a simple DFT-based diagnostic for nondynamical correlation effects. Aλ= (1-TAE [ΧλC]/TAE[XC])/λ where TAE is the total atomization energy, XC the “pure” DFT exchange-correlation functional, and ΧλC the corresponding hybrid with 100λ% HF-type exchange. The diagnostic is a good predictor for sensitivity of energetics to the level of theory, unlike most of the wavefunction-based diagnostics. For GGA functionals, Aλ values approaching unity indicate severe non-dynamical correlation. The diagnostic is only weakly sensitive to the basis set (beyond polarized double zeta) and can be applied to problems beyond practical reach of wavefunction ab-initio methods required for other diagnostics.

Early life is critical for the development of an organism. External factors alter placental efficiency which can lead to consequential effects. The objective of the current study was to (1) examine placental characteristics and molecular factors affected by nutrient restriction (2) evaluate the mitigating properties of melatonin in a nutrient restricted pregnancy in regard to circadian, myogenic and adipogenic factors in fetal muscle and (3) evaluate the effects of prenatal and postnatal melatonin supplementation on offspring performance. In study 1, cows were fed a control (CON) or a 60% restricted (RES) diet from day 140 to 240 of gestation. Animals were slaughtered for placentome collection. Nutrient restriction increased vessel perimeter, downregulated genes related to blood vessel development, and upregulated ribosomal and translation factor expression. In lieu of downregulated vessel development, a compensatory effect geared towards nutrient-transport apparent. The 2nd study utilized spring-calving and fall-calving heifers in two trials with a 2 x 2 x 2 factorial design. Treatments were adequately-fed (ADQ) or 60% restricted (RES) dams along with melatonin (MEL) or no melatonin (CON) from day 160 to 240 of gestation. Cesarean sections were performed either in the morning (AM) or afternoon (PM). Circadian, myogenic and adipose-related factors in fetal loin muscle (LM) were analyzed. Fetal LM from the spring-calving (fall) group experienced a downregulation of circadian genes, myogenic genes and tendency for downregulation in lipolysis genes. Fetal LM collected from the fall-calving (summer) group had interactions in myogenic expression. Results demonstrate photoperiod and seasonal effects on nutrient restriction and melatonin supplementation in regard to tissue prioritization. In study 3, melatonin was supplemented to calves during the prenatal and postnatal period in a 2 x 2 factorial design. Supplementation did not alter offspring performance. A lack of differences may be attributed to similar endogenous melatonin levels in dams. Despite no differences in calf performance, tendencies for decreased milk yield and fat were observed in MEL versus CON dams. This demonstrates that melatonin may influence feed efficiency in calves that receive less nutrients during early life. The programming effects of melatonin after birth appear to be low.

This thesis describes the application of the radioisotope 11C to the synthesis of two analogues of the neurohormone melatonin. The labelled compounds were intended to be used as tracers for the medical imaging technology Positron Emission Tomography (PET). [ 11C]Carbon dioxide, produced in a small on-site cyclotron by the nuclear reaction 14N(p,a)11C, was converted into [11C]CH3COC1 by reaction first with CH3MgBr, followed by reaction with phthaloyl dichloride. The labelled acid chloride was distilled into a solution of an amine, yielding the corresponding 11C-labelled amide, which was purified by a simple solid-phase extraction method. An apparatus was designed and built that allowed the remote synthesis with several hundred millicuries of [11C]C02• The apparatus was mounted in a hot cell and operated remotely with a Macintosh Powerbook programmed in Hypercard. The apparatus and software are generic for these acylation reactions. The individual reaction steps were optimized in terms of reaction time, solvents and equipment; radiosyntheses of a number of purified labelled acetamides were completed in 35 minutes. The radiochemical yields ranged from 15 to 20% with specific activities in the 500 mCi/J..tmol range at the end of the synthesis. 2-Iodo-[11C-acetyl]melatonin (11) and 7-methoxynaphthylenyl-1-ethyl-N-[11Cacetyl] acetamide (15) were synthesized for the first time for PET studies. It was shown that both compounds readily cross the blood-brain-barrier and penetrate into all brain tissues. Specific binding to the melatonin receptors in the suprachiasmatic nuclei of the hypothalamus could not be visualized with either one of the 11C-labelled ligands because of low specific activity and high nonspecific binding. / Thesis / Master of Science (MSc)

No / Synthesis of a melatonin agonist for treatment of sleep disorders produced a pair of enantiomers, of which one is biologically active. Two polymorphs were discovered using the inactive enantiomer, conserving the active enantiomer for toxicological testing. Later studies with the active enantiomer yielded only the metastable form, despite more than 1000 attempts to isolate the stable form. The difficulty is surprising, since the stable form is favored by 0.7 kcal mol–1, which is toward the extreme for stability differences between organic polymorphs. Study of individual enantiomers allowed the phase behavior of polymorphs of greatly different energy to be examined without interconversion. A number of unusual features are noted. After the stable polymorph of the inactive enantiomer was nucleated, the metastable form became very difficult to isolate. The metastable form converts into a less soluble monohydrate structure in water, whereas the stable polymorph does not due to its reduced activity. Both chiral polymorphs are denser than the racemic crystalline form at low temperature, the stable form being at the extreme for chiral-racemic pairs. Free energy-temperature relations predict “spontaneous resolution” of the racemic crystalline form into a conglomerate mixture of stable polymorph at low temperature. The unusual characteristics of the system are explained by hydrogen bonding and conformational flexibility of the molecule. Ab initio calculations aid in understanding the relative contributions of these interactions to the lattice energies and the role that conformational energy differences play in the polymorphic stability. This system highlights the importance of the creation of the very first nuclei of a crystalline form. The reluctance of the stable form to nucleate is attributed to a large energy difference between polymorphic forms. The large interfacial tension for primary nucleation reduces the probability of forming clusters of size sufficient for favorable growth in the absence of heterogeneous nucleation. This study highlights how nucleation of a new form can revise the readily “accessible” region of a compound’s crystal form landscape.