O sistema de expressão baseado nos promotores PL ou PR do fago lambda que usualmente é regulado pelo repressor termo lábil (cIts) é amplamente utilizado para produzir proteínas recombinantes em células procarióticas. No entanto, o aumento da temperatura requerido neste sistema para promover a inativação do repressor apresenta algumas limitações, como o aumento da expressão de proteínas de HSP (Heat Shock Proteins), por exemplo, proteases, que dependendo da natureza da proteína expressa podem ser prejudiciais ou não. Uma outra limitação é a ativação da resposta SOS, resultando na parada da replicação do DNA celular ou dependendo da cepa pode ocorrer lise celular. Nesse trabalho nós descrevemos o uso do promotor λPL para expressão constitutiva, isto é, sem a regulação do repressor. Nós otimizamos diferentes condições de cultura para aumentar a secreção no espaço periplásmico de Escherichia coli de cinco proteínas: o hormônio do crescimento humano (hGH), que tem sido amplamente utilizado no tratamento de crianças com deficiência e/ou resistência ao hGH, síndrome de Turner, entre outras desordens; prolactina humana (hPRL), um hormônio polipeptídico conhecido por estimular a lactação e por exercer ação regulatória no crescimento e na diferenciação da glândula mamária, dois antagonistas de hPRL, estudados como potenciais fármacos para o tratamento de alguns tipos de cânceres e por fim o interferon α2a (IFN-α2a), que é uma citocina produzida pelas células, em resposta a diferentes estímulos, incluindo ácidos nucléicos virais, células estranhas (particularmente as neoplásicas), antígenos de bactérias, protozoários e vírus. No caso do IFN-α2a, essa citocina de alto valor agregado e de importante aplicação terapêutica, foi desenvolvida em nosso laboratório como parte desse trabalho, incluindo o desenvolvimento e a validação da metodologia de análise por HPLC de fase reversa para determinação do IFN presente no fluído periplásmico bacteriano ou na sua forma pura. As principais estratégias utilizadas para melhorar a expressão foram iniciar a indução junto à densidade óptica máxima do crescimento bacteriano e otimizar a temperatura de indução para controlar a expressão da proteína heteróloga. Essa metodologia pode ser utilizada nos casos onde o produto não será tóxico para a célula hospedeira ou quando a instabilidade do plasmídeo não é problema. A possibilidade de cultivo em temperaturas mais baixas, já que o repressor termo-sensível não se encontra presente, colaborou para o aumento significativo da expressão, mesmo para proteínas menos sensíveis à temperatura de cultivo, como o hGH. / The expression system based on the PL or PR promoters of the lambda phage that is usually regulated by the term labile repressor (clts) is widely used to produce recombinant proteins in prokaryotic cells. However, the temperature increase required in this system to promote the repressor inactivation shows some limitations, like the increase of the HSP (Heat Shock Proteins) proteins expression, proteases e.g., that depending on the nature of the expressed protein can be harmful or not. Another limitation is the activation of SOS response, resulting on the stop of the DNA cell replication or depending on the strain can occur cell lysis. In this paper we describe the use of the λPL promoter for constitutive expression, without the repressor regulation. We optimized different cultivation conditions to increase the secretion in the periplasmic space of Escherichia coli of five proteins: the human growth hormone (hGH), that is being widely used in the treatment of children with disabilities and/or resistance to hGH, Turner syndrome, within another disorders; human prolactin (hPRL), a polypeptide hormone known for stimulating the lactation and for exercising regulatory action on growth and on the differentiation of the mammary gland; two hPRL antagonists, studied as potential medicine to the treatment of some kinds of cancers and finally the interferon α2a (IFN-α2a), that is a cytokine produced by the cells, in response to different actions, including viral nucleic acids, neoplastic cells, antigens of bacteria, protozoa and viruses. In the case of IFN-α2a, this high value added and important therapeutic application, was developed in our laboratory as a part of this paper, including the development and the validation of the analysis methodology by reversed-phase HPLC to determine the IFN present in the bacterial periplasmic fluid or in its pure form. The main strategies used to improve the expression were to start the induction with the maximum optical density of bacterial growth and optimize the induction temperature to control the expression of heterologous protein. This methodology can be used in cases where the product wont be toxic to the host cell or when the instability of the plasmid is not a problem. The possibility of cultivation in lower temperatures, since the heat-sensitive repressor is not present, contributed to the significant increase of the expression, even to proteins that are less sensitive to the cultivation temperature, like the hGH.

Immunological stressors, in the form of clinical and sub-clinical disease are currently controlled using both prophylactic antibiotics in-feed, and therapeutic antibiotic treatment. Respiratory disease, primarily Actinobacillus pleuropneumoniae (App) infection, is recognised as a major factor causing reduced productivity in pigs. This thesis reports investigations into the use of novel immunomodulators in particular Interleukin 4 (IL-4) and Interleukin 1 receptor antagonist (IL-1ra) as alternatives to antibiotics to treat App infection. Immunological and molecular biological assays were used to investigate and accumulate data. An in vitro study undertaken to find potential anti-inflammatory substances, revealed that Interleukin 8 (IL-8) mRNA production stimulated by PMA or LPS in whole pigs' blood was suppressed by IL-4. IL-1ra also suppressed stimulated IL-8 mRNA production by heat killed App bacteria (KB) in vitro. An acute LPS challenge in pigs in vivo however, showed no variation in illness or weight loss between pigs treated prophylactically with anti-inflammatory substance (IL-4 and IL-1ra) and saline treated pigs. The use of plasmids as a delivery system for anti-inflammatory substance did not show promise since it did not enhance growth or prolong the expression of the substances in the pigs. However, in the chronic App challenge model IL-4 and IL-1ra administered prophylactically in vivo showed an ability to improve growth. The therapeutic administration of IL-4 and IL-1ra to App challenged pigs showed no difference in pigs' growth, regardless of the treatment or control administered. To conclude, IL-4 and IL-1ra showed promise when administered prophylactically and improved growth and abrogated disease under conditions of App challenge. However when IL-4 and IL-1ra where administered therapeutically they did not perform as well. Moreover these compounds have potential as a commercial application to reduce the growth reduction caused by disease such as App.

Interleukin-1 (IL-1) is a prototypic multifunctional cytokine. IL-1 family include interleukin-1 a (IL-1 a), interleukin-1b (IL-1 b) and interleukin-1 receptor antagonist (IL-1 Ra). IL-1 b is the archetypeal pleiotropic cytokine which have been produced by many cells and exerting its biological effects on almost all cell types. IL-1 b is the most potent of known agents that are gastric cytoprotective, antiulcer, antisecretory and an inhibitor of gastric emptying. IL-1 Ra competes with IL-1 b for cell surface receptor occupancy. Host genetic factors that affect interleukin-1 (IL-1) have been reported to influence the susceptibility of Caucasians to gastric cancer. Whether Asians have the same genetic susceptibility remains unclear. In this study, the genetic associations of IL-1B and IL-1RN polymorphisms with gastric cancer and duodenal ulcer in Taiwan were evaluated. Genomic DNA from 140 unrelated Taiwanese patients with gastric adenocarcinoma, 94 with duodenal ulcer and 165 ethically matched healthy controls was typed for polymorphisms at positions ¡V31, -511, and +3954 in the IL-1B gene, and the variable number of tandem repeats polymorphisms in intron 2 of the IL-1RN gene. The allele frequencies of IL-1RN 2R in gastric cancer cases were much higher than those in healthy controls (9% vs. 3%, p = 0.781). The allele frequencies of IL-1B ¡V31, IL-1B ¡V511 and IL-1B +3954 did not differ. An increased risk of the development of intestinal type gastric carcinoma was found in IL-1RN 2R carriers with an odds ratio (OR) of 4.06 (95% confidence interval [CI]: 1.68 ¡V 9.79, p-value=0.085). And another increased risk of the development of diffuse type gastric carcinoma was found in IL-1RN 2R carriers with an odds ratio (OR) of 3.15 (95% confidence interval [CI]: 1.16 ¡V 8.56, p-value=0.061). A significant association was found in IL-1RN 2R/4R genotype and the risk of the development of duodenal ulcer, with an odds ratio (OR) of 2.57 (95% CI: 1.03 ¡V 6.38, p = 0.292). No significant relationship was noted in duodenal ulcer patients with IL-1B genotype examed in this study. Additionally, a synergistic interaction between blood type A and IL-1 RN 2R carriers existed in gastric cancer patients (OR= 4.51; 95% CI: 1.20 ¡V 16.88, p-value=0.516). The synergistic interaction was even stronger between blood type O and IL-1 RN 2R carriers of duodenal ulcer patients (OR= 10.3; 95% CI: 2.10 ¡V 50.61, p-value=0.160). In conclusion, the genetic polymorphisms of IL-1RN 2R and blood type A are associated with the development of gastric cancer. The genetic polymorphisms of IL-1RN 2R and blood type O are associated with the development of duodenal ulcer.

Zugl.: Berlin, Freie Univ., Diss., 2008

During adolescent brain maturation, there are likely sensitive periods where environmental conditions, including drug exposure, may influence development by modifying neuronal connections. Altering neuronal function may produce different phenotypes than expected under normal conditions that may influence subsequent responding to drugs of abuse after the brain is fully mature. Experiment one investigated the relationship between novelty preference and cocaine place preference in adolescent and adult rats. High responding adolescent rats displaying greater free choice novelty exploration (but not forced novelty locomotion) expressed decreased cocaine place conditioning compared to low responding rats. No relationship was found in adult rats. Experiment two evaluated novelty-induced behaviors in adulthood after adolescent cocaine exposure. Repeated cocaine administration produced greater stress and anxiogenic behavioral responses to novelty in adult rats. Repeated alcohol administration produced less-inhibited novelty-induced behaviors in adulthood. Experiment three and four evaluated the consequence of developmental cocaine exposure on the rewarding efficacy of cocaine in adolescence and adulthood. Additionally, the interaction of D2 receptors and the rewarding efficacy of cocaine were investigated. After developmental cocaine exposure, adolescent and adult rats demonstrate decreased rewarding efficacy to cocaine. Importantly, blockade of the D2 receptor prevents cocaine-induced neurochemical changes, potentially regulating the behavioral and neurochemical alterations that occur after repeated drug use that increases the likelihood of dependence. Together, these data implicate both short and long-term behavioral adaptations that occur after developmental cocaine exposure that may result in a predisposition to develop adulthood drug dependence.

Vitamin B6, or more specifically the active form pyridoxal 5ʹ-phosphate (PLP), plays a crucial role as a cofactor for numerous enzymes linked to carbohydrate, fatty acid, and amino acid metabolism. There is a high prevalence of moderate vitamin B6 deficiency in the population that may be further exacerbated through the ingestion of vitamin B6 antagonists present in the food supply. For example, flaxseed contains the anti-pyridoxine factor 1-amino D-proline (1ADP) in the form of a dipeptide called linatine. In order to address these issues, the current study was designed to: 1) characterize and quantify the total amount of anti-pyridoxine factors present in flaxseed through the use of UPLC/ESI-MS analysis, 2) investigate the in vivo effects of synthetic and flaxseed-derived 1ADP on amino acid metabolism using a rat model of moderate B6 deficiency, and 3) identify novel biomarkers of vitamin B6 inadequacy using a LC-Qtof-MS based non-targeted metabolomics approach. The total anti-pyridoxine content, measured as 1ADP equivalents, in the flaxseed extract was found to be 177-437 μg/g of whole flaxseed, depending on the variety tested. Plasma biochemical analyses revealed that B6 vitamers, particularly PLP concentrations were reduced (P≤0.001), due to 1ADP ingestion (10 mg/kg diet) irrespective of the sources. Oral ingestion of flaxseed-derived 1ADP in moderately vitamin B6-deficient rats increased plasma cystathionine (P≤0.001), and decreased plasma α-aminobutyric acid (P≤0.001) and glutamic acid (P=0.017) concentrations compared to the controls. However, the ingestion of synthetic 1ADP elicited greater perturbations in amino acid profile compared to the flaxseed-derived 1ADP, which was predominantly in the form of the dipeptide linatine. Additionally, oral ingestion of the synthetic as well as the flaxseed-derived 1ADP significantly (P≤0.05) inhibited the activities of hepatic PLP-dependent enzymes involved in transsulphuration reactions of methionine metabolism. The use of a non-targeted metabolomics approach identified ten potential lipophilic markers of vitamin B6-insufficiency: glycocholic acid, glycoursodeoxycholic acid, murocholic acid, N-docosahexaenoyl GABA, N-arachidonoyl GABA, lumula, nandrolone, orthothymotinic acid, cystamine and 3-methyleneoxindole. These data serve to highlight potential deleterious effects of anti-pyridoxine factors linked to flaxseed in a population at risk for moderate vitamin B6 deficiency. / October 2015

The endocannabinoid system is a novel pharmacological target in the treatment of metabolic syndrome. Antagonism of the endocannabinoid-1 receptor (CB1R) leads to a transient reduction in food intake, a sustained decrease in body weight and an improvement in metabolic parameters in animal models of obesity. Skeletal muscle is the primary tissue involved in glucose uptake in response to insulin, and insulin sensitivity of skeletal muscle is vital to the maintenance of whole-body euglycemia. Little is known regarding the effects of CB1R antagonism on skeletal muscle glucose transport activity. The purpose of this dissertation was to test the hypothesis that antagonism of the CB1R activates signaling molecules of the insulin signaling pathway to increase glucose transport activity in normal and insulin-resistant skeletal muscle, thereby improving whole-body glucose tolerance. CB1R antagonism with SR141716 directly enhanced basal and insulin-stimulated glucose transport activity in skeletal muscle from lean and obese Zucker while activation of the CB1R with ACEA, decreased glucose transport activity. Key proteins associated with regulation of glucose transport activity were not altered by either CB1R agonism (ACEA) or antagonism (SR141716). Chronic CB1R antagonist treatment (10 mg/kg SR141716 i.p. / 14 days) also enhanced insulin-stimulated glucose transport activity in skeletal muscle of both lean and obese animals, again with no alteration in relevant signaling factors. Plasma free fatty acids (FFAs) were decreased in chronically-treated lean and obese animals and whole-body insulin sensitivity was improved in obese Zucker rats. The enhanced insulin sensitivity seen in chronically-treated obese animals was associated with a dramatic reduction in insulin secretion following a glucose challenge. Acute CB1R antagonism in obese animals also elicited a reduction in insulin secretion following a glucose challenge; however, with no improvement of whole-body insulin sensitivity. Acute CB1R antagonist treatment did not alter skeletal muscle glucose transport activity or circulating FFAs for any animals. These data suggest that although CB1R antagonism directly enhances basal and insulin stimulated glucose transport in skeletal muscle of lean and obese rats, direct action on the skeletal muscle is not responsible for the improvement in insulin-stimulated glucose transport activity and whole-body insulin sensitivity seen in chronically-treated obese animals.

Introduction: Tobacco dependence is a chronic disorder that carries the risk of relapse at any time point during abstinence. It is a major health issue in the world and current pharmacotherapies have had limited efficacy. Therefore, development and validation of novel treatments are required. Objective: Investigate the novel neutral cannabinoid-1 receptor antagonist AM4113 on nicotine (main psychoactive ingredient in tobacco)-taking behaviour in animals. Methods: Using the nicotine intravenous- and food control- self administration paradigms, we tested the acute and chronic (10-days) effects of AM4113 on nicotine- and food-taking behaviour. Results: Acute AM4113 treatments (1-, 3-, 10-mg/kg) reduced nicotine self administration. Chronic AM4113 administration (10mg/kg) produced a sustained reduction of nicotine-taking behaviour during the course of the treatment. In the similar food control self administration experiments, AM4113 overall produced no effect. Conclusion: AM4113 can attenuate nicotine-taking behaviour and its effect is sustained under chronic treatment.

Introduction: Tobacco dependence is a chronic disorder that carries the risk of relapse at any time point during abstinence. It is a major health issue in the world and current pharmacotherapies have had limited efficacy. Therefore, development and validation of novel treatments are required. Objective: Investigate the novel neutral cannabinoid-1 receptor antagonist AM4113 on nicotine (main psychoactive ingredient in tobacco)-taking behaviour in animals. Methods: Using the nicotine intravenous- and food control- self administration paradigms, we tested the acute and chronic (10-days) effects of AM4113 on nicotine- and food-taking behaviour. Results: Acute AM4113 treatments (1-, 3-, 10-mg/kg) reduced nicotine self administration. Chronic AM4113 administration (10mg/kg) produced a sustained reduction of nicotine-taking behaviour during the course of the treatment. In the similar food control self administration experiments, AM4113 overall produced no effect. Conclusion: AM4113 can attenuate nicotine-taking behaviour and its effect is sustained under chronic treatment.

No description available.