La transmission sexuelle du VIH-1 constitue le mode majeur de contamination à travers le monde. Au cours d’un rapport sexuel avec un homme séropositif vis-à-vis du VIH-1, le virus transporté par le sperme est déposé à la surface des muqueuses sous forme libre et/ou associée aux cellules mononuclées du sperme. Les particules virales peuvent alors être captées par les cellules dendritiques qui les transportent jusqu’aux lymphocytes T CD4 des ganglions lymphatiques favorisant ainsi la dissémination du virus. Plus qu’un simple véhicule, il semblerait que le plasma séminal, fraction acellulaire du sperme, puisse influencer la transmission sexuelle du VIH-1. Cependant, son rôle en tant que facilitateur ou inhibiteur de l’infection est largement débattu. La première partie de cette thèse s’intéresse à l’influence directe du plasma séminal sur la transmission sexuelle du VIH-1 via les cellules dendritiques. Nous avons montré que le TGF-β1, une des cytokines majoritaires du sperme, favorise la capture du virus par les cellules dendritiques via l’induction de l’expression de CD169, un récepteur du VIH-1 récemment décrit. Cependant, ni l’augmentation de la capture du virus ni celle de l’expression de CD169 ne sont observées lorsque les cellules sont en présence de plasma séminal. Au contraire, le fluide semble avoir tendance à diminuer la capture des particules virales et s’avère même être capable de diminuer l’induction de l’expression de CD169 par le TGF-β1 ou le LPS, suggérant un effet protecteur du plasma séminal sur la transmission sexuelle du VIH-1, même en cas de co-infection bactérienne localisée. La seconde partie de cette thèse s’intéresse à l’effet indirect du plasma séminal sur les cellules dendritiques via l’induction de la sécrétion de facteurs par les cellules épithéliales de la muqueuse, premières cellules en contact avec le fluide. A partir de deux modèles in vitro d’épithéliums simples (colorectaux et endocervicaux), les résultats obtenus montrent que les sécrétions des cellules épithéliales induites par le plasma séminal ne provoquent pas l’expression de CD169 ni la maturation des cellules dendritiques. Ces résultats contribuent à une meilleure compréhension des mécanismes précoces impliqués dans la transmission sexuelle du VIH-1 et soulignent le rôle complexe du plasma séminal dans cette dernière / Sexual transmission of Human Immunodeficiency Virus type 1 (HIV-1) remains the major way of contamination worldwide. During male to female or male transmission, the virus is deposited on the mucosal surface as cell-free or cell-associated virions carried by semen. The virions can be captured by dendritic cells (DCs) located in the mucosae. DCs play a crucial role in disseminating virus by capturing virions at the contact site and bringing them to the principal target cells in the lymph nodes i.e. the lymphocytes. More than just a carrier for viral particles, semen may also play a role in modulating HIV-1 sexual transmission. The influence of seminal plasma, the acellular fraction of semen is particularly debated. In the first part of this thesis deals with the direct influence of seminal plasma on the HIV-1 sexual transmission mediated by dendritic cells. We demonstrated that TGF-β1, a cytokine present in high concentration in seminal plasma, increases HIV-1 capture by dendritic cells through the upregulation of CD169, an HIV-1 binding molecule recently described. However, these effects are not observed when dendritic cells are incubated with seminal plasma. On the contrary, seminal plasma tends toward a decrease of HIV-1 capture by dendritic cells. Furthermore, seminal plasma is able to counteract the CD169 upregulation observed following TGF-β1 or LPS exposure indicating that seminal plasma may display a protective effect against HIV-1 transmission in the case of bacterial local coinfection. The second part of this thesis deals with the indirect effect of seminal plasma on dendritic cells through the induction of soluble factors secretion by epithelial cells, first cells in contact with the contaminated fluid. Two different epithelial cell models mimicking the female genital and intestinal tracts by using well characterized epithelial cell lines (HEC-1A and CaCo-2 respectively) have been developed. Results demonstrated that the epithelial cells secretions induced by seminal plasma do not modulate CD169 expression nor induce dendritic cells maturation. All these data contribute to a better understanding of the various mechanisms allowing HIV-1 sexual transmission and underscore the complex role of the seminal plasma in the particular case of the transmission mediated by dendritic cells

Submitted by Adriana Martinez (amartinez@unoeste.br) on 2017-06-01T14:42:01Z No. of bitstreams: 1 Isamara.pdf: 1900151 bytes, checksum: bf2306ee5b36c025c99dadb0b3ed1c84 (MD5) / Made available in DSpace on 2017-06-01T14:42:01Z (GMT). No. of bitstreams: 1 Isamara.pdf: 1900151 bytes, checksum: bf2306ee5b36c025c99dadb0b3ed1c84 (MD5) Previous issue date: 2017-03-17 / The objective of this study was to study the influence of magnetized water intake on body weight gain, body morphometry and reproductive system, semen characteristics and body and scrotal area temperatures with digital infrared thermography in young Nellore bulls in extensive management and In confinement. Twenty bulls with initial age of 14 months were used, divided into two groups: Treated - water ingestion treated by magnetic field; And Control - drinking water intake. Four collections were made, 2 in extensive management (14 and 18 months of age) and 2 in confinement (20 and 21 months of age), in which they were measured: climatic factors, rectal temperature, body weight, wither height, Length, width and height of the testicles, semen collection and infrared digital thermography of the eyeball, mufla, flank, ischium and scrotum. It was concluded that the intake of water treated by magnetic field was not efficient in relation to weight gain in pasture and in confinement, as well as for body morphometry and reproductive system. In confinement, there was also no influence of water treated by magnetic field on the quantitative and qualitative characteristics of the semen. Digital infrared thermography proved to be effective for measuring body and scrotal areas, where the grazing group treated with magnetic field water showed higher temperatures for the ocular globe, mufla, flank and ischium. For the scrotal pouch, the pasture, the temperature of the spermatic cord and testis were higher in the treated group; And in confinement, the temperature of the testes and tail of the epididymis were higher in the group with drinking water intake. / Objetivou-se estudar a influência da ingestão da água magnetizada no ganho de peso corpóreo, morfometria corpórea e do aparelho reprodutor, características do sêmen e temperaturas de áreas do corpo e escroto com termografia digital de infravermelho em touros jovens da raça Nelore em manejo extensivo e em confinamento. Foram utilizados 20 touros com idade inicial de 14 meses, divididos em dois grupos: Tratado - ingestão de água tratada por campo magnético; e Controle - ingestão de água potável. Foram realizadas 4 coletas sendo 2 em manejo extensivo (14 e 18 meses de idade) e 2 em confinamento (20 e 21 meses de idade), nas quais foram aferidos: fatores climáticos, temperatura retal, peso corpóreo, altura de cernelha, mensuração do comprimento, largura e altura dos testículos, colheita de sêmen e termografia digital por infravermelho do globo ocular, mufla, flanco, ísquio e escroto. Conclui-se que a ingestão da água tratada por campo magnético não se apresentou eficiente em relação ao ganho de peso a pasto e em confinamento, assim como para morfometria corpórea e do aparelho reprodutor. Em confinamento, também não houve influência da água tratada por campo magnético sobre as características quantitativas e qualitativas do sêmen. A termografia digital por infravermelho se mostrou eficaz para mensuração das áreas do corpo e da bolsa escrotal, onde a pasto o grupo com ingestão de água tratada por campo magnético apresentou temperaturas superiores para globo ocular, mufla, flanco e ísquios. Para bolsa escrotal, a pasto, a temperatura do cordão espermático e testículo foram superiores no grupo tratado; e em confinamento, a temperatura do testículo e cauda dos epidídimos foram superiores no grupo com ingestão de água potável.

Previously, we described prostate cancer (PCa) detection (83% sensitivity; 67% specificity) in seminal plasma by CE-MS/MS. Moreover, advanced disease was distinguished from organ-confined tumors with 80% sensitivity and 82% specificity. The discovered biomarkers were naturally occurring fragments of larger seminal proteins, predominantly semenogelin 1 and 2, representing endpoints of the ejaculate liquefaction. Here we identified proteases putatively involved in PCa specific protein cleavage, and examined gene expression and tissue protein levels, jointly with cell localization in normal prostate (nP), benign prostate hyperplasia (BPH), seminal vesicles and PCa using qPCR, Western blotting and confocal laser scanning microscopy. We found differential gene expression of chymase (CMA1), matrix metalloproteinases (MMP3, MMP7), and upregulation of MMP14 and tissue inhibitors (TIMP1 and TIMP2) in BPH. In contrast tissue protein levels of MMP14 were downregulated in PCa. MMP3/TIMP1 and MMP7/TIMP1 ratios were decreased in BPH. In seminal vesicles, we found low-level expression of most proteases and, interestingly, we also detected TIMP1 and low levels of TIMP2. We conclude that MMP3 and MMP7 activity is different in PCa compared to BPH due to fine regulation by their inhibitor TIMP1. Our findings support the concept of seminal plasma biomarkers as non-invasive tool for PCa detection and risk stratification.

Les cellules dendritiques présentes dans les muqueuses comme les cellules de Langerhans, sont capables de présenter le Virus d'Immunodéficience Humaine (VIH) aux lymphocytes T CD4+, aboutissant à l'infection par le VIH lors de contacts hétérosexuels non protégés. Le recrutement des cellules de Langerhans dans la muqueuse vaginale est assuré par la chimiokine Quimiocin C-C motif ligant 20 (CCL20) produite par les cellules épithéliales de la muqueuse. L'objectif de ce travail a été d’étudier la capacité du plasma séminal, de la salive et de la sécrétion vaginale à stimuler la production de CCL20 par les cellules de l’épithélium endocervical (cellules HEC-1A), et de corréler cette production avec la présence de la lactoferrine (LF) et de ses produits de clivage dans ces différents fluides biologiques. Des cellules HEC-1A ont été cultivées avec les plasmas séminaux, les salives et les sécrétions vaginales de patients séronégatifs et séropositifs pour le VIH, et la production de CCL20 a été mesurée dans les surnageants cellulaires en ELISA. Le dosage de la Lf par ELISA a été réalisé sur tous les échantillons. La présence d’un petide de clivage de la Lf (G-12-I) a été analysée en western blot. Les résultats obtenus montrent que les plasmas séminaux des patients séropositifs ont une plus grande capacité à stimuler la sécrétion de CCL20 que les patients séronégatifs pour le VIH. La concentration de la Lf dans le plasma séminal est corrélée à la stimulation de cette sécrétion par les cellules HEC-1A. Il semble que les échantillons ayant la plus grande capacité à stimuler la sécrétion de CCL20 possèdent une plus forte concentration en petide de clivage de la Lf (G-12-I), bien que ce résultat, obtenu en western blot. La salive est également capable de stimuler de la production de CCL20 par les cellules HEC-1A, mais cette stimulation n'est pas corrélée avec sa concentration en Lf. Nous concluons que la sécrétion de CCL20 par l’épithélium génital féminin induite par le plasma séminal est corrélée à la présence de la Lf dans ce fluide. Bien que les mécanismes puissent être différents, la salive peut également induire la production de CCL20 par les cellules épithéliales de la muqueuse génitale féminine / Dendritic cells, among them Langerhans cells, are recognized as presenters of Human Immunodeficiency Virus (HIV) to CD4 + T lymphocytes. The recruitment of Langerhans cells in the vaginal mucosa is modulated by Chemokine CC motif ligand 20 (CCL20) produced by epithelial cells of the female genital mucosa. The aim of this study was to evaluated the capacity of seminal plasma, saliva and vaginal secretions to induce the production of CCL20 by monostratified endocervical epithelium cells culture (HEC-1A), also, the relation of this production with the presence of lactoferrin and its cleavage products (G- 12-I) in seminal plasma and saliva. Cultures of HEC-1A cells were stimulated with seminal plasma, saliva and vaginal secretions of HIV-seronegative and HIV-seropositive participants, and the CCL20 production measured by enzyme linked immunosorbent assay (ELISA) in its cellular supernatant. ELISA for Lf was performed on all samples, and the peptide cleavage Lf (G-12-I) observed in the samples which more and less induced the CCL20 secretion on western blot. We observed that the seminal plasma of seropositive participants were responsible for a higher stimulation of CCL20 production by HEC-1A cells, when compared to the seminal plasma of HIV-seronegative participants. Lf concentration in seminal plasma was positively related to the CCL20 production by HEC-1A cells, the higher production of CCL20 was also more associated with Lf peptide cleavage Lf (G-12-I), on western blot. Saliva stimulated CCL20 production by HEC-1A cells, and such stimulation was not correlated to Lf concentration. In conclusion, the presence of Lf in seminal plasma positively related to the production of CCL20 by HEC-1A cells, and saliva may induce the production of CCL20 in the female genital mucosa / As células dentríticas, dentre elas as células de Langerhans, são capazes de apresentar o vírus da imunodeficiência humana (HIV) aos linfócitos T CD4+, cujo processo culmina com a infecção por esse vírus. O recrutamento das células de Langerhans na mucosa vaginal é modulada pela Quimiocin C-C motif ligant 20 (CCL20), produzida pelas células epiteliais da mucosa genital feminina. O objetivo desse trabalho foi estudar a capacidade do plasma seminal, da saliva e do conteúdo vaginal de induzir a produção de CCL20 pela cultura de células do epitélio monoestratificado endocervical (HEC-1A), relacionando tal produção com a presença da lactoferrina (Lf) e de seu produto de clivagem (G-12-I) nestes fluidos. Culturas de células HEC-1A foram estimuladas com plasmas seminais, salivas e conteúdos vaginais de participantes soronegativos e soropositivos para o HIV, visando avaliar a produção de CCL20, a qual foi mensurada utilizando-se o Enzyme Linked Immunosorbent Assay (ELISA). A dosagem de Lf (ELISA) foi realizada em todas as amostras estudadas e a presença do peptídeo de clivagem da Lf (G-12-I) observada nas amostras que mais e que menos induziram a secreção de CCL20 pelas células HEC-1A por Western blot. Verificou-se que os plasmas seminais de participantes soropositivos foram responsáveis por maior estimulação da produção de CCL20 pelas células HEC-1A, quando comparados aos plasmas seminais de participantes soronegativos para o HIV. A concentração de Lf no plasma seminal associou-se à indução na produção de CCL20 pelas células HEC-1A, assim como as amostras que mais induziram a produção de CCL20 apresentaram presenças mais evidentes do peptídeo de clivagem da Lf (G-12-I). A saliva foi responsável pela estimulação na produção de CCL20 pelas células HEC-1A, no entanto, tal estimulação não se associou à concentração de Lf salivar. Concluímos que a presença de Lf no plasma seminal esteve correlacionada à produção de CCL20 pelas células HEC-1A, e que a saliva pode induzir a produção da CCL20 pelas células epiteliais endocervicais

Submitted by Edisangela Bastos (edisangela@ufpa.br) on 2014-06-09T18:26:34Z No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_EletroejaculacaoCaititusCaracteristicas.pdf: 2929601 bytes, checksum: 37f332b144d3af9572950f1922dcbe72 (MD5) / Approved for entry into archive by Ana Rosa Silva (arosa@ufpa.br) on 2014-07-02T12:50:20Z (GMT) No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_EletroejaculacaoCaititusCaracteristicas.pdf: 2929601 bytes, checksum: 37f332b144d3af9572950f1922dcbe72 (MD5) / Made available in DSpace on 2014-07-02T12:50:20Z (GMT). No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_EletroejaculacaoCaititusCaracteristicas.pdf: 2929601 bytes, checksum: 37f332b144d3af9572950f1922dcbe72 (MD5) Previous issue date: 2009 / CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico / Para implementar um sistema de criação de Tayassu tajacu é necessário estudar sua biologia reprodutiva, principalmente os aspectos referentes aos machos. O objetivo do trabalho foi testar a eficiência da eletroejaculação para obtenção de sêmen, descrever o perfil seminal e testar a capacidade de conservação de gametas masculinos sob refrigeração. Foram selecionados 11 machos adultos (76,8±37,8 meses, 19,5±2,7 kg) para a avaliação da biometria testicular, e 8 deles foram submetidos à eletroejaculação (45±13 estímulos, 12 volts, 3 segundos/pulso e descanso de 3 segundos). As amostras colhidas foram avaliadas quanto às características físicas (aspecto, cor, volume total, concentração, pH, motilidade e vigor espermáticos) e características morfológicas (viabilidade e defeitos espermáticos). As amostras foram divididas e diluídas em BTS (diluidor de curta duração) ou X-Cell® (longa duração) e mantidas sob refrigeração a 17º C. As amostras foram avaliadas imediatamente após a diluição (T0), após 24 horas (T24) e 48 horas (T48) de resfriamento. Os animais apresentaram testículo esquerdo com comprimento de 3,8±0,4 cm, largura de 2,6±0,3 cm e consistência de 2,3±0,2, e testículo direito com 3,8±0,5 cm de comprimento, 2,7±0,3 cm de largura e 2,3±0,2 de consistência. A taxa de sucesso obtida com a eletroejaculação foi de 75,21%. O sêmen apresentou: volume 0,81±0,86 mL, concentração 137,44±153 x 106 sptz/mL, pH 7,92±0,73, motilidade 52,66±28,79%, vigor 2,2±0,8, viabilidade 55,84±28,55%, defeitos maiores 22,87±12,93%, defeitos menores 9,11±5,88% e defeitos totais 31,52±13,81%. Não houve variações expressivas na produção e qualidade seminal ao longo dos meses do ano. Não houve diferença significativa entre os diluidores testados, com relação à capacidade de manutenção dos parâmetros seminais durante o período de estocagem, havendo influência significativa do tempo de armazenamento (P<0,05). Nos tempos T0, T24 e T48, o sêmen diluído em BTS apresentou motilidade espermática média de 56,1±17,6 %, 17,9±23,3 % e 7,7 ±19,6%, a viabilidade espermática média de 60,2±17,2%, 27,9±23,8% e 11,7±20,2%, e defeitos totais médios de 28,1±10,8%, 37,5±12,1% e 41,28±11,4%., respectivamente. Amostras seminais diluídas em X-Cell® apresentaram motilidade média de 54,2±22,1%, 27,0±28,0% e 14,0±24,2%, viabilidade média de 57,8±21,7%, 33,0±26,9% e 19,9±25,6% e defeitos totais médios de 30,8±11,2%, 41,2±13,4% e 44,2±14,4% nos três tempos avaliados. Assim, a eletroejaculação é eficiente e segura para a obtenção de ejaculados de caititus, o sêmen produzido possui potencial para estocagem, e pode ser conservado sob 17ºC até 24 horas. / Studies concerning reproductive biology of collared peccary (Tayassu tajacu) are worthy to improve creation system. The aims of this research were testing electroejaculation as safety method to collect semen; describing the seminal profile along the year; and testing the capacity for spermatozoa storage under refrigeration. Eleven adults males (76.8±37.8 months, 19.5±2.7 kg) were selected for the measurements of testicular length and width. Eight adults males were used to semen collection (45±13 stimuli, 12 volt, 3 seconds; and resting pulse of 3 seconds). Semen samples were evaluated for physic characteristics (appearance, color, volume, sperm concentration, pH, sperm motility and vigor), and morphological characteristics (sperm viability and sperm defects). Semen samples were split and diluted in two different extenders: BTS (short-term storage) or X-Cell ® (long-term storage). Diluted semen samples were maintained under refrigeration at 17ºC. The samples were evaluated immediately after dilution (T0), after 24 hours (T24) and 48 hours (T48) of cooling. Animals presented 3.8±0.4 cm on left testicles length, 2.6±0.3 cm on left testicles width and left testicles consistency of 2.3±0.2; right testicles presented 3.8±0.5 cm length, 2.7±0.3 cm width and 2.3±0.2 for consistency. The success rate obtained with electroejaculation was 75.21%. The seminal features observed were: volume 0.81±0.86 mL, concentration 137.44±153 x 106 sptz/mL, pH 7.92±0.73, motility 52.66±28.79%, vigor 2.2±0.8, viability 55.84±28.55%, primary abnormalities 22.87±12.93%, secondary abnormalities 9.11±5.88% and total abnormalities 31.52±13.81%. There were no significant changes in production and quality semen along the months of the year. There was no significant difference between extenders tested concerning to maintenance capacity of seminal parameters during the storage period of storage; however, significant influence of storage time was observed (P<0.05). Diluted semen in BTS presented at T0, T24 and T48, average sperm motility of 56.1±17.6%, 17.9±23.3% and 7.7±19.6%, the average sperm viability was 60.2±17.2%, 27.9±23.8% and 11.7±20.2%, and the average total defects were 28.1±10.8%, 37.5±12.1% and 41.28±11.4%, respectively. Semen samples diluted in X-Cell® presented at T0, T24 and T48 average motility of 54.2±22.1%, 27.0±28.0% and 14.0±24.2%, average viability of 57.8±21.7%, 33.0±26.9% and 19.9±25.6% and total defects were 30.8±11.2%, 41.2±13.4% and 44.2±14.4%. Thus, electroejaculation is an effective and safe method to obtain seminal samples from collared peccary, the semen of collared peccary has potential for storage and can be maintained under 17º C up to 24 hours.

Information on the biosynthetic origin and functional advantage of marine mollusc natural products is not only essential to our understanding of chemical ecology, but to the development and responsible production of therapeutic agents. As demonstrating in situ activity is methodologically hindered, functions inferred by in vitro activity have been assumed for many secondary metabolites. The anatomical and ontogenetic distribution of natural products can not only provide information on the biosynthesis and storage of metabolites, but identify selective pressures likely to affect survivorship at a specific life stage. Thus, dissection and chemical analysis of distinct tissues, in combination with histochemistry may offer a valuable approach. Marine gastropods of the Muricidae are renowned for the ancient dye Tyrian purple, which evolves from choline esters of bromoindoxyl sulphate in the hypobranchial gland through a series of enzymatic and photo-oxidative reactions. Prochromogen hydrolysis by arylsulphatase liberates neuromuscular active choline esters and cytotoxic bromoindole precursors, which also occur in muricid egg masses. Although visual accounts of dye pigments in the muricid gonoduct suggest precursors may be incorporated into egg masses from a maternal source, their biosynthetic origin and the evolutionary significance of the hypobranchial gland is unknown. Thus, the Muricidae, and in particular Dicathais orbita upon which most previous research has been focused, is an ideal model for this novel approach to natural product research. To confirm observations of dye pigments in muricid gonoducts and gain an understanding of their anatomical distribution, a liquid chromatography-mass spectrometry (LC-MS) method was developed to simultaneously quantify pigments, precursors and the prochromogen, tyrindoxyl sulfate. The prochromogen was not only detected in albumen and capsule gland extracts, but bioactive intermediates and the dye 6,6’-dibromoindigo were also present in the latter. These findings provided preliminary evidence for the maternal provision of prochromogens in egg masses of D. orbita and identified regions within which to conduct histochemical investigations. Tyrindoxyl sulphate was also detected in male prostate gland extracts, along with the dibromoindigo isomer, 6,6’-dibromoindirubin and its oxidative precursor, 6-bromoisatin. This not only implies physiological differences exist between male and female gonoducts, but that these secondary metabolites are not solely intended for egg masses and may hold significance throughout the life cycle. Histomorphological inspection of the pallial gonoduct-hypobranchial gland complex was conducted over the annual cycle to determine a mechanism for precursor transfer between these structures. Although an anatomical connection was not detected, the secretions of two hypobranchial cell types thought to be involved in Tyrian purple synthesis were of remarkable biochemical similarity to those of various capsule and albumen gland lobes. Together these findings implied the potential for natural product synthesis within the pallial gonoduct of D. orbita. To establish the role of these glandular lobes in the incorporation of intracapsular fluid and capsule laminae, identical histochemical techniques were applied to transverse capsule wall sections. Biochemical correlations not only provided a simple method of deciphering the complex process of encapsulation in neogastropods, but effectively identified the destination of gonoduct secretions in egg capsules of D. orbita. Comparisons of capsule and gonoduct biochemistry revealed that the intracapsular fluid and inner capsule wall are secreted by the posterior capsule gland lobe, the middle lamina by the lateral lobes and the outer layers by the dorsal lobe, albumen and pedal glands. Investigation into the location of regulatory enzymes and precursors was conducted to establish the biosynthetic origin of Tyrian purple prochromogens and mechanisms governing bioactive precursor synthesis. Novel histochemical techniques for the localization of bromoperoxidase, the enzyme thought to facilitate prochromogen bromination, and tyrindoxyl sulphate were developed and applied to gonoduct, hypobranchial gland, and encapsulated larvae sections. Standard staining reactions for the indole precursor, tryptophan, and arylsulphatase were also applied. The histochemical approach adopted revealed that tyrindoxyl sulphate is de novo biosynthesized through the post-translational bromination of dietary derived tryptophan. Two biosynthetic sites were identified, one related to hypobranchial secondary metabolism and the second of significance to the presence of bioactive precursors in muricid egg masses. Tryptophan is stored within secretory cells of the lateral hypobranchial epithelium and once exocytosed, is united with bromoperoxidase from supportive cells to form tyrindoxyl sulphate. Prochromogen synthesis also occurs in the subepithelial vascular sinus for storage and secretion by medial hypobranchial secretory cells. Bioactive precursor synthesis on the epithelial surface is regulated by the liberation of arylsulphatase from adjacent supportive cells. These findings not only provide evidence for de novo biosynthesis of Tyrian purple precursors, but are first account of natural product biosynthesis within the gastropod hypobranchial gland. Together these findings imply a naturally selected function for the synthesis of bioactive indoles in hypobranchial gland secretions of the Muricidae and Gastropoda. Tyrindoxyl sulphate is also transported within the vascular sinus to lateral and dorsal capsule gland lobes where bromoperoxidase and arylsulphatase also occur. Arylsulphatase was also detected within the albumen gland, which along with the posterior capsule gland lobe, acts as a storage site for dietary tryptophan. Thus, tyrindoxyl sulphate and the constituents for prochromogen and precursor biosynthesis are introduced to intracapsular fluid and capsule laminae by the capsule gland. Histochemistry in combination with LC-MS revealed an identical biosynthetic profile within larval vitellus, which is elaborated during oogenesis and may also receive secretions from the albumen gland. Due to the absence of a hypobranchial gland in veligers, it appears that pelagic larvae rely on vitelline natural products until settlement and metamorphous. These findings together with the in situ antimicrobial activity of bromoindoles suggest Tyrian purple precursors are incorporated into muricid egg masses as a maternal investment in larval defence against pathogens. The results of this investigation clearly highlight the benefits of adopting a histochemical approach to natural product research. This novel alternative to radioisotopes and in situ demonstration of bioactivity, can not only aid in the elucidation of secondary metabolic pathways and chemically mediated interactions, but identify mechanisms of metabolite regulation and differentiate between biosynthetic and storage tissues. Apart from providing insight into the ecological significance of muricid secondary metabolites, the biosynthetic information provided is valuable to our understanding of chemical phylogeny and biosynthetic enzyme sequencing for the environmentally sound development of natural products as biomedical agents.

International trade ties the world together and is hypothetically fair and equal. In reality, it is highly asymmetrical and poses a significant challenge for developing countries. A massive sea change occurred in the international trade regime during the Uruguay Round of negotiations of the General Agreement on Tariffs and Trade (GATT) from 1986 to 1994. Developing countries as a whole began to embrace liberal trade policies which seemed to be the only alternative to failing import substitution industrialization (ISI). An historical comparative account describing and explaining this transformation of developing countries’ attitudes toward the GATT is used in this dissertation to provide an alternative explanation for the transition of developing countries from having little interest in the Uruguay Round of GATT negotiations to sharply changing course and adopting neo-liberal policies which supported the conclusion of the Round. Three theoretical approaches seek to explain why this change occurred, including: liberal trade theory (economic reforms), dependency theory (external forces) and constructivism (the role epistemic communities). The Uruguay Round negotiations were dynamic and heavily influenced by two power-house developing economies, India and Brazil, who were initially opposed to the Round itself. Kenya found itself in a starkly different situation with minimal ability to participate or influence negotiations. These three countries constitute the study’s illustrative case studies. As negotiations progressed, India and Brazil changed course and agreed to the Round’s ‘single-undertaking’ and the ‘inequitable Grand Bargain’ between the developed and developing economies. This subsequently led to other developing countries following suit through a powerful demonstration effect in a trade-off between the inclusion of trade in services and intellectual property for reforms in agriculture and textiles & clothing. While economic reforms began to occur and attitudes began to change during the Uruguay Round itself, assessing developing countries during the Round found that no single theoretical approach can explain developing countries’ transformation; rather each had their own trajectory for their economic reforms. A multi-dimensional conclusion provides the most comprehensive account of this transformation of the global trade regime.

Tesis por compendio / [ES] La longevidad de las semillas, o el tiempo durante el cual permanecen las semillas viables, es de gran importancia para la conservación de la biodiversidad, la agricultura y la economía. Además, el estudio de este parámetro puede contribuir a conocer mejor los mecanismos moleculares comunes a todos los organismos para prevenir el envejecimiento. Una de las principales estrategias de las semillas para ralentizar su envejecimiento consiste en detener su metabolismo, a través de su deshidratación. Otros mecanismos moleculares para evitar daños son el aislamiento frente al entorno a través de la cubierta de la semilla, y la producción de antioxidantes y otras moléculas para evitar el daño oxidativo, uno de los principales causantes del envejecimiento de las semillas. Los mecanismos de reparación mitigan parte del daño acumulado. El organismo modelo de plantas Arabidopsis thaliana brinda la oportunidad de la realización de estudios genómicos para el estudio de, en este caso, la longevidad de las semillas para descubrir nuevos factores genéticos y mecanismos moleculares determinantes. Este conocimiento servirá para entender mejor los procesos de deterioro de las semillas y que también será clave para aumentar la longevidad de estas. Mediante el uso de variedades naturales genotipadas de Arabidopsis thaliana y un estudio de asociación del genoma conocido como GWAS, seguido de estudios de genética reversa, se han identificado 12 nuevos genes relacionados con la longevidad de las semillas, relacionados con la protección del embrión, el control del daño oxidativo, y la permeabilidad de la cubierta de la semilla. El desarrollo de la cubierta de la semilla está determinado por factores de transcripción. Plantas mutantes en diversos factores de transcripción involucrados en el desarrollo de la cubierta de la semilla presentan una longevidad alterada. La sobreexpresión de los factores de transcripción AtHB25 y COG1 provoca que las semillas presenten una mayor longevidad debido a una incrementada deposición de poliésteres lipídicos. Estas barreras de poliésteres lipídicos son la cutícula, formada por cutina, y la suberina. Ambas participan positivamente en la protección del embrión frente al ambiente exterior. Estudios genómicos de ambos factores de transcripción han demostrado que AtHB25 regula directamente a enzimas biosintéticos de los monómeros de suberina y cutina, y COG1 regula la expresión de enzimas relacionados con la polimerización de poliésteres lipídicos y lignina. La regulación en la que participa AtHB25 es muy importante debido a la alta conservación de las secuencias genómicas y funciones de AtHB25 en angiospermas, y parece involucrado en la respuesta a bajas temperaturas. Por otra parte, COG1, que está involucrado en la percepción de luz, regula parte del desarrollo del tegumento externo a través de la regulación de AP2, un factor clave en el establecimiento de la identidad de tejido de este tegumento de la cubierta de la semilla, donde se localiza la suberina. AtHB25 y COG1 están involucrados en la adaptación de la longevidad de la semilla a través de señales ambientales como la temperatura y la luz, respectivamente, regulando la deposición de poliésteres lipídicos. / [CAT] La longevitat de les llavors, o el temps que romanen les llavors viables, es de gran importància per la conservació de la biodiversitat, l'agricultura i l'economia. A més a més, l'estudi d'aquest paràmetre pot contribuir a conèixer millor els mecanismes moleculars comuns a tots els organismes per prevenir l'envelliment. Una de les principals estratègies de les llavor per retardar el seu envelliment consisteix detenir el seu metabolisme, mitjançant la seua deshidratació. Altres mecanismes moleculars per evitar danys son el seu aïllament de l'entorn per mitjan de la coberta de la llavor, i la producció d'antioxidants i altres molècules per evitar el dany oxidatiu, un dels principal causants del envelliment de les llavors. Els mecanismes de reparació mitiguen part del dany acumulat. L'organisme model Arabidopsis thaliana brinda la oportunitat de la realització d'estudis genòmics per a l'estudi de, en aquest cas, la longevitat de les llavors per descobrir nous factors genètics y mecanismes moleculars determinants. Aquest coneixement servirà per entendre millor els processos de deteriorament de les llavor i serà clau per augmentar la longevitat d'aquestes. Mitjançant l'ús de varietats naturals genotipades d'Arabidopsis thaliana i un estudi d'associació del genoma conegut com GWAS, seguits d'estudis de genètica inversa, s'han identificat 12 nous gens relacionats amb la longevitat de les llavors, relacionats amb la protecció de l'embrió, el control del dany oxidatiu, y la permeabilitat de la coberta de la llavor. El desenvolupament de la coberta de la llavor està determinada per factors de transcripció. Plantes mutants a diversos factors de transcripció involucrats al desenvolupament de la coberta de les llavors presenten una longevitat alterada. La sobreexpressió dels factors de transcripció AtHB25 i COG1 provoca que les llavors presenten una major longevitat degut a una deposició de polièsters lipídics incrementada. Aquestes barreres de polièsters lipídics son la cutícula, formada per cutina, i la suberina. Ambdues participen positivament la protecció de l'embrió enfront de l'entorn exterior. Estudis genòmics d'ambdós factors de transcripció han demostrat que AtHB25 directament regula a enzims biosintètics dels monòmers de suberina i cutina i COG1 regula enzims relacionats amb la polimerització de polièsters lipídics i lignines. La regulació en la que participa AtHB25 es molt important degut a l'alta conservació de les seqüències genòmiques i funcions de AtHB25 en angiospermes, i parteix estar involucrat en la resposta a baixes temperatures. Per altra banda, COG1, que està involucrat en la percepció de la llum, regula part del desenvolupament del integument extern mitjançant la regulació de AP2, un factor clau en l'establiment de la identitat de teixit de aquest integument de la coberta de la llavor, on es localitza la suberina. AtHB25 i COG1 estan involucrats en l'adaptació de la longevitat de la llavor per mitjan de senyals ambientals com la temperatura i la llum, respectivament, regulant la deposició de polièsters lipídics. / [EN] Seed longevity, or period that seeds remain viable, is important for biodiversity conservation, agriculture and economy. In addition, the study of this parameter could ease the knowledge about molecular mechanisms common to all organisms to prevent aging. One of the main strategies of seeds to reduce their aging consists to stop their metabolism, through drying. Other molecular mechanisms to avoid damages are the isolation from the environment with the seed coat, and the production of antioxidants and other molecules to avoid oxidative damage, one of the main seed aging causes. Repair mechanisms relieve part of the accumulated damage. The model plant Arabidopsis thaliana provides the opportunity to carry out genomic studies for the research of, in this case, seed longevity to discover determinant genetic factors and molecular mechanisms. This will serve to better understand seed deterioration processes and it will be key to increase seed longevity. Using natural genotyped varieties of Arabidopsis thaliana and a genome-wide association study (GWAS) followed by reverse genetic studies, 12 new genes related to seed longevity have been identified. They are related to embryo protection, oxidative damage control, and seed coat permeability. Seed coat development is determined by transcription factors. Mutant plants in some transcription factors involved in the seed coat development present altered seed longevity. The over-expression of the transcription factors AtHB25 and COG1 resulted in seeds with increased longevity due to an increased lipid polyester deposition. These lipid polyesters barriers are the cuticle, formed by cutin, and the suberin layer. Both participate positively in the embryo protection from the external environment. Genomic studies of both transcription factors have revealed that AtHB25 directly regulates biosynthetic enzymes of suberin and cutin monomers, and COG1 regulates the expression of enzymes related to the polymerization of lipid polyesters and lignin. The regulation involving AtHB25 is crucial due to the high conservation of genomic sequences and functions of AtHB25 in angiosperms, and it seems to be involved in the response to low temperatures. On the other hand, COG1, which is involved in light perception, regulates part of the development of the external integument through its regulation by AP2, a key factor in establishing the tissue identity of this seed coat integument, where suberin is located. AtHB25 and COG1 are involved in seed longevity adaptation through environmental signals such as temperature and light, respectively, regulating lipid polyesters deposition. / Agradezco a las instituciones públicas la inversión en investigación. Gracias a ella, los laboratorios, el personal y los distintos equipos se han podido financiar. Gaetano fue quien me ayudó enormemente conseguir la beca FPI del Ministerio de Economía y Competitividad BES-2015-072096, asociada al proyecto de investigación nacional BIO2014-52621-R-AR / Renard Meseguer, J. (2021). Identification of genes related to seed longevity in Arabidopsis thaliana using genomic molecular techniques [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/170554 / TESIS / Compendio

Glutamate carboxypeptidase II (GCPII) is a transmembrane glycoprotein, which consists of short intracellular and transmembrane domains, and a large extracellular domain possessing carboxypeptidase activity. In the human body, GCPII fulfils a neuromodulatory function in the brain and facilitates folate absorption in the small intestine. In addition to the brain and small intestine, high level of GCPII is also present in the prostate and kidney. However, GCPII function in these tissues has not been determined yet. To study the role of GCPII in detail, several research groups attempted to inactivate GCPII encoding gene Folh1 in mice. Surprisingly, the experiments led to rather conflicting results ranging from embryonic lethality to generation of viable GCPII-deficient mice without any obvious phenotype. This dissertation project aimed to dissect the discrepancy using alternative strategy for gene modification. For this purpose, we designed TALENs that specifically targeted exon 11 of Folh1 gene and manipulated mouse zygotes of C57BL/6NCrl genetic background. We analysed all genetically modified mice of F0 generation for presence of TALEN-mediated mutations and established 5 different GCPII-mutant mouse colonies from founder mice that altogether carried 2 frame-shift mutations and 3 small in-frame...

Tesis por compendio / [ES] La alcaparra (Capparis spinosa L.) es un arbusto característico de la región mediterránea, del que se aprovechan sus botones florales y frutos. Uno de los inconvenientes más importantes para su cultivo es la dificultad en su propagación. El fruto es una baya con muchas semillas, que presentan un porcentaje de germinación que no supera el 5%. Las semillas son de color marrón oscuro, de 2 a 3 mm y la cubierta tiene una capa lignificada con paredes radiales engrosadas. La baja germinación se relaciona con distintos tipos de latencia, que no ha sido científicamente atribuida a ningún tipo concreto. El objetivo de esta tesis es estudiar las distintas fases del proceso de germinación para determinar el tipo de latencia, cómo superarla, y establecer pautas para una propagación viable mediante semillas. Los ensayos se realizaron en el Departamento de Producción Vegetal de la Universitat Politècnica de València, utilizando semillas de producción propia y de lotes comerciales. Para determinar el tipo de latencia, se estudió el proceso de imbibición durante la germinación, la resistencia mecánica de la cubierta seminal y el endospermo frente a la protusión de la radícula, y el efecto de la aplicación de giberelinas. La absorción de agua sigue las dos primeras fases del modelo trifásico típico de absorción de agua en la germinación y la hidratación comienza por la región hilo-micropilar. La imbibición no es un factor limitante para su germinación, alcanzando un contenido de humedad del 32%, con el que se obtienen porcentajes de germinación superiores al 90%. Por tanto, no tienen una cubierta impermeable al agua, es decir, no muestran latencia física. Las semillas comerciales presentan menor viabilidad y germinación que las de producción propia, lo que se ha evidenciado con el deterioro en la cubierta seminal que se relaciona con los procesos de extracción, limpieza y almacenamiento. Para obtener altos porcentajes de germinación se requiere el uso de giberelinas, lo que indica la presencia de latencia fisiológica. Se recomienda el uso de 500 mg L-1 de ácido giberélico (AG) para humedecer el sustrato de germinación. El AG desencadena el incremento del contenido de giberelinas activas endógenas y la disminución del ácido abscísico. La germinación consta de dos eventos separados temporalmente, el agrietamiento de la cubierta, en el área hilo-micropilar y luego la perforación del endospermo micropilar, por donde emerge la radícula. Las giberelinas activas no disminuyen la fuerza de punción para perforar el endospermo, sino que aumentan el potencial de crecimiento del embrión, logrando una mayor germinación y en menor tiempo. La viabilidad y germinación de semillas recién extraídas de frutos recolectados en su dehiscencia alcanzan valores muy elevados. Si la recolección de los frutos se retrasa y la pulpa se seca, la viabilidad y la germinación disminuyen, por lo que, como regla general, se aconseja recolectar los frutos al menos una vez por semana, e inmediatamente extraer las semillas y ponerlas a germinar. En el ensayo de flotación, las semillas que flotan y que presentan buena germinación son muy pocas (0.24%), por lo que se recomienda desecharlas. La germinación no es afectada por la iluminación con diferentes longitudes de onda, por lo que puede realizarse en oscuridad, lo que puede suponer un ahorro económico para semilleros comerciales. La irradiación con láser He-Ne durante tiempos cortos de exposición mejora el porcentaje de germinación en semillas previamente humedecidas, pero no sustituye a la adición de AG al sustrato, complementando su efecto. Con la ultrasonicación se escarifica la testa sin afectar al tegmen, y se acelera la imbibición inicial, aunque la humedad alcanzada en las semillas es la misma que en el control. Existe una correlación lineal y negativa, entre la germinación y la temperatura alcanzada con estos tratamientos. / [CA] La tàpera (Capparis spinosa L.) és un arbust característic de la regió mediterrània, que s'aprofita pels seus botons florals i fruits. Un dels principals inconvenients que presenta el seu cultiu, es la dificultat que presenta la seua propagació. El fruit és una baia que té moltes llavors, que presenten una germinació que no sol superar el 5%. Les llavors són de color marró fosc, mesuren 2 - 3 mm, i la coberta té una capa lignificada amb parets radials engrossides. La baixa germinació s'ha relacionat amb diferents tipus de latència, però fins ara no ha estat científicament atribuïda a cap tipus concret. L'objectiu d'aquesta tesi és estudiar les diferents fases de la germinació per determinar el tipus de latència, com superar-la, i establir les pautes per a una propagació viable mitjançant les seves llavors. Els assajos s'han realitzat en el Dep. Producció Vegetal de la Universitat Politècnica de València, utilitzant llavors de producció pròpia, i de lots comercials. Per determinar el tipus de latència, s'ha estudiat el procés d' imbibició que es produeix durant la germinació, la resistència mecànica que exerceixen la coberta seminal i l'endosperma enfront de la protrusió de la radícula, i l' efecte de l'aplicació de gibberel·lines. L'absorció d'aigua segueix les dues primeres fases del model trifàsic típic d'absorció d'aigua en la germinació, i la hidratació comença per la regió micropilar. La imbibició no és un factor limitant per a la seua germinació, aconseguint un contingut d'humitat del 32%, permet percentatges de germinació superiors al 90%. Per tant, les llavors no tenen una coberta impermeable a l'aigua, és a dir, no mostren latència física. Les llavors comercials presenten menor viabilitat i germinació que les de producció pròpia, relacionat amb el seu deteriorament, que ha estat evident a la coberta; conseqüència dels processos d'extracció, neteja i emmagatzematge. Per obtenir alts percentatges de germinació es requereix l'ús de gibberel·lines, la qual cosa indica la presència de latència fisiològica. Es recomana l'ús d'una solució de 500 mg L-1 d'àcid gibberèl·lic (AG). L'AG desencadena l'augment del contingut de gibberel·lines actives endògenes i una disminució d'àcid abscísic. La germinació consta de dos esdeveniments separats temporalment, l'esquerdament de la coberta, que comença a l'àrea micropilar i a continuació es produeix la perforació de l'endosperma micropilar, per on emergeix la radícula. Les gibberel·lines actives no disminueixen la força de punció necessària per perforar l'endosperma, sinó que augmenten el potencial de creixement de l'embrió, aconseguint major germinació i en menor temps. La viabilitat i el percentatge de germinació de les llavors obtinguts de fruits recol·lectats en la seva dehiscència assoleixen valors molt elevats. Si la recol·lecció dels fruits es retarda i la polpa s'asseca, la viabilitat i la germinació disminueixen, per la qual cosa, com a regla general, per a la producció de llavor comercial s'aconsella recol·lectar els fruits almenys una vegada per setmana, i immediatament extreure les llavors i posar-les a germinar. En l 'assaig de flotació, les llavors que floten i que presenten capacitat germinativa són molt poques (0,24%), recomanant-se rebutjar les llavors flotants. La germinació no és afectada per la il·luminació amb llums de diferents longituds d'ona, per la qual cosa la germinació pot realitzar-se en foscor, i, pot suposar un estalvi econòmic en planters comercials. La irradiació amb làser He-Ne durant temps curts d'exposició millora el percentatge de germinació de les llavors prèviament humitejades, però no substitueix l' addició d'AG al substrat, sinó que complementa el seu efecte. Amb la ultrasonicació, s'escarifica la testa sense afectar el tegmen i s'accelera la imbibició inicial, però la humitat assolida en les llavors és la mateixa que el control. Hi ha una correlació lineal i negativa, entre la germinació i la temperatura assolida amb estos tractaments. / [EN] The caper (Capparis spinosa L.) is a shrub characteristic of the Mediterranean region. It is use for its flower buds and fruits. One of the main drawbacks of its cultivation, is the difficulty presented by its propagation, both vegetative and through seeds. The fruit is a berry containing many seeds, these have a very low germination percentage, which usually is at most 5%. The seeds are dark brown colour, measure 2 - 3 mm, and the cover has a lignified layer with thickened radial walls. Low germination power has been linked to different types of dormancies, but, it has not been scientifically attributed to any specific type. This thesis aims to study the different phases of the germination process to determine the type of dormancy, how to overcome it, and to establish guidelines for viable propagation through their seeds. The tests have been carried out in the Universitat Politècnica de València Plant Production Department, mainly using own-produced seeds, and seeds from commercial lots. To determine the type of dormancy, the imbibition process that occurs during germination has been studied, as well as the mechanical resistance exerted by the seed coat and the endosperm against the protrusion of the radicle and the effect of the external gibberellins application. Water uptake follows the first two phases of the typical three-phase water uptake pattern in seed germination, and seed hydration begins in the hilum-micropillar region. Imbibition is not a limiting factor for germination, reaching a moisture content of 32%, which allows germination percentages higher than 90%. Therefore, caper seeds do not have a waterproof coat; that is, they do not show physical dormancy. Commercial seeds showed lower viability and germination than those of own production, which has been related to seed deterioration. This deterioration, which has been evident in the seed coat, is related to their extraction, cleaning, and storage processes. Obtaining high percentages of germination requires gibberellins, which indicates the presence of physiological dormancy. The use of 500 mg L-1 of gibberellic acid (GA) is recommended to moisten the germination substrate. GA triggers an increase in endogenous active gibberellins content and a decrease in abscisic acid. The germination consists of two temporarily separated events: the cracking of the coat in the hilum-micropillar area, and then the perforation of the micropillar endosperm, where the radicle emerges. Active gibberellins do not decrease the puncture force needed to pierce the endosperm but increase the growth potential of the embryo, achieving higher germination percentages in less time. The viability and germination of seeds freshly extracted from fruits harvested in their dehiscence reach very high values. If the harvest of the fruits is delayed and the pulp dries, the viability and the percentage of germination decrease so, as a rule, for the production of commercial seed, it is advisable to collect the fruits at least once a week and immediately extract the seeds and put them to germinate. In the flotation test, floating seeds with germination capacity are very few (0.24%), so it is recommended to discard them. The germination is not affected by different wavelengths lights, so it can be carried out in darkness, which can mean economic savings in commercial nurseries. He-Ne laser irradiation during short exposure times improves the germination of previously moistened seeds but does not replace the addition of GA to the substrate but complements its effect. With the ultrasonication treatment, the testa is scarified without affecting the tegmen, and the initial imbibition is accelerated, although the seed moisture is the same as in the control. There is a linear and negative correlation between germination and the temperature reached with these treatments. / Foschi, ML. (2023). Mejora de la germinación para la propagación sexual de la alcaparra (Capparis spinosa L.) [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/202596 / Compendio