The abuse of and addiction to drugs of abuse, such as tobacco, alcohol, opioids, and illicit drugs, are growing global problems that affect the welfare of individuals and societies worldwide. The National Institute of Drug Abuse estimates the annual cost of substance abuse to be over $740 billion in costs related to drug intoxication, withdrawal and relapse. A primary challenge in the treatment of substance abuse is the tendency of users to relapse following acute or extended periods of abstinence; on average over 60% of substance abusers will return to drug use within a year of receiving treatment, many relapsing following stressful life events. Central to the successful treatment of drug addiction is understanding the cellular mechanisms by which relapse episodes occur. Current data suggest that the activation of pituitary adenylate cyclase activating peptide (PACAP) systems in the bed nucleus of the stria terminalis (BNST) is an important event underlying stress-induced reinstatement to drug-seeking in a rodent model of stress-induced relapse. In conjunction with immunohistochemistry and pharmacological treatments, we used this behavioral model of stress-induced relapse to evaluate PACAP and PACAP type-1 receptor (PAC1-R) signaling in stress-induced reinstatement to cocaine seeking. Activation of the PAC1 receptor appears to be critical to stress-induced reinstatement, as the selective PAC1-R agonist, maxadilan, produced reinstatement behaviors in the absence of stress. Moreover, BNST pretreatment with either mitogen activated protein kinase-ERK (MEK) or endocytosis inhibitors to block extracellular signal-related kinase (ERK) signaling attenuated stress-induced reinstatement. Furthermore, BNST phosphorylated ERK (pERK) expression, mediated by PAC1-R activation, is substantially potentiated in cocaine-experienced animals after stressor exposure, in a manner that is dependent on endosomal signaling and MEK activity. These data suggest that the activation of a PAC1 signaling cascade is a key event underlying stress-induced reinstatement. Furthermore, this data may suggest a permanent change in the BNST PACAP system (sensitization) following cocaine exposure.

In Chinese language, the smallest meaningful unit is a word which is composed of a sequence of characters. A Chinese sentence is composed of a sequence of words without any separation between them. In the area of information retrieval or data mining, the segmentation of a sequence of Chinese characters should be done before anyone starts to use these segments of characters. The process is called the Chinese word segmentation. The researches of Chinese word segmentation have been developed for many years. Although some recent researches have achieved very high performance, the recall of those words that are not in the dictionary only achieves sixty or seventy percent. An approach described in this paper makes use of the linear-chain conditional random fields (CRFs) to have a more accurate Chinese word segmentation. The discriminatively trained model that uses two of our proposed feature templates for deciding the boundaries between characters is used in our study. We also propose three other methods, which are the duplicate word repartition, the date representation repartition, and the segment refinement, to enhance the accuracy of the processed segments. In the experiments, we use several different approaches for testing and compare the results with those proposed by Li et al. and Lau and King based on three different Chinese word corpora. The results prove that the improved feature template which makes use of the information of prefix and postfix could increase both the recall and the precision. For example, the F-measure reaches 0.964 in the MSR dataset. By detecting repeat characters, the duplicated characters could also be better repartitioned without using extra resources. In the representation of date, the wrongly segmented date could be better repartitioned by using the proposed method which deals with numbers, date, and measure words. If a word is segmented differently from that of the corresponding standard segmentation corpus, a proper segment could be produced by repartitioning the assembled segment which is composed of the current segment and the adjacent segment. In the area of using the conditional random fields for Chinese word segmentation, we have proposed a feature template for better result and three methods which focus on other specific segmentation problems.

PhD / HIV-1 is the cause of the majority of global HIV infections. Not only being more virulent, and relatively easily transmitted than HIV-2, HIV-1 is also more extensively studied. HIV-1 is known for its highly recombinogenic nature, together with an extreme genetic variety, both attributable to an error-prone reverse transcriptase which gives rise to heterozygous virion. Sequence diversity of HIV-1 has resulted in identification of 9 subtypes of HIV-1 M group, as well as 43 circulating and a number of other unique recombinant forms of HIV-1. The extensive heterogeneity of HIV-1 has become the main consideration in vaccine development, mainly due to the inherent variability of HIV-1 and the frequent generation of new recombinant forms, which subsequently makes the effort to control the HIV-1 pandemic more challenging. The inter-subtype recombination event is a common phenomenon observed in Malaysia whereby there is a co-circulation of multiple HIV-1 subtypes; CRF01_AE and subtype B. Therefore, it becomes crucial to widen the knowledge of currently emerging CRF01_AE/B inter-subtype recombinants, in order to assist the future regional vaccine design and also to prevent wider spread of these strains. Concurrently, with a better understanding on the characteristics of HIV-1 CRF01_AE/B recombinant forms, further diversification of these strains can possibly be thwarted. The objectives of this study included, firstly to study the molecular epidemiology pattern of different HIV-1 strains, as well as to observe their frequency and distribution. Our second aim was to identify possible derivative from CRF33_01B, and also other new CRF01_AE/B inter-subtype recombinant forms in Malaysia. Thirdly, we aimed to identify possible biological advantages of the CRF33_01B isolates over its parental strains; CRF01_AE and subtype B. Currently, the HIV-1 epidemic in Malaysia is in a concentrated phase with evidence of predominance of both CRF01_AE and subtype B found among heterosexuals and injecting drug users, respectively. There is urgent necessity to apply a more detailed and continuous molecular characterization and epidemiological monitoring of these recombinant forms in Malaysia. We obtained plasma samples from 115 HIV-1-infected patients who attended HIV clinic at the University Malaya Medical Centre in Kuala Lumpur, Malaysia. The HIV-1 PR-RT, gp120-env and gp41-env genes were amplified and sequenced from 50 samples, while the remaining 65 samples were successfully studied at either one or two HIV-1 specific genomic regions. Cloning, phylogenetic analyses, together with bootscanning methods were employed to assign subtypes and to identify inter-subtype recombination based on all three genomic regions. From the plasma-derived sequences of 50 patients, 46% were found to harbour CRF01_AE, 10% and 6% had subtype B and B’, and a total of 18% of the patients were infected with CRF33_01B, while the remaining 18% of patients was found to have unique recombinant forms. As for the other 65 patients, majority of them harboured CRF01_AE and subtype B. This study shows that co-circulation of multiple HIV-1 subtypes and their recombinant strains are frequent in the Malaysian population, while capable of spreading to different HIV-1 risk groups. Possible recombination hotspots in CRF01_AE/B recombinants are suggested to be within the HIV-1 PR-RT gene region. Further, this study highlights the need to characterize and monitor the molecular epidemiology of these recombinant forms. The ideal environment for the inter-subtype recombination event to take place is created by the co-circulation and dual infections of both CRF01_AE and subtype B. With more HIV-1 CRF01_AE/B recombinant forms emerging and shaping the nature of HIV epidemic in Malaysia, certainly it will complicate the timely diagnosis of these molecularly altered HIV-1 forms. The recent identification of the novel CRF33_01B suggests the emergence of other new CRF01_AE/B inter-subtype recombinant forms in Malaysia, as preliminarily demonstrated in some HIV-1 patients identified in the first part of this study. The peripheral blood mononuclear cells (PBMCs) of these HIV-1 patients were co-cultured with those of healthy donors, which we then isolated the proviral genomic DNA. The nested long-range PCR was performed to obtain seven overlapping viral genome fragments that made up the whole viral genome. The detailed phylogenetic, as well as bootscan analyses confirmed the mosaic compositions and recombinant structures of the newly emerging CRF01_AE/B recombinant forms derived from CRF01_AE and subtype B. One of them in particular; HIV-1 isolate 06MYKLD46 is structurally similar to CRF33_01B, except for an extra subtype B fragment within the env region. It also has close phylogenetic relationship and similar breakpoints with CRF33_01B, mainly at the PR-RT region. Furthermore, the other three distinct HIV-1 recombinants; isolates 07MYKLD47, 07MYKLD48 and 07MYKLD49 also display near full-length genomes composed of the backbone of CRF01_AE, with insertions of subtype B fragments at different gene regions. These results indicate the high possibility of second generation of minor recombinant forms derived from CRF33_01B, as well as the continuous evolution and rapid dispersal of CRF01_AE/B recombinants in Malaysia. The high prevalence of newly emerging CRF33_01B (CRF01_AE/B inter-subtype recombinant) may cause a possible epidemiologic shift, attributable to its altered virologic characteristics and possible transmission advantages compared to its parental strains. Two major determinants; the viral factor and host factor have influenced the progress of a productive HIV-1 infection upon virus entry into the host cells. We have assessed the two main viral factors; the in vitro viral replication capacity and the viral fitness of the circulating HIV-1 strains in Malaysia. We have determined that CRF33_01B primary isolate (07MYKLVik) replicates better in activated whole PBMCs and CD4+ T-lymphocytes and is ‘fitter’ than one of its parental strain; CRF01_AE (07MYKLNBL) but not subtype B (07MYKLAfik). Subtype B has more advanced ability to produce a progressive infection in all cell types, including MDMs, and has a comparable viral fitness to that of CRF33_01B. We also investigated the role of host factors in a productive HIV-1 infection, by determining the viral effect on the host cell morphological features. We found that CRF33_01B (07MYKLVik) culture displayed more large syncytia (multinucleated giant cells) with multiple nuclei compared to subtype B (07MYKLAfik) culture, while no snycytia was observed in CRF01_AE (07MYKLNBL) culture. Generally, the cells within CRF33_01B and subtype B cultures appeared to be morphologically distinct from CRF01_AE cultures. This may indicate a more productive HIV-1 infection of CRF33_01B and subtype B, similar to our finding from the in vitro viral replicative capacity and viral fitness assays of these HIV-1 strains. We also studied the effect of different HIV-1 strain infections on host differential gene expression profiles, by using the PCR Array, which detects a total of 84 genes known to be involved in the host response to HIV-1 infection. It was observed that the in vitro infection with CRF33_01B isolates resulted in a more damaging effect on host cells and caused more apoptotic death within the infected cultures, compared to the isolates of its parental subtypes. Moreover, subtype B isolates resulted in a poorer cell response upon viral infection, compared to CRF01_AE/B isolate. Concurrently, it also gave less productive spread of viral infection within the infected cultures, in comparison to CRF01_AE/B isolate. We speculate that if the same scenario is reflected in vivo, CRF01_AE/B inter-subtype recombinant including CRF33_01B would have a better survival rate within the host upon their infection, in comparison to their parental strains. This again strengthens our presumption that CRF33_01B has potential ability to disseminate widely in the Malaysian population and gives a progressive change of the current molecular epidemiological trend by gradually replacing the current predominance of CRF01_AE in the country.

PhD / HIV-1 is the cause of the majority of global HIV infections. Not only being more virulent, and relatively easily transmitted than HIV-2, HIV-1 is also more extensively studied. HIV-1 is known for its highly recombinogenic nature, together with an extreme genetic variety, both attributable to an error-prone reverse transcriptase which gives rise to heterozygous virion. Sequence diversity of HIV-1 has resulted in identification of 9 subtypes of HIV-1 M group, as well as 43 circulating and a number of other unique recombinant forms of HIV-1. The extensive heterogeneity of HIV-1 has become the main consideration in vaccine development, mainly due to the inherent variability of HIV-1 and the frequent generation of new recombinant forms, which subsequently makes the effort to control the HIV-1 pandemic more challenging. The inter-subtype recombination event is a common phenomenon observed in Malaysia whereby there is a co-circulation of multiple HIV-1 subtypes; CRF01_AE and subtype B. Therefore, it becomes crucial to widen the knowledge of currently emerging CRF01_AE/B inter-subtype recombinants, in order to assist the future regional vaccine design and also to prevent wider spread of these strains. Concurrently, with a better understanding on the characteristics of HIV-1 CRF01_AE/B recombinant forms, further diversification of these strains can possibly be thwarted. The objectives of this study included, firstly to study the molecular epidemiology pattern of different HIV-1 strains, as well as to observe their frequency and distribution. Our second aim was to identify possible derivative from CRF33_01B, and also other new CRF01_AE/B inter-subtype recombinant forms in Malaysia. Thirdly, we aimed to identify possible biological advantages of the CRF33_01B isolates over its parental strains; CRF01_AE and subtype B. Currently, the HIV-1 epidemic in Malaysia is in a concentrated phase with evidence of predominance of both CRF01_AE and subtype B found among heterosexuals and injecting drug users, respectively. There is urgent necessity to apply a more detailed and continuous molecular characterization and epidemiological monitoring of these recombinant forms in Malaysia. We obtained plasma samples from 115 HIV-1-infected patients who attended HIV clinic at the University Malaya Medical Centre in Kuala Lumpur, Malaysia. The HIV-1 PR-RT, gp120-env and gp41-env genes were amplified and sequenced from 50 samples, while the remaining 65 samples were successfully studied at either one or two HIV-1 specific genomic regions. Cloning, phylogenetic analyses, together with bootscanning methods were employed to assign subtypes and to identify inter-subtype recombination based on all three genomic regions. From the plasma-derived sequences of 50 patients, 46% were found to harbour CRF01_AE, 10% and 6% had subtype B and B’, and a total of 18% of the patients were infected with CRF33_01B, while the remaining 18% of patients was found to have unique recombinant forms. As for the other 65 patients, majority of them harboured CRF01_AE and subtype B. This study shows that co-circulation of multiple HIV-1 subtypes and their recombinant strains are frequent in the Malaysian population, while capable of spreading to different HIV-1 risk groups. Possible recombination hotspots in CRF01_AE/B recombinants are suggested to be within the HIV-1 PR-RT gene region. Further, this study highlights the need to characterize and monitor the molecular epidemiology of these recombinant forms. The ideal environment for the inter-subtype recombination event to take place is created by the co-circulation and dual infections of both CRF01_AE and subtype B. With more HIV-1 CRF01_AE/B recombinant forms emerging and shaping the nature of HIV epidemic in Malaysia, certainly it will complicate the timely diagnosis of these molecularly altered HIV-1 forms. The recent identification of the novel CRF33_01B suggests the emergence of other new CRF01_AE/B inter-subtype recombinant forms in Malaysia, as preliminarily demonstrated in some HIV-1 patients identified in the first part of this study. The peripheral blood mononuclear cells (PBMCs) of these HIV-1 patients were co-cultured with those of healthy donors, which we then isolated the proviral genomic DNA. The nested long-range PCR was performed to obtain seven overlapping viral genome fragments that made up the whole viral genome. The detailed phylogenetic, as well as bootscan analyses confirmed the mosaic compositions and recombinant structures of the newly emerging CRF01_AE/B recombinant forms derived from CRF01_AE and subtype B. One of them in particular; HIV-1 isolate 06MYKLD46 is structurally similar to CRF33_01B, except for an extra subtype B fragment within the env region. It also has close phylogenetic relationship and similar breakpoints with CRF33_01B, mainly at the PR-RT region. Furthermore, the other three distinct HIV-1 recombinants; isolates 07MYKLD47, 07MYKLD48 and 07MYKLD49 also display near full-length genomes composed of the backbone of CRF01_AE, with insertions of subtype B fragments at different gene regions. These results indicate the high possibility of second generation of minor recombinant forms derived from CRF33_01B, as well as the continuous evolution and rapid dispersal of CRF01_AE/B recombinants in Malaysia. The high prevalence of newly emerging CRF33_01B (CRF01_AE/B inter-subtype recombinant) may cause a possible epidemiologic shift, attributable to its altered virologic characteristics and possible transmission advantages compared to its parental strains. Two major determinants; the viral factor and host factor have influenced the progress of a productive HIV-1 infection upon virus entry into the host cells. We have assessed the two main viral factors; the in vitro viral replication capacity and the viral fitness of the circulating HIV-1 strains in Malaysia. We have determined that CRF33_01B primary isolate (07MYKLVik) replicates better in activated whole PBMCs and CD4+ T-lymphocytes and is ‘fitter’ than one of its parental strain; CRF01_AE (07MYKLNBL) but not subtype B (07MYKLAfik). Subtype B has more advanced ability to produce a progressive infection in all cell types, including MDMs, and has a comparable viral fitness to that of CRF33_01B. We also investigated the role of host factors in a productive HIV-1 infection, by determining the viral effect on the host cell morphological features. We found that CRF33_01B (07MYKLVik) culture displayed more large syncytia (multinucleated giant cells) with multiple nuclei compared to subtype B (07MYKLAfik) culture, while no snycytia was observed in CRF01_AE (07MYKLNBL) culture. Generally, the cells within CRF33_01B and subtype B cultures appeared to be morphologically distinct from CRF01_AE cultures. This may indicate a more productive HIV-1 infection of CRF33_01B and subtype B, similar to our finding from the in vitro viral replicative capacity and viral fitness assays of these HIV-1 strains. We also studied the effect of different HIV-1 strain infections on host differential gene expression profiles, by using the PCR Array, which detects a total of 84 genes known to be involved in the host response to HIV-1 infection. It was observed that the in vitro infection with CRF33_01B isolates resulted in a more damaging effect on host cells and caused more apoptotic death within the infected cultures, compared to the isolates of its parental subtypes. Moreover, subtype B isolates resulted in a poorer cell response upon viral infection, compared to CRF01_AE/B isolate. Concurrently, it also gave less productive spread of viral infection within the infected cultures, in comparison to CRF01_AE/B isolate. We speculate that if the same scenario is reflected in vivo, CRF01_AE/B inter-subtype recombinant including CRF33_01B would have a better survival rate within the host upon their infection, in comparison to their parental strains. This again strengthens our presumption that CRF33_01B has potential ability to disseminate widely in the Malaysian population and gives a progressive change of the current molecular epidemiological trend by gradually replacing the current predominance of CRF01_AE in the country.

The aim of this study was to evaluate the influence of special fertilizers on the quality of nursery plants. The experiment was carried out in 2014 and lasted for one growing season. As a model wood was used kind of Weigela florida (Bunge) A. DC. 'Eva Rathke'. An attempt was made up of five different variants of fertilizers for the basic fertilization substrate and fertilization during vegetation. The influence of fertilizers Multicote, Osmocote, Polyon and Multicote in combination with fertilizer Vitality Complex. Control variant was fertilizing fertilizer YaraMila Complex. Best results showed the control variant. Among CRF fertilizers were tested (with some exceptions) in the monitored parameters statistically significant differences.

O CRF é um regulador do eixo HPA, gerando respostas neuroendócrinas, autônomicas, comportamentais ao estresse, assim como a Ucn-I. Os benzodiazepínicos agem por um efeito inibitório sobre o CRF e um efeito ainda discutido sobre a Ucn-I.Para análise das áreas relacionadas à resposta ao estresse pela imunorreatividade da proteína Fos, da expressão do mRNA do CRF e da Ucn-I, e dos efeitos do alprazolam, ratos foram submetidos aos estresses de restrição e eletrochoque, e ao alprazolam. No estresse de restrição as áreas com maior imunorreatividade da proteína Fos foram PVH e AMIme, e no estresse de eletrochoque foram AMIme e PAGdl. Com o alprazolam houve uma baixa imunorreatividade nas áreas PVH, PAGlat e CC no grupo restrição e, PVH, LSV e PAGlat no grupo choque, com alta imunorreatividade nas áreas EW e LSV no grupo restrição e PAGdl no grupo choque. O fármaco gerou uma diminuição da expressão do mRNA do CRF no PVH no grupo restrição e um aumento no grupo choque, e um aumento da expressão do mRNA da Ucn-I no EW no grupo restrição e uma diminuição no grupo choque. / The CRF is a regulator of the HPA axis, with neuroendocrine, autonomic and behavioral responses to the stress, such as the Ucn-I. The benzodiazepines performing an inhibitory effect on the CRF and a possibly effect on the Ucn-I. It was analyzed the stress related areas by Fos protein and the expression of the CRF and Ucn- I mRNA. Rats were submitted to the restraint and electroshock stress, and to the alprazolan. The restraint stress showed greater Fos-ir in the following areas PVH and AMIme and AMIme and PAGdl in the the electroshock stress. The alprazolam had an inhibition effect in the following areas PVH, PAGlat and CC in the restraint group, and PVH, LSV and PAGlat in the electroshock group. The alprazolan had an excitatory effect in the following areas EW and LSV in the restraint group, and PAGdl in the electroshock group. The alprazolan caused reduction of CRF mRNA in the PVH in the restraint group and increase in the electroshock group, and increase of Ucn-I mRNA in the EW in the restraint group, with reduction in the electroshock group.

O núcleo leito da estria terminal (NLET) é uma estrutura límbica envolvida na expressão de respostas relacionadas à ansiedade. O NLET é um dos principais alvos de inervação noradrenérgica no sistema nervoso central e evidências sugerem uma ativação desta neurotransmissão em situações aversivas, em particular aquelas associadas ao medo condicionado. Assim, o presente estudo investigou o envolvimento do sistema noradrenérgico do NLET na modulação de respostas comportamentais e autonômicas induzidas pelo medo condicionado contextual (MCC) em ratos. Animais condicionados apresentaram resposta comportamental (congelamento) e alterações autonômicas, como aumento da pressão arterial média e da frequência cardíaca e queda da temperatura cutânea após serem re-expostos ao contexto previamente pareado com choques nas patas. Esses efeitos foram atenuados pela injeção de L-propranolol e fentolamina, antagonistas não seletivos dos adrenoceptores e , respectivamente, no NLET. Adicionalmente, também foi observado que a administração de WB4101, antagonista seletivo de adrenoceptores 1, e de CGP20712, antagonista seletivo de adrenoceptores 1, no NLET, reduziu a resposta emocional condicionada (REC) contextual, sugerindo que a neurotransmissão noradrenérgica no NLET, através da ativação dos receptores 1 e 1 adrenérgicos estaria envolvida na expressão das respostas induzidas pelo MCC. Além disso, dados da literatura indicam que a ativação dos receptores adrenérgicos 1 no NLET aumenta a liberação de glutamato de forma dependente da ativação de receptores CRF1. Portanto, o presente estudo também investigou uma possível interação entre a neurotransmissão noradrenérgica e os sistemas CRFérgico, glutamatérgico e nitrérgico no NLET sobre a modulação da REC contextual. A administração de reboxetina, um inibidor da recaptação de noradrenalina, aumentou o tempo de congelamento e as respostas autonômicas após reexposição ao contexto aversivo. Esses efeitos foram bloqueados pela administração prévia de antagonista dos adrenoceptores 1 e 1, WB4101 e CGP20712 respectivamente, do antagonista CRF1, CP376395, do antagonista de receptores NMDA, AP7 e do inibidor da óxido nítrico sintase neuronal, NPLA. Similar à reboxetina, a urocortina, um agonista de receptores CRF, aumentou a REC após a re-exposição ao contexto aversivo. Esses efeitos foram bloqueados pelo CP376395 e também pelo AP7 e NPLA, mas não pelo WB4101 e CGP20712. O presente estudo demonstra que o sistema noradrenérgico no NLET está envolvido na REC observada no modelo do MCC de forma dependente da ativação de receptores CRF1 e dos sistemas glutamatérgico e nitrérgico. / The bed nucleus of the stria terminalis (BNST) is a limbic structure that has been associated to the expression of anxiety responses. BNST is one of the main targets of noradrenergic innervation in the central nervous system and evidence suggests an activation of this neurotransmission in aversive situations, particularly fear conditioning. Thus, the present study investigated the involvement of BNST noradrenergic system in modulating of behavioral and autonomic responses induced by contextual fear conditioning (CFC) in rats. The conditioned animals showed significant behavioral response (freezing) and autonomic changes such as increased in mean arterial pressure, heart rate and decreased in the tail temperature after re-exposure to context. These effects were attenuated by intra-BNST injection of L-propranolol and phentolamine, non-selective antagonists of and -adrenoceptors, respectively. Additionally, it was also observed that the administration of WB4101, a selective 1 adrenoceptor antagonist, and CGP20712, selective 1 adrenoceptor antagonist, in the BNST reduced conditioned emotional response (CER), suggesting that the BNST noradrenergic neurotransmission through the activation of adrenergic receptors, especially 1 and 1-adrenergic receptors, is involved in the expression of CER. Moreover, evidence shows that activation of 1 adrenoreceptor increases the release of glutamate by CRF1 receptor activation. Therefore, this study also investigated the involvement of CRF, glutamatergic and nitrergic systems in the noradrenergic neurotransmission modulation of CER induced by CFC. Administration of reboxetine, a norepinephrine reuptake inhibitor, significantly increased freezing behavior and induced autonomic responses after re-exposure to aversive context. These effects were blocked by previous administration of the antagonist of 1 and 1-adrenoceptor, WB4101 and CGP20712 respectively, CRF1 antagonist, CP376395, an NMDA receptor antagonist, AP7, and inhibitor of neuronal nitric oxide synthase, NPLA. Similar to reboxetine, urocortin significantly induced CER. These effects induced by urocortin were blocked by CP376395, NPLA and AP7, but not by CGP20712 and WB4101. The present study demonstrates that BNST noradrenergic system is involved in CER observed in CFC model, possibly interacting with CRF, nitrergic and glutamatergic systems.

Condições músculo-esqueléticas como as desordens da articulação temporomandibular (ATM) são as principais causas de dor não odontogênica na região orofacial e incluem um grupo de condições usualmente acompanhadas de dor na região das ATMs e dos músculos mastigatórios, limitação da abertura bucal e dores de cabeça. Pacientes com desordens da articulação temporomandibular (DTM) também podem apresentar desordens relacionadas ao estresse caracterizadas por alterações somáticas e psicológicas como fadiga, distúrbios do sono, ansiedade e depressão. A causa exata das DTMs ainda não é completamente entendida, mas acredita-se que envolva fatores fisiológicos, comportamentais e ambientais. É possível que alguns sintomas de DTM e dor orofacial sejam apenas manifestações somáticas de estresse emocional. De fato, situações de estresse promovem uma série de mudanças fisiológicas e comportamentais que podem afetar os sistemas hormonais e de neurotransmissores. Estudos demonstraram que o CRF e seus receptores medeiam respostas comportamentais, endócrinas e autonômicas para o estresse, além de estar envolvidos em outras funções, como na modulação nociceptiva. O objetivo deste trabalho foi avaliar a nocicepção e o comportamento emocional em ratos com inflamação persistente nas ATMs, induzida por administração de adjuvante completo de Freund (CFA) em diferentes situações de estresse (agudo - EA, crônico repetido - ECR e crônico variado - ECV), bem como a ação do antidepressivo tricíclico (inibidor da recaptação de catecolaminas), imipramina, nessas respostas. Ainda, investigou-se o possível envolvimento de receptores CRF do núcleo central da amígdala (CEA) na modulação nociceptiva de animais com inflamação persistente das ATMs. Os resultados mostraram que a inflamação persistente das ATMs promoveu alodinia mecânica orofacial e hiperalgesia secundária. O EA per se promoveu analgesia nos animais sem inflamação das ATMs, além de reverter a alodinia mecânica e a hiperalgesia secundária induzidos pelo CFA intra-articular. Ainda, o ECV e o ECR promoveram per se hiperalgesia nos animais sem inflamação, mas não alteraram o efeito da administração intra-articular de CFA. Em adição, a inflamação persistente das ATMs promoveu alterações comportamentais com efeitos ansiogênicos significativos que não foram alterados pela exposição dos animais ao EA ou ECR. Em contrapartida, o ECV demonstrou efeito ansiogênico nos animais sem inflamação, porém não houve alterações nos animais com inflamação persistente nas ATMs. Em adição, a administração de imipramina reverteu a hiperalgesia, exercendo, dessa forma, efeito analgésico, e o efeito ansiogênico causado pelo CFA intra-articular. Com relação ao envolvimento de receptores para CRF no CEA verificamos que sua ativação promoveu analgesia nos animais sem inflamação das ATMs e ainda reverteu a hiperalgesia decorrente da administração intra-articular do CFA. Assim, nossos resultados sugerem que a inflamação persistente nas ATMs induzida por CFA produz efeitos ansiogênicos e aumenta a sensibilidade nociceptiva, bem como situações de estresse agudo e crônico podem modular essas alterações. Ainda, os efeitos da inflamação persistente podem ser revertidas pelo tratamento sistêmico com imipramina, e pela administração de agonista de receptores CRF no CEA. / Musculoskeletal conditions such as disorders of the temporomandibular joint (TMJ) are the main cause of non-odontogenic pain in the orofacial region and include a group of conditions usually accompanied by pain in TMJ region and masticatory muscles, limitation of mouth opening and headache. Patients with temporomandibular joint disorders (TMD) may also show related stress disorders characterized by somatic and psychological changes such as fatigue, sleep disturbances, anxiety and depression. The exact cause of TMD is not yet completely clarified, but is believed to involve physiological, behavioral and environmental factors. It is possible that some symptoms of TMD and orofacial pain are only somatic manifestations of emotional stress. In fact, stressful situations promote a series of physiological and behavioral changes that may affect hormonal and neurotransmitter systems. Studies have shown that CRF and its receptors mediate behavioral, endocrine and autonomic responses to stress, in addition to being involved in other functions such as nociceptive modulation. The aim of this study was to evaluate emotional behavior and nociception in rats with persistent inflammation in TMJ induced by administration of complete Freund\'s adjuvant (CFA) in different stress situations (acute stress - AS, chronic restraint stress - CRS and unpredictable chronic stress - UCS) and the action of the tricyclic antidepressant (catecholamine reuptake inhibitor), imipramine, in these responses. In addition, we investigated the possible involvement of CRF receptors in the central nucleus of the amygdala (CEA) in nociceptive modulation in animals with persistent inflammation of the TMJs. The results have shown that persistent TMJ inflammation produced mechanical orofacial allodynia and widespread secondary hyperalgesia. AS induced analgesia in animals without TMJ inflammation and reversed both mechanical allodynia and secondary hyperalgesia induced by intra-articular CFA injection. In addition, CRS and UCS induced hyperalgesia in animals without inflammation, but did not alter the effect of intra-articular administration of CFA. Also, the persistent inflammation of the TMJ promoted behavioral changes with significant ansiogenic effects that were not altered by exposure of the animals to AS or CRS. In contrast, UCS showed ansiogenic effect in animals without inflammation, but no changes in animals with persistent TMJ inflammation. In addition, administration of imipramine had an ansiogenic and analgesic effect, reversing the hyperalgesia caused by intra-articular CFA administration. Regarding the involvement of CRF receptors in the CEA this study demonstrated that this activation promoted analgesia in animals without inflammation of the TMJ and was able to reverse the hyperalgesia caused by intra-articular CFA administration. Thus, our results suggest that persistent temporomandibular inflammation induced by CFA produces anxiogenic effects and decreases nociceptive sensitivity and situations of acute and chronic stress can modulate these changes. In addition, these changes can be reversed by systemic treatment with imipramine and by administration of CRF receptor agonist in CEA.

O núcleo leito da estria terminal (NLET) é uma estrutura límbica envolvida na expressão de respostas relacionadas à ansiedade. O NLET é um dos principais alvos de inervação noradrenérgica no sistema nervoso central e evidências sugerem uma ativação desta neurotransmissão em situações aversivas, em particular aquelas associadas ao medo condicionado. Assim, o presente estudo investigou o envolvimento do sistema noradrenérgico do NLET na modulação de respostas comportamentais e autonômicas induzidas pelo medo condicionado contextual (MCC) em ratos. Animais condicionados apresentaram resposta comportamental (congelamento) e alterações autonômicas, como aumento da pressão arterial média e da frequência cardíaca e queda da temperatura cutânea após serem re-expostos ao contexto previamente pareado com choques nas patas. Esses efeitos foram atenuados pela injeção de L-propranolol e fentolamina, antagonistas não seletivos dos adrenoceptores e , respectivamente, no NLET. Adicionalmente, também foi observado que a administração de WB4101, antagonista seletivo de adrenoceptores 1, e de CGP20712, antagonista seletivo de adrenoceptores 1, no NLET, reduziu a resposta emocional condicionada (REC) contextual, sugerindo que a neurotransmissão noradrenérgica no NLET, através da ativação dos receptores 1 e 1 adrenérgicos estaria envolvida na expressão das respostas induzidas pelo MCC. Além disso, dados da literatura indicam que a ativação dos receptores adrenérgicos 1 no NLET aumenta a liberação de glutamato de forma dependente da ativação de receptores CRF1. Portanto, o presente estudo também investigou uma possível interação entre a neurotransmissão noradrenérgica e os sistemas CRFérgico, glutamatérgico e nitrérgico no NLET sobre a modulação da REC contextual. A administração de reboxetina, um inibidor da recaptação de noradrenalina, aumentou o tempo de congelamento e as respostas autonômicas após reexposição ao contexto aversivo. Esses efeitos foram bloqueados pela administração prévia de antagonista dos adrenoceptores 1 e 1, WB4101 e CGP20712 respectivamente, do antagonista CRF1, CP376395, do antagonista de receptores NMDA, AP7 e do inibidor da óxido nítrico sintase neuronal, NPLA. Similar à reboxetina, a urocortina, um agonista de receptores CRF, aumentou a REC após a re-exposição ao contexto aversivo. Esses efeitos foram bloqueados pelo CP376395 e também pelo AP7 e NPLA, mas não pelo WB4101 e CGP20712. O presente estudo demonstra que o sistema noradrenérgico no NLET está envolvido na REC observada no modelo do MCC de forma dependente da ativação de receptores CRF1 e dos sistemas glutamatérgico e nitrérgico. / The bed nucleus of the stria terminalis (BNST) is a limbic structure that has been associated to the expression of anxiety responses. BNST is one of the main targets of noradrenergic innervation in the central nervous system and evidence suggests an activation of this neurotransmission in aversive situations, particularly fear conditioning. Thus, the present study investigated the involvement of BNST noradrenergic system in modulating of behavioral and autonomic responses induced by contextual fear conditioning (CFC) in rats. The conditioned animals showed significant behavioral response (freezing) and autonomic changes such as increased in mean arterial pressure, heart rate and decreased in the tail temperature after re-exposure to context. These effects were attenuated by intra-BNST injection of L-propranolol and phentolamine, non-selective antagonists of and -adrenoceptors, respectively. Additionally, it was also observed that the administration of WB4101, a selective 1 adrenoceptor antagonist, and CGP20712, selective 1 adrenoceptor antagonist, in the BNST reduced conditioned emotional response (CER), suggesting that the BNST noradrenergic neurotransmission through the activation of adrenergic receptors, especially 1 and 1-adrenergic receptors, is involved in the expression of CER. Moreover, evidence shows that activation of 1 adrenoreceptor increases the release of glutamate by CRF1 receptor activation. Therefore, this study also investigated the involvement of CRF, glutamatergic and nitrergic systems in the noradrenergic neurotransmission modulation of CER induced by CFC. Administration of reboxetine, a norepinephrine reuptake inhibitor, significantly increased freezing behavior and induced autonomic responses after re-exposure to aversive context. These effects were blocked by previous administration of the antagonist of 1 and 1-adrenoceptor, WB4101 and CGP20712 respectively, CRF1 antagonist, CP376395, an NMDA receptor antagonist, AP7, and inhibitor of neuronal nitric oxide synthase, NPLA. Similar to reboxetine, urocortin significantly induced CER. These effects induced by urocortin were blocked by CP376395, NPLA and AP7, but not by CGP20712 and WB4101. The present study demonstrates that BNST noradrenergic system is involved in CER observed in CFC model, possibly interacting with CRF, nitrergic and glutamatergic systems.

O CRF é um regulador do eixo HPA, gerando respostas neuroendócrinas, autônomicas, comportamentais ao estresse, assim como a Ucn-I. Os benzodiazepínicos agem por um efeito inibitório sobre o CRF e um efeito ainda discutido sobre a Ucn-I.Para análise das áreas relacionadas à resposta ao estresse pela imunorreatividade da proteína Fos, da expressão do mRNA do CRF e da Ucn-I, e dos efeitos do alprazolam, ratos foram submetidos aos estresses de restrição e eletrochoque, e ao alprazolam. No estresse de restrição as áreas com maior imunorreatividade da proteína Fos foram PVH e AMIme, e no estresse de eletrochoque foram AMIme e PAGdl. Com o alprazolam houve uma baixa imunorreatividade nas áreas PVH, PAGlat e CC no grupo restrição e, PVH, LSV e PAGlat no grupo choque, com alta imunorreatividade nas áreas EW e LSV no grupo restrição e PAGdl no grupo choque. O fármaco gerou uma diminuição da expressão do mRNA do CRF no PVH no grupo restrição e um aumento no grupo choque, e um aumento da expressão do mRNA da Ucn-I no EW no grupo restrição e uma diminuição no grupo choque. / The CRF is a regulator of the HPA axis, with neuroendocrine, autonomic and behavioral responses to the stress, such as the Ucn-I. The benzodiazepines performing an inhibitory effect on the CRF and a possibly effect on the Ucn-I. It was analyzed the stress related areas by Fos protein and the expression of the CRF and Ucn- I mRNA. Rats were submitted to the restraint and electroshock stress, and to the alprazolan. The restraint stress showed greater Fos-ir in the following areas PVH and AMIme and AMIme and PAGdl in the the electroshock stress. The alprazolam had an inhibition effect in the following areas PVH, PAGlat and CC in the restraint group, and PVH, LSV and PAGlat in the electroshock group. The alprazolan had an excitatory effect in the following areas EW and LSV in the restraint group, and PAGdl in the electroshock group. The alprazolan caused reduction of CRF mRNA in the PVH in the restraint group and increase in the electroshock group, and increase of Ucn-I mRNA in the EW in the restraint group, with reduction in the electroshock group.