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Photosynthesis in higher plants relies upon collection of light by chlorophyll molecules associated with light harvesting chlorophyll a/b-binding (LHC) proteins. The two most abundant of these are Lhcb1 and Lhcb2, which make up light harvesting complex (LHC) II trimers. They are also involved in facilitating state transitions, a process during which energy balancing between photosystem (PS) II and I is achieved. Overexcitation of PSII reduces the plastoquinone pool which activates STN7, a kinase, that phosphorylates a threonine residue on Lhcb1 and Lhcb2. In order to studythe kinetics of this we developed antibodies capable of recognizingphosphorylated forms of each of these proteins. This showed that Lhcb2 is more rapidly phosphorylated than Lhcb1, that there are no differences in the migration of phosphorylated and non-phosphorylated forms of Lhcb1 and Lhcb2 and that the majority of phosphorylated LHCII (P-Lhcb1 and PLhcb2) are associated with super- and megacomplexes. Furthermore, a state 2-specific LHCII-PSI-LHCI band contains P-Lhcb2 but almost no P-Lhcb1, and a band corresponding to M trimers (band 4 from sucrose gradients, composed of LHCII, CP24 and CP29), contains only P-Lhcb1 but no P-Lhcb2. We also developed artificial microRNA lines specifically depleted in either Lhcb1 or Lhcb2, amiLhcb1 and amiLhcb2 respectively. We show that the roles of Lhcb1 and Lhcb2 in state transitions are complementary. Lhcb1 modulate the size of grana stacks. In the absence of Lhcb1 only a few LHC trimers are formed, while in the absence of Lhcb2, the antenna looks like in the wild type although the plants cannot perform state transitions normally. Trimers containing P-Lhcb2 functionally detach from PSII and connect to PSI to balance the relative excitation pressure. State transitions only occur when both Lhcb1 and Lhcb2 are present, presumably in a (Lhcb1)2 Lhcb2 heterotrimer. In absence of Lhcb2, the LHCII-PSI-LHCI supercomplex is not formed indicting that P-Lhcb2 mediates attachment of LHCII to PSI. We tried complementing amiLhcb2 with modified Lhcb2 genes coding for proteins with altered amino acids, Arg2 to Lys or the phosphorylatable Thr3 residue to Asn or Ser. Introduction of the additional gene often causes loss of amiRNA-inhibition, however we could confirm that substitution of the Thr3 with Asn led to the absence of Lhcb2 phosphorylation and thus no state transition. / Klorofyll a/b-bindande proteiner (s k light harvesting chlorophyll a/b-binding proteins eller LHC proteiner) är viktiga för högre växters fotosyntes, då deras klorofyllmolekyler skördar solljuset. Två av dessa proteiner, Lhcb1 och Lhcb2, bygger upp ”LHCII trimerer” och finns i större mängd än de andra och dessa är även viktiga för s k ”state transtions”, en process som ser till att fotosystem (PS) I och PSII exciteras lika mycket. Om PSII exciteras för mycket reduceras plastoquinon-poolen som i sin tur aktiverar ett proteinkinas, STN7, som fosforylerar en av Lhcb1/Lhcb2s treoniner. För att studera denna fosforylering har vi utvecklat antikroppar som är specifika för dessa fosforylerade former av proteinerna, och vi använde dem för att visa att Lhcb2 fosforyleras snabbare än Lhcb1, och att största delen av det fosforylerade proteinerna (P-Lhcb1 och P-Lhcb2) finns i s k super- eller megakomplex. Ett komplex som bara finns finns i ”state 2” består av LHCII, PSI och LHCI, och det innehåller endast P-Lhcb2 men nästan inget P-Lhcb1, och ett band som består av LHCII, CP24 och CP29 innehåller endast PLhcb1. Vi skapade artificiella mikro-RNA-linjer, amiLhcb1 och amiLhcb2, som saknade antingen Lhcb1 eller Lhcb2. Lhcb1 påverkar höjden av grana stackarna. Med hjälp av dessa visade vi att Lhcb1 och Lhcb2 har komplementära roller för state transitions, saknas Lhcb1 gör växten bara få LHCII trimerer, medan om Lhcb2 gör växten antennener som liknar vildtypens, men den kan inte utföra state transitions som den. Mängden Lhcb1 påverkar storleken av ”grana stacks”. Trimerer som innehåller PLhcb2 kopplas över från PSII till PSI för att balansera excitationstrycket. Både Lhcb1 och Lhcb2, antagligen i trimerer bestående av en Lhcb2 och the Lhcb1, behövs för state transitions. Saknas Lhcb2 bildas inga komplex bestående av LHCII, PSI och LHCI, vilket visar att P-Lhcb2 antagligen möjliggör LHCIIs bindning till PSI. Vi försökte komplementera amiLhcb2 med Lhcb2 gener där amino syror bytts ut, Arg2 till Lys eller den fosforylerbara Thr3 till Asn eller Ser. När denna gen introducerades försvann dock ofta amiRNA-inhiberingen, men vi kunde visa att om Thr3 ersattes med Asn skedde inga state transitions.

<p>In photosynthesis, sunlight is converted into chemical energy that is stored mainly as carbohydrates and supplies basically all life on Earth with energy.</p><p>In order to efficiently absorb the light energy, plants have developed the outer light harvesting antenna, which is composed of ten different protein subunits (LHC) that bind chlorophyll a and b as well as different carotenoids. In addition to the light harvesting function, the antenna has the capacity to dissipate excess energy as heat (feedback de-excitation or qE), which is crucial to avoid oxidative damage under conditions of high excitation pressure. Another regulatory function in the antenna is the state transitions in which the distribution of the trimeric LHC II between photosystem I (PS I) and II is controlled. The same ten antenna proteins are conserved in all higher plants and based on evolutionary arguments this has led to the suggestion that each protein has a specific function.</p><p>I have investigated the functions of individual antenna proteins of PS II (Lhcb proteins) by antisense inhibition in the model plant Arabidopsis thaliana. Four antisense lines were obtained, in which the target proteins were reduced, in some cases beyond detection level, in other cases small amounts remained.</p><p>The results show that CP29 has a unique function as organising the antenna. CP26 can form trimers that substitute for Lhcb1 and Lhcb2 in the antenna structure, but the trimers that accumulate as a response to the lack of Lhcb1 and Lhcb2 cannot take over the LHC II function in state transitions. It has been argued that LHC II is essential for grana stacking, but antisense plants without Lhcb1 and Lhcb2 do form grana. Furthermore, LHC II is necessary to maintain growth rates in very low light.</p><p>Numerous biochemical evidences have suggested that CP29 and/or CP26 were crucial for feedback de-excitation. Analysis of two antisense lines each lacking one of these proteins clearly shows that there is no direct involvement of either CP29 or CP26 in this process. Investigation of the other antisense lines shows that no Lhcb protein is indispensable for qE. A model for feedback de-excitation is presented in which PsbS plays a major role.</p><p>The positions of the minor antenna proteins in the PS II supercomplex were established by comparisons of transmission electron micrographs of supercomplexes from the wild type and antisense plants.</p><p>A fitness experiment was conducted where the antisense plants were grown in the field and seed production was used to estimate the fitness of the different genotypes. Based on the results from this experiment it is concluded that each Lhcb protein is important, because all antisense lines show reduced fitness in the field.</p>

In photosynthesis, sunlight is converted into chemical energy that is stored mainly as carbohydrates and supplies basically all life on Earth with energy. In order to efficiently absorb the light energy, plants have developed the outer light harvesting antenna, which is composed of ten different protein subunits (LHC) that bind chlorophyll a and b as well as different carotenoids. In addition to the light harvesting function, the antenna has the capacity to dissipate excess energy as heat (feedback de-excitation or qE), which is crucial to avoid oxidative damage under conditions of high excitation pressure. Another regulatory function in the antenna is the state transitions in which the distribution of the trimeric LHC II between photosystem I (PS I) and II is controlled. The same ten antenna proteins are conserved in all higher plants and based on evolutionary arguments this has led to the suggestion that each protein has a specific function. I have investigated the functions of individual antenna proteins of PS II (Lhcb proteins) by antisense inhibition in the model plant Arabidopsis thaliana. Four antisense lines were obtained, in which the target proteins were reduced, in some cases beyond detection level, in other cases small amounts remained. The results show that CP29 has a unique function as organising the antenna. CP26 can form trimers that substitute for Lhcb1 and Lhcb2 in the antenna structure, but the trimers that accumulate as a response to the lack of Lhcb1 and Lhcb2 cannot take over the LHC II function in state transitions. It has been argued that LHC II is essential for grana stacking, but antisense plants without Lhcb1 and Lhcb2 do form grana. Furthermore, LHC II is necessary to maintain growth rates in very low light. Numerous biochemical evidences have suggested that CP29 and/or CP26 were crucial for feedback de-excitation. Analysis of two antisense lines each lacking one of these proteins clearly shows that there is no direct involvement of either CP29 or CP26 in this process. Investigation of the other antisense lines shows that no Lhcb protein is indispensable for qE. A model for feedback de-excitation is presented in which PsbS plays a major role. The positions of the minor antenna proteins in the PS II supercomplex were established by comparisons of transmission electron micrographs of supercomplexes from the wild type and antisense plants. A fitness experiment was conducted where the antisense plants were grown in the field and seed production was used to estimate the fitness of the different genotypes. Based on the results from this experiment it is concluded that each Lhcb protein is important, because all antisense lines show reduced fitness in the field.

This thesis provides a novel dynamic large-scale network perspective on brain activity of human sleep based on the analysis of unique human neuroimaging data. Specifically, I provide new information based on integrating spatial and temporal aspects of brain activity both in the transitions between and during wakefulness and various stages of non-rapid-eye movement (NREM) sleep. This is achieved through investigations of inter-regional interactions, functional connectivity (FC), between activity timecourses throughout the brain. Overall, the presented findings provide new important whole-brain insights for our current understanding of sleep, and potentially also of sleep disorders and consciousness in general. In Chapter 2 I present a robust global increase in similarity between the structural connectivity (SC) and the FC in slow-wave sleep (SWS) in almost all of the participants of two independent fMRI datasets. This could point to a decreased state repertoire and more rigid brain dynamics during SWS. Chapter 2 further identifies the changes in FC strengths between wakefulness and individual stages of NREM sleep across the whole-brain fMRI network. I report connectivity in posterior parts of the brain as particularly strong during wakefulness, while connections between temporal and frontal cortices are increased in strength during N1 and N2 sleep. SWS is characterised by a global drop in FC. In Chapter 3 I take advantage of rare MEG recordings of NREM sleep to show, for the first time, the feasibility of constructing source-space FC networks of sleep using power envelope correlations. The increased temporal information of MEG signals allows me to identify the specific frequencies underlying the FC differences identified in Chapter 2 with fMRI. The beta band (16 – 30 Hz) thus stands out as important for the strong posterior connectivity of wakefulness, while a range of frequency bands from delta (0.25 – 4 Hz) to sigma (13 – 16 Hz) all appear to contribute to N2-specific FC increases. Consistent with the fMRI results, slow-wave sleep shows the lowest level of FC. Interestingly, however, the MEG signals suggest a fronto-temporal network of high connectivity in the alpha band, possibly reflecting memory processes. In Chapter 4 I expand the within-frequency FC analysis of Chapter 3 to explore potential cross-frequency interactions in the MEG FC networks. It is shown that N2 sleep involves an abundance of frequency cross-talk, while SWS includes very little. A multi-layer network approach shows that the gamma band (30 – 48 Hz) is particularly integrated in wakefulness. Chapter 5 addresses the identified MEG FC findings from the perspective of traditional spectral sleep staging. By correlating temporal changes in spectral power at the sensor level to fluctuations in average FC, a specific type of transient events is found to underlie the strong N2-specific coupling in static FC values. Lastly, in Chapter 6 I make the leap out of the constraints of traditional low-resolution sleep staging, and extract dynamic states of FC from fMRI timecourses in a completely unsupervised fashion. This provides a novel representation of whole-brain states of sleep and the dynamics governing them. I argue that data-driven approaches like this are necessary to fully characterise the spatiotemporal principles underlying wakefulness and sleep in the human brain.

Nonphotochemical quenching and state transitions are an important photoprotective mechanism against excessive irradiation. In this work I studied changes in the size of the effective crosssection of photosystem II antennae in regard to the level of nonphotochemical quenching (state transitions) under different levels of light induced stress.

La chlororespiration a été initialement décrite chez Chlamydomonas reinhardtii. Cette voie alternative du transfert d’électrons, présente dans toutes des lignées photosynthétiques, est constituée par l’activité d’une NAD(P)H:plastoquinone oxidoreductase, et d’une plastoquinol oxydase (PTOX). Parce qu’elle utilise les plastoquinones comme transporteur d’électrons, la chlororespiration représente une voie potentiellement antagoniste au transfert photosynthétique de l’eau au CO2. Néanmoins, le faible flux autorisé par ces enzymes suggère que, au moins sous éclairement continu et en conditions stationnaires, leur contribution est limité. Je me suis donc concentré sur la rôle du PTOX pendant les transitions lumière-obscurité et vice-versa. J’ai observé qu’après une brève illumination, la relaxation redox du chloroplaste est entravée quand PTOX2, l’oxydase majeure chez Chlamydomonas, est absente. J’ai démontré la pertinence physiologique de cette observation par une étude des courbes de croissance de souches mutantes pour PTOX et de la souche sauvage: la croissance du mutant de PTOX2 est retardée en condition de la lumière intermittente – ce qui peux être expliqué par une diminution du flux d’électrons à partir du photosystème II. Je me suis également intéressé à l’impact de la chlororespiration sur le flux d’électrons cyclique en utilisant une nouvelle approche spectroscopique combinée à de la modélisation. Enfin, j’ai exploré, en collaboration avec Stefano Santabarbara, le mécanisme de redistribution de l’énergie lumineuse entre les deux photosystèmes, mécanisme gouverné par des changements d’état redox des plastoquinones. J’ai démontré que, contrairement à des récentes suggestions, un véritable transfert d’antennes se produit entre les deux photosystèmes. / Chlororespiration was initially described in Chlamydomonas reinhardtii. This electron transfer pathway, found in all photosynthetic lineages, consists of the action of a NAD(P)H:plastoquinone oxidoreductase and a plastoquinol oxidase (PTOX). Hence, because it uses plastoquinones for electron transport, chlororespiration constitutes an electron pathway potentially antagonistic to the linear photosynthetic electron flow from H2O to CO2 However, the limited flow these enzymes can sustain suggests that their relative contribution, at least in the light and in steady-state conditions, is limited. I thus focused on the involvement of PTOX in Chlamydomonas during transitions from dark to light and vice versa. I found that, following a brief illumination, the redox relaxation of the chloroplast in the dark was much affected when PTOX2, the major plastoquinol oxidase in Chlamydomonas, is lacking. Importantly, I show that this has a significant physiological relevance as the growth of a PTOX2- lacking mutant is markedly slower in intermittent light, which can be rationalized in terms of a decreased flux sustained by photosystem II. I also investigated the influence of chlororespiration on cyclic electron flow using novel experimental techniques combined with theoretical modelling. Last, I explored, in collaboration with Stefano Santabarbara, the mechanism for redistribution of light excitation energy between the two photosystems, a process triggered by changes in the redox state of plastoquinone pool. I showed that, contrarily to what has been suggested recently, this regulation mechanism corresponds to an actual transfer of light harvesting antenna between the two photosystems.

The aromatic amino acids tryptophan, tyrosine, and phenylalanine have been extensively used for different label-free protein studies. These investigations extract information on protein conformations and interactions from the emitted fluorescence's intensity, wavelength, and/or polarization. Like most fluorescent organic compounds, these amino acids also undergo transitions into dark meta-stable states, including triplet and photo-radical states. These transitions are notably sensitive to the surrounding environment, offering an additional set of parameters that reflect the protein's interactions, folding states, and immediate surroundings. Transient State (TRAST) monitoring has been developed to quantify fluorophore transition dynamics by recording the average fluorescence intensity in response to a modulated excitation. In this work, we performed TRAST experiments to investigate tyrosine autofluorescence and used it to detect conformational changes in calmodulin, a calcium-binding protein containing two tyrosine residues. A photophysical model for tyrosine was established, and it was revealed how tyrosine's dark state transitions changed with excitation intensity, solvent pH, and redox conditions. The TRAST experiments demonstrated that tyrosine's dark state transitions could serve as valuable information sources for label-free analyses of protein conformations and interactions. / De aromatiska aminosyrorna tryptofan, tyrosin och fenylalanin har använts i stor utsträckning för olika inmärkningsfria proteinstudier. Dessa undersökningar extraherar information om proteinkonformationer och interaktioner från den emitterade fluorescens intensiteten, dess våglängd och/eller polarisering. Liksom de flesta fluorescerande organiska föreningar genomgår dessa aminosyror också övergångar till mörka metastabila tillstånd, inklusive triplett- och fotoradikaltillstånd. Dessa övergångar är särskilt känsliga för den omgivande miljön, och erbjuder en extra uppsättning parametrar som återspeglar proteinets interaktioner, vikningstillstånd och omedelbara omgivningar. Transient State (TRAST) monitorering har utvecklats för att kvantifiera fluoroforövergångsdynamik genom att registrera den genomsnittliga fluorescensintensiteten som svar på en modulerad excitation. I detta arbete utförde vi TRAST-experiment för att undersöka tyrosinautofluorescens och använde den för att detektera konformationsförändringar i calmodulin, ett kalciumbindande protein som innehåller två tyrosiner. En fotofysikalisk modell för tyrosin etablerades, och hur tyrosins mörka tillståndsövergångar förändrades med excitationsintensitet, lösningsmedels pH och redoxförhållanden kunde faststållas. TRAST- experimenten visade att tyrosins mörka tillståndsövergångar kan fungera som värdefulla informationskällor för inmärkningsfria analyser av proteinkonformationer och interaktioner.

The GERDA experiment searches for the neutrinoless double beta (0𝜈𝛽𝛽) decay of Ge-76. The observation of this decay would prove the Majorana character of the neutrino, i.e. that it is its own antiparticle. This would clarify the question which neutrino mass ordering is realized in nature and give a hint of the effective Majorana neutrino mass. Furthermore, the existence of the 0𝜈𝛽𝛽 decay would imply the violation of lepton number conservation which is a key feature in some theories explaining the asymmetry of matter and antimatter in the universe. The effective Majorana neutrino mass is connected with the half life of the 0𝜈𝛽𝛽 decay via a nuclear matrix element (NME), which is predicted by various theoretical models that are afflicted by large uncertainties. The accuracy of the different NMEs and their internal model assumptions can be increased by considering experimental investigations. While the NMEs for the 0𝜈𝛽𝛽 decay and the neutrino accompanied double beta (2𝜈𝛽𝛽) decay are numerically different, they rely on similar model assumptions. Thus, experimental constraints can be given by the 2𝜈𝛽𝛽 decay into the ground state, which has been already measured by GERDA with unprecedented precision for Ge-76, but also by the investigation of the 2𝜈𝛽𝛽 decay into excited states, which has not yet been observed for Ge-76. GERDA operates enriched germanium detectors in liquid argon (LAr) which serves as an additional background veto using the scintillation light that is created when energy is deposited in LAr. The signal signature of the decay into excited states can be enhanced with the application of the LAr veto, however, for that the efficiency of the LAr veto needs to be determined. One of the key parameters of the LAr efficiency is the attenuation of the scintillation light in LAr, which is dependent on the impurity composition and concentration in LAr. Therefore, the attenuation length of the scintillation light in LAr has been measured in GERDA with a dedicated setup in the course of this work. The analysis of the acquired data required intense computer simulations in order to describe the background for the measurement sufficiently. This also involved the measurement of the steel reflectivity in the visible and the UV region, where LAr scintillates. Therewith, the search for excited states has been performed in this work for the data accumulated in GERDA Phase I, Phase II and Phase II+ including the LAr veto for the latter two data sets. New limits have been set on the investigated excited states decay modes and some of the corresponding theoretical half life predictions could be disfavored, i.e. the underlying NMEs models can be constrained. The successor experiment LEGEND will continue searching for the 0𝜈𝛽𝛽 decay of Ge-76 using more germanium detectors together with an improved LAr veto. The investigation of the decay of Ge-76 into excited states will also be further pursued in LEGEND.

Many normal and cancerous cell lines exhibit a stable composition of cells in distinct states which can, e.g., be defined on the basis of cell surface markers. There is evidence that such an equilibrium is associated with stochastic transitions between distinct states. Quantifying these transitions has the potential to better understand cell lineage compositions. We introduce CellTrans, an R package to quantify stochastic cell state transitions from cell state proportion data from fluorescence-activated cell sorting and flow cytometry experiments. The R package is based on a mathematical model in which cell state alterations occur due to stochastic transitions between distinct cell states whose rates only depend on the current state of a cell. CellTrans is an automated tool for estimating the underlying transition probabilities from appropriately prepared data. We point out potential analytical challenges in the quantification of these cell transitions and explain how CellTrans handles them. The applicability of CellTrans is demonstrated on publicly available data on the evolution of cell state compositions in cancer cell lines. We show that CellTrans can be used to (1) infer the transition probabilities between different cell states, (2) predict cell line compositions at a certain time, (3) predict equilibrium cell state compositions, and (4) estimate the time needed to reach this equilibrium. We provide an implementation of CellTrans in R, freely available via GitHub (https://github.com/tbuder/CellTrans).