Estrias vinosas de la piel: contribución al estudio y valor semiológico de las mismas

Tesis Universidad de Madrid, Facultad de Medicina, 1954.Fac. de MedicinaTRUEProQuestpu

Handling method affects measures of anxiety, but not chronic stress in mice.

Studies in mice have shown that less aversive handling methods (e.g. tunnel or cup handling) can reduce behavioural measures of anxiety in comparison to picking mice up by their tail. Despite such evidence, tail handling continues to be used routinely. Besides resistance to change accustomed procedures, this may also be due to the fact that current evidence in support of less aversive handling is mostly restricted to effects of extensive daily handling, which may not apply to routine husbandry practices. The aim of our study was to assess whether, and to what extent, different handling methods during routine husbandry induce differences in behavioural and physiological measures of stress in laboratory mice. To put the effects of handling method in perspective with chronic stress, we compared handling methods to a validated paradigm of unpredictable chronic mild stress (UCMS). We housed mice of two strains (Balb/c and C57BL/6) and both sexes either under standard laboratory conditions (CTRL) or under UCMS. Half of the animals from each housing condition were tail handled and half were tunnel handled twice per week, once during a cage change and once for a routine health check. We found strain dependent effects of handling method on behavioural measures of anxiety: tunnel handled Balb/c mice interacted with the handler more than tail handled conspecifics, and tunnel handled CTRL mice showed increased open arm exploration in the elevated plus-maze. Mice undergoing UCMS showed increased plasma corticosterone levels and reduced sucrose preference. However, we found no effect of handling method on these stress-associated measures. Our results therefore indicate that routine tail handling can affect behavioural measures of anxiety, but may not be a significant source of chronic husbandry stress. Our results also highlight strain dependent responses to handling methods

Človeška in ptičja gripa: zakaj ptičja gripa ogroža človeštvo?

Gripa je pogost vzrok obolenj dihal, ki povzroča pomembno obolevnost in smrtnost predvsem pri starejših osebah in osebah s kroničnimi obolenji. Gripe ni mogoče izkoreniniti in se v obliki epidemije pojavlja sezonsko vsako zimo, vendar se proti njej lahko uspešno zaščitimo s pravočasnim cepljenjem. Cepljenje zelo priporočamo zdravstvenim delavcem, da okužbe ne prenašajo na svoje bolnike. V zadnjem času so na voljo protivirusna zdravila iz skupine zaviralcev nevraminidaze, s katerimi lahko preprečimo ali omilimo gripo. Kljub napredku nas nedavna obsežna epidemija ptičje gripe med perutnino v azijskih državah opozarja na ranljivost človeške vrste in tveganje za pojav nove pandemije, ki bi zahtevala več milijonov človeških žrtev. Zadnjo epidemijo ptičje gripe so v Aziji uspeli omejiti (ne pa tudi izkoreniniti) in ptičji virus se ni pomembno širil med ljudmi. Oboleli so le posamezniki, ki so bili v tesnem stiku z okuženo perutnino. V prihodnosti je pomembno zgodnje prepoznavanje epidemij ptičje gripe in takojšnji poboj vse okužene in obolele perutnine. Potrebno je pripraviti krizni načrt za primer izbruha epidemije, pripraviti cepivo in zalogo protivirusnih zdravil. Članek povzema značilnosti človeške in ptičje gripe, načine širjenje in zaščito

Do multiple experimenters improve the reproducibility of animal studies?

The credibility of scientific research has been seriously questioned by the widely claimed "reproducibility crisis". In light of this crisis, there is a growing awareness that the rigorous standardisation of experimental conditions may contribute to poor reproducibility of animal studies. Instead, systematic heterogenisation has been proposed as a tool to enhance reproducibility, but a real-life test across multiple independent laboratories is still pending. The aim of this study was therefore to test whether heterogenisation of experimental conditions by using multiple experimenters improves the reproducibility of research findings compared to standardised conditions with only one experimenter. To this end, we replicated the same animal experiment in 3 independent laboratories, each employing both a heterogenised and a standardised design. Whereas in the standardised design, all animals were tested by a single experimenter; in the heterogenised design, 3 different experimenters were involved in testing the animals. In contrast to our expectation, the inclusion of multiple experimenters in the heterogenised design did not improve the reproducibility of the results across the 3 laboratories. Interestingly, however, a variance component analysis indicated that the variation introduced by the different experimenters was not as high as the variation introduced by the laboratories, probably explaining why this heterogenisation strategy did not bring the anticipated success. Even more interestingly, for the majority of outcome measures, the remaining residual variation was identified as an important source of variance accounting for 41% (CI95 [34%, 49%]) to 72% (CI95 [58%, 88%]) of the observed total variance. Despite some uncertainty surrounding the estimated numbers, these findings argue for systematically including biological variation rather than eliminating it in animal studies and call for future research on effective improvement strategies

The rearing environment persistently modulates mouse phenotypes from the molecular to the behavioural level.

The phenotype of an organism results from its genotype and the influence of the environment throughout development. Even when using animals of the same genotype, independent studies may test animals of different phenotypes, resulting in poor replicability due to genotype-by-environment interactions. Thus, genetically defined strains of mice may respond differently to experimental treatments depending on their rearing environment. However, the extent of such phenotypic plasticity and its implications for the replicability of research findings have remained unknown. Here, we examined the extent to which common environmental differences between animal facilities modulate the phenotype of genetically homogeneous (inbred) mice. We conducted a comprehensive multicentre study, whereby inbred C57BL/6J mice from a single breeding cohort were allocated to and reared in 5 different animal facilities throughout early life and adolescence, before being transported to a single test laboratory. We found persistent effects of the rearing facility on the composition and heterogeneity of the gut microbial community. These effects were paralleled by persistent differences in body weight and in the behavioural phenotype of the mice. Furthermore, we show that environmental variation among animal facilities is strong enough to influence epigenetic patterns in neurons at the level of chromatin organisation. We detected changes in chromatin organisation in the regulatory regions of genes involved in nucleosome assembly, neuronal differentiation, synaptic plasticity, and regulation of behaviour. Our findings demonstrate that common environmental differences between animal facilities may produce facility-specific phenotypes, from the molecular to the behavioural level. Furthermore, they highlight an important limitation of inferences from single-laboratory studies and thus argue that study designs should take environmental background into account to increase the robustness and replicability of findings

Systematic assessment of the replicability and generalizability of preclinical findings: Impact of protocol harmonization across laboratory sites.

The influence of protocol standardization between laboratories on their replicability of preclinical results has not been addressed in a systematic way. While standardization is considered good research practice as a means to control for undesired external noise (i.e., highly variable results), some reports suggest that standardized protocols may lead to idiosyncratic results, thus undermining replicability. Through the EQIPD consortium, a multi-lab collaboration between academic and industry partners, we aimed to elucidate parameters that impact the replicability of preclinical animal studies. To this end, 3 experimental protocols were implemented across 7 laboratories. The replicability of results was determined using the distance travelled in an open field after administration of pharmacological compounds known to modulate locomotor activity (MK-801, diazepam, and clozapine) in C57BL/6 mice as a worked example. The goal was to determine whether harmonization of study protocols across laboratories improves the replicability of the results and whether replicability can be further improved by systematic variation (heterogenization) of 2 environmental factors (time of testing and light intensity during testing) within laboratories. Protocols were tested in 3 consecutive stages and differed in the extent of harmonization across laboratories and standardization within laboratories: stage 1, minimally aligned across sites (local protocol); stage 2, fully aligned across sites (harmonized protocol) with and without systematic variation (standardized and heterogenized cohort); and stage 3, fully aligned across sites (standardized protocol) with a different compound. All protocols resulted in consistent treatment effects across laboratories, which were also replicated within laboratories across the different stages. Harmonization of protocols across laboratories reduced between-lab variability substantially compared to each lab using their local protocol. In contrast, the environmental factors chosen to introduce systematic variation within laboratories did not affect the behavioral outcome. Therefore, heterogenization did not reduce between-lab variability further compared to the harmonization of the standardized protocol. Altogether, these findings demonstrate that subtle variations between lab-specific study protocols may introduce variation across independent replicate studies even after protocol harmonization and that systematic heterogenization of environmental factors may not be sufficient to account for such between-lab variation. Differences in replicability of results within and between laboratories highlight the ubiquity of study-specific variation due to between-lab variability, the importance of transparent and fine-grained reporting of methodologies and research protocols, and the importance of independent study replication

Katja Pavlič Škerjanc septuagenaria

Ad multos anno

Effect of bioisosteric replacement of central phenyl group on the inhibitory activity of the tetrahydropiran type InhA inhibitors

Bakterijska okužba z M.tuberculosis lahko povzroči tuberkulozo, bolezen, ki v večini primerov prizadane pljuča, lahko pa tudi ostale organe. V nekaterih primerih je lahko tudi smrtna. Standardno zdravljenje poteka s protibakterijskimi učinkovinami, natančneje z izoniazidom ali več učinkovinami hkrati (rifampicin, etambutol). Za učinkovito zdravljenje tuberkuloze je potrebno sočasno jemanje več zdravil. Čeprav se je pojavnost in smrtnost bolezni v zadnjih letih močno zmanjšala, je ta napredek pogosto spregledan zaradi naraščanja razširjenosti okužb z večkrat odpornimi bakterijami tuberkuloze ali še hujše oblike rezistence. Zaradi odpornosti določenih sevov na obstoječe metode zdravljenja se razvijajo nove oblike zdravilnih učinkovin za zdravljenje tuberkuloze, gre za t.i. direktne zaviralce encima InhA: tetrahidropiranski derivati, triklosan, piridomicin, 4-hidroksi-2-piridoni, tiadiazoli. Pri eksperimentalnem delu smo se osredotočili na bioizosterno zamenjavo osrednje fenilne skupine na tetrahidropiranskem tipu InhA zaviralcev. S pomočjo obročnih ekvivalentov smo fenilni obroč zamenjali s tiofenom in tiazolom. Med eksperimentalnim delom smo se srečali z določenimi tipi reakcij, kot npr.: radikalno halogeniranje – bromiranjeredukcijo nitrilov do aminovsklopitvenimi reakcijami, kjer se je tvoril karboksamid. Uspeli smo sinetizirati dve končni spojini: spojino 8: 5-((3,5-dimetil-1H-pirazol-1-il)metil)-N-((4-(4-feniltiazol-2-il)tetrahidro-2H-piran-4-il)metil)tiofen-2-karboksamid in spojino 16: 2-((3,5-dimetil-1H-pirazol-1-il)metil)-N-((4-(4-feniltiazol-2-il)tetrahidro-2H-piran-4-il)metil)tiazol-4-karboksamid. Po končani sintezi smo analizirali končne spojine z jedrsko magnetno resonanco (1H,13C) in masno spektrometrijo visoke ločljivosti. Preverili smo tudi čistost dobljenih spojin s tekočinsko kromatografijo visoke ločljivosti. Končnim spojinam so na encimu InhA določili vrednost zaviralne koncentracije (IC50) na encimu. Glede na rezultate analiz se je tiofen izkazal za ustreznejšo zamenjavo fenila. Uspelo nam je pripraviti bolj čist tetrahidropiranski deriavat s tiofenom kot s tiazolom, poleg tega je izkazal tudi nižjo IC50 vrednost od tiazola.The bacterial infection with M.tuberculosis can cause tuberculosis, a disease that in most cases affects the lungs, but it can also affect other organs. Sometimes, it can also be deadly. The standard treatment uses antibacterial substances, specifically isoniazid or more substances at the same time (rifampicin, etambutol). For an effective treatment, it is necessary to take several medications simultaneously. Although the incidence and mortality of the disease lowered in the last years, this achievement is often overlooked because of increasing infections with multiple drug-resistant M. tuberculosis or an even worse form of resistance. Due to the resistance of strains to the existing methods of treatment, new forms of active substances for treatment of tuberculosis are being developed, these are direct inhibitors of enzyme InhA: tetrahydropyran derivatives, triclosan derivatives, pyridomycin, 4-hydroxy-2-pyridones, thiadiazoles. In the experimental part of our work, we focused on the effect of bioisosteric replacement of central phenyl group on the inhibitory activity of the tetrahydropiran type InhA inhibitors. With the help of ring equivalents, we replaced phenyl group with thiophene and thiazole. During this part of our work, we encountered a certain type of reactions, e.g. radical halogenation – bromination, reduction of nitriles to amines and coupling reactions. We synthesized two final compounds: compound 8: 5-((3,5-dimethyl-1H-pyrazol-1-yl)methyl)-N((4-(4-phenylthiazol-2-yl)tetrahydro-2Hpyran-4-yl)methyl)thiophene-2-carboxamide, and compound 16: 2-((3,5-dimethyl-1H-pyrazol-1yl)methyl)-N-((4-(4-phenylthiazol-2yl)tetrahydro-2H-pyran-4yl)methyl)thiazole-4-carboxamide. After the synthesis was completed, we analyzed the final compounds with nuclear magnetic resonance and high resolution mass spectrometry. We also checked the purity of the compounds with high performance liquid chomatography. The final compounds were tested on encyme InhA for the values of IC50. Based on the results of the analysis, tiophene proved to be the better replacement of phenyl. We also managed to prepare a purer tetrahydropyran derivative with thiophene than with thiazole and it showed a lower IC50 value than tiazol