C3 glomerulopathy (C3G) encompasses a group of severe renal diseases characterized by “dominant C3” deposition in the renal glomerulus. Patients typically present as nephritic nephrotics, with hematuria, hypertension, heavy proteinuria and edema. Within ten years of diagnosis, 50% of affected patients progress to end-stage renal disease and require dialysis or renal transplantation. No treatment is available to halt disease progression and thus both disease recurrence and allograft loss are common after transplantation. Genetic studies of C3G have firmly implicated dysregulation of the alternative pathway (AP) of complement in disease pathogenesis. In addition to genetic factors, acquired factors like autoantibodies can also exaggerate AP activity in the circulation to cause C3G. Although AP dysregulation in the circulation (i.e. fluid-phase dysregulation) has been well studied in these patients, AP activity in the glomerular microenvironment is not well understood. In this body of work, we used MaxGel, an ex-vivo surrogate for the glomerular extracellular matrix, to study AP activity and regulation. We showed that C3 convertase can be assembled on MaxGel and elucidated the dynamics of its formation and decay in the presence of complement regulators. We confirm that on MaxGel factor H (fH) inhibits C3 convertase formation and accelerates its decay, while properdin has a stabilizing effect. We also show that the complement factor H-related proteins (FHRs) are vital to the regulation of AP activity. Consistent with our MaxGel data, CFHR gene-fusion events have been reported as genetic drivers of disease in a few familial cases of C3G. One such familial case in which we identified and characterized the rearrangement event results from a novel CFHR5-CFHR2 fusion gene. The fusion gene is translated into a circulating FHR-5/-2 protein that consists of the first two SCRs of FHR-5 followed by all four SCRs of FHR-2. The structural repetition of SCR1-2 followed by another SCR1-2 motif facilitates the formation of complex FHR-1, FHR-2 and FHR-5 multimers, which have enhanced affinity for C3b and by out-competing fH, lead to impaired C3 convertase regulation in the glomerular microenvironment. Finally, we tested gene therapy as a tool to rescue the disease phenotype and restore fluid-phase AP complement control in a mouse model of C3G (Cfh-/-/huCR1-Tg mice). Using the piggyBac transposon system, we introduced a construct derived from complement regulator 1 (CR1) into Cfh-/-/huCR1-Tg mice. Delivery of sCR1-AC via hydrodynamic tail vein injection provided constitutive circulatory expression of sCR1-AC, and in animals followed for 6 months, we found that long-term expression of this complement regulator rescued the renal phenotype. These results suggest that sCR1 may be a potential therapy for patients with this disease.

Coral reefs are biologically and economically important ecosystems underpinned by corals that are able to flourish in oligotrophic waters due to their mutualistic association with dinoflagellate symbionts (genus Symbiodinium). Symbiodinium are strictly intracellular, residing within the gastrodermal tissues of the coral host, and contributing the majority of the coral’s energy requirements. Coral reefs are in rapid decline due to a range of threats such as local human influences, bleaching (loss of Symbiodnium and/or reduction of pigment), disease and ocean acidification, to which links to climate change have been made. The close association of corals and a diverse community of microbes led to development of the coral holobiont hypothesis, in which a range of microorganisms (e.g Bacteria) form a functionally-relevant mutualistic relationship with corals and Symbiodinium. This thesis aimed to fill knowledge gaps in the coral holobiont hypothesis and the host-microbe interactions within this system, including pathogen interactions and coral immune system functioning. This thesis revealed that host-microbe interactions in corals are complex, and that the underlying mechanisms of immunity and symbiosis may be similar. The findings corroborate the idea that corals maintain specific bacterial communities that have potential probiotic and nutritional value. In particular, a group of common coral associates were identified, and it is suggested that members of this group are globally occurring key associates. Corals affected by a disease previously described as “White Syndrome” were observed to undergo pronounced changes in their microbial community structure in comparison to healthy colonies. However, in contrast to previous findings, no single pathogen could be identified as the causative agent of the disease syndrome, and it is speculated that corals experiencing altered health status result in a breakdown of the resident associated microbial community structure. Culturable bacterial isolates from corals were shown to affect the growth of each other and in particular some species had great inhibitory properties. Hence, the presence of some bacterial species has the potential to influence the all over structure of the coral associated microbial community. It was also shown that changed environmental conditions may alter the growth conditions for coral associated bacteria in mucus. It is suggested that increased replication is needed in studies of bacterial assemblages on corals, as variability between coral species and sites were observed. In addition, studies of the role of coral microbial communities in health and disease should broaden their focus to more thoroughly consider the role of the coral holobiont, especially with regards to the coral host. This thesis identified the first functional Pattern Recognition Protein (PRP), a C-type lectin named Millectin, in scleractinian corals. Millectin was isolated by affinity chromatography and was shown to bind to bacterial pathogens as well as coral Symbiodinium symbionts. Gene expression of Millectin was upregulated in response to immune stimuli and the lectin was further abundantly expressed in the tissues of corals, suggesting a major role for this protein in system functioning and immunity. Further research into Millectin and a complement factor C3 homolog suggested that these molecules may have been co-opted into the equally important role of symbiont recruitment. Gene expression analysis of C3 also indicated this molecule may be involved in responses to tissue trauma. Millectin shows variability in the binding region, and hence, is the earliest evolutionary representative to date of a variable PRP. This finding, and the observed ancestral relation with vertebrate homologs, provided further information on the evolution of the innate immune system and gives further insight into invertebrate immunity.

The complement system plays an important role in innate and adaptive immunity. Central to all complement activities is the function of complement component 3 (C3). C3 contains a C-terminal extension of ~150 residues known as the NTR (or C345C) domain. To address the role of the NTR domain in binding and functional activities of C3, a C3/C5 chimera was engineered, in which the NTR domain of C3 was replaced by the homologous domain of the closely related protein C5. Functionally, the C3(C5NTR) was devoid of classical pathway-dependent hemolytic activity and deficient in factor H- and CR1-dependent factor I cleavability. Direct binding SPR assays, using chip bound methylamine treated His6-tagged C3(C5NTR), showed a complete loss of C5 binding while retaining wild type binding with CR1, factor H and factor B. These results present the first evidence for a major C5 binding site within C3 NTR.

The complement system plays an important role in innate and adaptive immunity. Central to all complement activities is the function of complement component 3 (C3). C3 contains a C-terminal extension of ~150 residues known as the NTR (or C345C) domain. To address the role of the NTR domain in binding and functional activities of C3, a C3/C5 chimera was engineered, in which the NTR domain of C3 was replaced by the homologous domain of the closely related protein C5. Functionally, the C3(C5NTR) was devoid of classical pathway-dependent hemolytic activity and deficient in factor H- and CR1-dependent factor I cleavability. Direct binding SPR assays, using chip bound methylamine treated His6-tagged C3(C5NTR), showed a complete loss of C5 binding while retaining wild type binding with CR1, factor H and factor B. These results present the first evidence for a major C5 binding site within C3 NTR.

Optically active polychlorinated bicyclic systems are important starting materials for the synthesis of complex target molecules. In the first part of the study, the syntheses of several racemic and meso hexachloronorbornene derivatives were executed successfully, starting from hexachlorocyclopentadiene. The enantio-enriched acetoxymethyl derivative (-)-2 and the hemiester (-)-6 were synthesized in high e.e. values by using several hydrolase type enzymes. The absolute configuration of (-)-2 was determined by transforming it to the corresponding norbornene derivative (-)-7 with known absolute configuration. In the second part of the study, C2 symmetric chiral ligand (-)-11 and C3 symmetric chiral triamide derivative (-)-12 were synthesized in high chemical yields starting from L-proline. In connection to these studies, the syntheses of the monoamide derivative (-)-14 and the C2 symmetric diamide derivative (-)-15 were achieved by using appropriate amounts of L-proline.

Το σύστημα του συμπληρώματος περιλαμβάνει περισσότερες από 30 πρωτεΐνες του ορού και της κυτταρικής επιφάνειας και αποτελεί έναν από τους αρχαιότερους φυλογενετικά μηχανισμούς ανοσίας του ξενιστή. Η ενεργοποίησή του, μέσω της κλασικής, της εναλλακτικής ή της λεκτινικής οδού, οδηγεί σε έναν καταρράκτη αντιδράσεων. Οι αντιδράσεις αυτές συγκλίνουν στην τελική λυτική οδό, οδηγώντας στη δημιουργία του συμπλόκου MAC, με αποτέλεσμα την ωσμωτική λύση του κυττάρου στόχου. Στην παρούσα εργασία, σχετικά με τη μελέτη της μοριακής εξέλιξης των γονιδίων της τελικής λυτικής οδού του συμπληρώματος, κλωνοποίηθηκε η αλληλουχία του cDNA του έκτου συστατικού του συμπληρώματος (C6). Στη συνέχεια, προκειμένου να εξαχθούν συμπεράσματα σχετικά με τη συντήρηση όλων των δομικών γονιδίων του συμπλόκου MAC κατά τη διάρκεια της φυλογενετικής εξέλιξης, δημιουργήθηκε φυλογενετικό δέντρο, στο οποίο συγκρίνονται τα παραπάνω γονίδια με τα αντίστοιχα ορθόλογα, σε διάφορους οργανισμούς. Από τη φυλογενετική ανάλυση φαίνεται ότι το C6 της όρνιθας ομαδοποιείται με τα αντίστοιχα ορθόλογα των θηλαστικών και του βατράχου, δημιουργώντας μια καλά καθορισμένη υποομάδα μέσα στην οικογένεια των πρωτεϊνών MACPF. Σύμφωνα με την υπάρχουσα βιβλιογραφία, εκτός από το ρόλο που διαδραματίζει το σύστημα του συμπληρώματος στην ανοσία, έχει δειχθεί η εμπλοκή του, μεταξύ άλλων, σε διαδικασίες αναγέννησης ιστών, ενεργοποίησης και πολλαπλασιασμού κυττάρων και ρύθμισης της απόπτωσης. Προκειμένου λοιπόν να διερευνηθεί περαιτέρω η πιθανότητα εμπλοκής του συστήματος του συμπληρώματος σε διαδικασίες που σχετίζονται με την ανάπτυξη, μελετήθηκε το αναπτυξιακό και ιστικό πρότυπο έκφρασης συστατικών του στην όρνιθα. Η ιστική μελέτη περιελάμβανε τη διερεύνηση και ημι-ποσοτική ανάλυση της έκφρασης, σε επίπεδο mRNA, σε οκτώ ιστούς της ενήλικης όρνιθας: εγκέφαλο, καρδιά, έντερο, νεφρό, ήπαρ, πνεύμονα, σπλήνα και στόμαχο. Η αναπτυξιακή μελέτη, περιελάμβανε τη διερεύνηση και ημι-ποσοτική ανάλυση της έκφρασης, σε επίπεδο mRNA, σε ολικό ομογενοποιημένο έμβρυο 4 και 6 ημερών, σε ήπαρ εμβρύου 12 και 17 ημερών και σε ήπαρ νεογένητου ατόμου 2 και 5 ημερών. Συγκεκριμένα, μελετήθηκε η έκφρασή του γονιδίου C3 σε διάφορα αναπτυξιακά στάδια, όπου και πιστοποιήθηκε η παρουσία του μορίου σε όλα τα στάδια. Επίσης, μελετήθηκε η ιστική και η αναπτυξιακή έκφραση των δομικών γονιδίων του συμπλόκου MAC (C6, C7, C8α, C8β, C8γ) και η αναπτυξιακή των ρυθμιστικών γονιδίων του (CD59, CLU,VTN). Η μελέτη του ιστοειδικού προτύπου έκφρασης για τα δομικά συστατικά του συμπλόκου, έδειξε ότι τα διάφορα συστατικά παρουσιάζουν ποικίλη έκφραση στους υπό μελέτη ιστούς, ενώ κύρια πηγή έκφρασής τους αποτελεί το ήπαρ. Κατά τη μελέτη του αναπτυξιακού προτύπου έκφρασης, δείχθηκε ότι τα δομικά συστατικά του συμπλόκου πρωτοεμφανίζονται τη 12η εμβρυική μέρα ενώ δεν παρατηρείται έκφρασή τους σε νεογέννητο άτομο 2 ημερών. Αντίθετα, η μελέτη του αναπτυξιακού προτύπου έκφρασης των ρυθμιστικών μορίων του συμπλόκου, έδειξε έκφραση όλων των συστατικών σε όλα τα υπό μελέτη στάδια. Το γεγονός αυτό αντανακλά πιθανώς την πλειοτροπική δράση των ρυθμιστικών αυτών πρωτεϊνών, καθώς εμπλέκονται σε πρόσθετες λειτουργίες οι οποίες δε σχετίζονται με τη δράση του συμπληρώματος, όπως είναι η ανάπτυξη και ωρίμανση οργάνων. Ανάλογα συμπεράσματα θα μπορούσαν να εξαχθούν και στην περίπτωση του μορίου C3, λόγω της έκφρασής του σε πρώιμα στάδια ανάπτυξης της όρνιθας, παρά την απουσία έκφρασης των γονιδίων του συμπλόκου MAC στα αντίστοιχα αναπτυξιακά στάδια. Συμπερασματικά, η παραπάνω εργασία αποτελεί την πρώτη αναφορά στη χωρική και χρονική μελέτη της έκφρασης γονιδίων του συμπληρώματος στην όρνιθα. Βάσει των παραπάνω, περαιτέρω διερεύνηση αναφορικά με τη συμμετοχή των συστατικών του συμπληρώματος σε αναπτυξιακές διεργασίες, παρουσιάζει πλέον ιδιαίτερο ενδιαφέρον. / The complement system includes more than 30 serum and membrane proteins and constitutes one of the most phylogenetically ancient immune mechanisms of the host. Its activation, via the classic, alternative or lectin pathway, leads to a cascade of reactions. All these reactions converge to the terminal lytic pathway, leading to the MAC complex creation, which results in osmotic lysis of the targeted cells. In the present work, concerning the study of the molecular evolution of the terminal complement pathway genes, the sixth complement component (C6) of the chicken (Gallus gallus) was cloned. Afterwards, a phylogenetic tree was created, in which the above genes are compared with the respective orthologs, in various organisms. As it seems from the phylogenetic analysis, chicken’s C6 clusters with the respective mammalian and frog orthologs, creating a well defined subgroup in the MACPF gene family. According to literature, besides the complement system’s role in immunity, it has been shown that it is also involved, in tissue regeneration, cell activation, proliferation and regulation of apoptosis. In order to further investigate the hypothesis of complement system’s involvement in developmental procedures, the developmental and tissue expression profile of complement components were studied in the chicken. The tissue study involved the investigation and semi-quantitative expression analysis, at the mRNA level, in eight tissues deriving from one adult chicken: brain, heart, intestine, kidney, liver, lung, spleen and stomach. The developmental study included the investigation and semi-quantitative expression analysis, at the mRNA level, of the whole homogenized 4 and 6 day embryo, in the liver from a 12 and 17 day embryo and the liver from a neonate chicken, 2 and 5 days old. In particular, the expression of C3 gene was studied in various developmental stages where the gene expression was certified in all stages. Moreover, the tissue and developmental expression of MAC complex structural genes (C6, C7, C8α, C8β, C8γ) was studied, as well as the developmental expression of the regulatory genes of MAC complex (CD59, CLU,VTN). The study of the tissue expression profile for the structural MAC components was shown that the various components exhibit a variable expression, with the liver being the major source of expression. In the developmental expression 117 profile study, it was shown that all structural MAC components initially appear in the 12th embryonic day, while no expression of them was detected in the 2 day old neonate chicken. On the contrary, the developmental expression profile study of the regulatory MAC genes was shown that they expressed throughout the developmental stages. This fact possibly reflects the regulatory proteins pleiotropic action, as they are involved in additional functions, which are not related to complement functions, such as development and organs maturation. Analogous conclusions could be drawn in the case of C3 gene, because of its expression in stages of chicken early development, despite the absence of expression of structural MAC genes in the respective developmental stages. Concluding, the above study is the first reference to the spatial and temporal expression of complement genes in the chicken. In accordance with the above mentioned, further investigation concerning the involvement of complement components in developmental procedures, appears to be of great interest.

Submitted by Izabel Franco (izabel-franco@ufscar.br) on 2016-10-26T11:43:36Z No. of bitstreams: 1 TeseLHGCB.pdf: 6352153 bytes, checksum: abc62a8dee4c17039eaf7c9c23ead40c (MD5) / Approved for entry into archive by Marina Freitas (marinapf@ufscar.br) on 2016-11-08T19:17:16Z (GMT) No. of bitstreams: 1 TeseLHGCB.pdf: 6352153 bytes, checksum: abc62a8dee4c17039eaf7c9c23ead40c (MD5) / Approved for entry into archive by Marina Freitas (marinapf@ufscar.br) on 2016-11-08T19:17:26Z (GMT) No. of bitstreams: 1 TeseLHGCB.pdf: 6352153 bytes, checksum: abc62a8dee4c17039eaf7c9c23ead40c (MD5) / Made available in DSpace on 2016-11-10T17:30:54Z (GMT). No. of bitstreams: 1 TeseLHGCB.pdf: 6352153 bytes, checksum: abc62a8dee4c17039eaf7c9c23ead40c (MD5) Previous issue date: 2016-08-17 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / This research was conducted to understand the climate responses of two tropical forages to a future scenario predicted for 2050, regardless of the season. We consider the hypothesis of the consortium between the two forages is a grazing alternative in a future climate. To test this hypothesis, we studied two forages under controlled atmospheric CO2 concentration and temperature in field conditions using a system named Trop-T-FACE. This system gave us the competence to mimic atmospheric conditions predicted for 2050 (600 ppm of CO2 in the atmosphere (eC) and an increase of 2 ºC in the canopy temperature). The C4 grass Panicum maximum and the C3 legume Stylosanthes capitata grew on current agricultural practice, in an area of 2500 m2 on the campus of the University of São Paulo in Ribeirão Preto – SP. We tested the hypothesis about the consortium between the two forages being a grazing alternative in a future climate. In the winter of 2013 and the autumn of 2014, the forage grew on irrigated consortium. In the autumn of 2015, S. capitata grew in monoculture without irrigation. Sixteen parcels in a ring form with 2 m of diameter were used for monitoring the growth and development of the species C3 and C4 species during growth periods of 35 days approximately after the initial cut. Several plant organization levels were accompanied under field conditions in Control plots, plots with elevated CO2 concentration (eC), under heating (eT), and under high CO2 concentration and heat (eC+eT). In the first chapter is the experiment conducted during the winter of 2013, with P. maximum in irrigated consortium with S. capitata. The climatic conditions of temperature were suboptimal for the C4 grass growth. Thus, the warming explicitly promoted the foliage development. The higher atmospheric CO2 concentration caused downregulation in leaf biomass accumulation. The changes resultant of the atmospheric alterations also caused modifications of leaf N concentration and biomass partition in the plant. Under combined treatment (eC+eT), the inhibitory effects of the CO2 increase were offset by the increment resultant of warming. Therefore, in the future climatic conditions, during the winter in the Brazilian Southeast region, the heating of the leaves will mitigate the inhibition by excess carbon, as long as the consortium is free of water and nutritional impediments. In the autumn of 2014, a new experiment was performed with P. maximum and S. capitata growing in irrigated consortium. This experiment is described in the second and third chapters. In the second chapter are the results of P. maximum in irrigated consortium. The purpose of conducting this experiment in the autumn was mainly to compare the influence of warming on the grass leaves in a warmer season. During the autumn, the treatments accelerated the leaf phenology of the C4 grass, including leaf senescence. The isolated increase in the atmospheric CO2 concentration (eC) or combined with warming (eC+eT) conditioned narrower leaves, probably by alterations in the leaf meristem formation process. Changes in leaf width may cause modifications in forage quality and affect the consumption by the cattle. However, the presence of narrower leaves was compensated by a greater number of leaves and the tiller biomass remained. On the other hand, the C3 legume growing in irrigated consortium during the autumn of 2014, presented several changes with no statistical differences in vegetative growth, despite the heat (eT) have been shown to be harmful to it. The results of this experiment are described in the third chapter. Being a legume adapted to warm climates, the main negative changes observed in S. capitata under warming (eT) were attributed to competition with P. maximum in the consortium. The separate heating (eT) stimulated further growth of the grass, which shadowed and softened the heat arrival in the C3 species. However, the warming (eT) significantly stimulated the flowering. In the treatment that simulated warming and CO2 concentration in the 2050 climate (eC+eT), there were more branches due intense flowering at the apex of the shoot and consequently interruption of apical dominance. The predicted future climate scenario is not favorable besides leaf biomass in this C3 species remaining the same among applied atmospheric regimes. Furthermore, the irrigation of extensive grazing warmed areas is economically and ecologically unviable and did not increase the availability of leaf biomass of S. capitata in the consortium in the year 2050. An experiment was conducted during the autumn of 2015, with the legume in monoculture without irrigation to identify the real influence of the irrigation on S. capitata growth. CO2 atmospheric enrichment increased neither biomass nor the leaf area. On the other hand, it occurred greater investment on flowers at the expense of vegetative shoot compartments. Nonetheless, enhancing flowering was only possible with soil water content greater than 0.3 m3 m-3. Warming combined with soil water shortage caused higher mortality of shoots. The rise in atmospheric CO2 concentration predicted for 2050 will not be enough to mitigate the damaging effects on leaf biomass production of the warming of about 2 ºC, in field conditions without irrigation, in this shrub C3 legume. Thus, the consortium, even irrigated, was not as an alternative pasture in climate predicted for 2050. The monoculture of the C3 legume without irrigation brought results even more concerning. The data obtained in these studies can base the development of new pasture management strategies. Also, they provide relevant information for the development of public policies to support the productive chain of meat and milk, the largest in Brazil and one of the largest in the world. / Essa pesquisa foi desenvolvida para entender as respostas climáticas de duas forrageiras tropicais a um cenário futuro previsto para 2050, independente da época do ano. Nós consideramos a hipótese do consórcio entre as duas forrageiras ser uma alternativa de pastagem em um clima futuro. Para testar essa hipótese, nós estudamos as forrageiras sob controle de concentração de CO2 e temperatura em condições de campo utilizando um sistema denominado Trop-TFACE. Esse sistema nos deu competência para mimetizar condições atmosféricas previstas para 2050 (600 ppm de CO2 na atmosfera e aquecimento de 2 ºC na temperatura da cobertura vegetal). A gramínea C4 Panicum maximum e a leguminosa arbustiva C3 Stylosanthes capitata foram cultivadas como na prática agrícola vigente, em uma área de 2500 m2 no campus da Universidade de São Paulo em Ribeirão Preto – SP, Brasil. No inverno de 2013 e outono de 2014 as forrageiras cresceram em consórcio irrigado. No outono de 2015, S. capitata cresceu em plantio solteiro sem irrigação. Dezesseis parcelas em forma de anéis com 2 m de diâmetro foram utilizadas para o acompanhamento do crescimento e do desenvolvimento das espécies C3 e C4 durante períodos de aproximadamente 35 dias de crescimento após poda inicial. Vários níveis de organização vegetal foram acompanhados em condições de campo nas parcelas Controle, nas parcelas com elevada concentração de CO2 (eC), sob aquecimento (eT) e sob a combinação de tratamentos (eC+eT). No primeiro capítulo está descrito o experimento realizado no inverno de 2013, com P. maximum em consórcio irrigado com S. capitata. As condições climáticas de temperatura foram sub-ótimas para o crescimento da gramínea C4. Sendo assim, o aquecimento promoveu claramente o desenvolvimento da folhagem. A maior concentração de CO2 atmosférico provocou uma “regulação para baixo” (downregulation) no acúmulo de biomassa foliar. As alterações provocadas pelas mudanças atmosféricas causaram também modificações na concentração de N na folha e na partição de biomassa no corpo da planta. Sob o tratamento combinado (eC+eT), os efeitos inibitórios do aumento de CO2 na folhagem foram compensados pelo incremento resultante do aquecimento. Portanto, em condições climáticas futuras, durante o inverno na região Sudeste do Brasil, o aquecimento das folhas irá mitigar a inibição por excesso de carbono, se o consórcio estiver livre de impedimentos hídricos e nutricionais. No outono de 2014, um novo experimento foi realizado com P. maximum e S. capitata crescendo em consórcio irrigado. Esse experimento está descrito nos capítulos segundo e terceiro. No segundo capítulo, estão os resultados de P. maximum no consórcio irrigado. O objetivo da realização desse experimento durante o outono foi principalmente comparar a influência do aquecimento nas folhas da gramínea em uma época mais quente do ano. Durante o outono, os tratamentos aceleraram os eventos fenológicos foliares da gramínea C4, incluindo o início da senescência. O aumento isolado na concentração atmosférica de CO2 (eC) ou em combinação com o aquecimento (eC+eT) condicionou folhas mais estreitas, provavelmente por alteração no processo de formação do meristema foliar. As alterações na largura da folha podem provocar mudanças na qualidade da forragem e afetar o consumo pelo gado. No entanto, a presença de folhas estreitas foi compensada por um maior número de folhas e a biomassa por perfilho se manteve. Por outro lado, a leguminosa C3 crescendo no consórcio irrigado durante o outono de 2014, apresentou várias alterações sem diferença estatística significativa para o crescimento vegetativo, apesar do aquecimento (eT) ter se mostrado prejudicial ao mesmo. Os resultados desse experimento estão descritos no terceiro capítulo. Por ser uma leguminosa adaptada a climas quentes, as principais alterações negativas observadas em S. capitata sob aquecimento (eT) foram atribuídas à competição com P. maximum no consórcio. O aquecimento isolado (eT) estimulou mais o crescimento da gramínea, que sombreou e atenuou a chegada de calor à leguminosa. No entanto, o aquecimento isolado (eT) estimulou significativamente o florescimento. No tratamento que simulou aquecimento e concentração de CO2 no clima de 2050 (eC+eT) ocorreu mais ramificações devido ao intenso florescimento no ápice do ramo e a consequente interrupção da dominância apical. O cenário previsto em clima futuro não é favorável apesar da biomassa foliar para essa espécie de C3 permanecer a mesma entre os regimes atmosféricos aplicados. Além disso, a irrigação de extensas áreas aquecidas de pastagem é inviável economicamente e ecologicamente e não aumentaria a disponibilidade de biomassa foliar de S. capitata crescendo em consórcio em 2050. Para identificar a real influência da irrigação no crescimento de S. capitata, um novo experimento foi realizado durante o outono de 2015, com S. capitata em plantio solteiro e sem irrigação. O enriquecimento atmosférico com CO2 não incrementou nem a biomassa e nem a área foliar. Por outro lado, ocorreu maior investimento em flores em detrimento dos compartimentos vegetativos no ramo. No entanto, o incremento do florescimento só foi possível com disponibilidade de água no solo superior a 0,3 m3 m-3. O aquecimento combinado com a reduzida disponibilidade de água no solo provocou elevada mortalidade dos ramos. A elevação da concentração de carbono atmosférico prevista para 2050 não será suficiente para compensar os efeitos negativos do aquecimento de cerca de 2 ºC na produção de biomassa foliar, em condições de campo sem irrigação, nessa leguminosa arbustiva C3. Sendo assim, o consórcio, mesmo irrigado, não se mostrou como alternativa de pastagem no clima previsto para 2050. O plantio solteiro da leguminosa, sem irrigação, trouxe resultados ainda mais preocupantes. Os dados obtidos nesses estudos podem embasar o desenvolvimento de novas estratégias de manejo do pasto. Além disso, trazem informações relevantes para o desenvolvimento de políticas públicas para sustentar a cadeia produtiva de carne e leite, a maior do Brasil e uma das maiores do mundo. / 2012/20847-5

Memoria para optar al Título Profesional de Médico Veterinario / Trypanosoma cruzi, protozoo perteneciente a la familia Trypanosomatidae, es el agente causal de la enfermedad de Chagas. El principal mecanismo de transmisión de esta enfermedad es vectorial, por medio de insectos hemípteros hematófagos de la familia Reduviidae. Los tripomastigotes, formas infectantes del parásito, son capaces de resistir la acción del Sistema del Complemento (C) del hospedero, importante componente efector de la respuesta inmune innata. Se describen 3 vías de activación: clásica (VC), alterna (VA) y de las lectinas (VL), las cuales se activan en forma de cascada, por lo cual el C debe ser finamente regulado, impidiendo el daño en células del hospedero. En mamíferos, una proteínas reguladoras del C es el factor acelerador del decaimiento de la convertasa (DAF). DAF actúa inactivando las proteínas C3 y C5 convertasas de la VC y VA, acelerando su decaimiento. Se ha reportado que T. cruzi expresa factores reguladores del C similares a los del hospedero mamífero, inhibiendo su activación. Entre estos, se ha identificado la expresión de una proteína con función similar a DAF humana (T-DAF), de la cual sólo se han expresado parcialmente algunos fragmentos y se desconoce gran parte de sus funciones. En esta memoria de título se amplificó e insertó la secuencia nucleotídica codificante para la proteína de T. cruzi T-DAF en un vector de expresión de Escherichia coli. Utilizando bacterias transformadas con el plasmidio generado, se expresó y purificó en condiciones nativas la proteína recombinante T-DAF y se caracterizó su estructura tridimensional mediante un modelamiento in silico. / Trypanosoma cruzi, protozoan belonging to the Trypanosomatidae family, is the causative agent of Chagas disease. The main mechanism of transmission of this disease is through blood-sucking insects belonging to the Reduviidae family. Trypomastigotes, infectious forms of the parasite, are able to resist the action of the complement system (C), an important effector of the innate immune response in mammals. C has 3 activation pathways: classical (CP), alternative (AP) and lectin (LP) pathways. These 3 pathways are activated sequentially. For this reason, the activation of the C should be finely regulated in order to prevent damage on the host cells. In mammals, an important C regulatory proteins is the decay accelerating factor (DAF). DAF acts by inhibiting the C3 and C5 convertase proteins activation of the CP and AP, accelerating its decay. It has been reported that the parasite expresses C regulatory proteins similar to those present in the mammalian host, inhibiting C. One of these C regulatory proteins has a similar function of human DAF (T-DAF), however, it has only been expressed partially and most of their functions are unknown. In the present work, the nucleotide sequence coding for the protein of T. cruzi T-DAF was amplified and inserted into an expression vector for Escherichia coli. Using bacteria transformed with this vector, the recombinant protein T-DAF was expressed and purified under native conditions, and its three dimensional structure was characterized by an in silico modeling. / Financiamiento: Proyectos FONDECYT de Iniciación: Nð11110251 y Nð1130113.

The seasonal pattern of the carbon isotope content (delta C-13) of atmospheric CO2 depends on local and nonlocal land-atmosphere exchange and atmospheric transport. Previous studies suggested that the delta C-13 of the net land-atmosphere CO2 flux (delta(source)) varies seasonally as stomatal conductance of plants responds to vapor pressure deficit of air (VPD). We studied the variation of (source) at seven sites across the United States representing forests, grasslands, and an urban center. Using a two-part mixing model, we calculated the seasonal delta(source) for each site after removing background influence and, when possible, removing delta C-13 variation of nonlocal sources. Compared to previous analyses, we found a reduced seasonal (March-September) variation in delta(source) at the forest sites (0.5 parts per thousand variation). We did not find a consistent seasonal relationship between VPD and delta(source) across forest (or other) sites, providing evidence that stomatal response to VPD was not the cause of the global, coherent seasonal pattern in (source). In contrast to the forest sites, grassland and urban sites had a larger seasonal variation in (source) (5) dominated by seasonal transitions in C-3/C-4 grass productivity and in fossil fuel emissions, respectively. Our findings were sensitive to the location used to account for atmospheric background variation within the mixing model method that determined (source). Special consideration should be given to background location depending on whether the intent is to understand site level dynamics or regional scale impacts of land-atmosphere exchange. The seasonal amplitude in delta C-13 of land-atmosphere CO2 exchange (delta(source)) varied across land cover types and was not driven by seasonal changes in vapor pressure deficit. The largest seasonal amplitudes of delta(source) were at grassland and urban sites, driven by changes in C-3/C-4 grass productivity and fossil fuel emissions, respectively. Mixing model approaches may incorrectly calculate delta(source) when background atmospheric observations are remote and/or prone to anthropogenic influence.

Lors de ce travail de thèse nous nous sommes intéressés à deux thèmes principaux dans le but de fabriquer des matériaux conducteurs chiraux : la synthèse de TTF-hélicènes présentant une chiralité hélicoïdale moléculaire, ainsi que l’obtention de molécules de symétrie 3 à base de TTF ayant la capacité à former des auto-assemblages supramoléculaires hélicoïdaux. Dans la partie TTF-hélicène, plusieurs tailles d’hélicènes ont été envisagées avec pour objectif d’obtenir des énantiomères stables et séparables. Ils ont été caractérisés par diffraction de rayons X, de même que les sels obtenus par électrocristallisation. De plus, une étude théorique de la réaction de photocyclisation des stilbènes en hélicènes a été menée pour comprendre sa régiosélectivité. Les molécules de symétrie 3 sont composées d’un coeur BTA-DABP sur lequel sont fixés des TTF avec leurs substituants chiraux. L’assemblage colonnaire des molécules est étudié par CD et les fibres formées sont observées par microscopie ce qui permet de déterminer leur hélicité. On varie le cœur ainsi que les groupements fixés dessus. / In this work, we have been interested in two different topics to make chiral conducting materials: the synthesis of TTF-helicenes, with molecular helical chirality, as well as the formation of three-fold symmetry molecules based on TTF units able to self-assemble into supramolecular helical aggregates. For the TTF-helicenes, several lengths of helicenes are considered in order to form stable and separable enantiomers. They are characterized by X-ray diffraction just as the salts that are obtained by electrocrystallization. Moreover, a theoretical study of the photocyclisation reaction of stilbenes into helicenes was performed to understand its regioselectivity. The three-fold symmetry molecules are composed of a BTA-DABP core on which TTF with chiral substituents are fixed. The assembly into columnar aggregates is followed by CD and the fibers formed are observed by microscopy in order to determine their helicity. Variation of the core and the substituents are made.