Thylakoid Protein Phosphorylation in Regulation of Photosynthesis

Once the seed has germinated, the plant is forced to face all the environmental changes in its habitat. In order to survive, plants have evolved a number of different acclimation systems. The primary reaction behind plant growth and development is photosynthesis. Photosynthesis captures solar energy and converts it into chemical form. Photosynthesis in turn functions under the control of environmental cues, but is also affected by the growth, development, and metabolic state of a plant. The availability of solar energy fluctuates continuously, requiring non-stop adjustment of photosynthetic efficiency in order to maintain the balance between photosynthesis and the requirements and restrictions of plant metabolism. Tight regulation is required, not only to provide sufficient energy supply but also to prevent the damage caused by excess energy. The very first reaction of photosynthesis is splitting of water into the form of oxygen, hydrogen, and electrons. This most fundamental reaction of life is run by photosystem II (PSII), and the energy required for the reaction is collected by the light harvesting complex II (LHCII). Several proteins of the PSII-LHCII complex are reversibly phosphorylated according to the energy balance between photosynthesis and metabolism. Thylakoid protein phosphorylation has been under extensive investigation for over 30 years, yet the physiological role of phosphorylation remains elusive. Recently, the kinases behind the phosphorylation of PSII-LHCII proteins (STN7 and STN8) were identified and the knockout mutants of these kinases became available, providing powerful tools to elucidate the physiological role of PSII-LHCII phosphorylation. In my work I have used the stn7 and stn8 mutants in order to clarify the role of PSII-LHCII phosphorylation in regulation and protection of the photosynthetic machinery according to environmental cues. I show that STN7- dependent PSII-LHCII protein phosphorylation is required to balance the excitation energy distribution between PSII and PSI especially under low light intensities when the excitation energy transfer from LHC to PSII and PSI is efficient. This mechanism differs from traditional light quality-induced “state 1” – “state 2” transition and ensures fluent electron transfer from PSII to PSI under low light, yet having highest physiological relevance under fluctuating light intensity. STN8-dependent phosphorylation of PSII proteins, in turn, is required for fluent turn-over of photodamaged PSII complexes and has the highest importance upon prolonged exposure of the photosynthetic apparatus to excess light.

The Importance of Semantics: Visual World Studies on Drawing Inferences and Resolving Anaphors

The present thesis investigated the importance of semantics in generating inferences during discourse processing. Three aspects of semantics, gender stereotypes, implicit causality information and proto-role properties, were used to investigate whether semantics is activated elaboratively during discourse comprehension and what its relative importance is in backward inferencing compared to discourse/structural cues. Visual world eye-tracking studies revealed that semantics plays an important role in both backward and forward inferencing: Gender stereotypes and implicit causality information is activated elaboratively during online discourse comprehension. Moreover, gender stereotypes, implicit causality and proto-role properties of verbs are all used in backward inferencing. Importantly, the studies demonstrated that semantic cues are weighed against discourse/structural cues. When the structural cues consist of a combination of cues that have been independently shown to be important in backward inferencing, semantic effects may be masked, whereas when the structural cues consist of a combination of fewer prominent cues, semantics can have an earlier effect than structural factors in pronoun resolution. In addition, the type of inference matters, too: During anaphoric inferencing semantics has a prominent role, while discourse/structural salience attains more prominence during non-anaphoric inferencing. Finally, semantics exhibits a strong role in inviting new inferences to revise earlier made inferences even in the case the additional inference is not needed to establish coherence in discourse. The findings are generally in line with the Mental Model approaches. Two extended model versions are presented that incorporate the current findings into the earlier literature. These models allow both forward and backward inferencing to occur at any given moment during the course of processing; they also allow semantic and discourse/structural cues to contribute to both of these processes. However, while the Mental Model 1 does not assume interactions between semantic and discourse/structural factors in forward inferencing, the Mental Model 2 does assume such a link.

Percepciones de distintos agentes sociales acerca de la educación formal de los chicos y chicas tutelados

This article examines the education of children and young people in public care and the available data about their situation from several conceptual perspectives.We present a qualitative empirical study on how a sample of care leavers perceives this situation as well as the stakeholders involved in the public care process: managers of services and nominated adults by young people. We present results from a total of 96 interviews conducted in Catalonia. The young people interviewed (N = 35) were between 19 and 22 years old and were selected among those who at the age of 16 were still in care and had good academic results and the capacity andmotivation to continue studying. These young people were interviewed twice, the second a year after to follow the achievement of their training plans. The resultsshow their perception concerning the circumstances that make it easier or difficult to continue studying. Moreover, the results show the matches, mismatches anddiversity among their answers and those of other stakeholders interviewed about what factors facilitate and difficult the education. These results suggest the need for an in-depth review on the representations about the formal education of children in care from professionals and care policies, and how they address the support they need to participate in education

Fiktiivisen kirjallisuuden maailmasta monikulttuuriseen Suomeen. Ennakointikertomus kirjallisuudenopetuksen ja monikulttuurisuuskasvatuksen välineenä

From the world of fiction literature into multi-cultural Finland. Anticipatory story as an educational tool in teaching of literature and multiculturalism The research clarifies the relationship between reading fiction literature and multicultural value education in basic education. The research focuses on the subject didactics of mother tongue and literature and on the literature teaching in particular. The objective is to develop a method that is easily transferable into a teaching context so as to intensify the educational discussion based on fiction literature. In essence, understanding fiction literature and the ethical and moral thinking resemble one another, because both of them aim at empathizing with the thinking of a person or with a situation foreign to oneself. For this reason fiction literature is ideally suited for the discussion on ethical and moral values within a subject entity in the basic education. The empirical unit of the research consists of two parts. The first part explains how youth novels published in the years 1993 – 2007 describe multiculturalism in Finnish society. Books on multiculturalism are still few in number within youth literature, and people with a foreign background are mainly minor or background characters in such literary works. Nevertheless, youth novels serve well as a starting point for an educational discussion about multicultural issues. More often than not characters in youth novels are stereotypes and even opposite to each other. The juxtaposing makes a young reader question the stereotypes associated with immigrants. Besides the stereotype, reference to a prototype or a model is possible. The second part tests the usefulness of an anticipatory story based on a fictive text for an educational discussion about multicultural issues. The empirical material was collected from the eighth-grade teaching groups in basic education as follows: one teaching group was an immigrant group, the second one a group of Finns with experience of immigrants while the third group of students had hardly any experience of immigrants. The anticipatory stories were written on the basis of extracts from youth novels with multicultural themes. The material collected for a total of 120 anticipatory stories was analysed by using meaning cue analysis. Using the meaning cue analysis, the anticipatory stories were divided into three groups: stories with predominantly positive meaning cues of interculturalism, ambivalent stories with both positive and negative meaning cues of interculturalism and the stories with predominantly negative meaning cues of interculturalism. The meaning cues produced by girls and boys differ from one another, in particular, by the negative meaning cues of interculturalism. For girls, the predominant meaning cue is fear whereas for boys, it is that of violence. It would also seem that the students, in particular, boys with little experience of immigrants produce more negative meaning cues of interculturalism than do immigrants or Finnish students with experience of immigrants. Further still, it seems that active reading of fiction literature affects the meaning cues of interculturalism in an ambivalent direction. In the way of youth novels this is understandable, because youth novels in general are made up of opposite characters and meaning cues. The less the student takes an interest in reading, the more he used meaning cues from outside the parent text for his anticipatory story. No doubt it would be possible to use fiction literature in the literature education to a much higher extent than it is being used today whereby the literature could be used in basic education for reviewing subject entities or study contents of other study subjects. By way of an anticipatory story and the meaning cue analysis, it is possible to intensify the educational discussions based on fiction literature. However, using fiction literature in the literature education requires consideration of the specific genre of fiction literature.

Kehkeytyvät teokset

Kirjallisuusarvostelu

Anti-predator adaptations in aquatic environments

Predation is an important selective force that has led to the evolution of a variety of fascinating anti-predator adaptations, such as many types of protective coloration and prey behaviours. Because the evolution of life has begun in the aquatic environment and many anti-predator adaptations are found already in relative primitive taxa, it is likely that many of these adaptations evolved initially in the aquatic environment. Yet, there has been surprisingly little research on the mechanisms and function of antipredator adaptations in aquatic systems. To understand the function of anti-predator adaptations and natural selection imposed on prey appearance and behaviour, I have investigated how protective coloration can be used, either as such or together with behavioural adaptations, to manipulate predator behaviour and decrease predation risk. To this end I conducted a series of behaviour ecological laboratory experiments in which I manipulated the visual appearance of artificial backgrounds and prey items. In paper I of this thesis, I investigated background choice as an anti-predator strategy, by observing the habitat choice of the least killifish (Heterandria formosa) between pairs of artificial backgrounds, both in the presence and absence of predation threat. It has been suggested that prey could decrease their risk of being detected by predators either by preferring backgrounds into which they blend or by preferring visually complex backgrounds. The least killifish preferred a background that matched their patterning to a background that mismatched it, showing that they are able to respond to cues of visual similarity between their colour pattern and the surrounding environment. Interestingly however, in female least killifish visual complexity of the background was a more important cue for habitat safety and may override or act together with background matching when searching for a safe habitat. It is possible that in females, preference for visually complex backgrounds is associated with lower opportunity costs than preference for matching backgrounds would be. Generally, the least killifish showed stronger preference while under predation threat, indicating that their background choice behaviour is an antipredator adaptation. Many aquatic prey species have eyespots, which are colour patterns that consist of roughly concentric rings and have received their name because they for humans often resemble the vertebrate eye. I investigated the anti-predator function of eyespots against predation by fish in papers II, III and IV. Some eyespots have been suggested to benefit prey by diverting the strikes of predators away from vital parts of the prey body or towards a direction that facilitates prey escape. Although proposed over a century ago, the divertive effect of eyespots has proven to be difficult to show experimentally. In papers II and III, I tested for divertive effect of eyespots towards attacking fish by presenting artificial prey with eyespots to laboratory reared three-spined sticklebacks (Gasterosteus aculeatus). I found that eyespots strongly influenced the behaviour of attacking sticklebacks and effectively drew their strikes towards the eyespots. To further investigate this divertive effect and whether the specific shape of eyespots is important for it, I tested in paper III the response of fish also to other markings than eyespots. I found that eyespots were generally more effective in diverting the first strikes of attacking fish compared to other prey markings. My findings suggest that the common occurrence of eyespots in aquatic prey species can at least partly be explained by the divertive effect of the eyespot shape, possibly together with the relative simple developmental mechanisms underlying circular colour patterns. An eyebar is a stripe that runs through the eye, and this pattern has been suggested to obscure the real eyes of the prey by visually blending parts of the eyes and head of the prey and by creating false edges. In paper III, I show that an eyebar effectively disrupts an eyelike shape. This suggests that eyebars provide an effective way to conceal the eyes and consequently obstruct detection and recognition of prey. This experiment also demonstrates that through concealment of the eyes, eyebars could be used to enhance the divertive effect of eyespots, which can explain the common occurrence of eyebars in many species of fish that have eyespots. Larger eyespots have been shown to intimidate some terrestrial predators, such as passerine birds, either because they resemble the eyes of the predator’s own enemy or because highly salient features may have an intimidating effect. In papers II and IV, I investigated whether the occurrence of eyespots in some aquatic prey could be explained by their intimidating effect predatory fish. In paper IV, I also investigated the reason for the intimidating effect of eyelike prey marks. In paper II, I found no clear intimidating effect of eyespots, whereas in paper IV, using a different approach, I found that sticklebacks hesitated to attack towards eyelike but not towards non-eyelike marks. Importantly, paper IV therefore presents the first rigorous evidence for the idea that eye mimicry, and not merely conspicuousness, underlies the intimidating effect. It also showed that the hesitation shown by fish towards eyelike marks is partly an innate response that is reinforced by encounters with predators. Collectively, this thesis shows that prey colour pattern and the visual appearance of the habitat influence the behaviour of fish. The results demonstrate that protective coloration provides numerous distinctive ways for aquatic prey to escape predation. Thus, visual perception and behaviour of fish are important factors shaping the appearance and behaviours of aquatic prey.

Perspectives for using light quality knowledge as an advanced ecophysiological weed management tool

The current knowledge of light quality effects on plant morphogenesis and development represents a new era of understanding on how plant communities perceive and adjust to available resources. The most important consequences of light quality cues, often mediated by decreasing in red far-red ratios with respect to the spectral composition of incident sunlight radiation, affecting weed-crop interaction are the increased plant height and shoot to root ratio in anticipation of competition by light quantity, water or nutrients. Although the concepts related to light quality have been extensively studied and several basic process of this phenomenon are well known, little applications of photomorphogenic signaling currently are related to agricultural problems or weed management. The objectives of this review are to describe how light quality change can be a triggering factor of interspecific interference responses, to analyze how this phenomenon can be used to predict weed interference, to reevaluate the critical periods of interference concept, and to discuss its potential contribution towards developing more weed competitive crop varieties. Knowledge on light quality responses involved in plant sensing of interspecific competition could be used to identify red/far-red threshold values, indicating when weed control should be started. Light quality alterations by weeds can affect grain crop development mainly in high yielding fields. Unlike the traditional concept or the critical period of competition, light quality mediated interference implies that the critical period for weed control could start before the effects of direct resource (water, nutrients and available light) limitation actually occur. The variability in light quality responses among crop genotypes and the identification of mutants insensitive to light quality effects indicate that this characteristic can be selected or modified to develop cultivars with enhanced interspecific interference ability. Knowledge on light quality-elicited responses represents a new possibility to understand the underlying biology of interspecific interference, and could be used in the development of new weed management technologies.

Tuning cell motility : roles of nestin and vimentin in cancer cell invasion and motility

The cytoskeleton is a key feature of both prokaryotic and eukaryotic cells. Itis comprised of three protein families, one of which is the intermediate filaments (IFs). Of these, the IFs are the largest and most diverse. The IFs are expressed throughout life, and are involved in the regulation of cell differentiation, homeostasis, ageing and pathogenesis. The IFs not only provide structural integrity to the cell, they are also involved in a range of cellular functions from organelle trafficking and cell migration to signalling transduction. The IFs are highly dynamic proteins, able to respond and adapt their network rapidly in response to intra- and extra- cellular cues. Consequently they interact with a whole host of cellular signalling proteins, regulating function, and activity, and cellular localisation. While the function of some of the better-known IFs such as the keratins is well studied, the understanding of the function of two IFs, nestin and vimentin, is poor. Nestin is well known as a marker of differentiation and is expressed in some cancers. In cancer, nestin is primarily described as is a promoter of cell motility, however, how it fulfils this role remains undefined. Vimentin too is expressed in cancer, and is known to promote cell motility and is used as a marker for epithelial to mesenchymal transition (EMT). It is only in the last decade that studies have addressed the role that vimentin plays in cell motility and EMT. This work provides novel insight into how the IFs, nestin and vimentin regulate cell motility and invasion. In particular we show that nestin regulates the cellular localisation and organisation of two key facilitators of cell migration, focal adhesion kinase and integrins. We identify nestin as a regulator of extracellular matrix degradation and integrin-mediated cell invasion. Two further studies address the specific regulation of vimentin by phosphorylation. A detailed characterisation study identified key phosphorylation sites on vimentin, which are critical for proper organisation of the vimentin network. Furthermore, we show that the bioactive sphingolipids are vimentin network regulators. Specifically, the sphingolipids induced RhoA kinasedependent (ROCK) phosphorylation at vimentin S71, which lead to filament reorganisation and inhibition of cell migration. Together these studies shed new light into the regulation of nestin and vimentin during cell motility.

Physical Attractiveness as a Signal of Biological Quality

It has been commonly thought that standards of beauty are arbitrary cultural conventions that vary between cultures and time. In my thesis I found that it is not so. Instead, I show that attractiveness and preferred traits serve as cues to phenotypic qualities that provide selective benefits for those who choose their mates based on these criteria. In the first study I show that attractive men have a stronger antibody response to the hepatitis b vaccine and higher levels of testosterone than their less attractive peers. Men with low levels of testosterone also tend to have high levels of the stress hormone cortisol, suggesting that their immune responses may have been inhibited by stress hormones. Thus, facial attractiveness may serve as an honest cue of the strength of immune defence in men. In the second study, I show that the attractiveness of the male body is also a cue of better immunity. In addition, I show that adiposity, both in men’s faces and bodies, is a better cue of the strength of immunity and attractiveness than of masculinity. In the third study, I test the preferences of women from 13 countries for facial cues of testosterone and cortisol. I show that there is cross-cultural variation in women’s preference for cues of testosterone and cortisol in male faces. I found a relationship between the health of a nation and women’s preferences for cues of testosterone in the male face and the interaction between preferences for cues of testosterone and cortisol. I show also a relationship between preferences for cues of testosterone and a societal-level measure of parasite stress. Thus, it seems that societal-level ecological factors influence the relative value of traits as revealed by combinations of testosterone and stress hormones. In the fourth study, I show that women’s immune responsiveness (amount of antibodies produced) does not predict facial attractiveness. Instead, plasma cortisol level is negatively associated with attractiveness, indicating that stressed women look less attractive. Fat percentage is curvilinearly associated with facial attractiveness, indicating that being too thin or too fat reduces attractiveness. This study suggests that in contrast to men, facial attractiveness in women does not indicate the strength of immune defence, but is associated with other aspects of long-term health and fertility: circulating levels of the stress hormone cortisol and the percentage of body fat. In the last study I show that the attractiveness of men’s body odor is positively correlated with stress hormone levels, suggesting also that the attractiveness of body odors may signal the phenotypic quality of males to females. However, the attractiveness of men’s body odor was not associated with testosterone levels. My thesis suggests that the standard of beauty is not in the eye of the beholder. Instead, our standard of beauty is hardwired in our brains by genes that are selected by natural selection and also influenced by current environmental conditions.

Guilt, shame, emotion regulation, and social cognition: Understanding their associations with preadolescents' social behavior

In this thesis, two negatively valenced emotions are approached as reflecting children’s self-consciousness, namely guilt and shame. Despite the notable role of emotions in the psychological research, empirical research findings on the links between guilt, shame, and children’s social behavior – and particularly aggression – have been modest, inconsistent, and sometimes contradictory. This thesis contains four studies on the associations of guilt, shame, emotion regulation, and social cognitions with children’s social behavior. The longitudinal material of the thesis was collected as a survey among a relatively large amount of Finnish preadolescents. In Study I, the distinctiveness of guilt and shame in children’s social behavior were investigated. The more specific links of emotions and aggressive behavior were explored in Study II, in which emotion regulation and negative emotionality were treated as the moderators between guilt, shame, and children’s aggressive behavior. The role of emotion management was further evaluated in Study III, in which effortful control and anger were treated as the moderators between domain-specific aggressive cognitions and children’s aggressive behavior. In the light of the results from the Studies II and III, it seems that for children with poor emotion management the effects of emotions and social cognitions on aggressive behavior are straight-forward, whereas effective emotion management allows for reframing the situation. Finally, in Study IV, context effects on children’s anticipated emotions were evaluated, such that children were presented a series of hypothetical vignettes, in which the child was acting as the aggressor. Furthermore, the identity of the witnesses and victim’s reactions were systematically manipulated. Children anticipated the most shame in situations, in which all of the class was witnessing the aggressive act, whereas both guilt and shame were anticipated the most in the situations, in which the victim was reacting with sadness. Girls and low-aggressive children were more sensitive to contextual cues than boys and high-aggressive children. Overall, the results of this thesis suggest that the influences of guilt, shame, and social cognition on preadolescents’ aggressive behavior depend significantly on the nature of individual emotion regulation, as well as situational contexts. Both theoretical and practical implications of this study highlight a need to acknowledge effective emotion management as enabling the justification of one’s own immoral behavior.

Food and the circadian activity of the hypothalamic-pituitary-adrenal axis

Temporal organization is an important feature of biological systems and its main function is to facilitate adaptation of the organism to the environment. The daily variation of biological variables arises from an internal time-keeping system. The major action of the environment is to synchronize the internal clock to a period of exactly 24 h. The light-dark cycle, food ingestion, barometric pressure, acoustic stimuli, scents and social cues have been mentioned as synchronizers or" zeitgebers". The circadian rhythmicity of plasma corticosteroids has been well characterized in man and in rats and evidence has been accumulated showing daily rhythmicity at every level of the hypothalamic-pituitary-adrenal (HPA) axis. Studies of restricted feeding in rats are of considerable importance because they reveal feeding as a major synchronizer of rhythms in HPA axis activity. The daily variation of the HPA axis stress response appears to be closely related to food intake as well as to basal activity. In humans, the association of feeding and HPA axis activity has been studied under physiological and pathological conditions such as anorexia nervosa, bulimia, malnutrition, obesity, diabetes mellitus and Cushing's syndrome. Complex neuroanatomical pathways and neurochemical circuitry are involved in feeding-associated HPA axis modulation. In the present review we focus on the interaction among HPA axis rhythmicity, food ingestion, and different nutritional and endocrine states

Extracellular matrix molecules play diverse roles in the growth and guidance of central nervous system axons

Axon growth and guidance represent complex biological processes in which probably intervene diverse sets of molecular cues that allow for the appropriate wiring of the central nervous system (CNS). The extracellular matrix (ECM) represents a major contributor of molecular signals either diffusible or membrane-bound that may regulate different stages of neural development. Some of the brain ECM molecules form tridimensional structures (tunnels and boundaries) that appear during time- and space-regulated events, possibly playing relevant roles in the control of axon elongation and pathfinding. This short review focuses mainly on the recognized roles played by proteoglycans, laminin, fibronectin and tenascin in axonal development during ontogenesis.

The brain decade in debate: III. Neurobiology of emotion

This article is a transcription of an electronic symposium in which active researchers were invited by the Brazilian Society of Neuroscience and Behavior (SBNeC) to discuss the advances of the last decade in the neurobiology of emotion. Four basic questions were debated: 1) What are the most critical issues/questions in the neurobiology of emotion? 2) What do we know for certain about brain processes involved in emotion and what is controversial? 3) What kinds of research are needed to resolve these controversial issues? 4) What is the relationship between learning, memory and emotion? The focus was on the existence of different neural systems for different emotions and the nature of the neural coding for the emotional states. Is emotion the result of the interaction of different brain regions such as the amygdala, the nucleus accumbens, or the periaqueductal gray matter or is it an emergent property of the whole brain neural network? The relationship between unlearned and learned emotions was also discussed. Are the circuits of the former the underpinnings of the latter? It was pointed out that much of what we know about emotions refers to aversively motivated behaviors, like fear and anxiety. Appetitive emotions should attract much interest in the future. The learning and memory relationship with emotions was also discussed in terms of conditioned and unconditioned stimuli, innate and learned fear, contextual cues inducing emotional states, implicit memory and the property of using this term for animal memories. In a general way it could be said that learning modifies the neural circuits through which emotional responses are expressed.

The brain decade in debate: VI. Sensory and motor maps: dynamics and plasticity

This article is an edited transcription of a virtual symposium promoted by the Brazilian Society of Neuroscience and Behavior (SBNeC). Although the dynamics of sensory and motor representations have been one of the most studied features of the central nervous system, the actual mechanisms of brain plasticity that underlie the dynamic nature of sensory and motor maps are not entirely unraveled. Our discussion began with the notion that the processing of sensory information depends on many different cortical areas. Some of them are arranged topographically and others have non-topographic (analytical) properties. Besides a sensory component, every cortical area has an efferent output that can be mapped and can influence motor behavior. Although new behaviors might be related to modifications of the sensory or motor representations in a given cortical area, they can also be the result of the acquired ability to make new associations between specific sensory cues and certain movements, a type of learning known as conditioning motor learning. Many types of learning are directly related to the emotional or cognitive context in which a new behavior is acquired. This has been demonstrated by paradigms in which the receptive field properties of cortical neurons are modified when an animal is engaged in a given discrimination task or when a triggering feature is paired with an aversive stimulus. The role of the cholinergic input from the nucleus basalis to the neocortex was also highlighted as one important component of the circuits responsible for the context-dependent changes that can be induced in cortical maps.

Anatomical connections of the periaqueductal gray: specific neural substrates for different kinds of fear

The periaqueductal gray (PAG) has been traditionally considered to be an exit relay for defensive responses. Functional mapping of its subdivisions has advanced our knowledge of this structure, but synthesis remains difficult mainly because results from lesion and stimulation studies have not correlated perfectly. After using a strategy that combined both techniques and a reevaluation of the available literature on PAG function and connections, we propose here that freezing could be mediated by different PAG subdivisions depending on the presence of immediate danger or exposure to related signaling cues. These subdivisions are separate functional entities with distinct descending and ascending connections that are likely to play a role in different defensive responses. The existence of ascending connections also suggests that the PAG is not simply a final common path for defensive responses. For example, the possibility that indirect ascending connections to the cingulate cortex could play a role in the expression of freezing evoked by activation of the neural substrate of fear in the dorsal PAG has been considered.