MACIEL, V. T. Caracterização fisica, fisico-química e enzimática de frutos de seis cultivares de coqueiro anão em diferentes estadios de desenvolvimento. 2008. 102 f. Dissertação (Mestrado em Agronomia/Fitotecnia) - Centro de Ciências Agrárias, Universidade Federal do Ceará, Fortaleza, 2008. / Submitted by Francisco Lacerda (lacerda@ufc.br) on 2014-08-04T23:21:10Z No. of bitstreams: 1 2008_dis_vtmaciel.pdf: 1225013 bytes, checksum: 21456a791e7d0734e3e07a7553cf1169 (MD5) / Approved for entry into archive by José Jairo Viana de Sousa(jairo@ufc.br) on 2014-08-06T20:25:22Z (GMT) No. of bitstreams: 1 2008_dis_vtmaciel.pdf: 1225013 bytes, checksum: 21456a791e7d0734e3e07a7553cf1169 (MD5) / Made available in DSpace on 2014-08-06T20:25:22Z (GMT). No. of bitstreams: 1 2008_dis_vtmaciel.pdf: 1225013 bytes, checksum: 21456a791e7d0734e3e07a7553cf1169 (MD5) Previous issue date: 2008 / This study aimed at characterizing in fruit and physico-chemical, and sensory enzyme in the coconut water the of six dwarf coconut cultivars at seven stages of development. The studied cultivars were: Green dwarf from Jequi (GDJ), yellow dwarf from Gramame (YDG), yellow dwarf from Malaysia (YDM), Red dwarf from Cameroon (YDC), Red dwarf from Malaysia (RDM), Red dwarf from Gramame (RDG). Fruits were harvested from an experimental field, Embrapa Meio Norte (Parnaiba city, Piauí State), at the following stages of development: 126, 147, 168, 189, 210, 231, and 252 days after the opening of the inflorescences. Upon harvest, fruits were taken to the Laboratory of Postharvest physiology and technology at Embrapa Agroindustria Tropical, located in Fortaleza, Ceará, were the physical and physic-chemical analyses were performed. Part of the samples were stored at -85°C and then transported to the Laboratory of Plant Physiology, Biochemistry and Molecular Biology Department from The Federal University of Ceará, were the enzymatic study was conducted. The experimental designed was a completely randomized one in a factorial scheme 6 x 7. First physical characterization (mass, length, diameter, water volume, and color of the shell) was individually performed for each fruit, then the physic-chemical analyses of coconut water: total soluble solids (TSS), total soluble sugar (TSSu), reducing sugars (RS), total titratable acidity (TTA), sugar to acid ratio (TSS/TTA), pH, turbidity, and protein. It was also evaluated the activities of enzymes that oxide phenols [polyphenoloxidase (PFO) and peroxidase specific for the guaiacol (G-POD)], as well as those of the cycle of ascorbate-glutathione [superoxide dismutase (SOD), catalase (CAT) and the ascorbate peroxidase (APX). Moreover some of the sensory aspects of the water were evaluated. The results showed that the YDR, YDG, and RDG may be commercially exploited, due to their physical and physic-chemical characteristics. It was also concluded that, the best time for harvesting is somewhat between 189 to 210 days after fruit developed set, since right at this time the studied cultivars presented the best quality attributes. There was detected activity of G-POD in the coconut water from any of the cultivars. The activity of SOD in the coconut water increased in the early stages of development of the fruits of all the cultivars, such as RDG and YDG cultivars such as those that showed higher enzyme activity. A CAT proved to be the main enzyme eliminating of hydrogen peroxide of coconut water and its activity varied greatly it the development of the fruit and also between the cultivars. / O presente trabalho objetivou caracterizar as alterações físicas nos frutos e físico-químicas, enzimáticas e sensoriais na água de coco de seis cultivares de coqueiro anão em sete estádios de desenvolvimento. As cultivares analisadas foram: Anão Verde de Jequi (AVeJ), Anão Amarelo de Gramame (AAG), Anão Amarelo da Malásia (AAM), Anão Vermelho de Camarões (AVC), Anão Vermelho da Malásia (AVM), sendo os frutos colhidos em um campo experimental da Embrapa Meio Norte (Parnaíba, PI) nos seguintes estádios de desenvolvimento: 126, 147, 168, 189, 210, 231, 252 dias após a abertura da inflorescência. Logo após a colheita, os frutos foram conduzidos para o laboratório de Fisiologia e Tecnologia de Pós-colheita na Embrapa Agroindústria Tropical, em Fortaleza, Ceará, onde foram feitas as análises físicas e físico-químicas. Parte das amostras foram armazenadas a -85°C e, em seguida, transportadas para o Laboratório de Fisiologia Vegetal, do Departamento de Bioquímica e Biologia Molecular da Universidade Federal do Ceará, onde foram feitas as análises enzimáticas. O experimento seguiu um delineamento inteiramente casualizado, em esquema fatorial do tipo 6 x 7,. Foram feitas as caracterizações físicas individuais dos frutos (massa, comprimento maior, diâmetro, volume da água e cor da casca), e em seguida, as avaliações físico-químicas da água de coco: sólidos solúveis totais (SS), açúcares solúveis totais (AST) e redutores (AR), acidez total titulável (ATT), relação SS/ATT, pH, turbidez e proteína). Foram, também, avaliadas as atividades das enzimas que oxidam fenóis [polifenoloxidase (PFO) e peroxidase específica para o guaiacol (G-POD)], bem como aquelas do ciclo do ascorbato-glutationa [dismutase do superóxido (SOD), catalase (CAT) e peroxidase do ascorbato (APX)], sendo também determinados alguns atributos sensoriais da água de coco. Os resultados deste experimento mostraram que as cultivares AVM, AAG e AAM, podem ser exploradas comercialmente, devido às qualidade físicas e físico-químicas que apresentaram. Pode-se concluir também que, independente da cultivar, o melhor período para colheita encontra-se na faixa de 189 a 210 dias de desenvolvimento dos frutos, pois nesse período as cultivares analisadas apresentaram os melhores atributos de qualidade.. Não foi detectada atividade da G-POD na água de coco de nenhuma das cultivares. A atividade da SOD na água de coco aumentou nos estádios iniciais de desenvolvimento dos frutos de todas as cultivares, destacando-se as cultivares AVG e AVeJ como as que apresentaram maiores atividades dessa enzima. A CAT mostrou-se a principal enzima eliminadora de peróxido de hidrogênio da água de coco e sua atividade variou muito com o desenvolvimento dos frutos e também entre as cultivares.

Made available in DSpace on 2014-06-11T19:32:12Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-02-19Bitstream added on 2014-06-13T19:02:35Z : No. of bitstreams: 1 correa_lc_dr_botib.pdf: 1179525 bytes, checksum: beffa33d099cc5e0f66952e183424a29 (MD5) / A cultura da goiabeira ocupa importante espaço no agronegócio do país, um dos maiores produtores mundiais de goiaba. O araçazeiro, nativo do Brasil, embora não apresente a mesma importância econômica da goiaba, tem sido explorado em algumas regiões como alternativa de plantio devido à boa aceitação do seu fruto e também por apresentar características de interesse comercial e social. Dentre as características compartilhadas por goiabas e araçás estão a diversidade de uso, desde o consumo in natura até diferentes formas processadas, e a riqueza em nutrientes e em compostos fenólicos. Este trabalho visou à caracterização química e bioquímica de frutos de araçazeiros e goiabeiras do banco ativo de germoplasma (BAG) da Embrapa Semiárido, provenientes de diferentes regiões do Brasil, bem como a caracterização genética de parte dos acessos. Foram determinados os teores de açúcares, proteínas, sólidos solúveis, acidez titulável, “ratio” e umidade, além dos minerais: cálcio, magnésio, ferro, fósforo e potássio. Foram também analisados os antioxidantes: ácido ascórbico, fenóis totais, flavonóides totais, licopeno e β-caroteno, bem como a atividade antioxidante. Para a caracterização genética,, utilizou-se o marcador molecular AFLP (Amplified Fragment Length Polymorphism). Frutos de cinco acessos de goiabeira provenientes do Maranhão (G01MA, G12MA, G14MA, G16MA e G20MA), e dos acessos A08MA, A42PE, A45PE, A80RO e A100AM, de araçazeiros, se destacaram nas avaliações bioquímicas por apresentarem, no conjunto, os maiores teores de sólidos solúveis, açúcares, proteínas e minerais, além de estarem entre aqueles com menores teores de acidez titulável, no caso das goiabas, e maiores no caso dos araçás. Quanto aos antioxidantes, verificou-se que goiabas e araçás são ricos em compostos fenólicos, os quais apresentaram forte correlação... / The culture of guava has an important position in the agribusiness in the country, one of the largest producers of guava of the world. Brazilian guava tree, native of Brazil, although not present the same economic importance of guava has been explored in some regions as an alternative planting due to the high acceptance of its fruit and also present characteristics of commercial and social interest. The diversity of use, since the fresh market to different forms processed forms, and wealth of nutrients and phenolic compounds are among the characteristics shared by guavas and Brazilian guava. This work aimed at the chemical and biochemical characterization of Brazilian guava and guava fruit of the active germplasm bank (AGB) of Embrapa Semiarid, from different regions of Brazil and the genetic characterization of some accessions. The levels of sugars, proteins, soluble solids, titratable acidity, ratio and moisture, and minerals calcium, magnesium, iron, phosphorus and potassium were analyzed. There were also analyzed antioxidants: Ascorbic acid, total phenols, total flavonoids, lycopene and β-carotene, besides antioxidant activity. For the genetic characterization, was used the molecular marker AFLP (Amplified Fragment Length Polymorphism). Fruits of five accessions of guava from Maranhão state (G01MA, G12MA, G14MA, G16MA and G20MA), and the accessions A08MA, A42PE, A45PE, A80RO and A100AM of Brazilian guava highlighted in the biochemical assessment by presenting, in general, the larger soluble solids, sugars, proteins and minerals, and are among those with lower levels of acidity, in the case of guavas, and higher in the case of Brazilian guava. As for antioxidants, it was found that guavas and Brazilian guavas are rich in phenolic compounds, which showed strong correlation with antioxidant activity, showing be your main contributor. It was also observed that part of accesses... (Complete abstract click electronic access below)

Made available in DSpace on 2014-06-11T19:27:15Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-06-28Bitstream added on 2014-06-13T19:55:37Z : No. of bitstreams: 1 fonzar_jf_me_botfmvz.pdf: 256476 bytes, checksum: dd03be98e81f06c668a8d913775d5bef (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Em função da ausência de dados na literatura veterinária consultada a respeito do ritmo de piscar espontâneo em cães adultos, o presente trabalho teve por objetivos determinar o ritmo de piscar espontâneo e os tempos de abertura e fechamento palpebrais. Tais parâmetros foram avaliados no estado de repouso e atenção, assim como se verificou a possibilidade de haver diferenças entre faixas de peso distintas. Foram estudados 93 animais, sendo 40 machos e 53 fêmeas, de diferentes raças, com idade média de 4,76 anos e com peso corporal médio de 20,26kg. Para a obtenção das imagens foi utilizada uma filmadora Sony modelo Digital 8 (DCR-TRV110) e as mesmas foram processadas pelo programa iMovie 2.0.1. O ritmo de piscar, o número de piscadas incompletas e completas, o tempo de abertura e fechamento palpebral são menores no estado de atenção. O número de piscadas incompletas foi maior do que o número de piscadas completas, bem como o tempo de abertura palpebral foi maior do que o tempo de fechamento palpebral. O grupo de cães com peso inferior a 10kg apresenta valores mais elevados do ritmo de piscar / Due to the lack of data in the veterinary literature consulted about the spontaneous blinking in adult dogs, this study aimed at determining the spontaneous blink rate and the eyelid opening and closing times. These parameters were assessed in the resting state and attention, and the possibility of differences between different weight ranges was verified. Ninety-three animals, 40 males and 53 females, of different races, with an average age of 4.76 years and mean body weight of 20.26 kg were studied. Images were taken with a Sony Digital 8 camcorder (DCR-TRV110), and have been processed by the program iMovie 2.0.1. The blink rate, the number of incomplete and complete blinks, eyelid opening and closing times are smaller in the state of attention. The number of incomplete blinks was greater than the number of complete blinks, as well as the eyelid opening time was greater than the eyelid closing time. The group of dogs weighing less than 10kg had higher values of the blink rate

Orientador: José Joaquim Titton Ranzani / Banca: Cláudia Valéria Seullner Brandão. / Banca: Silvana Artioli Schellini / Resumo: Em função da ausência de dados na literatura veterinária consultada a respeito do ritmo de piscar espontâneo em cães adultos, o presente trabalho teve por objetivos determinar o ritmo de piscar espontâneo e os tempos de abertura e fechamento palpebrais. Tais parâmetros foram avaliados no estado de repouso e atenção, assim como se verificou a possibilidade de haver diferenças entre faixas de peso distintas. Foram estudados 93 animais, sendo 40 machos e 53 fêmeas, de diferentes raças, com idade média de 4,76 anos e com peso corporal médio de 20,26kg. Para a obtenção das imagens foi utilizada uma filmadora Sony modelo Digital 8 (DCR-TRV110) e as mesmas foram processadas pelo programa iMovie 2.0.1. O ritmo de piscar, o número de piscadas incompletas e completas, o tempo de abertura e fechamento palpebral são menores no estado de atenção. O número de piscadas incompletas foi maior do que o número de piscadas completas, bem como o tempo de abertura palpebral foi maior do que o tempo de fechamento palpebral. O grupo de cães com peso inferior a 10kg apresenta valores mais elevados do ritmo de piscar / Abstract: Due to the lack of data in the veterinary literature consulted about the spontaneous blinking in adult dogs, this study aimed at determining the spontaneous blink rate and the eyelid opening and closing times. These parameters were assessed in the resting state and attention, and the possibility of differences between different weight ranges was verified. Ninety-three animals, 40 males and 53 females, of different races, with an average age of 4.76 years and mean body weight of 20.26 kg were studied. Images were taken with a Sony Digital 8 camcorder (DCR-TRV110), and have been processed by the program iMovie 2.0.1. The blink rate, the number of incomplete and complete blinks, eyelid opening and closing times are smaller in the state of attention. The number of incomplete blinks was greater than the number of complete blinks, as well as the eyelid opening time was greater than the eyelid closing time. The group of dogs weighing less than 10kg had higher values of the blink rate / Mestre

Dissertação (mestrado)—Universidade de Brasília, 2011. / Submitted by wiliam de oliveira aguiar (wiliam@bce.unb.br) on 2011-06-22T15:10:17Z No. of bitstreams: 1 2011_CinthiaBeatrizdaSilvaDumont.pdf: 2671980 bytes, checksum: e97a6843613e36eecd66b567682e8f97 (MD5) / Approved for entry into archive by Elna Araújo(elna@bce.unb.br) on 2011-06-22T19:11:50Z (GMT) No. of bitstreams: 1 2011_CinthiaBeatrizdaSilvaDumont.pdf: 2671980 bytes, checksum: e97a6843613e36eecd66b567682e8f97 (MD5) / Made available in DSpace on 2011-06-22T19:11:50Z (GMT). No. of bitstreams: 1 2011_CinthiaBeatrizdaSilvaDumont.pdf: 2671980 bytes, checksum: e97a6843613e36eecd66b567682e8f97 (MD5) / Este estudo foi dividido em três etapas, buscando o emprego de uma metodologia mais direcionada e organizada visando com isso a obtenção de resultados expressivos a respeito de equinos praticantes de enduro equestre. Na primeira etapa foram estabelecidos os parâmetros eletrocardiográficos de 20 equinos Puro Sangue Árabe em repouso e após exercício prolongado de enduro. Foi verificado durante o repouso que a frequência cardíaca média foi de 35,85bpm com predominância do ritmo sinusal e de 53,78bpm com taquicardia sinusal após o exercício. Como variação fisiológica observou-se marcapasso migratório e como não fisiológica, complexo ventricular prematuro. No pós-exercício ocorreu aumento da amplitude das ondas P, R, S e T, com onda P tendendo a se tornar única em 35% dos animais e bífida positiva em 65%, e a onda T monofásica positiva. Houve redução da duração das ondas, intervalos e segmentos, e alongamento do QTc, porém o complexo QRS quase não alterou. O eixo elétrico no plano frontal apresentou desvios à direita e à esquerda em repouso, sugestivo de aumento de câmara e hipertrofia secundárias ao treinamento, ao passo que após o exercício demonstrou desvio extremo para a direita indicando alterações eletrolíticas. O escore cardíaco médio foi de 128,45ms, caracterizando os animais como atletas, reduzindo a 118,60ms após o exercício. Já na segunda etapa, foram examinados por meio de exame eletrocardiográfico 14 equinos Puro Sangue Árabe desclassificados por exaustão em provas de enduro entre 60 e 160 km, imediatamente após a desclassificação, antes da instituição de qualquer tipo de tratamento. Houve predomínio de taquicardia sinusal, seguido de arritmia sinusal e complexo atrial prematuro com a frequência cardíaca variando de 48 a 78 bpm e 93 a 111 bpm, respectivamente. Ocorreu aumento da amplitude das ondas P, R, S e T e redução nas suas durações, bem como redução nos intervalos e segmentos, porém o complexo QRS quase não se alterou. O alongamento observado do QTc indicou fadiga miocárdica moderada em resposta ao exercício e o supradesnível ST foi indicativo de hipovolemia. O eixo elétrico no plano frontal apresentou desvio à direita, sugerindo aumento de câmara e hipertrofia secundárias ao treinamento. Por fim, na terceira etapa, foi avaliado o comportamento da osmolaridade, do ânion gap, do Ph e dos íons plasmáticos mensuráveis de 14 equinos, 09 machos e 05 fêmeas, Puro Sangue Árabe finalistas em provas de enduro de 90 km. Para tanto, foram colhidas em dois momentos, repouso e após o exercício prolongado, amostras de sangue venoso para a mensuração das variáveis de interesse. Diante dos achados pode-se observar a partir do repouso, aumento com diferença significante dos valores do Hct, das PPT e do AG, sugerindo perda de fluidos e discreto grau de desidratação. O Ph e o íon H+ quase não se alteraram, indicando ausência de distúrbios metabólicos. Reduções, com diferença siginificativas foram observadas para os íons Cl-, HCO3-, K+, Ca++, assim como do EB. Pode-se observar que os parâmetros avaliados permaneceram dentro dos valores de referência descritos na literatura consultada, somente com elevação discreta do Hct e do AG acima desses valores, sobretudo sem manifestações clínicas. _________________________________________________________________________________ ABSTRACT / This study was divided into three stages, aiming the use of a methodology more focused and organized, in order to obtain expressive results concerning equines submitted to endurance training. In the first stage we established the electrocardiographic parameters of 20 Purebred Arabian horses at rest and after prolonged endurance exercise. During rest, we verified that the average heart rate was 35.85 bpm with predominance of sinus rhythm, and 53.78 bpm with sinus tachycardia after exercise. As physiological variation, a wandering pacemaker was observed, while as a non physiological variation, a premature ventricular complex. In the post-exercise, there was an increase in the amplitude of waves P, R, S and T, with P-wave tending to become unique in 35% of the animals and bifid positive in 65%, meantime the T-wave was monophasic and positive in all animals. A reduction in the duration, intervals and segments of the waves was recorded, and lengthening of QTc, however the QRS complex has hardly changed. The electrical axis in frontal plane presented deviations to the right and left at rest, suggesting increased chamber, and hypertrophy secondary to training, whereas after the exercise, presented extreme deviation to the right, indicating electrolyte abnormalities. The average heart score was 128.45ms, characterizing animals as athletes, reducing to 118.60ms after exercise. In the second stage, 14 Purebred Arabian horses were examined through electrocardiographic examination. They were disqualified due to exhaustion in endurance races between 60 and 160 km, immediately after disqualification, before any kind of treatment. There was prevalence of sinus tachycardia, followed by sinus arrhythmia and atrial premature complex, with the heart rate ranging from 48 to 78 bpm, and 93 to 111 bpm, respectively. We also registered an increase in the amplitude of waves P, R, S and T, and decrease in their durations, as well as reduction in the intervals and segments, nevertheless, the QRS complex has hardly changed. The lengthening observed of QTc pointed a moderate myocardial fatigue in response to exercise, and the elevation of ST was indicative of hypovolemia. The electrical axis in frontal plane presented deviation to the right, chamber increase and hypertrophy secondary to training. Finally, in the third stage, we assessed the behavior of osmolarity, anion gap, pH, and measurable plasma ions of 14 Purebred Arabian horses, 09 males and 05 females, finalists in an endurance race of 90 km. To this end, venous blood samples were taken at two times, at rest and after prolonged exercise, in order to measure the interest variables. Considering those findings, one can see from the rest, an increase with significant difference in the values of Hct, PPT and AG, suggesting a loss of fluid and a slight degree of dehydration. The pH and the H+ ion almost had no changes, indicating absence of metabolic disorders. Reductions, with significant difference, were verified for the ions Cl-, HCO3-, K+, Ca++, as well as BE. The examined parameters remained within the reference values described in the literature, only with slight increase of Hct and AG above these values, but without clinical manifestations.

Since the publication of the book of Russell and Burch in 1959, scientific research has never stopped improving itself with regard to the important issue of animal experimentation. The European Directive 2010/63/EU “On the protection of animals used for scientific purposes” focuses mainly on the animal welfare, fixing the Russell and Burch’s 3Rs principles as the foundations of the document. In particular, the legislator clearly states the responsibility of the scientific community to improve the number of alternative methods to animal experimentation. The swine is considered a species of relevant interest for translational research and medicine due to its biological similarities with humans. The surgical community has, in fact, recognized the swine as an excellent model replicating the human cardiovascular system. There have been several wild-type and transgenic porcine models which were produced for biomedicine and translational research. Among these, the cardiovascular ones are the most represented. The continuous involvement of the porcine animal model in the biomedical research, as the continuous advances achieved using swine in translational medicine, support the need for alternative methods to animal experimentation involving pigs. The main purpose of the present work was to develop and characterize novel porcine alternative methods for cardiovascular translational biology/medicine. The work was mainly based on two different models: the first consisted in an ex vivo culture of porcine aortic cylinders and the second consisted in an in vitro culture of porcine aortic derived progenitor cells. Both the models were properly characterized and results indicated that they could be useful to the study of vascular biology. Nevertheless, both the models aim to reduce the use of experimental animals and to refine animal based-trials. In conclusion, the present research aims to be a small, but significant, contribution to the important and necessary field of study of alternative methods to animal experimentation.

Obiettivo del lavoro è stato lo sviluppo e la validazione di nuovi bioassay e biomarker quali strumenti da utilizzare in un approccio ecotossicologico integrato per il biomonitoraggio di ambienti marino-costieri interessati da impatto antropico negli organismi che vivono in tali ambienti. L’ambiente reale impiegato per l’applicazione in campo è la Rada di Augusta (Siracusa, Italia). Una batteria di bioassay in vivo e in vitro è stata indagata quale strumento di screening per la misura della tossicità dei sedimenti. La batteria selezionata ha dimostrato di possedere i requisiti necessari ad un applicazione di routine nel monitoraggio di ambienti marino costieri. L’approccio multimarker basato sull’impiego dell’organismo bioindicatore Mytilus galloprovincialis in esperimenti di traslocazione ha consentito di valutare il potenziale applicativo di nuovi biomarker citologici e molecolari di stress chimico parallelamente a biomarker standardizzati di danno genotossico ed esposizione a metalli pesanti. I mitili sono stati traslocati per 45 giorni nei siti di Brucoli (SR) e Rada di Augusta, rispettivamente sito di controllo e sito impattato. I risultati ottenuti supportano l’applicabilità delle alterazioni morfometriche dei granulociti quale biomarker di effetto, direttamente correlato allo stato di salute degli organismi che vivono in un dato ambiente. Il significativo incremento dell’area dei lisosomi osservato contestualmente potrebbe riflettere un incremento dei processi degradativi e dei processi autofagici. I dati sulla sensibilità in campo suggeriscono una valida applicazione della misura dell’attività di anidrasi carbonica in ghiandola digestiva come biomarker di stress in ambiente marino costiero. L’utilizzo delle due metodologie d’indagine (bioassay e biomarker) in un approccio ecotossicologico integrato al biomonitoraggio di ambienti marino-costieri offre uno strumento sensibile e specifico per la valutazione dell’esposizione ad inquinanti e del danno potenziale esercitato dagli inquinanti sugli organismi che vivono in un dato ambiente, permettendo interventi a breve termine e la messa a punto di adeguati programmi di gestione sostenibile dell’ambiente. / The aim of the work was the development and validation of new bioassays and biomarkers as tools in an integrated ecotoxicological approach for the biomonitoring of impacted coastal marine environment environments. The Rada of Agusta (Syracuse, Sicily) was used as real environment for the field application of the proposed integrated approach. A battery of in vivo and in vitro bioassays was investigated as screening tool of the assessment of marine sediment toxicity. The battery has proven to have the necessary requirement for a routine application in marine coastal environment biomonitoring. The multimarker approach based on the use of bioindicator organism Mytilus galloprovincialis in translocation experiments allowed to evaluate the field application potential of new cytological and molecular biomarkers in parallel to standardized biomarkers of genotoxicity and heavy metal exposure. Mussels were caged for 45 days in Brucoli (SR) and Rada di Augusta, reference site and impacted site respectively. Results support the applicability of granulocytes morphometric alterations as effect biomarker, directly correlated to the health of the organism. Morphometric alterations were accompanied by a significative increase of the lysosomal compartment, which in turn could reflect the pollutant induced increase of the degradative and autophagic processes. Carbonic anhydrase activity in digestive gland proved to be a valuable biomarker of chemical stress in marine coastal environment. The functional role of carbonic anhydrase in the lysosomal compartment functioning was evaluated. The combined use of the two methodologies (bioassays and biomarkers) in an integrated ecotoxicological approach provides a sensitive and specific tool for the assessment of pollutant exposure and pollutant effects in biomonitoring of coastal marine environment, facilitating the application of monitoring data in risk-based decision making

The study of optic flow on postural control may explain how self-motion perception contributes to postural stability in young males and females and how such function changes in the old falls risk population. Study I: The aim was to examine the optic flow effect on postural control in young people (n=24), using stabilometry and surface-electromyography. Subjects viewed expansion and contraction optic flow stimuli which were presented full field, in the foveral or in the peripheral visual field. Results showed that optic flow stimulation causes an asymmetry in postural balance and a different lateralization of postural control in men and women. Gender differences evoked by optic flow were found both in the muscle activity and in the prevalent direction of oscillation. The COP spatial variability was reduced during the view of peripheral stimuli which evoked a clustered prevalent direction of oscillation, while foveal and random stimuli induced non-distributed directions. Study II was aimed at investigating the age-related mechanisms of postural stability during the view of optic flow stimuli in young (n=17) and old (n=19) people, using stabilometry and kinematic. Results showed that old people showed a greater effort to maintain posture during the view of optic flow stimuli than the young. Elderly seems to use the head stabilization on trunk strategy. Visual stimuli evoke an excitatory input on postural muscles, but the stimulus structure produces different postural effects. Peripheral optic flow stabilizes postural sway, while random and foveal stimuli provoke larger sway variability similar to those evoked in baseline. Postural control uses different mechanisms within each leg to produce the appropriate postural response to interact with extrapersonal environment. Ageing reduce the effortlessness to stabilize posture during optic flow, suggesting a neuronal processing decline associated with difficulty integrating multi-sensory information of self-motion perception and increasing risk of falls.

La pèrdua de funció del sistema vestibular causa vertigen, pèrdua d’equilibri i pèrdua de mirada fixa durant el moviment, sovint acompanyats de marejos i nàusees. Els trastorns de l’equilibri són comuns a les persones grans i alteren el seu patró de marxa, sent un important factor de risc per a les caigudes. En els éssers humans, una de les causes de pèrdua de funció vestibular és la toxicitat d’alguns fàrmacs, com antibiòtics aminoglicòsids, medicaments antipalúdics, diürètics de nansa, agents quimioterapèutics com el cisplatí, així com una sèrie de productes químics d’exposició laboral. La diana principal de la toxicitat són les cèl·lules ciliades. Estudis d’exposició aguda en animals han demostrat clarament que els components ototòxics poden causar discapacitat permanent perquè causen la degeneració de moltes o totes les cèl·lules ciliades. Aquestes cèl·lules no poden regenerar o ho poden fer de manera limitada en algunes espècies de mamífers com el conill d’Índies i la xinxilla i en qualsevol cas la recuperació funcional és limitada o nul·la. En peixos, amfibis i ocells les cèl·lules ciliades es produeixen i es poden regenerar al llarg de tota la vida. Les noves cèl·lules ciliades procedeixen de les cèl·lules de suport seguides de dos possibles mecanismes: mitosis o transdiferenciació. Aquestes evidències d’estudis experimentals no serveixen per explicar la recuperació funcional que té lloc en els humans després de la interrupció de l’exposició crònica ototòxica. Quan en pacients a qui s’ha diagnosticat ototoxicitat es para el tractament, els resultats són molt variables: des de la persistència completa dels símptomes fins una recuperació incompleta o completa. Encara que la recuperació comportamental es pot explicar en part per fenòmens de compensació al sistema nerviós central, es coneix molt poc sobre els mecanismes cel·lulars i moleculars involucrats en el dany vestibular crònic i la seva possible recuperació, així como la seva rellevància a nivell comportamental. Per a estudiar els processos involucrats en el dany i reparació vestibulars associats amb la ototoxicitat crònica i la subsegüent recuperació, es van exposar rates Long Evans mascles a IDPN (3,3’-iminodipropionitrilo) via l’agua de beguda durant vàries setmanes i es van avaluar al final del període d’exposició i al final d’un període de recuperació. Per a identificar les etapes de dany que són susceptibles de reparació i que poden ser la base de la recuperació funcional, es van examinar les característiques ultraestructurals de les crestes i els utricles. La intoxicació crònica per IDPN provoca pèrdua de les cèl·lules ciliades per extrusió. Per a revelar més característiques ultraestructurals del dany ototòxic i la seva posterior recuperació, així com dels efectes ciliars, es van examinar les crestes per microscòpia electrònica de transmissió. Per a caracteritzar la dinàmica del dany aferent i del procés de reparació, es van estudiar mitjançant immunohistoquímica i microscòpia de fluorescència confocal l’expressió de proteïnes prèviament identificades en el contacte entre el calze aferent i la cèl·lula ciliada de tipus I. El següent pas va ser estudiar que passava amb les cintes de les sinapsis aferents de les cèl·lules ciliades de tipus I i de tipus II. El model d’ototoxicitat crònica induït per IDPN a l’aigua de beguda, causa retracció i fragmentació del calze aferent abans que tingui lloc la mort de les cèl·lules ciliades per extrusió. En aquest treball es presenten les evidències que el desmantellament de les unions septades és un procés anterior al dany aferent durant la ototoxicitat crònica i que l’extensa reparació de les unions septades i del calze aferent són possibles després d’aturar-se l’agressió. / Acute toxic insults to the mammalian vestibular system cause loss of the sensory hair cells and subsequent loss of equilibrium. In animal models, many studies have demonstrated apoptosis, sometimes necrosis, of the hair cells. In most mammalian species the functional loss is permanent because the hair cells do not regenerate. Human vestibular dysfunction most commonly arises progressively as a result of chronic exposure to ototoxic compounds such as the aminoglycoside antibiotics. If the exposure is discontinued, a highly variable degree of recovery is observed, being either complete, only partial, or minimal with persistent dysfunction. The cellular and molecular bases of the vestibular dysfunction associated with chronic toxicity and of its remarkable variability in the degree of recovery are not established. One reason for this limited knowledge is the scarcity of animal studies of chronic vestibular toxicity, owing to, at least in part, the resiliency of rats and mice to aminoglycoside ototoxicity. Ototoxic nitriles offer an alternative model to study vestibular toxicity in these species. A particularly dependable model is the one of chronic drinking water exposure of rats to 3,3’-iminodipropionitrile (IDPN). We have established a protocol to study the vestibular sensory epithelia simultaneously by scanning electron microscopy, transmission electron microscopy and confocal immunofluorescence microscopy in rats previously assessed for vestibular dysfunction during chronic ototoxic exposure followed by variable times of recovery. This allows identifying the structural and molecular events associated with the loss and recovery of vestibular function. Initial data indicate that a deep but fully reversible functional loss is associated with synaptic and afferent damage and intact ciliary structure. With continued IDPN exposure, hair cell damage and more persistent functional are observed. We hypothesize that similar events occur in humans and are relevant to chronic damage, including that associated with age.

Los ácidos araquidónico (AA), eicosapentaenoico (EPA) y linoleico (LA) son tres ácidos grasos poliinsaturados (PUFAs) que se encuentran formando parte de los fosfolípidos de las membranas celulares. El AA, EPA y LA, después de ser liberados por las fosfolipasas, pueden ser metabolizados por la vía de las ciclooxigenasas (COXs), de las lipoxigenasas (LOXs) y la del citocromo P450 (CYP450), que constituyen la cascada del AA. Los metabolitos que se generan son los eicosanoides y los ácidos hidroxioctadecanoicos (HODEs), todos ellos con efectos pleitrópicos que incluyen su efecto sobre la proliferación celular, sobre todo en tejidos de renovación rápida como el epitelio intestinal, por ello podrian estar involucrados en el cáncer y concretamente en el cáncer colorrectal (CRC). El CRC principalmente se origina por mutaciones en el epitelio colorrectal que dan lugar a una hiperproliferación celular que puede originar adenomas y finalmente CRC. En el 85% de los adenomas y CRC existe una sobrexpresión de las COXs. Por este motivo, el objetivo de este trabajo ha sido estudiar el papel de la cascada del AA en el control de la proliferación de células epiteliales intestinales humanas. Como modelo experimental se han utilizado células epiteliales intestinales no diferenciadas Caco-2 procedentes de un adenocarcinoma. Los cultivos de células Caco-2 se mantuvieron en condiciones estándar y finalmente se incubaron en las diferentes condiciones experimentales durante 48 horas. Los recuentos celulares se realizaron con microscopia de fluorescencia con tinción de naranja de acridina y bromuro de etidio; la síntesis de DNA por incorporación de timidina tritiada o BrdU; el estudio del ciclo celular por citometria de flujo; estudio de la degradación de DNA cromosómico con el método TUNEL; la determinación de eicosanoides por espectometria de masas; el análisis de activación de las vías de señalización por ELISA y la síntesis de PGE2 por inmunoensayo enzimático. Los resultados indican que las células Caco-2 sintetizan prostaglandina E2 (PGE2), leucotrieno B4 (LTB4) y los ácidos hidroxieicosatetraenoicos 5-HETE, 12-HETE y 15-HETE. Estos eicosanoides, junto con LTD4 y 13-R-HODE, inducen el crecimiento celular y la síntesis de ADN. Estos efectos son consecuencia de la interacción de la PGE2 con los receptores EP1 y EP4, el LTB4 con los receptores BLT1 y BLT2 y el LTD4 con el receptor Cis-LT1R. Hasta el momento no se han identificado receptores celulares para la mayoría de HETEs y HODEs , en esta tesis observamos que el 13-R-HODE interacciona con BLT1 y BLT2 y el 12-HETE con BLT2. Además, cuando se unen a sus receptores activan diferentes vías de señalización implicadas en la regulación de la proliferación celular tales como ERK, p38, CREB, GSK-3β y β-catenina. Cabe destacar que los dos enantiómeros del 13-HODE presentan efectos contrarios. Así, mientras que el 13-R-HODE presenta un efecto proliferativo, el 13-S-HODE inhibe el crecimiento e induce apoptosis al unirse a PPARγ. En relación a los derivados del EPA, tanto la PGE3 como el 12-HEPE inducen la proliferación de las células Caco-2 de forma similar a la PGE2 y al 12-HETE, mientras que el LTB5 no es mitogénico. Finalmente, en este trabajo se propone que el efecto proliferativo de LTB4, LTD4 ,12-HETE, y 13-R-HODE podría deberse principalmente a la activación de la vía COXs y la subsecuente síntesis de PGE2, que a su vez activaría múltiples vías de señalización implicadas en la proliferación celular. Además estos estudios proporcionan nuevas evidencias sobre el papel de diversos eicosanoides y sus receptores en el control del crecimiento de las células epiteliales intestinales. Resultados que pueden ser considerados en futuras investigaciones sobre el CRC y en el planteamiento de su tratamiento. / Arachidonic acid (AA), eicosapentaenoic acid (EPA) and linoleic acid (LA) are three polyunsaturated fatty acids (PUFAs) which are part of the phospholipids of cell membranes. After being released, all of them are metabolized by cyclooxygenase (COXs), lipoxygenase (LOXs) and cytochrome P450 (CYP450) pathways that form the AA cascade. The metabolites generated are eicosanoids and hidroxioctadecanoicos acids (HODEs), all of them with pleiotropic effects. One function that these metabolites regulate is cell proliferation and, as a result, they may be involved in cancer and particularly in colorectal cancer (CRC). Therefore, the objective of this work is to study the role of AA cascade in controlling the proliferation of human intestinal epithelial cells. We have used undifferentiated intestinal epithelial cells Caco-2 derived from an adenocarcinoma, as the experimental model. The results indicate that the Caco-2 cells synthesize PGE2, LTB4 5-HETE, 12-HETE and 15-HETE. These eicosanoids, together with LTD4 and 13-R-HODE, induce cell growth and DNA synthesis. These effects are due to the interaction of PGE2 with EP1 and EP4 receptors, LTB4 and 13-R-HODE with BLT1 and BLT2 receptors, 12-HETE with BLT2 and LTD4 with Cys- LT1R receptor. Also, when they bind to their receptors, they activate different signaling pathways involved in the regulation of Caco-2 cell proliferation. Note that the two enantiomers of 13-HODE have opposite effects. Thus, while 13-R-HODE has a proliferative effect, 13-S-HODE inhibits growth and induces apoptosis by binding to PPARγ. Regarding the metabolites derived from EPA, both PGE3 and 12-HEPE induce cell proliferation similar to PGE2 and 12-HETE, whereas LTB5 is not mitogenic. Finally, this Thesis proposes that the proliferative effect of LTB4, LTD4, 12-HETE, and 13-R-HODE could be due mainly to the activation of the COXs pathway and subsequent synthesis of PGE2 which in turn activate multiple signaling pathways involved in cell proliferation. Taken together, these studies provide new evidence that could be considered in future research on the role of eicosanoids in the pathogenesis of CRC as well as in the development of new pharmacological strategies.