Cytoplasmic incompatibility (CI) is an intriguing, widespread, symbiont-induced reproductive failure that decreases offspring production of arthropods through crossing incompatibility of infected males with uninfected females or with females infected with a distinct symbiont genotype. For years, the molecular mechanism of CI remained unknown. Recent genomic, proteomic, biochemical, and cell biological studies have contributed to understanding of CI in the alphaproteobacterium Wolbachia and implicate genes associated with the WO prophage. Besides a recently discovered additional lineage of alphaproteobacterial symbionts only moderately related to Wolbachia, Cardinium (Bacteroidetes) is the only other symbiont known to cause CI, and genomic evidence suggests that it has very little homology with Wolbachia and evolved this phenotype independently. Here, we present the first transcriptomic study of the CI Cardinium strain cEper1, in its natural host, Encarsia suzannae, to detect important CI candidates and genes involved in the insect-Cardinium symbiosis. Highly expressed transcripts included genes involved in manipulating ubiquitination, apoptosis, and host DNA. Female-biased genes encoding ribosomal proteins suggest an increase in general translational activity of Cardinium in female wasps. The results confirm previous genomic analyses that indicated that Wolbachia and Cardinium utilize different genes to induce CI, and transcriptome patterns further highlight expression of some common pathways that these bacteria use to interact with the host and potentially cause this enigmatic and fundamental manipulation of host reproduction. IMPORTANCE The majority of insects carry maternally inherited intracellular bacteria that are important in their hosts' biology, ecology, and evolution. Some of these bacterial symbionts cause a reproductive failure known as cytoplasmic incompatibility (CI). In CI, the mating of symbiont-infected males and uninfected females produces few or no daughters. The CI symbiont then spreads and can have a significant impact on the insect host population. Cardinium, a bacterial endosymbiont of the parasitoid wasp Encarsia in the Bacteroidetes, is the only bacterial lineage known to cause CI outside the Alphaproteobacteria, where Wolbachia and another recently discovered CI symbiont reside. Here, we sought insight into the gene expression of a CI-inducing Cardinium strain in its natural host, Encarsia suzannae. Our study provides the first insights into the Cardinium transcriptome and provides support for the hypothesis that Wolbachia and Cardinium target similar host pathways with distinct and largely unrelated sets of genes.

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第22065号 / 工博第4646号 / 新制||工||1724(附属図書館) / 京都大学大学院工学研究科マイクロエンジニアリング専攻 / (主査)教授 井上 康博, 教授 中部 主敬, 教授 横川 隆司 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM

<p>Plants naturally deposit a significant amount of carbon towards lignin, a polymer that imparts mechanical strength to cell walls but impedes our utilization of the polysaccharides in lignocellulosic biomass. Genetic engineering of lignin has demonstrated profound success in improving the processing of the biomass. Lignin is derived from the phenylpropanoid pathway, the architecture of which is well understood based upon the biochemical and genetic studies conducted to date. In contrast, we lack a systematic and quantitative view of the factors that determine carbon flux into and within this branched metabolic pathway in plants. To explore the control of carbon allocation for phenylalanine and lignin biosynthesis, we have developed a kinetic model of the pathway in Arabidopsis to test the regulatory role of several key enzymatic steps. We first established a ¹³C isotope feeding system for the measurement of flux using excised wild-type Arabidopsis stems. The excised stems continued to grow and lignify in our feeding system. When ring ¹³C₆-labeled phenylalanine ([¹³C₆]-Phe) was supplied to excised stems, isotope label was rapidly incorporated into soluble intermediates and lignin. Using this approach, we then analyzed metabolite pool sizes and isotope abundances of the pathway intermediates in a time course from stems fed with [¹³C₆]-Phe of different concentrations, and used these data to parameterize a kinetic model constructed with Michaelis-Menten kinetics. Our model of the general phenylpropanoid pathway captured the dynamic trends of metabolite pools <i>in vivo</i>and predicted the metabolic profiles of an independent feeding experiment. Based on the model simulation, we found that subcellular sequestration of pathway intermediates is necessary to maintain lignification homeostasis when metabolites are over-accumulated. Both the measurements and simulation suggested that theavailability of substrate Phe is one limiting factor for lignin flux in developing stems. This finding indicates new gene targets for lignin manipulation in plants. To extend our kinetic model to simulate flux distribution in response to genetic perturbations, we conducted an RNA-sequencing experiment in wild type and 13 plants with modified lignification, and integrated the transcriptional data with the metabolic profiles. We found that the biosynthesis of Phe and lignification are tightly coordinated at transcriptional level. The coregulation of the shikimate and phenylpropanoid pathways involves transcriptional and post-translational regulatory mechanisms to maintain pathway homeostasis. Our results also indicate that induction of Phe supply and enhancement of PAL activity are both effective strategies to increase carbon flux into the phenylpropanoid network.</p><p>In this interdisciplinary project, we have taken various system biology approaches to understand metabolic flux towards lignin, the second most abundant carbon sink in nature. We have combined isotope labeling aided flux measurements and mathematical simulation, and have integrated metabolome data with transcriptome profiles. The experiments and analysis have been conducted in both wild-type Arabidopsis and those with perturbed lignification. The novel work not only provides insight into our knowledge of phenylpropanoid metabolism, but also creates a framework to systematically assemble gene expression, enzyme activity, and metabolite accumulation to study metabolic fluxes, the ultimate functional phenotypes of biochemical networks.</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>

In situ hybridization (ISH) has played a crucial role in developing a spatial transcriptomic understanding of emerging model organisms in the past, but advancing high-throughput RNA-sequencing (RNA-seq) technology has pushed this method into the shadows, leading to a loss of data resolution. This shift in research towards the exclusive use of RNA-seq neglects essential considerations for transcriptomic studies including the spatial and temporal expression of transcripts, available budget, experimental design needs, and validation of data. A synergy of spatiotemporal transcriptomic techniques is needed, using the bulk and unbiased analysis of RNA-seq and the visual validation and spatiotemporal resolution of ISH. Integration of this synergistic approach can improve our molecular understanding of non-model organisms and establish the background data needed for advancing research techniques. A prime example lies within an emerging model of the marine science and symbiosis fields, where I present a case study on a threatened coral reef keystone – the cnidarian-dinoflagellate symbiosis. Establishing a whole-mount ISH protocol for the emerging cnidarian model Aiptasia (sea anemone) will help future studies reveal the gene regulation underpinning the establishment, persistence, and breakdown of this complex symbiotic relationship.

Although DNA repair capacity has been correlated with lifespan in terrestrial vertebrate species, it remains unknown how evolutionarily conserved the process is across all vertebrate taxa. In particular, chondrichthyan fishes have lifespans that range from 3-350 years and they are evolutionarily separated from modern humans Homo sapiens by approximately 400 million years. We hypothesized that chondrichthyan fishes would show significant homology in nuclear excision repair (NER) genes with humans, and that the expression of NER genes will correlate with the lifespan of the respective assessed species. For this study, DNA repair gene homology and expression was performed on the nurse shark Ginglymostoma cirratum (n=3) and yellow stingray Urobatis jamaicensis (n=3). The five main NER pathways were analyzed and compared to see the differences in both elasmobranch species, then compared with human foreskin fibroblast samples (n=3). RNA sequencing was used to determine the extent of gene expression in each species, comparing the read counts in each gene and comparing between the two species. The elephant shark Callorhinchus milii reference genome was used to align the nurse shark and yellow stingray samples. Homology of each gene of the NER pathways was assessed by the NCBI BLAST software. Results show that the MMR pathway has all the significant genes in higher frequencies in the nurse shark than in human. Within elasmobranchs in the five DNA repair pathways, the longer-lived species (nurse shark) has a significant higher gene expression than shorter-lived species (yellow stingray). Genes involved in the NER and BER pathways showed significantly lower expression in elasmobranch than in humans. However, there were significantly higher expression of more genes for the HR and MMR pathways in elasmobranchs than in humans.

Indiana University-Purdue University Indianapolis (IUPUI) / Metabolic syndrome (MetS) affects 1/3 of all Americans and is the clustering of three or more of the following cardiometabolic risk factors: obesity, hypertension, dyslipidemia, glucose intolerance, and insulin resistance. MetS drastically increases the incidence of coronary artery disease (CAD), which is the leading cause of mortality globally. A cornerstone of CAD is arterial remodeling associated with coronary smooth muscle (CSM) cytodifferentiation from a contractile phenotype to proliferative and osteogenic phenotypes. This cytodifferentiation is tightly coupled to changes in intracellular Ca2+ handling that regulate several key cellular functions, including contraction, transcription, proliferation, and migration. Our group has recently elucidated the time course of Ca2+ dysregulation during MetS-induced CAD development. Ca2+ transport mechanisms, including voltage-gated calcium channels, sarcoplasmic reticulum (SR) Ca2+ store, and sarco-endoplasmic reticulum Ca2+ ATPase (SERCA), are enhanced in early, mild disease and diminished in late, severe disease in the Ossabaw miniature swine. Using this well-characterized large animal model, I tested the hypothesis that this Ca2+ dysregulation pattern occurs in multiple etiologies of CAD, including diabetes and aging. The fluorescent intracellular Ca2+ ([Ca2+]i) indicator fura-2 was utilized to measure [Ca2+]i handling in CSM from lean and diseased swine. I found that [Ca2+]i handling is enhanced in mild disease with minimal CSM phenotypic switching and diminished in severe disease with greater phenotypic switching, regardless of CAD etiology. We are confident of the translatability of this research, as the Ca2+ influx, SR Ca2+ store, and SERCA functional changes in CSM of humans with CAD are similar to those found in Ossabaw swine with MetS. Single-cell RNA sequencing revealed that CSM cells from an organ culture model of CAD exhibited many different phenotypes, indicating that phenotypic modulation is not a discreet event, but a continuum. Transcriptomic analysis revealed differential expression of many genes that are involved in the osteogenic signaling pathway and in cellular inflammatory responses across phenotypes. These genes may be another regulatory mechanism common to the different CAD etiologies. This study is the first to show that CSM Ca2+ dysregulation is common among different CAD etiologies in a clinically relevant animal model.

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