Learning and memory

Memory is one of the most fundamental mental processes. Neuroscientists study this process by using extremely diverse strategies. Two different approaches aimed at understanding learning and memory were introduced in this symposium. The first focuses on the roles played by synaptic plasticity, especially in long-term depression in the cerebellum in motor learning, and its regulatory mechanism. The second approach uses an elegant chick-quail transplantation system on defined brain regions to study how neural populations interact in development to form behaviorally important neural circuits and to elucidate neurobiological correlates of perceptual and motor predispositions.

Notch signaling in the development of the inner ear: Lessons from Drosophila

The sensory patches in the ear of a vertebrate can be compared with the mechanosensory bristles of a fly. This comparison has led to the discovery that lateral inhibition mediated by the Notch cell–cell signaling pathway, first characterized in Drosophila and crucial for bristle development, also has a key role in controlling the pattern of sensory hair cells and supporting cells in the ear. We review the arguments for considering the sensory patches of the vertebrate ear and bristles of the insect to be homologous structures, evolved from a common ancestral mechanosensory organ, and we examine more closely the role of Notch signaling in each system. Using viral vectors to misexpress components of the Notch pathway in the chick ear, we show that a simple lateral-inhibition model based on feedback regulation of the Notch ligand Delta is inadequate for the ear just as it is for the fly bristle. The Notch ligand Serrate1, expressed in supporting cells in the ear, is regulated by lateral induction, not lateral inhibition; commitment to become a hair cell is not simply controlled by levels of expression of the Notch ligands Delta1, Serrate1, and Serrate2 in the neighbors of the nascent hair cell; and at least one factor, Numb, capable of blocking reception of lateral inhibition is concentrated in hair cells. These findings reinforce the parallels between the vertebrate ear and the fly bristle and show how study of the insect system can help us understand the vertebrate.

Suppression of O-Methyltransferase Gene by Homologous Sense Transgene in Quaking Aspen Causes Red-Brown Wood Phenotypes1

Homologous sense suppression of a gene encoding lignin pathway caffeic acid O-methyltransferase (CAOMT) in the xylem of quaking aspen (Populus tremuloides Michx.) resulted in transgenic plants exhibiting novel phenotypes with either mottled or complete red-brown coloration in their woody stems. These phenotypes appeared in all independent transgenic lines regenerated with a sense CAOMT construct but were absent from all plants produced with antisense CAOMT. The CAOMT sense transgene expression was undetectable, and the endogenous CAOMT transcript levels and enzyme activity were reduced in the xylem of some transgenic lines. In contrast, the sense transgene conferred overexpression of CAOMT and significant CAOMT activity in all of the transgenic plants' leaves and sclerenchyma, where normally the expression of the endogenous CAOMT gene is negligible. Thus, our results support the notion that the occurrence of sense cosuppression depends on the degree of sequence homology and endogene expression. Furthermore, the suppression of CAOMT in the xylem resulted in the incorporation of a higher amount of coniferyl aldehyde residues into the lignin in the wood of the sense plants. Characterization of the lignins isolated from these transgenic plants revealed that a high amount of coniferyl aldehyde is the origin of the red-brown coloration—a phenotype correlated with CAOMT-deficient maize (Zea mays L.) brown-midrib mutants.

Light-induced phase-delay of the chicken pineal circadian clock is associated with the induction of cE4bp4, a potential transcriptional repressor of cPer2 gene

The chicken pineal gland contains the autonomous circadian oscillator together with the photic-input pathway. We searched for chicken pineal genes that are induced by light in a time-of-day-dependent manner, and isolated chicken homolog of bZIP transcription factor E4bp4 (cE4bp4) showing high similarity to vrille, one of the Drosophila clock genes. cE4bp4 was expressed rhythmically in the pineal gland with a peak at very early (subjective) night under both 12-h light/12-h dark cycle and constant dark conditions, and the phase was nearly opposite to the expression rhythm of cPer2, a chicken pineal clock gene. Luciferase reporter gene assays showed that cE4BP4 represses cPer2 promoter through a E4BP4-recognition sequence present in the 5′-flanking region, indicating that cE4BP4 can down-regulate the chick pineal cPer2 expression. In vivo light-perturbation studies showed that the prolongation of the light period to early subjective night maintained the high level expression of the pineal cE4bp4, and presumably as a consequence delayed the onset of the induction of the pineal cPer2 expression in the next morning. These light-dependent changes in the mRNA levels of the pineal cE4bp4 and cPer2 were followed by a phase-delay of the subsequent cycles of cE4bp4/cPer2 expression, suggesting that cE4BP4 plays an important role in the phase-delaying process as a light-dependent suppressor of cPer2 gene.

Female mating preference for bold males in the guppy, Poecilia reticulata.

Although females prefer to mate with brightly colored males in numerous species, the benefits accruing to such females are virtually unknown. According to one hypothesis of sexual selection theory, if the expression of costly preferred traits in males (such as conspicuous colors) is proportional to the male's overall quality or reveals his quality, a well-developed trait should indicate good condition and/or viability for example. A female choosing such a male would therefore stand to gain direct or indirect fitness benefits, or both. Among potential phenotypic indicators of an individual's quality are the amount and brightness of its carotenoid-based colors and its boldness, as measured by its willingness to risk approaching predators without being killed. Here, we show experimentally that in the Trinidadian guppy (Poecilia reticulata) the visual conspicuousness of the color pattern of males correlates positively with boldness toward, and with escape distance from, a cichlid fish predator. Bold individuals are thus more informed about nearby predators and more likely to survive encounters with them. Mate-choice experiments showed that females prefer colorful males as mates, but prefer bolder males irrespective of their coloration when given the opportunity to observe their behavior toward a potential fish predator. By preferentially mating with colorful males, female guppies are thus choosing on average, relatively bold, and perhaps more viable, individuals. In doing so, and to the extent that viability is heritable, they potentially gain indirect fitness benefits by producing more viable offspring than otherwise.

BEN/SC1/DM-GRASP, a homophilic adhesion molecule, is required for in vitro myeloid colony formation by avian hemopoietic progenitors.

BEN/SC1/DM-GRASP is a membrane glycoprotein of the immunoglobulin superfamily isolated in the chick by several groups, including ours. Its expression is strictly developmentally regulated in several cell types of the nervous and hemopoietic systems and in certain epithelia. Each of these cell types expresses isoforms of BEN which differ by their level of N-glycosylation and by the presence or absence of the HNK-1 carbohydrate epitope. In the present work, the influence of glycosylation on BEN homophilic binding properties was investigated by two in vitro assays. First, each BEN isoform was covalently coupled to microspheres carrying different fluorescent dyes and an aggregation test was performed. We found that homophilic aggregates form indifferently between the same or different BEN isoforms, showing that glycosylation does not affect BEN homophilic binding properties. This was confirmed in the second test, where the BEN-coated microspheres bound to the neurites of BEN- expressing neurons, irrespective of the isoform considered. The transient expression of the BEN antigen on hemopoietic progenitors prompted us to see whether it might play a role in their proliferation and differentiation. When added to hemopoietic progenitor cells in an in vitro colony formation assay anti-BEN immunoglobulin strongly inhibited myeloid, but not erythroid, colony formation although both types of precursors express the molecule.

Innovative analytical strategies for the development of sensor devices and mass spectrometry methods

Il lavoro presentato in questa tesi di Dottorato è incentrato sullo sviluppo di strategie analitiche innovative basate sulla sensoristica e su tecniche di spettrometria di massa in ambito biologico e della sicurezza alimentare. Il primo capitolo tratta lo studio di aspetti metodologici ed applicativi di procedure sensoristiche per l’identificazione e la determinazione di biomarkers associati alla malattia celiaca. In tale ambito, sono stati sviluppati due immunosensori, uno a trasduzione piezoelettrica e uno a trasduzione amperometrica, per la rivelazione di anticorpi anti-transglutaminasi tissutale associati a questa malattia. L’innovazione di questi dispositivi riguarda l’immobilizzazione dell’enzima tTG nella conformazione aperta (Open-tTG), che è stato dimostrato essere quella principalmente coinvolta nella patogenesi. Sulla base dei risultati ottenuti, entrambi i sistemi sviluppati si sono dimostrati una valida alternativa ai test di screening attualmente in uso per la diagnosi della celiachia. Rimanendo sempre nel contesto della malattia celiaca, ulteriore ricerca oggetto di questa tesi di Dottorato, ha riguardato lo sviluppo di metodi affidabili per il controllo di prodotti “gluten-free”. Il secondo capitolo tratta lo sviluppo di un metodo di spettrometria di massa e di un immunosensore competitivo per la rivelazione di prolammine in alimenti “gluten-free”. E’ stato sviluppato un metodo LC-ESI-MS/MS basato su un’analisi target con modalità di acquisizione del segnale selected reaction monitoring per l’identificazione di glutine in diversi cereali potenzialmente tossici per i celiaci. Inoltre ci si è focalizzati su un immunosensore competitivo per la rivelazione di gliadina, come metodo di screening rapido di farine. Entrambi i sistemi sono stati ottimizzati impiegando miscele di farina di riso addizionata di gliadina, avenine, ordeine e secaline nel caso del sistema LC-MS/MS e con sola gliadina nel caso del sensore. Infine i sistemi analitici sono stati validati analizzando sia materie prime (farine) che alimenti (biscotti, pasta, pane, etc.). L’approccio sviluppato in spettrometria di massa apre la strada alla possibilità di sviluppare un test di screening multiplo per la valutazione della sicurezza di prodotti dichiarati “gluten-free”, mentre ulteriori studi dovranno essere svolti per ricercare condizioni di estrazione compatibili con l’immunosaggio competitivo, per ora applicabile solo all’analisi di farine estratte con etanolo. Terzo capitolo di questa tesi riguarda lo sviluppo di nuovi metodi per la rivelazione di HPV, Chlamydia e Gonorrhoeae in fluidi biologici. Si è scelto un substrato costituito da strips di carta in quanto possono costituire una valida piattaforma di rivelazione, offrendo vantaggi grazie al basso costo, alla possibilità di generare dispositivi portatili e di poter visualizzare il risultato visivamente senza la necessità di strumentazioni. La metodologia sviluppata è molto semplice, non prevede l’uso di strumentazione complessa e si basa sull’uso della isothermal rolling-circle amplification per l’amplificazione del target. Inoltre, di fondamentale importanza, è l’utilizzo di nanoparticelle colorate che, essendo state funzionalizzate con una sequenza di DNA complementare al target amplificato derivante dalla RCA, ne permettono la rivelazione a occhio nudo mediante l’uso di filtri di carta. Queste strips sono state testate su campioni reali permettendo una discriminazione tra campioni positivi e negativi in tempi rapidi (10-15 minuti), aprendo una nuova via verso nuovi test altamente competitivi con quelli attualmente sul mercato.

Microenvironment and microvascular wall modulate cancer cell progression

Tumour progression is a complex process that frequently brings to cancer metastasis, the first cause of poor prognosis of cancer affected patients. Metastasis are generated by cells escaped from a primary mass and able to enter in the circulation, survive and proliferate in a new, distant site of the organism. To reach all these goal, many different phenomena had occur within both the cancer cells and the surrounding microenvironment. In the first part of this thesis, the focus was pointed on the metastatic potential of a leiomyosarcoma cell model. The studied cancer cells demonstrated a strong invasive capacity of the ECM in vitro, principally by production of matrix metalloproteinases 2 and 9, and robust pro-angiogenic activity in the chick CAM model, that facilitate its dissemination through same chick embryo internal organs. This study, with the title “MMPs and angiogenesis affect the metastatic potential of a human vulvar leiomyosarcoma cell line”, is presented in the published form. In the second part of this work, the emphasis was given to the microvascular element of the tumour microenvironment and specifically to the perivascular pericytes. These are intriguing cells due to their uncertain involvement in the biology of cancer. It is not clear how pericytes change within the tumour microenvironment and which is their contribute during the tumour dissemination. After the characterization of the chosen pericytic cell model, an in vitro study of the interaction between pericytes and different cancer cell lines where performed. Indirect and direct cell-cell interaction as well as movement of cancer cells in presence of pericytes conditioned media was analysed, in order to investigate the reciprocal influence of pericytes and tumour cells in the context of cancer progression.

Preparação e caracterização de nanopartículas de prata para aplicação no desenvolvimento de imunoensaio para imunoglobulina G humana

Neste trabalho, anticorpos anti-IgGh foram conjugados às nanopartículas de prata (NPAg) para detectar imunoglobulina G humana (IgGh). Um imunoensaio colorimétrico baseado na diminuição da agregação devido ao aumento da repulsão eletrostática após a interação ligante-alvo. A agregação é induzida pela variação da força iônica e uma mudança da coloração da suspensão coloidal de amarelo para vermelho pode ser observada. Na presença de IgGh, a agregação é inibida e a coloração da suspensão coloidal não se altera. As nanopartículas foram obtidas por meio de cinco procedimentos diferentes e caracterizadas por espectroscopia UV-Vis, espalhamento dinâmico de luz, difração de raios-X e microscopia eletrônica. Glicose e borohidreto de sódio foram utilizados como agentes redutores, enquanto CTAB e β-ciclodextrina foram utilizados como estabilizantes. Citrato de sódio foi utilizado como agente redutor e/ou estabilizante. Nanoesferas de carbono foram obtidas por tratamento hidrotérmico de uma solução aquosa de glicose e também foram utilizadas no preparo das nanopartículas. As nanopartículas foram funcionalizadas com ácido mercaptossuccínico e a conjugação ocorreu devido à interação entre grupos aminas e grupos carboxílicos ionizados, presentes no anticorpo e agente de acoplamento, respectivamente. A estabilidade dos conjugados e o efeito da adição de IgGh foram avaliados para todos os sistemas preparados. As nanopartículas de prata preparadas com borohidreto de sódio e citrato de sódio foram selecionadas para serem aplicadas no desenvolvimento do imunoensaio e as condições experimentais foram avaliadas. Em condições ótimas, observou-se uma correlação linear entre a diminuição da agregação do sistema (NPAg-anti-IgGh) e a concentração de IgGh (0 a 200 ng mL-1). O limite de detecção foi estimado em 25 ng mL-1. O método colorimétrico apresentou boa seletividade para a detecção de IgGh. Além disso, foi obtido um resultado satisfatório ao aplicar o método para determinação do fator IX de coagulação. Foi desenvolvido também um método para determinação de ATP baseado na agregação de nanopartículas de ouro. Aptâmeros foram utilizados como elemento de reconhecimento. Em princípio, o método pode ser aplicável à determinação de outros analitos, por meio da substituição do aptâmero utilizado neste trabalho pelo oligonucleotídeo específico para o alvo de interesse.

Estudo das táticas reprodutivas em duas espécies de Coenagrionidae (Odonata: Zygoptera)

Nos estudos sobre a teoria da Seleção Sexual, as libélulas têm sido amplamente estudadas devido à grande variedade de padrões comportamentais, de coloração e táticas reprodutivas. Como forma de demonstrar táticas reprodutivas adotadas por duas espécies de libélulas, esta dissertação teve como objetivos principais: i) investigar o papel de traços secundários como a coloração corporal na competição intra-sexual de uma espécie territorial e ii) analisar se os traços corporais como tamanho e morfologia das asas predizem a tática de acasalamento adotada por machos de uma espécie nãoterritorial. Sugere-se que a coloração corporal pode predizer o resultado de lutas e também se correlacionar positivamente com a condição física dos machos territoriais de Tigriagrion aurantinigrum. Ademais, traços corporais como o tamanho e a morfologia das asas influenciam na tática reprodutiva utilizada por machos não-territoriais de Epipleoneura williamsoni. Portanto, com os resultados obtidos, conclui-se que a variação nos sinais visuais exerce um papel essencial na comunicação animal e na resolução de conflitos, indicando ainda que pode haver uma sinalização da condição física dos machos. Além disso, os resultados mostraram evidências em como diferentes táticas reprodutivas se relacionam com traços corporais como agilidade e tamanho corporal, os quais podem influenciar no sucesso reprodutivo dos indivíduos.

Ciliary muscle in avian is derived from mesenchymal and epithelial cells

It has long been maintained that the ciliary muscle derives from mesenchymal cells. The embryonic development of the avian ciliary muscle was studied in chick embryos from stage 25 HH to the time of hatching. Serial sections of the eye were stained routinely or immunocytochemically using the monoclonal antibody 13F4, which recognizes a cytoplasmic antigen specific for all types of muscle cells. We found that the mesenchymal immunoreactive cells, at stage 37 HH, are arranged in two distinct orientations forming the anterior and posterior portions of the ciliary muscle. At stages 38 and 39 HH the pigmented epithelium contained 13F4 positive cells, which detach from the epithelium and apparently migrate into stroma. These epithelial cells may differentiate into muscle cells. Within this same time period a progressive accumulation of myoblasts was detected between the pigmented epithelium and the ciliary muscle. Some myoblasts containing melanin were also observed. At stage 40 HH the internal portion of the ciliary muscle was visible. These findings indicate that the immunopositive epithelial cells participate in the formation of the internal portion of the muscle. We conclude that the ciliary muscle derives not only from the mesenchymal cells but also from the pigmented epithelium.

Potentiostatic Reversible Photoelectrochromism: An Effect Appearing in Nanoporous TiO2/Ni(OH)2 Thin Films

In the field of energy saving, finding composite materials with the ability of coloring upon both illumination and change of the applied electrode potential keeps on being an important goal. In this context, chemical bath deposition of Ni(OH)2 into nanoporous TiO2 thin films supported on conducting glass leads to electrodes showing both conventional electrochromic behavior (from colorless to dark brown and vice versa) together with photochromism at constant applied potential. The latter phenomenon, reported here for the first time, is characterized by fast and reversible coloration upon UV illumination. The bleaching kinetics shows first order behavior with respect to the NiIII centers in the film, and an order 1.2 with respect to electrons in the TiO2 film. From a more applied point of view, this study opens up the possibility of having two-mode smart windows showing not only conventional electrochromism but also reversible darkening upon illumination.

Ectopic Meis1 expression in the mouse limb bud alters P-D patterning in a Pbx1-independent manner

During limb development, expression of the TALE homeobox transcription factor Meis1 is activated by retinoic acid in the proximal-most limb bud regions, which give rise to the upper forelimb and hindlimb. Early subdivision of the limb bud into proximal Meis-positive and distal Meis-negative domains is necessary for correct proximo-distal (P-D) limb development in the chick, since ectopic Meis1 overexpression abolishes distal limb structures, produces a proximal shift of limb identities along the P-D axis, and proximalizes distal limb cell affinity properties. To determine whether Meis activity is also required for P-D limb specification in mammals, we generated transgenic mice ectopically expressing Meis1 in the distal limb mesenchyme under the control of the Msx2 promoter. Msx2:Meis1 transgenic mice display altered P-D patterning and shifted P-D Hox gene expression domains, similar to those previously described for the chicken. Meis proteins function in cooperation with PBX factors, another TALE homeodomain subfamily. Meis-Pbx interaction is required for nuclear localization of both proteins in cell culture, and is important for their DNA-binding and transactivation efficiency. During limb development, Pbx1 nuclear expression correlates with the Meis expression domain, and Pbx1 has been proposed as the main Meis partner in this context; however, we found that Pbx1 deficiency did not modify the limb phenotype of Msx2:Meis1 mice. Our results indicate a conserved role of Meis activity in P-D specification of the tetrapod limb and suggest that Pbx function in this context is either not required or is provided by partners other than Pbx1.

Bibliography on corrosion by liquid metals (1957-September 1962)

Mode of access: Internet.