High-throughput sequencing has greatly influenced the amount of data produced and biological questions asked and answered. Sequencing approaches have also enabled rapid development of related technological fields such as single-cell and spatially resolved expression profiling. The introductory parts of this thesis give an overview of the basic molecular and technological apparatus needed to analyse the transcriptome in cells and tissues. This is succeeded by a summary of present investigations that report recent advancements in RNA profiling. RNA integrity needs to be preserved for accurate gene expression analysis. A method providing a low-cost alternative for RNA preservation was reported. Namely, a low concentration of buffered formaldehyde was used for fixation of human cell lines and peripheral blood cells (Paper I). The results from bulk RNA sequencing confirmed gene expression was not negatively impacted with the preservation procedure (r2&gt;0.88) and that long-term storage of such samples was possible (r2=0.95). However, it is important to note that a small population of cells overexpressing a limited amount of genes can skew bulk gene expression analyses making them sufficient only in carefully designed studies. Therefore, gene expression should be investigated at the single cell resolution when possible. A method for high-throughput single cell expression profiling termed microarrayed single-cell sequencing was developed (Paper II). The method incorporated fluorescence-activated cell sorting, sample deposition and profiling of thousands of barcoded single cells in one reaction. After sample attachment to a barcoded array, a high-resolution image was taken which linked the position of each array barcode sequence to each individual deposited cell. The cDNA synthesis efficiency was estimated at 17.3% while detecting 27,427 transcripts per cell on average. Additionally, spatially resolved analysis is important in cell differentiation, organ development and pathological changes. Current methods are limited in terms of throughput, cost and time. For that reason, the spatial transcriptomics method was developed (Paper III). Here, the barcoded microarray was used to obtain spatially resolved expression profiles from tissue sections using the same imaging principle. The mouse olfactory bulb was profiled on a whole-transcriptome scale and the results showed that the expression correlated well (r2=0.94-0.97) as compared to bulk RNA sequencing. The method was 6.9% efficient, reported signal diffusion at ~2 μm and accurately deconvoluted layer-specific transcripts in an unbiased manner. Lastly, the spatial transcriptomics concept was applied to profile human breast tumours in three dimensions (Paper IV). Unbiased clustering revealed previously un-annotated regions and classified them as parts of the immune system, providing a detailed view into complex interactions and crosstalk in the whole tissue volume. Spatial tumour classification divulged that certain parts of the tumour clearly classified as other subtypes as compared to bulk analysis providing useful data for current practice diagnostics. The last part of the thesis discusses a look towards the future, how the presented methods could be used, improved upon or combined in translational research. / <p>QC 20170109</p>

Photodynamic therapy (PDT) currently utilizes drug and light doses which are primarily based on clinical experience. This can lead to a dose which is not sufficient to destroy the entire tumor, or alternatively, it can lead to the undesirable destruction of healthy tissue around the treatment area. PDT of topically applied photosensitizers is one focus of this research. This concerns the diffusion of an externally applied drug into the tissue, as well as its subsequent destruction during the irradiation procedure. This work involves the non-invasive measurement of the inherent fluorescence of the photosensitizer, allowing the determination of the concentration and distribution of drug within the tissue, and thus optimizing this treatment. To do this, one must be able to describe the propagation of light within the tissue. Consequently, a photon diffusion model has been developed to calculate the steady-state spatially resolved fluorescence from a pencil beam excitation in a depth dependent medium. The validity of this model was then verified by comparison with Monte Carlo simulations and measurements made on phantoms with optical properties similar to those of human tissue. Theoretical conditions were then explored, and potential uses of the model were demonstrated. / Thesis / Master of Science (MS)

It is well known that cells in tissues display a large heterogeneity in gene expression due to differences in cell lineage origin and variation in the local environment at different sites in the tissue, a heterogeneity that is difficult to study by analyzing bulk RNA extracts from tissue. Recently, genome-wide transcriptome analysis technologies have enabled the analysis of this variation with single-cell resolution. In order to link the heterogeneity observed at molecular level with the morphological context of tissues, new methods are needed which achieve an additional level of information, such as spatial resolution. In this thesis I describe the development and application of padlock probes and rolling circle amplification (RCA) as molecular tools for spatially-resolved transcriptome analysis. Padlock probes allow in situ detection of individual mRNA molecules with single nucleotide resolution, visualizing the molecular information directly in the cell and tissue context. Detection of clinically relevant point mutations in tumor samples is achieved by using padlock probes in situ, allowing visualization of intra-tumor heterogeneity. To resolve more complex gene expression patterns, we developed in situ sequencing of RCA products combining padlock probes and next-generation sequencing methods. We demonstrated the use of this new method by, for the first time, sequencing short stretches of transcript molecules directly in cells and tissue. By using in situ sequencing as read-out for multiplexed padlock probe assays, we measured the expression of tens of genes in hundreds of thousands of cells, including point mutations, fusions transcripts and gene expression level. These molecular tools can complement genome-wide transcriptome analyses adding spatial resolution to the molecular information. This level of resolution is important for the understanding of many biological processes and potentially relevant for the clinical management of cancer patients. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.</p>

Sequencing technologies and applications have pushed the limits and enabled novel studies of biological mechanisms, evolutionary relationships and communication networks between cells. The technical developments leading to single cell RNA-sequencing have enabled detection of rare cell populations while spatial resolution added insights into larger biological environments, like tissues and organs. Massively parallel sequencing has paved the way for integrated high-throughput analyses including that of studying gene expression, protein expression and mapping of microbial communities. This thesis starts with an introduction describing the technical and biological advancements made in recent years with focus on spatially resolved approaches. Then, a summary of recent accomplishments is presented, which enabled ongoing work in a novel field of spatial hostmicrobiome profiling. Lastly, the concluding remarks include both a future perspective and a short reflection on the current developments in the spatial multi-omics field. 16S sequencing is often used for taxonomic classification of bacteria. In Paper I, this sequencing technique was used to study the aerodigestive microbiome in pediatric lung transplant recipients. Many of these patients regretfully reject the organ after transplant, but the underlying cause is, in many cases, unknown. In this paper, multiple factors influencing rejection were examined including that of the aerodigestive microbiome. Pediatric lung transplant recipients often suffer from gastrointestinal dysmotility and the focus of this study was also to analyze changes in the microbiome in relation to irregular gastric muscle movements. The results showed that lung transplant recipients had, in general, lower microbial diversity in the gastric fluid and throat and also that the microbial overlap between lung and gastric sampling sites was significantly less in transplant recipients compared to controls. In addition, gastrointestinal dysmotility was shown to influence the gastric microbiome in lung transplant recipients, but, given the small sample size available in this study, the correlation to patient outcome could not be examined. Integrated analysis of the transcriptome and the antibody-based proteome in the same tissue section was enabled using the method developed in Paper II. Spatial Multi- Omics (SM-Omics) uses a barcoded glass array to capture mRNA and antibody-based expression of selected proteins in the same section. The antibody-based profiling of the tissue section was enabled by either immunofluorescence or DNA-barcoded antibodies that were then decoded by sequencing. The protocol was scaled-up using an automated liquidhandling system. Using this method, simultaneous profiling of the transcriptome and multiplexed protein values was determined in both the mouse brain cortex and mouse spleen. Results showed a high correlation in spatial pattern between gene expression and antibody measurements, independently of the antibody labelling technique. SM-Omics generates a high-plex multi-omics characterization of the tissue in a high throughput manner while exhibiting low technical variation. / Tekniker och applikationer som använder sekvensering har flyttat fram gränsernaoch tillåtit nya undersökningar av biologiska mekanismer, evolutionära släktskap ochkommunikationsnätverk mellan celler. De tekniska utvecklingarna som har lett fram tillRNA-sekvensering av enskilda celler har möjliggjort upptäckten av sällsynta cellpopulationer medan den rumsliga upplösningen har inneburit en ökad förståelse av störrebiologiska miljöer, såsom vävnader och organ. Massively parallel sequencing har banat vägför integrerade analyser med hög kapacitet, vilket inkluderar analys av genuttryck,proteinuttryck och kartläggning av bakteriella samhällen. Den här avhandlingen börjar meden introduktion som beskriver tekniska och biologiska framsteg som gjorts de senaste åren,med fokus på den rumsliga upplösningen. Sedan följer en summering av de senasteprestationerna som har möjliggjort det pågående arbetet i ett nytt fält som avhandlarrumslig profilering av bakterien och dess värd. Slutligen innehåller slutordet både ettframtida perspektiv samt en kort reflektion av den nuvarande utvecklingen inom fälten förrumslig mång-omik. 16S-sekvensering används ofta för att taxonomiskt klassificera bakterier. Dennasekvenseringsteknik användes i artikel I för att studera mikrobiomet i luft- ochmatspjälkningskanalen hos barn med transplanterad lunga. Dessvärre är det vanligt medavstötning av lungan efter transplantationen hos många av dessa patienter, men denunderliggande orsaken till avstötningen är, i många fall, okänd. I denna studie undersöktesflertalet faktorer, inklusive mikrobiomet i luft- och matspjälkningskanalen, som kan tänkaspåverka bortstötningen. Barn med transplanterad lunga lider ofta av störningar i magtarmkanalens rörelser och artikelns fokus var därmed även att analysera förändringar imikrobiomet i relation till dessa avvikande rörelser i mag-tarmkanalen. Resultatet visade attpatienter med transplanterad lunga generellt hade lägre bakteriell mångfald i magsaft ochhals, samt att det bakteriella överlappet mellan lunga och magsaft var signifikant mindre ipatienter med transplanterad lunga jämfört med kontrollerna. För övrigt visade det sig attstörningar i mag-tarmkanalens rörelser påverkade magsaftens mikrobiom hos patientermed transplanterad lunga, men på grund av studiens storlek på urvalet, kunde det inteundersökas hur detta korrelerade till utfallet hos patienterna. Integrerad analys av transkriptomet och antikroppsbaserad analys av proteomet isamma vävnadssnitt har möjliggjorts genom metoden som utvecklats i artikel II. SpatialMulti-Omics (SM-Omics) använder ett avkodningsbart mönster av korta DNA-segment påen glasyta för att fånga mRNA och antikroppsbaserat uttryck av utvalda proteiner frånsamma vävnadssnitt. Den antikroppsbaserade profileringen av vävnadssnittet uppnåddesgenom antingen immunofluorescens eller antikroppar märkta med DNA-segment somkunde avkodas genom sekvensering. Protokollet skalades upp genom ett automatiseratsystem för att behandla vätskor. Genom användning av denna metod kunde simultanprofilering av transkriptomet och flertalet proteiner uppnås i både hjärnbarken och mjältenhos en mus. Resultaten visade en hög korrelation i det rumsliga mönstret mellangenuttrycket och de antikroppsbaserade mätningarna, oberoende av hur antikropparnahade märkts. SM-Omics genererar en storskalig karaktärisering av vävnaden av flera omikermed hög kapacitet samtidigt som den har låg teknisk variation. / <p>QC 2021-02-02</p>

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第21756号 / 工博第4573号 / 新制||工||1713(附属図書館) / 京都大学大学院工学研究科機械理工学専攻 / (主査)教授 蓮尾 昌裕, 教授 木村 健二, 教授 江利口 浩二 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM

In the 21st century the safe disposal of highly radioactive nuclear waste remains a major challenge to humanity. For the first 300 years nuclear fission products will be the main source of large amounts of the radioactivity, however, over a period of 100,000s of years most of the radiotoxicity of nuclear waste will originate from transuranium elements such as Pu, Am, and Cm. Because of the reducing conditions within a potential deep underground repository Am and Cm will exclusively – and Pu at least partially – be present in their trivalent state. For the safety case of a repository, it will be of importance to understand the mobility and retention behavior of those trivalent actinides within the host rock environment. Here, the focus is set on crystalline rock, a host rock under consideration in many countries such as Finland, Sweden, Czech Republic, and also Germany. Research in the last two decades has focused on determining the chemical speciation behavior of radionuclides on the molecular scale using primarily pure, individual mineral phases in their pulverized form. Real crystalline rock systems, on the km scale of a repository, are however much more complex, physically large and bulky, and heterogeneous. Parameters, such as heterogeneity of topography or composition, and competing surface processes have to be considered adequately in transport modelling as well as the safety assessment and thus large-scale experiments without the loss of molecular level information are needed. In this work, the focus was set on the assessment of parameters affecting chemical speciation and retention of Cm(III) and its chemical homologue Eu(III) at 5·10-7 M to 10-4 M concentrations on surfaces of crystalline rocks, relevant to potential future repositories. Correlative spectromicroscopy as a combination of spatially resolved techniques, was applied to study mineralogy, surface topography, quantitative metal ion uptake, and surface metal speciation on the same thin sections in the mm to cm scale with μm resolution. Cm(III) sorption experiments on cleaved K-feldspar crystals at pH 5.5 and 6.9 using autoradiography in conjunction with micro-focus time-resolved laser-induced fluorescence spectroscopy (μTRLFS), and vertical scanning interferometry, showed that sorption uptake and speciation is dependent on pH and surface roughness. In regions expressing a high surface roughness quantitatively more Cm(III) sorbed to the surface than in smoother regions. Further, the formed Cm(III) surface complexes are more strongly bound in regions with a high roughness, since they possess a higher number of strong sorption sites. The speciation between smooth and rough surfaces does not differ at pH 5.5. In contrast, at pH 6.9 in rougher areas, stronger species form favorably in addition to weaker surface sorption species. For comparison, Cm(III) sorption at pH 7.3 was conducted on a granodiorite crystalline rock thin section from the Grimsel Test Site, Switzerland with an inclusion of a large K-feldspar mineral grain. The spectromicroscopy approach was extended by Raman-microscopy to identify the surface mineralogy. From calibrated autoradiography of the entire thin section, it could be assessed that most sorption occurs on mica minerals rather than on feldspar. However, the K-feldspar grain was investigated in detail for a comparison of Cm(III) speciation on crystals and natural grains. On the grain, the sorption was heterogeneously distributed mainly occurring in rough surface regions. Since the overall sorption uptake was lower, the dominant species formed are bound strongly to the surface occupying strong sorption sites. Complexes were observed that were either the result of ternary complexation on the surface or incorporation. While sorption on K-feldspar crystals was higher than on the mineral grain, no ternary complexation could be observed, therefore the formation of this species is likely a result of the heterogeneous mineralogy. The formation of those complexes with carbonate is likely, whereas silicate does not seem to contribute. The exact complex structure determination will be the focus of future work. Increasing the complexity of the system, the next step was to study Cm(III) sorption at pH 8.0 on entire crystalline rock thin sections and analyzing speciation within and between different mineral gains. The focus was set on granite from Eibenstock, Germany and gneiss from Bukov, Czech Republic. On granite, sorption was highest on mica as well as feldspar and smallest on quartz as measured on the sub-mm to cm scale by quantitative autoradiography. On quartz regions, where surface roughness was higher, sorption quantities close to those on feldspar and mica could be observed. A detailed μTRLFS analysis shows highest sorption quantities on topaz, followed by feldspar, and only minor uptake was detected on quartz. Assessment of Cm(III) uptake on mica was not possible due to strong quenching of the Cm(III) luminescence through Fe as part of the mineral structure. Sorption on topaz, feldspar, and quartz occurred preferably in regions with a high surface roughness, such as surface pits, cracks, or mineral grain boundaries between the grains. Surface complexes in high roughness regions are bound more strongly than in smoother regions. A process that could either be Cm(III) ternary complexation or incorporation was exclusively observed on feldspar and quartz, likely because more sorption sites allowed for surface incorporation after which ternary complexation can occur. The experiments showed that mineralogy is the most important parameter when it comes to surface sorption, however it is closely followed by the surface roughness. On gneiss the overall mineralogy was different, which in turn affected the surface Cm(III) uptake. Combining the results from Raman microscopy and autoradiography, sorption was found almost exclusively on amphibole and mica, while little to no sorption was observed on feldspar and quartz. Due to the high Fe content of amphibole and mica, quenching hindered Cm(III) luminescence detection using μTRLFS. However, on feldspar and quartz μTRLFS allowed for uptake and speciation analysis. While no uptake was seen in smooth regions, uptake was increased in rougher regions. This highlights that in a competitive sorption environment, on low sorbing mineral phases, sorption is controlled by surface roughness. Cm(III) uptake and speciation analysis on mica using μTRLFS proved to be difficult because of luminescence quenching induced by structurally incorporated Fe in the minerals. However, to tackle this challenge, Cm(III) sorption experiments were performed on a granitic pegmatite from Olkiluoto, Finland at adjusted experimental settings, i.e., using a five times higher metal concentration than in previous experiments. Autoradiography as well as μTRLFS showed that Cm(III) sorption mainly occurred on mica, while uptake on feldspar and quartz was minor. Mica itself can be found as part of small cracks and pits, or as large grains. Inherently mica showed a high surface roughness, however Cm(III) uptake and speciation differed between the differently sized grains. On smaller grains, uptake was lower than on the larger grains, resulting in primarily stronger inner-sphere sorption species that formed. The high uptake on larger grains lead additionally to the formation of weaker inner-sphere sorption species. To compare the obtained Cm(III) results Eu(III) sorption was performed on a granite thin section. It was observed that Eu(III) uptake and speciation not only occurs heterogeneously between different minerals, but also within single grains particularly close to mineral grain boundaries. This implies that surface roughness, next to mineralogy, influences the sorption process. However, the detection of Eu(III) was hindered by surface precipitation, naturally incorporated Eu(III), and thus vague luminescence peak analysis. Spatially resolved correlative spectroscopy workflows that were further optimized in this thesis proved to be universally applicable in the range of mineral crystals to different crystalline rock thin sections and luminescent metals (Cm, Eu) and can be taken in the future as references for further studies with other luminescence metals such as Am(III) or U(VI). The derived findings show that in future assessments of the mobility of trivalent radionuclides in reactive transport modelling, parameters need to be selected carefully. Additional processes and parameters not considered before, like the surface roughness, will influence the retention of radionuclides within the geosphere. Those processes and parameters need to be quantified and implemented in the models to represent the deep repository system more reliable.

Abstract In this report I present a study of possible age gradients in the Green Pea galaxy J145735.13+223201.8 to be able to conclude if there is an extended star forming history in such a galaxy. Data are coming from two different sources, highly resolved images in four different wavelengths of stars in the galaxy, and of nebular gas in a narrow band Ha Balmer line filter, from the Hubble Space Telescope (HST), as well as spectral line information from the Sloan Digital Sky Survey (SDSS). I compare the observations with stellar population models from two different libraries, Yggdrasil and Starburst99. Due to the highly resolved images from HST this is one of the first studies of spatially resolved stellar populations in a Green Pea galaxy. With the help from these spatially resolved images it was possible to study star clumps independently from each other. This would not be possible when using only data from SDSS. In this way it was possible to conclude an age difference between the centre of the galaxy and its outskirts. I found that the galaxy has an age gradient at a confidence level greater than 95%.

The use of optical fiber is of great interest in developing extensive, high-speed networking infrastructures. Optical fiber provide many advantages over traditional copper cables and wireless links. Among them are high security, low electromagnetic interference, extremely low loss and high bandwidths, light weight and manageability. However, the very small wavelengths associated with optical radiation requires very small waveguide dimensions. Waveguide dimension of single mode fiber (SMF) are < 10µm, resulting in relatively poor yield in device manufacturing. For residential and other last-mile networks topologies, cost constraints limit the appeal of SMF. Multimode fiber (MMF) allow for less restrictive manufacturing tolerances; however, the distortion that results from the dispersion in propagation among the many modes can be prohibitively large for data rates approaching and exceeding 1 Gb/s. To improve the deployability of MMF, a method of dispersion compensation that maintains the ease-of-use characteristic of MMF is required This dissertation demonstrates an opto-electronic method of dispersion compensation by the use of a multisegment photodetector. It is shown the modes of the fiber can be seperated such that when the individual photodetector signals are combined, the resulting temporal response of the fiber link is improved from that of a conventional fiber link. This method is extremely robust to system variation and is independent of data rate and transmission format, allowing it to be employed in a wide variety of optical links. More importantly, the implementation demonstrated is comparable, in simplicity and alignment tolerance, to a conventional photodetector. System performance is shown using both temporal and frequency response as well as real bit error rate and eye diagram measurements.

Abstract Light scattering diagnostics of turbid media containing both structural and dynamic inhomogeneities is currently of significant importance. One of the important directions in modern light scattering diagnostics is the development of methods for probing biological media with visible- and near-infrared radiation allowing for visualization of the biotissue structure. Optical methods for studying the biotissue structure and characterization of its optical properties are very promising and have been rapidly developing during the past decade. The present work is aimed at improving and discovering new potentials of currently existing methods of laser diagnostics of biological tissues containing both structural and dynamic inhomogeneities. In particular, the feasibilities of spatially resolved reflectometry and time-of-flight techniques for the problem of noninvasive determination of glucose level in human blood and tissues were examined both numerically and experimentally. The relative sensitivities of these methods to changes in glucose level were estimated. Time-of-flight technique was found to be more sensitive. The possibilities of Doppler optical coherence tomography for imaging of dynamic inhomogeneities with high resolution were considered. This technique was applied for the first time for the imaging of complex autowave cellular motility and cytoplasm shuttle flow in the slime mold Physarum polycephalum. The effect of multiple scattering on the accuracy of the measured flow velocity profiles for the case of single flow and for the case of the flow embedded into the static medium with strong scattering was studied. It was shown that this effect causes significant distortion to the measured flow velocity profiles and it is necessary to take this into account while making quantitative measurements of flow velocities.

Einzelzellomics bieten unvoreingenommene Einblicke in Transkriptionsprogramme und Genom-Zugänglichkeiten auf zellulärer Ebene, auch wenn der zelluläre Kontext verloren geht. Wir haben einen virtuellen Multi-omic Embryo der Drosophila melanogaster erstellt, basierend auf den Datentypen RNA (Transkriptom) und ATAC (Zugänglichkeit der DNA), welche gleichzeitig auf Einzelzell Ebene erhoben wurden. Mithilfe des Tools novoSpaRc, welches den räumlichen Ursprung der Zellen rekonstruiert, konnte ein regulatorischen Bauplan erstellt werden, der die Genexpression und die Zugänglichkeit von Enhancern widerspiegelt. Diese Ressource hilft beim Verständnis der regulatorischen Dynamik in der Entwicklung. Bei der Untersuchung von ATAC-Peaks konnten wir Überschneidungen zwischen den Mustern der Chromatin Zugänglichkeit und der Aktivität unabhängiger getesteter Enhancer feststellen, was die Bedeutung der Zugänglichkeit unterstreicht. Die nicht-negative Matrixfaktorisierung identifizierte Archetypen der Genexpression und der Chromatin-Zugänglichkeit. Archetypen, die möglicherweise durch Transkriptionsfaktoren (TFs) reguliert werden, wurden einer Motiv-Anreicherungsanalyse für Archetyp-assoziierte CRMs unterzogen. Ein Ansatz zur Vorhersage von Enhancern, ordnete die Enhancer den Genen auf der Grundlage partieller Ähnlichkeit der Muster zu. Zusammenfassend dient unser multimodaler virtueller Embryo als Ressource und präsentiert zum ersten Mal räumliche Chromatin-Zugänglichkeiten für genomische Regionen für einen ganzen Organismus. Die Ergebnisse geben Aufschluss über die Prinzipien der Genregulation und zeigen den regulatorischen Einfluss von Transkriptionsfaktoren auf den Chromatinzustand von Enhancern. / Single-cell-omics techniques provide unbiased insights into transcriptional programs and genomic accessibility patterns at the cellular level despite sacrificing spatial information. We created a multi-omic virtual Drosophila melanogaster stage 6 embryo by simultaneously assessing genome accessibility and transcriptional states in individual cells. Using novoSpaRc, a spatial mapping tool, we accurately reconstructed the spatial origin of cells, yielding a regulatory blueprint reflecting gene expression and enhancer accessibilities. This resource aids in understanding developmental regulatory dynamics. Examining ATAC-peaks, we observed overlapping chromatin accessibility patterns with the activity of independently testes enhancers, emphasizing accessibility's importance. Non-negative matrix factorization identified archetypes in gene expression and chromatin accessibility. Accessibility archetypes, potentially regulated by transcription factors (TFs), were subjected to motif enrichment analysis for archetype-associated CRMs. An enhancer prediction approach, utilizing a generalized linear model, assigned enhancers to genes based on partial pattern similarity. In summary our multi-modal virtual embryo serves as a resource and presents for the first time single-cell chromatin accessibilities for genomic regions reconstructed in space for a whole organism in a single developmental stage. The results shed light on gene regulatory principles, highlighting the regulatory impact of TFs on chromatin states of enhancers.