Papel da proteína de reparo XPC na regulação das proteínas de reparo APE1, OGG1 e PARP-1 em células humanas e de camundongos

studies using UV as a source of DNA damage. However, even though unrepaired UV-induced DNA damages are related to mutagenesis, cell death and tumorigenesis, they do not explain phenotypes such as neurodegeneration and internal tumors observed in patients with syndromes like Xeroderma Pigmentosum (XP) and Cockayne Syndrome (CS) that are associated with NER deficiency. Recent evidences point to a role of NER in the repair of 8-oxodG, a typical substrate of Base Excision Repair (BER). Since deficiencies in BER result in genomic instability, neurodegenerative diseases and cancer, it was investigated in this research the impact of XPC deficiency on BER functions in human cells. It was analyzed both the expression and the cellular localization of APE1, OGG1 e PARP-1, the mainly BER enzymes, in different NER-deficient human fibroblasts. The endogenous levels of these enzymes are reduced in XPC deficient cells. Surprisingly, XP-C fibroblasts were more resistant to oxidative agents than the other NER deficient fibroblasts, despite presenting the highest of 8-oxodG. Furthermore, subtle changes in the nuclear and mitochondrial localization of APE1 were detected in XP-C fibroblasts. To confirm the impact of XPC deficiency in the regulation of APE1 and OGG1 expression and activity, we constructed a XPC-complemented cell line. Although the XPC complementation was only partial, we found that XPC-complemented cells presented increased levels of OGG1 than XPC-deficient cells. The extracts from XPC-complemented cells also presented an elevated OGG1 enzimatic activity. However, it was not observed changes in APE1 expression and activity in the XPCcomplemented cells. In addition, we found that full-length APE1 (37 kDa) and OGG1- α are in the mitochondria of XPC-deficient fibroblasts and XPC-complemented fibroblasts before and after induction of oxidative stress. On the other hand, the expression of APE1 and PARP-1 are not altered in brain and liver of XPC knockout mice. However, XPC deficiency changed the APE1 localization in hypoccampus and hypothalamus. We also observed a physical interaction between XPC and APE1 proteins in human cells. In conclusion, the data suggest that XPC protein has a role in the regulation of OGG1 expression and activity in human cells and is involved mainly in the regulation of APE1 localization in mice. Aditionally, the response of NER deficient cells under oxidative stress may not be only associated to the NER deficiency per se, but it may include the new functions of NER enzymes in regulation of expression and cell localization of BER proteins

Estudo comportamental, hormonal e histopatológico de animais com perda de peso progressina (SEP) e da variável peso associada a uma nova classificação ontogenética em Callithrix jacchus

The use of non-human primates in scientific research has contributed significantly to the biomedical area and, in the case of Callithrix jacchus, has provided important evidence on physiological mechanisms that help explain its biology, making the species a valuable experimental model in different pathologies. However, raising non-human primates in captivity for long periods of time is accompanied by behavioral disorders and chronic diseases, as well as progressive weight loss in most of the animals. The Primatology Center of the Universidade Federal do Rio Grande do Norte (UFRN) has housed a colony of C. jacchus for nearly 30 years and during this period these animals have been weighed systematically to detect possible alterations in their clinical conditions. This procedure has generated a volume of data on the weight of animals at different age ranges. These data are of great importance in the study of this variable from different perspectives. Accordingly, this paper presents three studies using weight data collected over 15 years (1985-2000) as a way of verifying the health status and development of the animals. The first study produced the first article, which describes the histopathological findings of animals with probable diagnosis of permanent wasting marmoset syndrome (WMS). All the animals were carriers of trematode parasites (Platynosomum spp) and had obstruction in the hepatobiliary system; it is suggested that this agent is one of the etiological factors of the syndrome. In the second article, the analysis focused on comparing environmental profile and cortisol levels between the animals with normal weight curve evolution and those with WMS. We observed a marked decrease in locomotion, increased use of lower cage extracts and hypocortisolemia. The latter is likely associated to an adaptation of the mechanisms that make up the hypothalamus-hypophysis-adrenal axis, as observed in other mammals under conditions of chronic malnutrition. Finally, in the third study, the animals with weight alterations were excluded from the sample and, using computational tools (K-means and SOM) in a non-supervised way, we suggest found new ontogenetic development classes for C. jacchus. These were redimensioned from five to eight classes: infant I, infant II, infant III, juvenile I, juvenile II, sub-adult, young adult and elderly adult, in order to provide a more suitable classification for more detailed studies that require better control over the animal development

Influência de fatores individuais e sociais sobre as respostas endócrina e comportamental de callithrix jacchus a desafios ambientais físicos e sociais

Regarding the growing number of human beings with physical and mental pathologies associated to different stressor agents, attempts are being made to validate animal models with a close phylogenetic resemblance to man, to study stress response. Callithrix jacchus has been widely used in biomedical research, including on stress, but there is scarce information in the literature about how individual and social factors modulate stressor response in this species. This study uses 4 approaches to investigate the response of male and female adult C. jacchus, under situations of stress, and in the first we show evidence of the importance of this animal as an experimental model in research involving the hypothalamus-pituitary-adrenal axis. And we investigate if sex and baseline cortisol levels modulate the behavioral and hormonal response to separation. In two additional approaches investigate if type of social support (co-specific parent or non-parent) and social rank interfere in behavioral and hormonal when the animal are exposure to a new environment, paired with a co-specific (F2), exposure of the animal to a new environment, isolated (F3) or during reunion (F4). Finally, we also investigated the androgen levels in the males, with a focus on the challenge hypothesis, referring to environmental responsiveness and male-male exposure to relatives and non-relatives of C. jacchus. It was observed that: (1) the baseline cortisol of the animal is predictive of cortisol reactivity at separation; (2) males and females do not show dimorphism in the response of cortisol to stressors, although the females have higher baseline levels of this hormone and exhibit higher frequencies of anxiety-related behaviors; (3) only social support provided by relatives proved to be effective in buffering the cortisol response. In behavioral terms this response was dimorphic, showing that only the male dyads displayed an attenuated response to stress; (4) the males showed differences in cortisol levels as a function of social rank and study phases, whereas in the females no such alterations were observed. The males with indefinite dominance hierarchy (IDH) had reduced cortisol in F2 and F4, while the IDH females showed an increase in F3 and F4; (5) the males of relative and non-relative dyads did not exhibit variations in androgen levels as a function of a new environment. These results, taken together, (a) corroborate the use of C. jacchus as a good animal model for stress-related studies, given that they exhibit similar behavioral and physiological alterations to those of human beings in response to stressor agents; (b) point to the importance of considering individual and social modulating factors during experiments with stressors; (c) provide more reliable comparison parameters in studies where these primates are used as animal models, and (d) show that androgens vary as a function of genetic proximity (relative or non-relative) when the animals are faced with physical and social environmental challenges, thus providing important information for studying the challenge hypothesis in this species

Expressão de fos após pulso de escuro no núcleo pré- geniculado do tálamo do sagui (Callithrix jacchus)

The pregeniculate nucleus (PGN) of the primate s thalamus is an agglomerate neuronal having a cap shaped located dorsomedially to the main relay visual information to the cerebral cortex, the dorsal lateral geniculate nucleus (GLD). Several cytoarchitectonic, neurochemical and retinal projections studies have pointed PGN as a structure homologous to intergeniculate leaflet (IGL) of rodents. The IGL receives retinal terminals and appears to be involved in the integration of photic and non-photic information relaying them, through geniculo-hypothalamic tract (TGH), to the main circadian oscillator in mammals, the suprachiasmatic nucleus (SCN) of the hypothalamus. Thus, the IGL participates in the control of the biological rhythm by modulating the activity of the SCN. Pharmacological and IGL injury studies conclude that it is critical in the processing of non-photic information which is transmitted to the SCN. Other studies have found that especially neurons immunoreactive to neuropeptide Y (NPY) respond to this type of stimulation, determined by its colocation with the FOS protein. Has not been determined if the PGN responds, expressing the FOS protein, to the non-photic stimulus nor the neurochemical nature of these cells. Thus, we apply a dark pulse in the specifics circadian phases and analyze the pattern of expression of FOS protein in PGN of the marmoset (Callithrix jacchus). We found that in all animals analyzed the FOS expression was higher in the experimental than in the control group. There was a higher expression of FOS when the dark pulse was applied during the subjective day between the groups. Still, a subregion of the PGN, known by immunoreactive to NPY, had a greater number of FOS-positive cells in relation to his other just close dorsal region. Our data corroborate the theory that the PGN and IGL are homologous structures that were anatomically modified during the evolutionary process, but kept its main neurochemical and functional characteristics. However, injury and hodological studies are still needed for a more accurate conclusion

Efeitos da senescência no núcleo supraquiasmático do sagui (Callithrix Jacchus): plasticidade morfológica e neuroquímica

The circadian timing system (CTS) is responsible for the generation and synchronization and the suprachiasmatic nucleus (SCN) of the hypothalamus has been described as the major circadian pacemaker in many mammalian species. The internal temporal organization managed by SCN is disturbed with aging bringing many pathological disorders that range from loss of complex cognitive performance to simple physiological functions. Therefore, our aim was perform a comparative study of the morphological aspects and neurochemical composition in the SCN of marmosets (Callithrix jacchus) adults and older using immunohistochemical techniques. We found morphometric and neurochemical changes in th SCN o folder animals in comparison to adults, among these a possible decreased in retinal projection to the SCN of older animals, found through a decline in CTB immunostaining, which can occur due atrophy and/or decreasing of fibers from the retinohypothalamic tract (RHT). The Klüver-Barrera histological technique strongly suggests a decrease in those fibers from RHT. Also, by means of a morphometric study, it is found a atrophy and numerical decline of neurons in SCN of aged animals, investigated by Nissl technique, and immunostaining with NeuN and calbindin. Relative optical density (ROD) analysis were used to evaluate the expression of some neurochemical components in SCN, such as GFAP expression, which was increased in older, result that indirectly reinforces that morphological changes occurs due the aging; the vasoactive intestinal polipeptide (VIP) showed no expression alteration in SCN of older animals; the serotonin (5-HT) was descreased in the dorsomedial portion of the SCN, and neurpeptide Y (NPY) apparently also decrease due to the increase of age. Many of these modifications were seen in other animals, such as rodents, human primates and non-human primates. These data about marmoset comes to add new information of the effect of aging on structures responsibles for the circadian rhytmicity, and that some behavioral changes controlled by th SCN, and founded in aged animals, may be caused by these morphological and neurochemical changes. Although some results have been quantitatively negative, qualitatively all analysis show significant change comparing adult and older animals, perhaps due to a low sampling number. In conclusion, the marmoset presents several morphological and neurochemical changes in the SCN of aged animals compared to adults, which may result in behavioral changes that favor pathology aging related

O trato retino-hipotalâmico no mocó (Kerodon rupestris): Um estudo de traçado anterógrado com a subunidade b da toxina colérica

The light, besides the vision stimuli, controls other process completely independent of image formation, such as the synchronization of the organismic circadian rhythms to the enviromental light/dark cycle. In mammals, this adjust occurs through the retinohypothalamic tract, a direct retinal projection to the suprachiasmatic nucleus, considered to be the major circadian pacemaker. Early studies have identified only the suprachiasmatic nucleus as a retinal target in the hypothalamus. However, using more sensitive neuroanatomic tracers, other retinorecipient hypothalamic regions outside to suprachiasmatic nucleus were pointed in a great number of mammalian species. In this study, the retinohypothalamic tract was shown in the rock cavy (Kerodon rupestris), an endemic rodent of the semiarid region of the Brazilian Northeast, using unilateral intravitreal injections of cholera toxin subunit b as a neuronal tracer. The results reveal that in the rock cavy, besides the suprachiasmatic nucleus, several hypothalamic regions receive direct retinal projection, such as the ventrolateral preoptic nucleus, medial and lateral preoptic areas, the supraoptic nucleus and bordering areas, anterior, lateral and rectrochiasmatic hypothalamic areas, and the subparaventricular zone. The results are discussed by comparing with those of the literature, into a functional context

A Zona Incerta no sagüi (Callithrix jacchus): Análise Citoarquitetônica, Neuroquímica e Projeção Retiniana

The retinal projections in mammals usually reach, classically, three major functional systems: the primary visual system, the accessory optic system, and the circadian timing system. But the retinal projections also reach areas classically considered non-visual, one of which groups the neurons of the zona incerta (ZI), target this study. The primary visual system includes thalamic lateral geniculate complex is formed by the dorsal lateral geniculate nucleus, intergeniculate leaflet and the ventral lateral geniculate nucleus and other Components. The accessory optic system is composed of the small nuclei: nuclei terminal dorsal, lateral, medial and the interstitial nucleus of the superior posterior fasciculus. These nuclei are involved in visuo-motor activities. The circadian timing system is comprised of the suprachiasmatic nucleus of the hypothalamus, that act as master circadian pacemaker, entraining pathways and efferents pathways to the efectors, and the intergeniculate leaflet, that seems to act as a modulator of the pacemaker. The retinal projections too reach classically considered non-visual areas, including the zona incerta. This region is localized in the ventral thalamus and has been implicated in various functional properties including nociceptive and somatosensory processing, motor response, sociosexual behaviour, feeding and drinking, in symptoms of neurodegenerative diseases, arousal and attention. It also displays connection with several areas of central nervous system. The aim of this study was characterize the retinal projection in the zona incerta of Callithrix jacchus (sagüi), a primate of the New World through the anterograde axonal transport of the cholera toxin subunit b and analyze the citoarchicteture using Nissl and NeuN, and neurochemical substances such as serotonin, GABA, VIP, VP, GFAP and binding-calcium proteins. The zona incerta showed a different division of the literature in citoarquitetura, both by means of Nissl as neurochemical by NeuN, with a subdivision ventrolateral and dorsomedial. The neurochemical to the other substances corroborate with this subdivision. The GFAP was almost completely negative for the zona incerta, result non evidenced in previous studies yet. The 16 retinal projection in sagüi, unlike other primates and rodents, reached the caudal portion only. This work helps to make further studies are conducted based on this subdivision and the localization of the neurochemical substances associated with possible behaviors that the zona incerta is involved

Avaliação do cortisol, dehidroepiandrosterona sulfato e dos perfis lipídico e imunológico e suas correlações com a sensibilidade dolorosa, depressão e funcionalidade em mulheres com fibromialgia na pós-menopausa

Fibromyalgia (FM) is a non-inflammatory rheumatic syndrome characterized by widespread musculoskeletal pain with palpable tender points, muscle stiffness, fatigue, and sleep disturbances. Patients with FM have hormonal changes that are directly correlated with symptoms of the syndrome. The neuroendocrine regulation may be impaired, with abnormalities in the hypothalamus-pituitary-adrenal (HPA) axis with various hormones showing changes in their levels. In women in fertile period, various gonadal hormones are associated with symptoms of the syndrome, but studies focusing only a population of women in post-menopausal period who do not use hormone replacement are rare. We developed an analytical cross sectional study to assess the plasma levels of cortisol and dehidroepiandrosterona sulfate (DHEA-S) with quimioluminescence method in a group of 17 women with FM and 19 healthy women in post-menopause who do not use hormone replacement and observe the correlation with the symptoms of pain through algometry, depression and physical functional capacity measured from the Beck Depression Index (BDI) and the Fibromyalgia Impact Questionnaire (FIQ). Three blood samples were collected in the morning (between 8:00 9:30) with an interval of 24 hours for the measurements of hormonal levels and biochemical profile. There were no immunological or lipid changes in patients with FM. Comparing the two groups, there is no difference in levels of cortisol and a tangential effect for DHEA-S (p=0,094) with the lowest levels in the FM. DHEA-S also correlated with pain threshold (r=0,7) and tolerance (r=0,65) in group FM. We found the presence of depressive state and low physical functional capacity in FM. It was also evident that women in post-menopausal period, DHEA-S should influence the symptoms of increased sensitivity to pain, but not the presence of depressive status and low physical functional

Análise imunoistoquímica da distribuição de Serotonina, transportador de serotonina e receptores de serotonina no hipotálamo do sagui (callithrix jacchus)

The hypothalamus is a diencephalic portion located around the third ventricle below the hypothalamic sulcus, limited by the optic chiasm, and by the mammillary bodies, acting as a center that integrates behavioral and homeostatic functions. Serotonin is a neurotransmitter produced in limited sites in the midbrain and brain stem, but is distributed throughout the central nervous system and has many functions, acting through specific receptors that are also distributed throughout the nervous system. Using immunohistochemical techniques, the aim of this study was to delineate the hypothalamic nuclei of the marmoset (Callithrix jacchus) and study the distribution of serotonin transporter and serotonin receptors in the hypothalamus of this species. We used the Nissl method to determine the cytoarchitecture of the hypothalamic nuclei, and immunohistochemistry to reveal the presence of NeuN as a method to determine the contours of the hypothalamic nuclei. As a result, we found serotonin containing fibers and terminals throughout the rostrocaudal extent of the hypothalamus, more concentrated in some nuclei, and even absent in some. Like serotonin, serotonin transporter was observed between pre-optic area and tuberal region of the hypothalamus, in densities and distribution similar to serotonin. The 5-HT1A and 5-HT1B receptors were found with minor differences among itselves regarding the disposition and intensity of staining.

Análise citoarquitetônica dos componentes do sistema de temporização circadiana do sagui (Calithrix jacchus), comparada com a inervação das suas principais aferências

The suprachiasmatic nucleus (SCN) of the anterior hypothalamus, together with the intergeniculate leaflet (IGL) of the thalamus are considered the central components of the circadian timing system (CTS) of mammals. This system is responsible for the generation and regulation of circadian rhythms by establishing a temporal organization of physiological processes and behaviors. The neuronal specific nuclear protein (NeuN) has been widely used as a neuronal marker in several studies. Since glial fibrillary acidic protein (GFAP) is a component of intermediate filaments found in the cytoplasm of astrocytes and is commonly used as a specific marker for these cells. This study aims to identify, in the marmoset, the NeuN immunoreactive neurons and glial cells immunoreactive to GFAP, as well as map the major route of photic synchronization of the STC, retinohypothalamic tract (RHT), and identify the indirect pathway to the SCN and pregeniculate nucleus (PGN) - structure homologous to IGL rodents, using immunohistochemical and cytoarchitectonic techniques. Observed in SCN the presence of neurons immunoreactive to NeuN and terminals immunoreactive subunit b of cholera toxin (CTb), neuropeptide Y (NPY) and serotonin (5- HT). In the PGN noted the presence of the NeuN and NPY immunoreactive neurons and the immunoreactive terminals CTb and 5-HT. Astrocytes are present throughout the extent of the SCN and the PGN this New World primate

Efeitos da senescência no folheto intergeniculado do tálamo de ratos: análises morfológicas e neuroquímicas

The circadian timing system (CTS), in rodents, consists of interconnected neural structures such as the suprachiasmatic nucleus (SCN) of the hypothalamus, Intergeniculate Leaflet (IGL) of the thalamus, synchronous pathways and behavioral effectors. The SCN has been described as the major circadian pacemaker in several species of mammals, while the IGL appears to be involved in integration of photic and non-photic clues relaying them to SCN. The CTS allows an ordered internal temporal organization to the organism, providing the proper execution of physiological and behavioral mechanisms, which brings homeostasis. However, this stability is disrupted with aging process causing numerous pathological disorders, ranging from simple loss of physiological functions to decreases in cognitive performance. Therefore, is fundamental understanding the effects of senescence in this system. In this context, is proposed in this study to check if there are changes in IGL cytoarchitecture, neurochemical and retinal afferent markers with aging and their possible morpho-functional implications. To achieve this goal wistar rats were divided into 3 groups: young (3 months); Middle Age (13 months); Old (23 months). They were submitted to paraformaldhyde (4%) transcardiac perfusion to tissue fixation. Then, they had their brain removed and sectioned in 30 µm slices, which every sixth section were collected. This sections were processed by nissl method and immunostaining for GFAP, GAD, ENK, NPY and CTb in order to analyze the IGL features. It was observed a cell loss in middle age and old animals at Nissl, NPY and CTb stains. In addition, it was shown a increase in GFAP in middle aged animals compared to young and old ones. No differences were found in other neurochemichal stains. These data suggests IGL loss retinal afferents and neurons, in special the NPY-IR ones, likely having a compensatory gliogenesis. This supports the correlations between the CTS functional deficits and an anatomical deterioration of its components with the aging process.

Influência dos odores de gato e de cobra no comportamento defensivo e expressão de fos do sistema hipotalâmico de defesa de camundongo

Studies using neuronal tract-tracer in rat have shown that the anterior hypothalamic nucleus, dorsomedial division of the ventromedial nucleus of the hypothalamus and dorsal premammillary nucleus are highly connected. When the rat is exposed to predator or its odor these nuclei have shown a expression of Fos and their lesion reduces defensive behavior against predator. This set of nuclei was named the Hypothalamic Defense System. However, little is known about the response of this system to the odor of different predators or its role in mice. In this work, we exposed Swiss mice to two different predators odor (cat and snake) to verify the Fos expression in the Hypothalamic Defense System, as well as the defensive behaviors displayed. The analysis showed that the mice exposure to cat odor had an increased expression of Fos protein compared to control, while those exposed to snake odor showed no rise in Fos expression, which was corroborated by the behavioral data. Our results indicate that this distinct circuit in mice seems to act differentially to odorous stimuli of different predators, causing distinct behavioral responses of mice and that the odor of snake seems not to be perceived by Swiss mice as a threatening stimulus.

Papel da proteína de reparo XPC na regulação das proteínas de reparo APE1, OGG1 e PARP-1 em células humanas e de camundongos

studies using UV as a source of DNA damage. However, even though unrepaired UV-induced DNA damages are related to mutagenesis, cell death and tumorigenesis, they do not explain phenotypes such as neurodegeneration and internal tumors observed in patients with syndromes like Xeroderma Pigmentosum (XP) and Cockayne Syndrome (CS) that are associated with NER deficiency. Recent evidences point to a role of NER in the repair of 8-oxodG, a typical substrate of Base Excision Repair (BER). Since deficiencies in BER result in genomic instability, neurodegenerative diseases and cancer, it was investigated in this research the impact of XPC deficiency on BER functions in human cells. It was analyzed both the expression and the cellular localization of APE1, OGG1 e PARP-1, the mainly BER enzymes, in different NER-deficient human fibroblasts. The endogenous levels of these enzymes are reduced in XPC deficient cells. Surprisingly, XP-C fibroblasts were more resistant to oxidative agents than the other NER deficient fibroblasts, despite presenting the highest of 8-oxodG. Furthermore, subtle changes in the nuclear and mitochondrial localization of APE1 were detected in XP-C fibroblasts. To confirm the impact of XPC deficiency in the regulation of APE1 and OGG1 expression and activity, we constructed a XPC-complemented cell line. Although the XPC complementation was only partial, we found that XPC-complemented cells presented increased levels of OGG1 than XPC-deficient cells. The extracts from XPC-complemented cells also presented an elevated OGG1 enzimatic activity. However, it was not observed changes in APE1 expression and activity in the XPCcomplemented cells. In addition, we found that full-length APE1 (37 kDa) and OGG1- α are in the mitochondria of XPC-deficient fibroblasts and XPC-complemented fibroblasts before and after induction of oxidative stress. On the other hand, the expression of APE1 and PARP-1 are not altered in brain and liver of XPC knockout mice. However, XPC deficiency changed the APE1 localization in hypoccampus and hypothalamus. We also observed a physical interaction between XPC and APE1 proteins in human cells. In conclusion, the data suggest that XPC protein has a role in the regulation of OGG1 expression and activity in human cells and is involved mainly in the regulation of APE1 localization in mice. Aditionally, the response of NER deficient cells under oxidative stress may not be only associated to the NER deficiency per se, but it may include the new functions of NER enzymes in regulation of expression and cell localization of BER proteins