Metastatic bone cancers cause severe symptoms including pain that compromises patient functional status, quality of life, and survival. Current treatment strategies have limited efficacy and dose-limiting side effects. Cancer-induced bone pain (CIBP) is a unique pain state that shares features with but is distinct from the pathology of neuropathic and inflammatory pain. This dissertation investigates how CIBP is generated and maintained by the direct effects of cancer cells on their metastatic microenvironment and the peripheral nervous system, including unique signaling properties and gene expression changes. In particular, we found that genetic knockdown of the functional subunit xCT of the system xC- cystine/glutamate antiporter can reduce CIBP, further elucidating this as a therapeutic of interest. We found that the neuroprotective voltage-gated calcium channel inhibitors progesterone and pregabalin markedly reduce mechanical hypersensitivity and excitability in sensory neurons of the dorsal root ganglion (DRG) in male rat models of neuropathic pain, but that these effects and less pronounced in females. In cancer pain, these sex differences are reversed, with females but not males demonstrating a delay in time-to-onset of mechanical hypersensitivity. We also analyzed gene expression at the DRG by RNA-Sequencing of rat models of CIBP. Our findings uncovered differential gene expression between CIBP and sham controls and between ipsilateral and contralateral DRGs in CIBP model rats. These studies have identified several promising avenues for therapeutic research for CIBP. / Dissertation / Doctor of Philosophy (PhD) / The tools we have right now to manage severe and chronic pain are insufficient. Patients with advanced cancers including bone cancer can suffer from very severe pain. This pain is generated in a number of ways including by the tumour itself releasing chemicals that activate pain-sensing nerves, by the destruction of the bone in and around the tumour, and by the sensitization of the nervous system, which can make pain worse and longer lasting. We have taken three approaches to researching cancer pain and to investigating new treatments. We have found that by reducing the amount of glutamate that cancer cells can release into their environment, we can reduce cancer pain in mice. We also found that treating rats with pregabalin and progesterone can change nerve signaling and reduce neuropathic pain, but that this effect is most pronounced in male rats with neuopathic pain and smaller in female rats with neuropathic pain, and even smaller in rats with cancer pain. We also analyzed expression of all the protein-coding genes in dorsal root ganglia from rats with cancer pain and found that there are many differences from rats without pain. Some of these differences may be promising new research targets. Going forward this research has provided important evidence necessary for next steps to develop new therapies and research strategies for cancer pain.

Droplet-based microfluidics is a powerful tool permitting massive-scale single-cell analysis in pico-/nano-liter water-in-oil droplets. It has been integrated into various library preparation techniques to accomplish high-throughput scRNA-seq, scDNA-seq, scATAC-seq, scChIP-seq, as well as scMulti-omics-seq. These advanced technologies have been providing unique and novel insights into both normal differentiation and disease development at single-cell level. In this thesis, we develop four new droplet-based tools for single-cell omics profiling. First, the developed Drop-BS is the first droplet-based platform to construct single-cell bisulfite sequencing libraries for DNA methylome profiling and allows production of BS library of 2,000-10,000 single cells within 2 d. We applied the technology to separately profile mixed cell lines, mouse brain tissues, and human brain tissues to reveal cell type heterogeneity. Second, the new Drop-ChIP platform only requires two steps of droplet generation to achieve multiple steps of reactions in droplets such as single-cell lysis, chromatin fragmentation, ChIP, and barcoding. Third, we aim to establish a droplet-based platform to accomplish high-throughput full-length RNA-seq (Drop-full-seq), which both current tube-based and droplet-based methods cannot realize. Last, we constructed an in-house droplet-based tool to assist single-cell ATAC-seq library preparation (Drop-ATAC), which provided a low-cost and facile protocol to conduct scATAC-seq in laboratories without the expensive instrument. / Doctor of Philosophy / Microfluidics is a collection of techniques to manipulate fluids in the micrometer scale. One of microfluidic techniques is called "droplet-based microfluidics". It can manipulate (i.e., generate, merge, sort, split, etc) pico-/nano-liter of water-in-oil droplets. First, since the water phase is separated by the continuous oil phase, these droplets are discrete and individual reactors. Second, droplet-based microfluidics can achieve highly parallel manipulation of thousands to millions of droplets. These two advantages make droplet-based microfluidics an ideal tool to perform single-cell assays. Over the past 10 years, various droplet-based platforms have been developed to study single-cell transcriptome, genome, epigenome, as well as multi-ome. To expand droplet-based tools for single-cell analysis, we aim to develop four novel platforms in this thesis. First, Drop-BS, by integrating droplet generation and droplet fusion techniques, can achieve high-throughput single-cell bisulfite sequencing library preparation. It can generate 10,000 single-cell BS libraries within 2 days which is difficult to achieve for conventional library preparation in tubes/microwells. Second, we developed a novel and facile Drop-ChIP platform to prepare single-cell ChIP-seq library. It is easy to operate since it only requires two steps of droplet generation. It also generates higher quality of data compared to previous work. In addition, we are working on the development and characterization of the other two droplet-based tools to achieve full-length single-cell RNA-seq and single-cell ATAC-seq.

Chronic Obstructive Pulmonary Disease (COPD) is the 3rd leading cause of death in the US, and idiopathic pulmonary fibrosis (IPF), a type of Interstitial Lung Disease (ILD), is a fast acting, irreversible disease that leads to mortality within 3-5 years. RNA-sequencing provides the opportunity to quantitatively examine the sequences of millions mRNAs, and offers the potential to gain unprecedented insights into the structure of chronic non-malignant lung disease transcriptome. By identifying changes in splicing and novel loci expression associated with disease, we may be able to gain a better understanding of their pathogenesis, identify novel disease-specific biomarkers, and find better targets for therapy. Using RNA-seq data that our group generated on 281 human lung tissue samples (47=Control, 131=COPD, 103=ILD), I initially defined the transcriptomic landscape of lung tissue by identifying which genes were expressed in each tissue sample. I used a mixture model to separate genes into reliable and not reliable expression. Next, I employed reads that overlapped splice junctions in a linear model interaction term to identify disease-specific differential splicing. I identified alternatively spliced genes between control and disease tissues and validated three (PDGFA, NUMB, SCEL) of these genes with qPCR and nanostring (a hybridization-based barcoding technique used to quantify transcripts). Finally, I implemented and improved a pipeline to perform transcriptome assembly using Cufflinks that led to the identification of 1,855 novel loci that did not overlap with UCSC, Vega, and Ensembl annotations. The loci were classified into potential coding and non-coding loci (191 and 1,664, respectively). Expression analysis revealed that there were 120 IPF-associated and 10 emphysema-associated differentially expressed (q < 0.01) novel loci. RNA-seq provides a high-resolution transcript-level view of the pulmonary transcriptome and its modification in lung disease. It has enabled a new understanding of the lung transcriptome structure because it measures not only the transcripts we know but also the ones we do not know. The approaches and improvements I have employed have identified these novel targets and make possible further downstream functional analysis that could identify better targets for therapy and lead to an even better understanding of chronic lung disease pathogenesis. / 2999-01-01T00:00:00Z

RNA-Seq is an increasingly popular approach to transcriptome profiling that uses the capabilities of next generation sequencing technologies and provides better measurement of levels of transcripts and their isoforms. In this thesis, we apply RNA-Seq protocol and transcriptome quantification to estimate gene expression and pathway activity levels. We present a novel method, called IsoDE, for differential gene expression analysis based on bootstrapping. In the first version of IsoDE, we compared the tool against four existing methods: Fisher's exact test, GFOLD, edgeR and Cuffdiff on RNA-Seq datasets generated using three different sequencing technologies, both with and without replicates. We also introduce the second version of IsoDE which runs 10 times faster than the first implementation due to some in-memory processing applied to the underlying gene expression frequencies estimation tool and we also perform more optimization on the analysis. The second part of this thesis presents a set of tools to differentially analyze metabolic pathways from RNA-Seq data. Metabolic pathways are series of chemical reactions occurring within a cell. We focus on two main problems in metabolic pathways differential analysis, namely, differential analysis of their inferred activity level and of their estimated abundance. We validate our approaches through differential expression analysis at the transcripts and genes levels and also through real-time quantitative PCR experiments. In part Four, we present the different packages created or updated in the course of this study. We conclude with our future work plans for further improving IsoDE 2.0.

Next-generation sequencing has generated enormous amount of DNA and RNA sequences that potentially carry volumes of genetic information, e.g. protein-coding genes. The thesis is divided into three main parts describing i) GeneMarkS-2, ii) GeneMarkS-T, and iii) MetaGeneTack. In prokaryotic genomes, ab initio gene finders can predict genes with high accuracy. However, the error rate is not negligible and largely species-specific. Most errors in gene prediction are made in genes located in genomic regions with atypical GC composition, e.g. genes in pathogenicity islands. We describe a new algorithm GeneMarkS-2 that uses local GC-specific heuristic models for scoring individual ORFs in the first step of analysis. Predicted atypical genes are retained and serve as ‘external’ evidence in subsequent runs of self-training. GeneMarkS-2 also controls the quality of training process by effectively selecting optimal orders of the Markov chain models as well as duration parameters in the hidden semi-Markov model. GeneMarkS-2 has shown significantly improved accuracy compared with other state-of-the-art gene prediction tools. Massive parallel sequencing of RNA transcripts by the next generation technology (RNA-Seq) provides large amount of RNA reads that can be assembled to full transcriptome. We have developed a new tool, GeneMarkS-T, for ab initio identification of protein-coding regions in RNA transcripts. Unsupervised estimation of parameters of the algorithm makes unnecessary several steps in the conventional gene prediction protocols, most importantly the manually curated preparation of training sets. We have demonstrated that the GeneMarkS-T self-training is robust with respect to the presence of errors in assembled transcripts and the accuracy of GeneMarkS-T in identifying protein-coding regions and, particularly, in predicting gene starts compares favorably to other existing methods. Frameshift prediction (FS) is important for analysis and biological interpretation of metagenomic sequences. Reads in metagenomic samples are prone to sequencing errors. Insertion and deletion errors that change the coding frame impair the accurate identification of protein coding genes. Accurate frameshift prediction requires sufficient amount of data to estimate parameters of species-specific statistical models of protein-coding and non-coding regions. However, this data is not available; all we have is metagenomic sequences of unknown origin. The challenge of ab initio FS detection is, therefore, twofold: (i) to find a way to infer necessary model parameters and (ii) to identify positions of frameshifts (if any). We describe a new tool, MetaGeneTack, which uses a heuristic method to estimate parameters of sequence models used in the FS detection algorithm. It was shown on several test sets that the performance of MetaGeneTack FS detection is comparable or better than the one of earlier developed program FragGeneScan.

Deux phénomènes liés au TFIIH ont été étudiés : l'influence des mutations spécifiques dans la sous-unité XPD de TFIIH sur la réponse transcriptionnelle de certains gènes après l'irradiation UV, et l'interaction entre le TFIIH et la transcription des gènes de classe III. Une analyse détaillée de la dynamique du transcriptome a été effectuée pour la réponse des cellules humaines mutantes XP-D/CS à l'UV. Il a été démontré que la dysrégulation sélective observée de l'expression des gènes était liée à l'incapacité pour la ré-initiation transcriptionnelle et à l'hétérochromatinisation suivante, où l'histonedésacétylase SIRT1 a été identifiée comme le principal facteur. Son inhibition a permis de recouvrer l'expression normale d'un nombre substantiel des gènes affectés. Une étude de la participation pangénomique du coeur de TFIIH dans latranscription a découvert son association avec les gènes actifs de classe III. Cette association a été démontrée être indépendante de Pol II. Le coeur de TFIIH a été montré participer directement à la transcription effectuée in vitro par Pol III.

Les interactions mito-nucléaire sont au centre des fonctions de la mitochondrie, telles que la production d’énergie. Or, peu d’informations sont connues sur comment le génome mitochondrial peut influencer la réponse nucléaire. Les cybrides (hybrides cytoplasmiques) peuvent présenter des modifications phénotypiques (adaptatives ou non) dûe à ces interactions. Dans cette étude, les différents mitotypes (haplotype mitochondriale) de l'espèce Chrosomus eos furent utilisés comme modèle afin d’étudier plus en profondeur les interactions entre les différents génomes d’une cellule. Ce complexe présente en plus du type sauvage, deux types de cybrides, dont les mitochondries proviennent de deux refuges glaciaires différents : Mississippien et Atlantique. Cette étude fut effectuée sur des individus en sympatrie afin de prendre en compte l’influence environnementale. Des populations en allopatrie furent également mesurés séparément afin de complémenter les études précédentes. Afin de répondre à ces objectifs, plusieurs approches furent employées, adressant ainsi divers niveaux d’intégration : la méthylation, la transcription, la protéomique et l’activité enzymatique. Des différences entre les divers mitotypes furent observées, mais de magnitude inférieure à l’influence environnementale. Ce résultat suggère donc que les différences associées au mitotype en sympatrie affecte leur répartition et leur habileté à coloniser différents types d’environnements, créant ainsi la similarité intra-mitotype inter-lac observé dans les études précédentes. Pour adresser l’analyse transcriptomique, une référence a été produite. Cette référence offre beaucoup d’informations pour des applications futures. / The mito-nuclear interactions are at the center of the mitochondrial functions, such as energy production. Despite their importance, little is known about how the influence of the mitochondrial genome on the nuclear genes expressions. The cybrids (cytoplasmic hybrids) can present phenotype modification (adaptive or not) caused by the mito-nuclear interactions. In this study, different mitotype of Cybrids of the Chrosomus eos species were used as model to further study the interaction between a cell’s genomes. This complex of species has two types of cybrids, the mitochondria of which originates from two different glacial refugia : Atlantic and Mississippian. This set up of this study was a sympatric lake, to minimise the environmental factor. Allopatric population were also separately analysed to supplement past studies. To accomplish these goals, multiple methods were used, assessing also multiple levels of gene expression: Methylation, transcription, protein and enzymatic activity. Differences between mitotypes were observed, but of lesser magnitude then the environmental factor. Thus, this result suggest that the differences associated to the sympatric mitotypes drive a deference in environment colonization, which would create the inter-lake intra-mitotype similarity observed in past studies. To assess the transcription of mitotype, a transcriptomic reference was produced. This reference offers a lot of information and future applications.

Lignocellulosic plants are the most abundant source of terrestrial biomass and are one of the potential sources of renewable energy that can replace the use of fossil fuels. For a country such as Sweden, where the forest industry accounts for 10% of the total export, there would be large economical benefits associated with increased biomass yield. The availability of research on wood development conducted in conifer tree species, which represent the majority of the forestry in Sweden, is limited and the majority of research has been conducted in model angiosperm species such as Arabidopsis thaliana. However, the large evolutionary distance between angiosperms and gymnosperms limits the possibility to identify orthologous genes and regulatory pathways by comparing sequence similarity alone. At such large evolutionary distances, the identification of gene similarity is, in most cases, not sufficient and additional information is required for functional annotation. In this thesis, two high-spatial resolution datasets profiling wood development were processed; one from the angiosperm tree Populus tremula and the other from the conifer species Picea abies. These datasets were each published together with a web resource including tools for the exploration of gene expression, co-expression and functional enrichment of gene sets. One developed resource allows interactive, comparative co-expression analysis between species to identify conserved and diverged co-expression modules. These tools make it possible to identifying conserved regulatory modules that can focus downstream research and provide biologists with a resource to identify regulatory genes for targeted trait improvement. / Lignocellulosa är den vanligast förekommande källan till markburen biomassa och är en av de förnybara energikällor som potentiellt kan ersätta användningen av fossila bränslen. För ett land som Sverige, där skogsindustrin som står för 10 \% av den totala exporten, skulle därför en ökad produktion av biomassa kunna ge stora ekonomiska fördelar. Forskningen på barrträd, som utgör majoriteten av svensk skog är begränsad och den huvudsakliga forskningen som har bedrivits på växter, har skett i modell organismer tillhörande gruppen gömfröiga växter som till exempel i Arabidopsis thaliana. Det evolutionära avståndet mellan gömfröiga (blommor och träd) och nakenfröiga (gran och tall) begränsar dock möjligheten att identifiera regulatoriska system mellan dessa grupper. Vid sådana stora evolutionära avstånd krävs det mer än att bara identifiera en gen i en modellorganism utan ytterligare information krävs som till exempel genuttrycksdata. I denna avhandling har två högupplösta experiment som profilerar vedens utveckling undersökts; ett från gömfröiga träd Populus tremula och det andra från nakenföriga träd (barrträd) Picea abies. Datat som behandlats har publicerats tillsammans med webbsidor med flera olika verktyg för att bland annat visa genuttryck, se korrelationer av genuttryck och test för anrikning av funktionella gener i en grupp. En resurs som utvecklats tillåter interaktiva jämförelser av korrelationer mellan arter för att kunna identifiera moduler (grupper av gener) som bevaras eller skilts åt mellan arter över tid. Identifieringen av sådana bevarade moduler kan hjälpa att fokusera framtida forskning samt ge biologer en möjlighet att identifiera regulatoriska gener för en riktad förbättring av egenskaper hos träd.

Les impacts environnementaux dues à l'extraction minière sont considérables. C'est l'action des microorganismes, en utilisant leur métabolisme du soufre sur les déchets miniers, qui engendre les plus grands défis. Jusqu'à présent, peu de recherches ont été effectués sur les microorganismes environnementaux pour la compréhension globale de l'action du métabolisme du soufre dans une optique de prévention et de rémédiation des impacts environnementaux de l'extraction minière. Dans cette étude, nous avons étudié une bactérie environnementale, Acidithiobacillus thiooxidans, dans le but de comprendre le métabolisme du soufre selon le milieu de culture et le niveau d'acidité du milieu. Nous avons utilisé la transcriptomique à haut débit, RNA-seq, en association avec des techniques de biogéochimie et de microscopie à électrons pour déterminer l'expression des gènes codants les enzymes du métabolisme du soufre. Nous avons trouvé que l'expression des gènes des enzymes du métabolisme du soufre chez ce microorganisme sont dépendantes du milieu, de la phase de croissance et du niveau d'acidité présent dans le milieu. De plus, les analyses biogéochimiques montrent la présence de composés de soufre réduits et d'acide sulfurique dans le milieu. Finalement, une analyse par microscopie électronique révèle que la bactérie emmagasine des réserves de soufre dans son cytoplasme. Ces résultats permettent une meilleure compréhension de son métabolisme et nous rapprochent de la possibilité de développer une technique de prédiction des réactions ayant le potentiel de causer des impacts environnementaux dus à l'extraction minière. / The environmental impact of mining extraction is important. The action of microorganisms using their sulfur metabolism to metabolise compounds in mining waste contributes to reactions that may impact water quality and the environment. Few studies have been conducted on environmental microorganisms to advance the global comprehension of their sulfur metabolism in an attempt to study their impact on the environment. In this study, we cultivate an environmental bacterium, Acidithiobacillus thiooxidans, in an attempt to understand its sulfur metabolism in different growth media and at different levels of acidity. We used high-throughput RNA sequencing in association with sulfur biogeochemistry and electron microscopy to determine the expression of the genes encoding sulfur metabolism enzymes. The expression of genes encoding sulfur metabolism enzymes was media and pH-dependent. Also, the biogeochemical analysis showed the presence of reduced sulfur intermediates and of sulfuric acid in the medium. Finally, an electron microscopic analysis revealed that the bacteria stock sulfur in the cytoplasm. These results resulted in a better comprehension of its sulfur metabolism and it opens the possibility to predict reactions in mining operations that have impact on the environment.

Odorantes são detectados por centenas de receptores olfatórios (ORs) que pertencem à superfamília dos receptores acoplados à proteína G. Estes receptores são expressos nos neurônios sensoriais olfatórios localizados na cavidade nasal. Cada neurônio sensorial olfatório expressa um único alelo de gene OR de uma grande família de genes OR. Este padrão característico da expressão de genes OR resulta na formação de um mapa olfatório espacial no bulbo olfatório, que é necessário para a discriminação de odorantes pelo sistema olfatório. Os mecanismos envolvidos nesta regulação ainda não são bem conhecidos. O DNA genômico em neurônios olfatórios é coberto com marcas repressivas de metilação de histonas, indicando que a regulação da estrutura da cromatina deve desempenhar um papel importante na regulação da expressão de genes OR. Trabalhos anteriores demonstraram que RNAs não codificadores (ncRNAs) estão envolvidos na deposição de marcas de histonas em determinados genes. No entanto, os ncRNAs expressos no epitélio olfatório ainda não são conhecidos. Neste trabalho, identificamos e catalogamos o repertório completo de ncRNAs anotados, incluindo os miRNAs, expressos no epitélio olfatório de camundongos recémnascidos e adultos. Muitos destes, apesar de já anotados como ncRNAs, ainda não foram descritos na literatura como expressos no MOE. Identificamos ao todo 1161 miRNAs e 295 lincRNAs expressos no epitélio olfatório, e pudemos verificar como os níveis de expressão destes RNAs variam durante o desenvolvimento. A partir deste repertório, selecionamos lincRNAs que são preferencialmente expressos no epitélio olfatório quando comparados a outros tecidos de camundongo. Dez destes lincRNAs foram selecionados para validação utilizando-se RT-PCR. Cinco lincRNAs foram validados e analisados quanto à sua expressão em diferentes tecidos. Nosso trabalho estabelece uma plataforma de dados que permitirá o estudo do papel desempenhado por ncRNAs no epitélio olfatório. Além disto, os nossos resultados mostram que a abordagem utilizada permite a identificação de novos lincRNAs que apresentam expressão restrita ou preferencial no epitélio olfatório, e que, portanto, devem apresentar uma função relevante para o olfato. / Odorants are detected by hundreds of odorant receptors (ORs) which belong to the superfamily of G protein-coupled receptors. These receptors are expressed in the olfactory sensory neurons of the nose. Each olfactory sensory neuron expresses one single OR gene allele from a large family of OR genes. This characteristic pattern of OR gene expression results in the formation of a spatial olfactory map in the olfactory bulb, which is required for odorant discrimination by the olfactory system. The mechanisms involved in this regulation are unknown. OR genomic DNA in olfactory neurons is covered with repressive histone methylation marks, indicating that the chromatin structure should play an important role in the regulation of OR gene expression. Previous studies suggest that noncoding RNAs (ncRNAs) are involved in the deposition of histone marks in certain genes. However, the ncRNAs expressed in the olfactory epithelium are completely unknown. In this work, we used RNA-seq to identify and catalogue the complete repertoire of ncRNAs, including miRNAs, expressed in the olfactory epithelium from newborn and adult mice. In this way, we were able to identify 1161 miRNAs and 295 lincRNAs and analyze how their levels of expression varies during development. Out of these repertoire, we selected lincRNAs that are preferentially expressed in the olfactory epithelium when compared to other mouse tissues. Ten out of these lincRNAs were selected for validation by using RTPCR, and five of them could be validated and further analyzed. Our work establishes a data platform which will enable the study of the role played by ncRNAs in the olfactory epithelium. In addition, our results show that our approach can be successfully used to identify ncRNAs that are restrictedly or preferentially expressed in the olfactory epithelium, and which therefore must be relevant for olfaction.