The role of learning and environmental variables on the acquisition of ethanol self-administration in outbred rodents

For several decades, the field of alcohol research has been grappling with a seemingly straightforward question: what factors contribute to or mediate the voluntary intake of a licit yet "addictive" substance such as alcohol? Nevertheless, the search for and description of the mechanisms underlying voluntary alcohol consumption has been plagued with difficulties, in part due to the multiplicity of factors believed to influence alcohol consumption. As alcohol use appears to be a multi-determined behavior, it is suggested that the variability underlying it is apt to be attributed to both pharmacological, as well as, non-pharmacological variables. The present dissertation was designed to assess the role of non-pharmacological variables in the acquisition of voluntary ethanol self-administration in unselected rodents. More specifically, we aimed to determine the role played by the learning ability of individual animals in influencing subsequent ethanol preference. Furthermore, we also wanted to evaluate the role of environmental variables in the acquisition of ethanol intake. The results of Experiment 1 and Experiment 2 suggested that individual differences in learning ability, as assessed by performance in two different spatial tasks, might be related to the affinity to ingest ethanol. Specifically, it appears that individual differences in spatial ability and its components, such as spatial working memory are likely to be related to ethanol intake. The results of Experiment 3 demonstrated that lesions to the hippocampus, an area largely associated with spatial learning, disrupted the acquisition of ethanol intake in both a limited and continuous access paradigm. Experiment 4 demonstrated that superimposing an operant procedure for access to ethanol and providing animals with a distinct environment during acquisition, elevated ethanol consumption as compared to a standard voluntary intake home-cage procedure. Experiment 5 showed that providing animals with a distinct environment and transport as part of the procedures used during ethanol acquisition increased ethanol intake. Taken together, the studies reported within this dissertation provide support for the role of non-pharmacological factors such as learning ability and environment, in the development of ethanol self-administration within outbred rodents. It is argued that in order to understand the tremendous variability inherent in ethanol self-administration both pharmacological and non-pharmacological factors must be considere

The effects of a mixed dopamine D2/D3 agonist and antagonist on ethanol intake within a limited and a continuous access paradigm

The effects of the mixed dopamine D2/D3 agonist, quinpirole and antagonist, raclopride on ethanol consumption were examined within both a limited and a continuous access paradigm. The goals of these studies were to determine if decreases in ethanol intake after administration of the dopaminergic agents were the result of the type of paradigm employed, reductions of DA-mediated reinforcement or rather to non-specific drug effects, such as locomotor suppression. Experiment 1 demonstrated that quinpirole (0.1 mg/kg) failed to alter ethanol intake in rats exposed to a 1hr-limited access period. Raclopride-treated (0.5 mg/kg) rats significantly decreased their ethanol intake but were ataxic for the entire 1-hr limited access period. These results suggested that manipulations of the D2/D3 receptors at the dose tested had non-specific effects on the consumption of ethanol. Experiment 2 assessed whether the effects obtained within a limited access paradigm were comparable to a 22-hr continuous access paradigm. Similar, to Experiment 1, quinpirole (0.1 mg/kg) failed to attenuate ethanol consumption within a continuous access paradigm. Contrary to Experiment 1, raclopride (0.5 mg/kg) significantly increased ethanol intake during the treatment and post-treatment periods. Taken together, the findings of Experiment 1 and 2 demonstrated that the D2/D3 agents employed did not produce reductions in ethanol intake specific to ethanol reinforcement. Further, the raclopride induced increase in ethanol intake in Experiment 2 is contradictory to previous reports that systemic injections of DA antagonists produce reductions in ethanol intake. The results suggested that the D2/D3 receptors are not likely to be the primary mediators of ethanol intake

Proteolytic maturation of α 2 δ represents a checkpoint for activation and neuronal trafficking of latent calcium channels

The auxiliary α2δ subunits of voltage-gated calcium channels are extracellular membrane-associated proteins, which are post-translationally cleaved into disulfide-linked polypeptides α2 and δ. We now show, using α2δ constructs containing artificial cleavage sites, that this processing is an essential step permitting voltage-dependent activation of plasma membrane N-type (CaV2.2) calcium channels. Indeed, uncleaved α2δ inhibits native calcium currents in mammalian neurons. By inducing acute cell-surface proteolytic cleavage of α2δ, voltage-dependent activation of channels is promoted, independent from the trafficking role of α2δ. Uncleaved α2δ does not support trafficking of CaV2.2 channel complexes into neuronal processes, and inhibits Ca2+ entry into synaptic boutons, and we can reverse this by controlled intracellular proteolytic cleavage. We propose a model whereby uncleaved α2δ subunits maintain immature calcium channels in an inhibited state. Proteolytic processing of α2δ then permits voltage-dependent activation of the channels, acting as a checkpoint allowing trafficking only of mature calcium channel complexes into neuronal processes

Proteolytic maturation of alpha(2)delta represents a checkpoint for activation and neuronal trafficking of latent calcium channels

The auxiliary a2d subunits of voltage-gated calcium channels are extracellular membrane-associated proteins, which are post-translationally cleaved into disulfide-linked polypeptides a2 and d. We now show, using a2d constructs containing artificial cleavage sites, that this processing is an essential step permitting voltage-dependent activation of plasma membrane N-type (CaV2.2) calcium channels. Indeed, uncleaved a2d inhibits native calcium currents in mammalian neurons. By inducing acute cell-surface proteolytic cleavage of a2d, voltage-dependent activation of channels is promoted, independent from the trafficking role of a2d. Uncleaved a2d does not support trafficking of CaV2.2 channel complexes into neuronal processes, and inhibits Ca2+ entry into synaptic boutons, and we can reverse this by controlled intracellular proteolytic cleavage. We propose a model whereby uncleaved a2d subunits maintain immature calcium channels in an inhibited state. Proteolytic processing of a2d then permits voltage-dependent activation of the channels, acting as a checkpoint allowing trafficking only of mature calcium channel complexes into neuronal processes

Morphological analysis and description of the ovaries of female silky sharks, Carcharhinus falciformis (Müller & Henle, 1839)

This work aims to study the female reproductive tract of silky sharks, Carcharhinus falciformis, captured in the South and Equatorial Atlantic Ocean. Samples were collected between January 2008 and March 2010 through oceanic commercial vessels that targeted tuna and swordfish, with a total of 17 females collected. The methodologies followed for analyzing the ovaries of those females included both macroscopic and histological analysis. Macroscopically, it was possible to determine that the ovaries on these sharks is suspended by mesenteries in the anterior section of the body cavity, heavily irrigated by blood vessels, and contains a wide range of oocytes. Ovaries were found in three distinct maturational stages: Stage I (Immature), Stage II (Maturing) and Stage III (Mature). Immature ovaries were small, with widths ranging from 1.0 to 3.1 cm, and had a gelatinous or granulose internal structure; maturing ovaries were slightly larger, ranging in width between 5.2 and 6.0 cm; mature ovaries ranged in width between 6.5 and 7.8 cm, and had a more rounded shape and the presence of large and well developed oocytes. Under microscopic examination, it was observed that the ovaries were covered with simple epithelial tissue during the early development stages and a simple cubic epithelium in the final stages of maturation. During the initial maturation stages the epigonal organ was not differentiated from the ovary. In mature specimens, the ovary showed a simple cubic epithelium and just below this epithelium there was a layer of dense connective tissue and muscle with the presence of vitellogenic oocytes and fat cells. A thin yolk membrane enclosing the oocytes was also evident. Finally, it was possible to distinguish a zona pellucida, separating the oocytes from the follicle wall and a basal lamina between the granular layers and the teak layer.info:eu-repo/semantics/publishedVersio

RAM: A Conserved Signaling Network That Regulates Ace2p Transcriptional Activity and Polarized Morphogenesis

In Saccharomyces cerevisiae, polarized morphogenesis is critical for bud site selection, bud development, and cell separation. The latter is mediated by Ace2p transcription factor, which controls the daughter cell-specific expression of cell separation genes. Recently, a set of proteins that include Cbk1p kinase, its binding partner Mob2p, Tao3p (Pag1p), and Hym1p were shown to regulate both Ace2p activity and cellular morphogenesis. These proteins seem to form a signaling network, which we designate RAM for regulation of Ace2p activity and cellular morphogenesis. To find additional RAM components, we conducted genetic screens for bilateral mating and cell separation mutants and identified alleles of the PAK-related kinase Kic1p in addition to Cbk1p, Mob2p, Tao3p, and Hym1p. Deletion of each RAM gene resulted in a loss of Ace2p function and caused cell polarity defects that were distinct from formin or polarisome mutants. Two-hybrid and coimmunoprecipitation experiments reveal a complex network of interactions among the RAM proteins, including Cbk1p–Cbk1p, Cbk1p–Kic1p, Kic1p–Tao3p, and Kic1p–Hym1p interactions, in addition to the previously documented Cbk1p–Mob2p and Cbk1p–Tao3p interactions. We also identified a novel leucine-rich repeat-containing protein Sog2p that interacts with Hym1p and Kic1p. Cells lacking Sog2p exhibited the characteristic cell separation and cell morphology defects associated with perturbation in RAM signaling. Each RAM protein localized to cortical sites of growth during both budding and mating pheromone response. Hym1p was Kic1p- and Sog2p-dependent and Sog2p and Kic1p were interdependent for localization, indicating a close functional relationship between these proteins. Only Mob2p and Cbk1p were detectable in the daughter cell nucleus at the end of mitosis. The nuclear localization and kinase activity of the Mob2p–Cbk1p complex were dependent on all other RAM proteins, suggesting that Mob2p–Cbk1p functions late in the RAM network. Our data suggest that the functional architecture of RAM signaling is similar to the S. cerevisiae mitotic exit network and Schizosaccharomyces pombe septation initiation network and is likely conserved among eukaryotes

Vaccine Safety Surveillance Using Routinely Collected Healthcare Data—An Empirical Evaluation of Epidemiological Designs

Background: Routinely collected healthcare data such as administrative claims and electronic health records (EHR) can complement clinical trials and spontaneous reports to detect previously unknown risks of vaccines, but uncertainty remains about the behavior of alternative epidemiologic designs to detect and declare a true risk early. Methods: Using three claims and one EHR database, we evaluate several variants of the case-control, comparative cohort, historical comparator, and self-controlled designs against historical vaccinations using real negative control outcomes (outcomes with no evidence to suggest that they could be caused by the vaccines) and simulated positive control outcomes. Results: Most methods show large type 1 error, often identifying false positive signals. The cohort method appears either positively or negatively biased, depending on the choice of comparator index date. Empirical calibration using effect-size estimates for negative control outcomes can bring type 1 error closer to nominal, often at the cost of increasing type 2 error. After calibration, the self-controlled case series (SCCS) design most rapidly detects small true effect sizes, while the historical comparator performs well for strong effects. Conclusion: When applying any method for vaccine safety surveillance we recommend considering the potential for systematic error, especially due to confounding, which for many designs appears to be substantial. Adjusting for age and sex alone is likely not sufficient to address differences between vaccinated and unvaccinated, and for the cohort method the choice of index date is important for the comparability of the groups. Analysis of negative control outcomes allows both quantification of the systematic error and, if desired, subsequent empirical calibration to restore type 1 error to its nominal value. In order to detect weaker signals, one may have to accept a higher type 1 error

Current Approaches to Vaccine Safety Using Observational Data: A Rationale for the EUMAEUS (Evaluating Use of Methods for Adverse Events Under Surveillance-for Vaccines) Study Design

Post-marketing vaccine safety surveillance aims to detect adverse events following immunization in a population. Whether certain methods of surveillance are more precise and unbiased in generating safety signals is unclear. Here, we synthesized information from existing literature to provide an overview of the strengths, weaknesses, and clinical applications of epidemiologic and analytical methods used in vaccine monitoring, focusing on cohort, case-control and self-controlled designs. These designs are proposed to be evaluated in the EUMAEUS (Evaluating Use of Methods for Adverse Event Under Surveillance-for vaccines) study because of their widespread use and potential utility. Over the past decades, there have been an increasing number of epidemiological study designs used for vaccine safety surveillance. While traditional cohort and case-control study designs remain widely used, newer, novel designs such as the self-controlled case series and self-controlled risk intervals have been developed. Each study design comes with its strengths and limitations, and the most appropriate study design will depend on availability of resources, access to records, number and distribution of cases, and availability of population coverage data. Several assumptions have to be made while using the various study designs, and while the goal is to mitigate any biases, violations of these assumptions are often still present to varying degrees. In our review, we discussed some of the potential biases (i.e., selection bias, misclassification bias and confounding bias), and ways to mitigate them. While the types of epidemiological study designs are well established, a comprehensive comparison of the analytical aspects (including method evaluation and performance metrics) of these study designs are relatively less well studied. We summarized the literature, reporting on two simulation studies, which compared the detection time, empirical power, error rate and risk estimate bias across the above-mentioned study designs. While these simulation studies provided insights on the analytic performance of each of the study designs, its applicability to real-world data remains unclear. To bridge that gap, we provided the rationale of the EUMAEUS study, with a brief description of the study design; and how the use of real-world multi-database networks can provide insights into better methods evaluation and vaccine safety surveillance

Plasmodium vivax cell traversal protein for Ookinetes and Sporozoites (PvCelTOS) gene sequence and potential epitopes are highly conserved among isolates from different regions of Brazilian Amazon

This work was supported by Brazilian National Research Council–CNPq/PAPES, (Conselho Nacional de Desenvolvimento Científico e Tecnológico/Programa de Apoio e Pesquisa Estratégica em Saúde) Fiocruz. JdCLJ is recipient of a FAPERJ APQ1 (E-26/210.653/2015), Jovem Cientista do Nosso Estado (E26/203.255/2016).Fundação Oswaldo Cruz. Oswaldo Cruz Institute. Laboratory of Immunoparasitology. Rio de Janeiro, RJ, Brazil.Fundação Oswaldo Cruz. Oswaldo Cruz Institute. Laboratory of Clinical Immunology. Rio de Janeiro, RJ, Brazil.Fundação Oswaldo Cruz. Oswaldo Cruz Institute. Laboratory of Immunoparasitology. Rio de Janeiro, RJ, Brazil.Fundação Oswaldo Cruz. Computational Modeling Group. Fortaleza, CE, Brazil.University of Campinas. Department of Genetics, Evolution and Bioagents. Laboratory of Tropical Diseases - Prof. Luiz Jacintho da Silva. Campinas, SP, BrazilMinistério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Laboratório de Imunogenética da Malária. Ananindeua, PA, Brasil.Fundação Oswaldo Cruz. Oswaldo Cruz Institute. Laboratory of Malaria Research. Rio de Janeiro, RJ, Brazil.Fundação Oswaldo Cruz. Oswaldo Cruz Institute. Laboratory of Clinical Immunology. Rio de Janeiro, RJ, Brazil.Fundação Oswaldo Cruz. Oswaldo Cruz Institute. Laboratory of Immunoparasitology. Rio de Janeiro, RJ, Brazil.The Plasmodium vivax Cell-traversal protein for ookinetes and sporozoites (PvCelTOS) plays an important role in the traversal of host cells. Although essential to PvCelTOS progress as a vaccine candidate, its genetic diversity remains uncharted. Therefore, we investigated the PvCelTOS genetic polymorphism in 119 field isolates from five different regions of Brazilian Amazon (Manaus, Novo Repartimento, Porto Velho, Plácido de Castro and Oiapoque). Moreover, we also evaluated the potential impact of non-synonymous mutations found in the predicted structure and epitopes of PvCelTOS. The field isolates showed high similarity (99.3% of bp) with the reference Sal-1 strain, presenting only four Single-Nucleotide Polymorphisms (SNP) at positions 24A, 28A, 109A and 352C. The frequency of synonymous C109A (82%) was higher than all others (p<0.0001). However, the non-synonymous G28A and G352C were observed in 9.2% and 11.7% isolates. The great majority of the isolates (79.8%) revealed complete amino acid sequence homology with Sal-1, 10.9% presented complete homology with Brazil I and two undescribed PvCelTOS sequences were observed in 9.2% field isolates. Concerning the prediction analysis, the N-terminal substitution (Gly10Ser) was predicted to be within a B-cell epitope (PvCelTOS Accession Nos. AB194053.1) and exposed at the protein surface, while the Val118Leu substitution was not a predicted epitope. Therefore, our data suggest that although G28A SNP might interfere in potential B-cell epitopes at PvCelTOS N-terminal region the gene sequence is highly conserved among the isolates from different geographic regions, which is an important feature to be taken into account when evaluating its potential as a vaccine candidate