Development of an ex vivo organ culture model using human gastro-intestinal tissue and Campylobacter jejuni.

Campylobacter jejuni is an important food-borne pathogen. However, relatively little is understood regarding its pathogenesis, and research is hampered by the lack of a suitable model. Recently, a number of groups have developed assays to study the pathogenic mechanisms of C. jejuni using cell culture models. Here, we report the development of an ex vivo organ culture model, allowing for the maintenance of intestinal mucosal tissue, to permit more complex host-bacterium interactions to be studied. Ex vivo organ culture highlights the propensity for C. jejuni to adhere to mucosal tissue via the flagellum, either as discrete colonies or as multicellular units.

Mechanism of transport of saquinavir-loaded nanostructured lipid carriers across the intestinal barrier

[EN] The aims of this work were (i) to evaluate the potential of nanostructured lipid carriers (NLCs) as a tool to 24 enhance the oral bioavailability of poorly soluble compounds using saquinavir (SQV), a BCS class IV drug 25 and P-gp substrate as a model drug, and (ii) to study NLC transport mechanisms across the intestinal barrier. 26 Three different NLC formulations were evaluated. SQV transport across Caco-2 monolayers was enhanced up 27 to 3.5-fold by NLCs compared to SQV suspension. M cells did not enhance the transport of NLCs loaded with 28 SQV. The size and amount of surfactant in the NLCs influenced SQV's permeability, the transcytosis pathway 29 and the efflux of SQV by P-gp. An NLC of size 247 nm and 1.5% (w/v) surfactant content circumvented P-gp 30 efflux and used both caveolae- and clathrin-mediated transcytosis, in contrast to the other NLC formulations, 31 which used only caveolae-mediated transcytosis. By modifying critical physicochemical parameters of the 32 NLC formulation, we were thus able to overcome the P-gp drug efflux and alter the transcytosis mechanism 33 of the nanoparticles. These findings support the use of NLCs approaches for oral delivery of poorly 34 water-soluble P-gp substrates.

1-42 beta-Amyloid peptide requires PDK1/nPKC/Rac 1 pathway to induce neuronal death

1-42 beta-Amyloid (A beta(1-42)) peptide is a key molecule involved in the development of Alzheimer's disease. Some of its effects are manifested at the neuronal morphological level. These morphological changes involve loss of neurites due to cytoskeleton alterations. However, the mechanism of A beta(1-42) peptide activation of the neurodegenerative program is still poorly understood. Here, A beta(1-42) peptide-induced transduction of cellular death signals through the phosphatidylinositol 3-kinase (PI3K)/phosphoinositol- dependent kinase (PDK)/novel protein kinase C (nPKC)/Rac 1 axis is described. Furthermore, pharmacological inhibition of PDK1 and nPKC activities blocks Rac 1 activation and neuronal cell death. Our results provide insights into an unsuspected connection between PDK1, nPKCs and Rac 1 in the same signal-transduction pathway and points out nPKCs and Rac 1 as potential therapeutic targets to block the toxic effects of A beta(1-42) peptide in neurons.

Intestinal absorption by Sarotherodon galilaeus (syn Tilapia galilaea ) of Lake Kainji, Nigeria

Carbohydrates, protein, lipid and crude fibre were found to reduce in amount from the anterior to posterior regions along the gut of Sarotherodon galilaeus collected from Lake Kainji. Different regions of the gut exhibited different absorptive powers and all the compounds were differently absorbed in amount. Different sizes of fish showed different absorptive capacity

Cytokine control of neuronal phenotype

Diffusible proteins regulate neural development at a variety of stages. Using a novel neuronal culture assay, I have identified several cytokines that regulate the expression of neurotransmitters and neuropeptides in sympathetic neurons. These cytokines fall into two families. The first group is termed the neuropoietic cytokines, while including CDF/LIF, CNTF, OSM and GPA, induces expression of the same set of neuropeptide mRNAs in cultured sympathetic neurons. These four factors not only exhibit similar biological activities; they also share a predicted secondary structure and bind to a signal-transducing receptor subunit in common with IL-6 and IL-11. The latter two cytokines display a weaker activity in this assay. In addition, I find that several members of the TGF-β superfamily, activin A, BMP-2, and BMP-6, have a selective overlap with the neuropoietic family in the spectrum of neuropeptides that these cytokines induce in sympathetic neurons. Different patterns of neuropeptides induced by the TGF-β family members, however, demonstrate that the activities of these cytokines are distinct from those of the neuropoietic family. Another 30 cytokines are without detectable effect in this neuronal assay.

Activin A induces a set of neurotransmitters and neuropeptides that is somewhat similar to the phenotype of sympathetic neurons innervating sweat glands in rat footpads. In situ hybridization and RNase protection were carried out to test whether activins were involved in the phenotypic transition when sympathetic neurons contact sweat glands. I find that activin mRNA is present in both cholinergic and noradrenergic targets. Moreover, homogenates of footpads do not contain activin-like activity in the neuronal assay in vitro. Taken together, these data do not support activins as the best candidates for the sweat gland factor.

Several novel factors that regulate neuropeptide expression exist in heart cell conditioned medium. I attempted to purify these factors in collaboration with Dr. Jane Talvenheimo. Our results suggest that these factors are sensitive to the storage conditions used. Several modifications of purification strategy are discussed.

State-dependent modulation of neuronal circuits in C. elegans sleep

C. elegans is a compact system of 302 neurons with identifiable and mapped connections that makes it ideal for systems analysis. This work is a demonstration of what I have been able to learn about the nature of state-specific modulation and reversibility during a state called lethargus, a sleep-like state in the worm. I begin with description about the nervous system of the worm, the nature of sleep in the worm, the questions about behavior and its apparent circuit properties, the tools available and used to manipulate the nervous system, and what I have been able to learn from these studies. I end with clues that the physiology helps to teach us about the dynamics of state specific modulation, what makes sleep so different from other states, and how we can use these measurements to understand which modulators, neurotransmitters, and channels can be used to create different dynamics in a simple model system.

Nonlinear dynamic transfer functions for certain retinal neuronal systems

The applicability of the white-noise method to the identification of a nonlinear system is investigated. Subsequently, the method is applied to certain vertebrate retinal neuronal systems and nonlinear, dynamic transfer functions are derived which describe quantitatively the information transformations starting with the light-pattern stimulus and culminating in the ganglion response which constitutes the visually-derived input to the brain. The retina of the catfish, Ictalurus punctatus, is used for the experiments.

The Wiener formulation of the white-noise theory is shown to be impractical and difficult to apply to a physical system. A different formulation based on crosscorrelation techniques is shown to be applicable to a wide range of physical systems provided certain considerations are taken into account. These considerations include the time-invariancy of the system, an optimum choice of the white-noise input bandwidth, nonlinearities that allow a representation in terms of a small number of characterizing kernels, the memory of the system and the temporal length of the characterizing experiment. Error analysis of the kernel estimates is made taking into account various sources of error such as noise at the input and output, bandwidth of white-noise input and the truncation of the gaussian by the apparatus.

Nonlinear transfer functions are obtained, as sets of kernels, for several neuronal systems: Light → Receptors, Light → Horizontal, Horizontal → Ganglion, Light → Ganglion and Light → ERG. The derived models can predict, with reasonable accuracy, the system response to any input. Comparison of model and physical system performance showed close agreement for a great number of tests, the most stringent of which is comparison of their responses to a white-noise input. Other tests include step and sine responses and power spectra.

Many functional traits are revealed by these models. Some are: (a) the receptor and horizontal cell systems are nearly linear (small signal) with certain "small" nonlinearities, and become faster (latency-wise and frequency-response-wise) at higher intensity levels, (b) all ganglion systems are nonlinear (half-wave rectification), (c) the receptive field center to ganglion system is slower (latency-wise and frequency-response-wise) than the periphery to ganglion system, (d) the lateral (eccentric) ganglion systems are just as fast (latency and frequency response) as the concentric ones, (e) (bipolar response) = (input from receptors) - (input from horizontal cell), (f) receptive field center and periphery exert an antagonistic influence on the ganglion response, (g) implications about the origin of ERG, and many others.

An analytical solution is obtained for the spatial distribution of potential in the S-space, which fits very well experimental data. Different synaptic mechanisms of excitation for the external and internal horizontal cells are implied.

Neuronal mechanism of state control in Drosophila melanogaster

The changes in internal states, such as fear, hunger and sleep affect behavioral responses in animals. In most of the cases, these state-dependent influences are “pleiotropic”: one state affects multiple sensory modalities and behaviors; “scalable”: the strengths and choices of such modulations differ depending on the imminence of demands; and “persistent”: once the state is switched on the effects last even after the internal demands are off. These prominent features of state-control enable animals to adjust their behavioral responses depending on their internal demands. Here, we studied the neuronal mechanisms of state-controls by investigating energy-deprived state (hunger state) and social-deprived state of fruit flies, Drosophila melanogaster, as prototypic models. To approach these questions, we developed two novel methods: a genetically based method to map sites of neuromodulation in the brain and optogenetic tools in Drosophila.

These methods, and genetic perturbations, reveal that the effect of hunger to alter behavioral sensitivity to gustatory cues is mediate by two distinct neuromodulatory pathways. The neuropeptide F (NPF) – dopamine (DA) pathway increases sugar sensitivity under mild starvation, while the adipokinetic hormone (AKH)- short neuropeptide F (sNPF) pathway decreases bitter sensitivity under severe starvation. These two pathways are recruited under different levels of energy demands without any cross interaction. Effects of both of the pathways are mediated by modulation of the gustatory sensory neurons, which reinforce the concept that sensory neurons constitute an important locus for state-dependent control of behaviors. Our data suggests that multiple independent neuromodulatory pathways are underlying pleiotropic and scalable effects of the hunger state.

In addition, using optogenetic tool, we show that the neural control of male courtship song can be separated into probabilistic/biasing, and deterministic/command-like components. The former, but not the latter, neurons are subject to functional modulation by social experience, supporting the idea that they constitute a locus of state-dependent influence. Interestingly, moreover, brief activation of the former, but not the latter, neurons trigger persistent behavioral response for more than 10 min. Altogether, these findings and new tools described in this dissertation offer new entry points for future researchers to understand the neuronal mechanism of state control.

Effects of TI-299423 on Neuronal Nicotinic Acetylcholine Receptors

Nicotinic acetylcholine receptors (nAChRs) are pentameric, ligand-gated, cation channels found throughout the central and peripheral nervous system, whose endogenous ligand is acetylcholine, but which can also be acted upon by nicotine. The subunit compositions of nAChR determine their physiological and pharmacological properties, with different subunits expressed in different combinations or areas throughout the brain. The behavioral and physiological effects of nicotine are elicited by its agonistic and desensitizing actions selectively on neuronal nAChRs. The midbrain is of particular interest due to its population of nAChRs expressed on dopaminergic neurons, which are important for reward and reinforcement, and possibly contribute to nicotine dependence. The α6-subunit is found on dopaminergic neurons but very few other regions of the brain, making it an interesting drug target. We assayed a novel nicotinic agonist, called TI-299423 or TC299, for its possible selectivity for α6-containing nAChRs. Our goal was to isolate the role of α6-containing nAChRs in nicotine reward and reinforcement, and provide insight into the search for more effective smoking cessation compounds. This was done using a variety of in vitro and behavioral assays, aimed dually at understanding TI-299423’s exact mechanism of action and its downstream effects. Additionally, we looked at the effects of another compound, menthol, on nicotine reward. Understanding how reward is generated in the cholinergic system and how that is modulated by other compounds contributes to a better understand of our complex neural circuitry and provides insight for the future development of therapeutics.

Binding Studies of Neuronal Nicotinic Acetylcholine Receptors Expressing Unnatural Amino Acids

Nicotinic acetylcholine receptors are pentameric ligand-gated ion channels mediating fast synaptic transmission throughout the peripheral and central nervous systems. They have been implicated in various processes related to cognitive functions, learning and memory, arousal, reward, motor control and analgesia. Therefore, these receptors present alluring potential therapeutic targets for the treatment of pain, epilepsy, Alzheimer’s disease, Parkinson’s disease, Tourette’s syndrome, schizophrenia, anxiety, depression and nicotine addiction. The work detailed in this thesis focuses on binding studies of neuronal nicotinic receptors and aims to further our knowledge of subtype specific functional and structural information.

Chapter 1 is an introductory chapter describing the structure and function of nicotinic acetylcholine receptors as well as the methodologies used for the dissertation work described herein. There are several different subtypes of nicotinic acetylcholine receptors known to date and the subtle variations in their structure and function present a challenging area of study. The work presented in this thesis deals specifically with the α4β2 subtype of nicotinic acetylcholine receptor. This subtype assembles into 2 closely related stoichiometries, termed throughout this thesis as A3B2 and A2B3 after their respective subunit composition. Chapter 2 describes binding studies of select nicotinic agonists on A3B2 and A2B3 receptors determined by whole-cell recording. Three key binding interactions, a cation-π and two hydrogen bonds, were probed for four nicotinic agonists, acetylcholine, nicotine, smoking cessation drug varenicline (Chantix®) and the related natural product cytisine.

Results from the binding studies presented in Chapter 2 show that the major difference in binding of these four agonists to A3B2 and A2B3 receptors lies in one of the two hydrogen bond interactions where the agonist acts as the hydrogen bond acceptor and the backbone NH of a conserved leucine residue in the receptor acts as the hydrogen bond donor. Chapter 3 focuses on studying the effect of modulating the hydrogen bond acceptor ability of nicotine and epibatidine on A3B2 receptor function determined by whole-cell recording. Finally, Chapter 4 describes single-channel recording studies of varenicline binding to A2B3 and A3B2 receptors.

Avaliação do estado nutricional de pacientes com doença inflamatória intestinal

A doença Inflamatória Intestinal (DII) é uma desordem caracterizada pela inflamação crônica do trato gastrointestinal. Os dois principais tipos de DII são a Retocolite Ulcerativa (RCU) e a Doença de Crohn (DC) e ambas cursam com importantes alterações no estado nutricional (EN). O objetivo deste estudo foi identificar as diferenças na composição corporal entre pacientes com DC, RCU e indivíduos saudáveis, além de comparar o estado nutricional dos três grupos de pacientes, ajustando para fatores que podem interferir no EN, como o uso atual de corticosteróides, a atividade física, a atividade de doença, a idade e o sexo. Foi realizado um estudo transversal que incluiu 101 pacientes com DII (50 com DC e 51 com RCU) e 35 indivíduos saudáveis, selecionados no Ambulatório do Hospital Universitário Pedro Ernesto (HUPE) da Universidade do Estado do Rio de Janeiro (UERJ). Foram colhidas informações sócio-demográficas e pessoais, tais como: prática de atividade física, tabagismo, doenças pregressas e procedimentos cirúrgicos prévios. Outras informações necessárias à pesquisa foram coletadas em prontuário médico. A avaliação antropométrica foi realizada por meio das seguintes medidas: peso corporal; altura; circunferências do braço, da cintura (CC) e do quadril; dobras cutâneas do tríceps, subescápula, supra-ilíaca e da coxa; e circunferência muscular do braço (CMB). A análise da composição corporal foi realizada por meio da bioimpedância elétrica (BIA), utilizando-se o aparelho Biodynamics modelo 450. As variáveis laboratoriais analisadas foram: glicose, hemograma completo, perfil lipídico, proteínas totais, albumina, globulina, velocidade de hemossedimentação e proteína C reativa. O peso, o índice de massa corporal, a CC e o percentual de gordura corporal calculado a partir da aferição das dobras cutâneas, foram menores nos pacientes com DC, quando comparados aos indivíduos saudáveis e/ou aos pacientes com RCU. A CMB foi menor nos pacientes com DC e RCU quando comparados aos indivíduos saudáveis, porém sem apresentar diferenças entre os dois grupos de pacientes. Por BIA, verificou-se que os pacientes com DC apresentaram valores de massa magra, massa celular corpórea, massa extracelular, água corporal total e água extracelular menores quando comparados aos indivíduos saudáveis. Os níveis séricos de colesterol total, proteínas totais e albumina, e a contagem total de hemácias foram menores nos indivíduos com DC quando comparados aos indivíduos do grupo controle e/ou aos indivíduos do grupo da RCU. Os pacientes com RCU exibem composição corporal semelhante à da população saudável. Em contraposição, os pacientes com DC apresentam EN amplamente comprometido com depleção de gordura corporal e massa magra em relação aos demais indivíduos

Condição oral em pacientes com doença inflamatória intestinal

O objetivo deste estudo foi avaliar a prevalência de periodontite e a presença de lesões cariosas, restauradas e elementos perdidos por cárie em pacientes com Doença de Crohn (DC) e Retocolite Ulcerativa (RCUI), comparado-os a pacientes saudáveis sistemicamente. Como objetivo secundário, avaliamos a condição clínica da mucosa oral nos três grupos. Foram examinados 99 pacientes com DC (39.0 DP 12.9 anos), 80 com RCUI (43.3 13.2 anos) e 74 no grupo C (40.3 12.9 anos). A condição periodontal foi avaliada através do índice de placa visível, do sangramento gengival à sondagem, da profundidade de bolsa à sondagem (PBS) e do nível de inserção à sondagem (NIS). Indivíduos que apresentavam pelo menos quatro sítios com NIS ≥3 forma considerados como portadores de periodontite. As condições dentárias foram avaliadas pelo índice de dentes com lesões cariosas, restaurados e perdidos por cárie (CPOD). A condição clínica da mucosa oral foi investigada através da presença de lesões no tecido mole. A porcentagem de placa foi significativamente menor no grupo DC (44.0 30.5) que no C (54.1 26.4), p= 0.017. O sangramento gengival a sondagem era significativamente menor nos pacientes com DC (22.5 18.0) comparado ao grupo C (29.2 22.1), p= 0.038. A quantidade total de sítios com PBS ≥ 4mm foi significativamente menor no grupo DC (5.4 6.6), comparado ao grupo C (12.9 17.7), p= 0.02. A porcentagem de pacientes portadores de periodontite foi significativamente maior nos grupos RCUI (92.6%, p= 0.004) e DC (91.9%, p=0.019), comparado ao grupo C (79.7%). O índice de CPOD foi significativamente maior nos grupos RCUI (16.4 6.6; p< 0.0001) e no DC (15.1 7.3; p= 0.016) quando comparados ao C (12.5 6.8). Foram observadas significativamente mais lesões bucais nos grupos DC (17.2%; p= 0.0041) e RCUI (28.7%; p < 0.0001) quando comparadas ao grupo C (6.7%). Assim, conclui-se que os pacientes com Doença de Crohn e Retocolite Ulcerativa apresentam maior prevalência de periodontite, e maior índice de CPOD quando comparados aos indivíduos do grupo controle. A perda de inserção foi significativamente maior no grupo da Retocolite Ulcerativa quando comparado a Doença de Crohn. Além disso, os pacientes com comprometimento intestinal apresentam significativamente mais lesões bucais que os pacientes do grupo controle.

Composição corporal em pacientes com doença inflamatória intestinal: antropometria versus bioimpedância

A Doença Inflamatória Intestinal (DII) é uma desordem caracterizada pela inflamação difusa do trato gastrointestinal. Os dois principais tipos de DII são a Retocolite Ulcerativa (RCU) e a Doença de Crohn (DC) e ambas cursam com alterações no estado nutricional (EN). O objetivo deste estudo foi comparar a composição corporal, obtida por meio de diferentes métodos, em pacientes com DC e RCU em atendimento ambulatorial, avaliando possíveis diferenças nos grupos de doentes entre si e quando comparados a indivíduos saudáveis. Foi realizado um estudo transversal incluindo 101 pacientes com DII, sendo 50 com DC (GDC) e 51 com RCU (GRCU), além de 35 indivíduos saudáveis (GCON), selecionados no Ambulatório do Hospital Universitário Pedro Ernesto (HUPE) da Universidade do Estado do Rio de Janeiro (UERJ). Informações sócio-demográficas e pessoais, como prática de exercício físico, tabagismo, doenças pregressas e procedimentos cirúrgicos prévios foram coletadas. A avaliação antropométrica consistiu de: peso; altura; circunferências do braço, da cintura e do quadril; circunferência muscular do braço (CMB) e pregas cutâneas do tríceps, bíceps, peitoral, axilar, subescapular, suprailíaca, supraespinhal, abdominal, perna e coxa. O percentual de gordura corporal (% GC) foi estimado a partir de equações que utilizam o somatório de pregas cutâneas e por meio de bioimpedância elétrica (BIA). Para estimar o percentual de gordura subcutânea foi utilizado o somatório de dez dobras. As variáveis laboratoriais analisadas foram: hemograma completo, proteínas totais, albumina, globulina, velocidade de hemossedimentação e proteína C reativa. As análises estatísticas foram realizadas utilizando-se o software STATA versão 10.0. A classificação do EN, por meio do índice de massa corporal (IMC), evidenciou baixa prevalência de desnutrição nos três grupos avaliados. Ao analisar diretamente as medidas antropométricas de peso e IMC, observou-se que os pacientes com DC apresentaram valores significativamente menores do que os indivíduos do grupo controle. A avaliação da CMB mostrou que os pacientes do GDC e GRCU apresentaram depleção de massa magra em comparação aos indivíduos do GCON, porém sem apresentar diferenças entre os dois grupos de pacientes com DII. Em relação ao %GC obtido por BIA não foram verificadas diferenças entre os três grupos de estudo. Ao se verificar o %GC com a utilização das fórmulas de Peterson, Durnin & Womersley e Jackson & Pollock (que utiliza o somatório de três dobras) observou-se que os pacientes com DC apresentaram tecido adiposo significativamente depletado em relação aos indivíduos do GCON e do GRCU. Ao compararmos os %GC obtidos por diferentes métodos de estimativa, observou-se que as equações de Jackson & Pollock (que utilizam o somatório de três e sete dobras) apresentaram resultados significativamente menores quando comparados aos das equações de Peterson e Durnin & Womersley, nos dois grupos de pacientes. Os níveis séricos de proteínas totais e albumina, e a contagem total de hemácias foram menores nos indivíduos com DC quando comparados aos indivíduos do grupo controle e/ou aos indivíduos do grupo com RCU. Os pacientes com DC apresentaram comprometimento importante do EN em comparação aos pacientes com RCU e, notadamente, em relação aos indivíduos saudáveis.