The mechanisms involved in desiccation tolerance of lichens and their photobionts are still poorly understood. To better understand these mechanisms we have studied dehydration rate and desiccation ...time in Trebouxia, the most abundant chlorophytic photobiont in lichen. Our findings indicate that the drying rate has a profound effect on the recovery of photosynthetic activity of algae after rehydration, greater than the effects of desiccation duration. The basal fluorescence (F'(o)) values in desiccated algae were significantly higher after rapid dehydration, than after slow dehydration, suggesting higher levels of light energy dissipation in slow-dried algae. Higher values of PSII electron transport were recovered after rehydration of slow-dried Trebouxia erici compared to rapid-dried algae. The main component of non-photochemical quenching after slow dehydration was energy dependent (q (E)), whereas after fast dehydration it was photoinhibition (q (I)). Although q (E) seems to play a role during desiccation recovery, no significant variations were detected in the xanthophyll cycle components. Desiccation did not affect PSI functionality. Classical antioxidant activities like superoxide dismutase or peroxidase decreased during desiccation and early recovery. Dehydrins were detected in the lichen-forming algae T. erici and were constitutively expressed. There is probably a minimal period required to develop strategies which will facilitate transition to the desiccated state in this algae. In this process, the xanthophyll cycle and classical antioxidant mechanisms play a very limited role, if any. However, our results indicate that there is an alternative mechanism of light energy dissipation during desiccation, where activation is dependent on a sufficiently slow dehydration rate.
En el presente trabajo se estudian caracteres fenológicos como la longitud de las acículas (LA) y entrenudos (LE) y la longevidad de las acículas (LG) de Pinus canariensis Ch. Sm. ex DC. en 19 ...estaciones de las islas de Tenerife, El Hierro y La Palma, por campañas de recolección de muestras realizadas durante 5 años; así como, la relación entre su crecimiento, el clima y la concentración de nutrientes en las acículas. Se ha observado una elevada variabilidad interpoblacional. Esta variabilidad fenotípica podría relacionarse principalmente con las variadas condiciones climáticas existentes en cada localidad. Se han obtenido correlaciones significativas positivas entre las precipitaciones, LA y LE, mientras que la LG se correlaciona negativamente. Por otra parte, la concentración de nutrientes en las acículas fue menor en aquellos árboles con una mayor retención foliar. Así pues, en aquellos lugares donde el crecimiento está limitado por las condiciones ambientales una mayor retención foliar permitiría que, por un lado, la baja capacidad fotosintética sea compensada con el incremento de las superficies foliares activas y, por otro, que se optimice el uso de los nutrientes mediante un incremento del tiempo de retención de las acículas en el árbol.
The study of desiccation tolerance of lichens, and of their phycobionts in particular, has been mainly focused on the antioxidant system that protects the cell against photo-oxidative stress produced ...during dehydration and rehydration. However, desiccation tolerance cannot be achieved in lichen phycobionts with antioxidant activity alone. The main objective of the thesis was to expand the knowledge of the poorly understood desiccation tolerant mechanisms.
The first chapter presents an introduction to the subject of the thesis, based on the available background information, and explains the rationale of the thesis.
The aim of the chapter 2 was to establish the optimal growing conditions for the isolated phycobiont Trebouxia erici Ahmadjian (SAG 32.85 = UTEX 911) and to determine how these working procedures could modify the results of further experiments. We concluded that cellulose-acetate discs for agar cultures should be inoculated with 14-day-old liquid cultures, and growth under irradiance of 30 mol m-2 s-1 PAR at 20 ºC. Experiments should be carried out with 21day-old algal discs.
The experiments carried out in third chapter were designed on one hand, to determine the grade of desiccation tolerance of the isolated lichen phycobiont T. erici, and on the other hand, we studied the response of some protective mechanisms. The results showed that both long desiccations and rapid drying produced greater membrane damage and lower recovery of metabolic and photosynthetic activity than after brief desiccations and slow drying, respectively. Photosynthesis was never recovered totally neither after rapid nor slow drying. Classical mechanisms involved in the protection of the cell against oxidative stress were present in hydrated algae although were not enhanced during dehydration/rehydration. Likewise, in T. erici, dehydrins were also constitutively expressed. However, a slow drying time was required for activating the conformational change in the photosystem II which protect against photoinhibition in desiccation state.
In the fourth chapter we decided to carry out proteomic and genetic expression analyses of the changes associated with desiccation and rehydration in the isolated phycobiont Trebouxia erici, in order to find out other proteins that may be involved on desiccation tolerance mechanisms of phycobionts. Proteomic analysis showed that desiccation caused up-regulation of around 19 proteins and down-regulation of 43 proteins. Among the proteins up-regulated during drying were found: a putative cation transport protein; a protein with an F-box domain; a cell division cycle 48 protein; -tubulin protein; a 90 kDa Heat shock protein. We observed that five Hsp90 and two -tubulin genes were activated during dehydration and mRNA was accumulated until the cell was completely dried.
In the fifth chapter we investigated the role of the NO in the biochemical interaction between lichen symbionts and whether the NO may increase the endurance of lichenized phycobionts under desiccation. Rehydration of the lichen R. farinacea caused the release of NO together with a high production of ROS. The amount of NO detected decreased significantly with the addition of c-PTIO. In the isolated phycobionts, scavenging of the self produced NO caused a decrease in the recovery of photosynthetic activity after dehydration, probably due to the higher levels of photo-oxidative stress.
The aim of the work presented in the sixth chapter was to determine the effect of cell ageing in desiccation resistance of T. erici. Phycobiont capacity to recover photosynthesis activity after desiccation was inversely related with cell ageing, suggesting that desiccation tolerance is age-dependent.
The results presented in this thesis demonstrate that desiccation tolerance of T. erici is achieved by a complex system of constitutive and induced mechanisms. Moreover, some factors such as drying rate, desiccation duration, lichenization and ageing may alter the recovery capacity of T. erici after desiccation.
El estudio de la tolerancia a la desecación de los líquenes, y de sus fotobiontes en particular, ha sido principalmente enfocado hacia los sistemas antioxidantes que protegen a la célula frente al estrés fotoxidativo. Sin embargo, la tolerancia a la desecación no puede ser alcanzada solamente con sistemas antioxidantes. Por esta razón, el objetivo principal de esta tesis fue profundizar en el conocimiento de los mecanismos de tolerancia a la desecación en fotobiontes liquénicos.
El fotobionte liquénico Trebouxia erici Ahmadjian mostró ser tolerante a la desecación incluso cuando es cultivado separado de su compañero micobionte. Sin embargo, la resistencia a la desecación dependió de la velocidad de deshidratación, del tiempo de desecación y del envejecimiento del cultivo. Tasas de deshidratación altas, largos periodos de desecación y cultivos viejos redujeron la capacidad de recuperación. Esto es debido a que la tolerancia a la desecación del fotobionte se consigue por un lado mediante mecanismos de protección celular que son constitutivamente expresados como, enzimas antioxidantes, xantofilas o proteínas LEA, junto con la activación Mientras que por otro, existen mecanismos que se activan durante la deshidratación, como son de la síntesis de proteínas implicadas en el transporte, la protección de membranas y proteínas, el citoesqueleto, el ciclo celular y el marcaje y la degradación de proteínas.
Aunque los fotobiontes liquénicas tienen sus propios mecanismos para resistir la desecación, en la relación simbióntica la tolerancia a la desecación podría ser aumentada por otros mecanismos. Nuestros estudios con el liquen Ramalina farinacea Ach. y fotobiontes aislados mostraron que el gas bioactivo oxido de nitrógeno (NO) es liberado principalmente por el hongo durante la rehidratación del talo, el cual podría tener un papel importante en la maquinaria antioxidante del fotobionte durante las primeras fases de la rehidratación.
A new styracosternan ornithopod genus and species is here described based on a partial postcranial skeleton and an associated dentary tooth of a single specimen from the Arcillas de Morella Formation ...(Early Cretaceous, late Barremian) at the Morella locality, (Castellón, Spain). Morelladon beltrani gen. et sp. nov. is diagnosed by eight autapomorphic features. The set of autapomorphies includes: very elongated and vertical neural spines of the dorsal vertebrae, midline keel on ventral surface of the second to fourth sacral vertebrae restricted to the anterior half of the centrum, a posterodorsally inclined medial ridge on the postacetabular process of the ilium that meets its dorsal margin and distal end of the straight ischial shaft laterally expanded, among others. Phylogenetic analyses reveal that the new Iberian form is more closely related to its synchronic and sympatric contemporary European taxa Iguanodon bernissartensis and Mantellisaurus atherfieldensis, known from Western Europe, than to other Early Cretaceous Iberian styracosternans (Delapparentia turolensis and Proa valdearinnoensis). The recognition of Morelladon beltrani gen. et sp. nov. indicates that the Iberian Peninsula was home to a highly diverse medium to large bodied styracosternan assemblage during the Early Cretaceous.
A new medium-sized theropod dinosaur from the Arcillas de Morella Formation (upper Barremian) in Vallibona (Castellón, Spain) is described based on a partial skeleton. Vallibonavenatrix cani, gen. et ...sp. nov., is diagnosed by five autapomorphies: moderately high dorsal neural spines relative to the centrum height, the presence of deep pleurocoelous fossae and pneumatic foramina in the sacral vertebrae, a strongly pneumatic ilium with large internal cameras, the presence of a broad and flat platform on the ventromedial surface of the postacetabular blade of the ilium, and the strong ventral torsion of the ischium diaphysis. Phylogenetic analysis recovered Vallibonavenatrix cani as a spinosaurid megalosauroid and revealed that the new Iberian form is more closely related to Gondwanan spinosaurines, such as Spinosaurus, Irritator or Angaturama and the Asian taxon Ichthyovenator than it is to its synchronic contemporary European taxon Baryonyx walkeri. The Gondwanan or Asian affinities of Vallibonavenatrix indicate a complex palaeobiogeographic pattern and may be interpreted simultaneously as evidence for contact between Europe and North Africa and between Europe and Asia before the Aptian. This specimen is currently the most complete evidence of a spinosaurid theropod known in the fossil record of the Iberian Peninsula.
Iguanodon bernissartensis is the most frequently and widely cited styracosternan ornithopod in Western Europe during the Early Cretaceous, although some of these assignments likely need to be revised ...to establish the true distribution of the taxon. Here, we describe a new specimen of I. bernissartensis from the upper Barremian of the Iberian Peninsula. Based on the unique combination of shared characters, the new specimen from the Arcillas de Morella Formation at Morella locality (Castellón, Spain) can be confidently referred to Iguanodon bernissartensis. These characters include parallel-sided anterior and posterior margins of the dorsal and the caudal neural spines as well as the presence of a ventral keel in the posterior dorsal centra and a broad ventral sulcus in the midline of the central surface of the most posterior sacral vertebrae. This new evidence of Iguanodon bernissartensis reinforces the knowledge about styracosternan ornithopods as the most frequently recorded dinosaur group in the Arcillas de Morella Formation.
A new dryosaurid ornithopod, Eousdryosaurus nanohallucis, gen. et sp. nov., is described here based on a single specimen from the Late Jurassic Alcobaça Formation of Portugal. Eousdryosaurus ...nanohallucis is distinguished from all other dryosaurids by eight autapomorphic features and an unique combination of characters, some of which are also shared by other dryosaurids. Eousdryosaurus is linked with Dryosauridae, because the fourth trochanter is proximally placed and widely separated from the scar for the insertion of the M. caudifemoralis longus, which is restricted to the medial surface of the femoral shaft. Phylogenetic analysis nests Eousdryosaurus in an unresolved polytomy at the base of Dryosauridae together with Callovosaurus, Dryosaurus, and Kangnasaurus. The complete pes of Eousdryosaurus, which has a phalangeal formula of 1-3-4-5-0, supports the putative autapomorphic reduction of the dryosaurid pes that also occurs in parallel in more derived ornithopods.
The yeast Pichia pastoris is a cost-effective and easily scalable system for recombinant protein production. In this work we compared the conformation of the receptor binding domain (RBD) from severe ...acute respiratory syndrome coronavirus-2 (SARS-CoV-2) Spike protein expressed in P. pastoris and in the well established HEK-293T mammalian cell system. RBD obtained from both yeast and mammalian cells was properly folded, as indicated by UV-absorption, circular dichroism and tryptophan fluorescence. They also had similar stability, as indicated by temperature-induced unfolding (observed T
were 50 °C and 52 °C for RBD produced in P. pastoris and HEK-293T cells, respectively). Moreover, the stability of both variants was similarly reduced when the ionic strength was increased, in agreement with a computational analysis predicting that a set of ionic interactions may stabilize RBD structure. Further characterization by high-performance liquid chromatography, size-exclusion chromatography and mass spectrometry revealed a higher heterogeneity of RBD expressed in P. pastoris relative to that produced in HEK-293T cells, which disappeared after enzymatic removal of glycans. The production of RBD in P. pastoris was scaled-up in a bioreactor, with yields above 45 mg/L of 90% pure protein, thus potentially allowing large scale immunizations to produce neutralizing antibodies, as well as the large scale production of serological tests for SARS-CoV-2.
The record of spinosaurids currently known in Spain is scarce and represented mainly by isolated teeth and other fragmentary skeletal elements. Most of these previously described specimens have been ...tentatively attributed to Baryonyx, but there are some differences on dental morphology that may indicate the presence of more than one spinosaurid taxon. A set of postcranial osteological remains of theropod dinosaurs from the upper Barremian Arcillas de Morella Formation of Spain is here described. These elements show a combination of characters compatible with Spinosauridae. The specimens from Morella are interpreted as closely related to other Early and early Late Cretaceous spinosaurids, in particular to Baryonyx walkeri from the Barremian of England, Suchomimus tenerensis from the Aptian of Niger, and Sigilmassasaurus from the Cenomanian of Morocco. However, it was identified a combination of features distinct from other spinosaurids, supporting the presence of a distinct form, adding to the knowledge of the diversity and distribution of this clade of theropod dinosaurs in the Lower Cretaceous of the Iberian Peninsula.
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