The dissemination of tumor cells to distant organs i.e. metastasis is an exceedingly complex process leading to 90% of all cancer deaths. Despite being so clinically important, little is known about this process that requires tumor cells to leave the primary tumor site, intravasate and transport through the blood stream, extravasate and colonize at secondary sites leading to distant metastases. Survivin, a member of the IAP (Inhibitor of Apoptosis) family with known functions in apoptosis and mitosis, is highly expressed in aggressive tumors and is associated with poor prognosis and adverse clinical outcome. But the mechanistic role of survivin in metastatic dissemination has not been investigated. In this study, we demonstrate an important and novel role of survivin in activating a broad gene expression program in tumor cells. Of particular importance is the upregulation of a distinct class of cell adhesion molecules, particularly fibronectin. This IAP mediated gene regulation requires synergistic intermolecular cooperation between survivin and its related cofactor molecule, XIAP that results in activation of NF-κB dependent fibronectin gene expression. The binding of fibronectin with its cognate cell surface receptors initiates outside–in signaling leading to the autocrine and paracrine activation of cell motility kinases, FAK and Src, in turn leading to enhanced tumor invasion and metastasis. The importance of survivin and XIAP in the process of metastasis has also been demonstrated in vivousing intrasplenic injections in mouse models. Overall this study is the first to place survivin upstream of transcriptional activation of gene expression particularly fibronectin. In addition, it also demonstrates the importance of survivin-XIAP complex in mediating NF-κB activation which in turn switches on the expression of various target genes involved in tumor metastasis. Hence this study dissects the upstream and downstream requirements of survivin- XIAP complex mediated tumor dissemination and metastasis. Significance of this Study The hallmark of end-stage cancer is metastasis, an incurable condition almost invariably associated with death from disease. Despite a better understanding of the metastatic process, and the identification of key gene expression requirements of this pathway, the development of anti-metastatic therapies has lagged behind, with no viable options being currently offered in the clinical setting. Our findings that Inhibitor of Apoptosis (IAP) proteins functions as metastasis-promoting genes independently of cell survival, but through activation of cell motility could have important ramifications for the broader application of IAP antagonists currently in early clinical trials, as novel anti-metastatic therapies.

Le médulloblastome est la tumeur du système nerveux central la plus répandue et la plus meurtrière chez l’enfant. Malgré de bons taux de survie globale, le traitement du médulloblastome est confronté à deux problèmes cliniques majeurs: les survivants présentent souvent des séquelles graves et irréversibles causées par les traitements, et certains sous-groupes de la maladie répondent mal au traitement. Dans ce travail, nous avons cherché à traiter les deux problèmes en améliorant l'efficacité/tolérance de la radiothérapie dans des modèles précliniques de médulloblastome grâce à l’utilisation d’AsiDNA, un inhibiteur de la réparation de l'ADN déjà testé dans des essais cliniques. Nous avons caractérisé l'effet des molécules AsiDNA sur la réponse à l’irradiation des cellules tumorales, in vitro et in vivo. Nous avons observé qu'AsiDNA peut pénétrer dans les jeunes cerveaux et des tumeurs cérébrales après administration systémique. Aucune preuve d’augmentation de la toxicité liée à l‘irradiation n’a été observée. De plus, l’addition d'AsiDNA aux doses létales de radiations délivrées dans le cerveau murin en développement semble protéger la toxicité radio-induite. Nos résultats démontrent que la combinaison d'AsiDNA avec l’irradiation augmente la survie des animaux greffés avec un modèle de médulloblastome sur le flanc ou dans le cervelet. Les analyses de transcriptome indiquent qu’AsiDNA amplifie les modifications de la transcription induites par les radiations d’une manière similaire à une augmentation de dose. Ses effets sont indépendants de la cellule d'origine ou du statut de TP53. Les données obtenues suggèrent qu’AsiDNA est une drogue qui pourrait potentiellement améliorer la prise en charge des enfants ayant des tumeurs cérébrales et leur qualité de vie. / Medulloblastoma is the most common central nervous system malignancy in pediatric oncology. Despite good overall survival rates, medulloblastoma treatment plans face two major clinical problems: survivors of medulloblastoma often present severe and irreversible sequela caused by the treatments and, certain subgroups of the disease will respond poorly to the treatments. In this work we aimed at tackling both clinical issues by improving radiotherapy efficacy in medulloblastoma preclinical models using a DNA repair inhibitor in clinical trials, AsiDNA. We have characterized the effect of AsiDNA in combination with radiation in vitro and in vivo studies. We have observed that AsiDNA can penetrate young brains and brain tumors through systemic delivery. No evidences of additional radiation-associated brain toxicity were observed. In addition, combination of AsiDNA treatments to lethal doses of radiation delivered in developing murine brain seem to protect from the radiation-induced toxicity. Our results show that the combination of AsiDNA to radiation improves survival of subcutaneous and orthotopic models of medulloblastoma. Transcriptome analysis on medulloblastoma cell lines indicate that AsiDNA intensifies the transcriptional response to radiation resembling the increase in radiation doses. This effect is independent of the cell of origin or TP53 status of the cells. The obtained results indicate that AsiDNA may be a good candidate to improve the efficacy of treatment protocols and quality of life of medulloblastoma patients.

1 Abstract Diabetes mellitus is characterized by the dysfunction and reduction of insulin-producing cells, resulting in hyperglycemia, which in long term harms the organism. For future therapy, it is crucial to understand the function of various factors participating in the differentiation and maturation of endocrine pancreatic cells. The aim of this study was to unravel the functional role of ISL1 during the development of the pancreas. ISL1 is expressed in all endocrine cells of the islets of Langerhansbut its function remains unclear, especially during early pancreatogenesis. As the global deletion of this gene is embryonically lethal, we used the tissue specific deletion of Isl1 in Neurod1 possitive cells using the Cre-loxP system. In this work we studied the effect of this deletion on the structure of islets of Langerhans, the formation of endocrine cell types and relative expression of genes during early pancreatic development. A defective achitecture of islets together with postnatal absence of α-cells was found in the Isl1 deletion mutant. Also, the expression of genes important for the specification of α-cell lineage and their subsequent function was decreased. The secondary outcome was the optimalization of a protocol for effective sorting of endocrine cells using fluorescent flow cytometry, which...

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Indiana University-Purdue University Indianapolis (IUPUI) / SIMPL is a transcriptional co-activator that alters the activity of transcription factor, NF-κB. In response to pathogens, cytokines such as Interleukin-1 (IL-1) and Tumor Necrosis Factor (TNF) signal through the IL-1 and TNF-α receptors, respectively, which are found on various cell types. Activation of these receptors can result in the nuclear localization of NF-κB where it enables the transcription of several different genes key in the innate immune response. Endogenous co-localization of the SIMPL protein with NF-κB in response to these same cytokine signals has yet to be demonstrated. Polyclonal antibody generated against a truncated version of the SIMPL protein was purified from the sera obtained from immunized rabbits using affinity chromatography. The antibody was found to have a high specificity for both the native and denatured form of the protein as demonstrated by the lack of nonspecific bands observed in immunoprecipitations and Western blotting. The antibody was utilized in immunofluorescence experiments on mouse endothelial cells that were either unstimulated or were stimulated (IL-1 or TNF-α). In the absence of cytokine, SIMPL was localized in both the cytoplasm and the nucleus as opposed to NF-κB which was almost exclusively localized in the cytoplasm. In the presence of IL-1, the concentration of SIMPL in the nucleus was increased, and in the presence of TNF-α, the concentration of SIMPL in the nucleus was even greater. Results of this study identified future routes for SIMPL antibody isolation as well as to demonstrate that endogenous SIMPL protein nuclear localization may not be solely dependent upon TNF-α signaling.

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Anti-CD19 CAR-T cell immunotherapy is a hopeful treatment option for patients with B cell lymphomas, however it copes with partly severe adverse effects like neurotoxicity. Single-cell resolved molecular data sets in combination with clinical parametrization allow for comprehensive characterization of cellular subpopulations, their transcriptomic states, and their relation to the adverse effects. We here present a re-analysis of single-cell RNA sequencing data of 24 patients comprising more than 130,000 cells with focus on cellular states and their association to immune cell related neurotoxicity. For this, we developed a single-cell data portraying workflow to disentangle the transcriptional state space with single-cell resolution and its analysis in terms of modularly-composed cellular programs. We demonstrated capabilities of single-cell data portraying to disentangle transcriptional states using intuitive visualization, functional mining, molecular cell stratification, and variability analyses. Our analysis revealed that the T cell composition of the patient’s infusion product as well as the spectrum of their transcriptional states of cells derived from patients with low ICANS grade do not markedly differ from those of cells from high ICANS patients, while the relative abundancies, particularly that of cycling cells, of LAG3-mediated exhaustion and of CAR positive cells, vary. Our study provides molecular details of the transcriptomic landscape with possible impact to overcome neurotoxicity.

<p>  </p> <p>Plant complex traits are controlled by multi-layer of dynamic and complicated gene networks regulated at different levels. To better inform crop breeding to promote desired traits, a comprehensive and fundamental understanding of their genetic basis is much needed. With the rapid developments of <em>omics</em> planforms and next generation sequencing technology, we now have large-scale data from genome, epigenome, transcriptome, metabolome, and others for the crop plants. Integration of those multiple <em>omics</em> data together with computational approaches led to the establishment of a novel science known as system biology. Research described in this thesis used system biology approaches to dissect complex crop traits such as disease response of tomato (Chapter2 and Chapter3) and the heterosis of nitrogen use efficiency of maize (Chapter4).</p> <p>Plant disease response is an elaborate, multilayered complex trait involving several lines of defense signaling. In the past decades, progress in molecular analyses of plant immune system has revealed key elements of a complex response network in Arabidopsis, a model species. Histone modifications, a type of epigenetic regulation, have emerged as key modulators that regulate defense responses, while our understanding of the role of histone-modifying enzymes in this process is still in its infancy. Here, we described the immune function of two histone methyltransferases SDG33 and SDG34 in tomato. We found the single mutants in <em>sdg33</em> and <em>sdg34</em> showed increased susceptibility to hemibiotrophic bacterial pathogen <em>Pseudomonas syringae</em> whereas the double mutant <em>sdg33sdg34</em> is comparable to wild type. Using RNA-seq and histone ChIP-seq approaches, we investigated the possible underlying mechanisms and found that the expression of a set of immune-related genes is misregulated by <em>P. syringae</em> only in the single mutants but not in the double mutant. Integrating with epigenomic data, we found that the misexpression of those SDG33/SDG34 dependent immune-response genes was associated with altered histone methylation status in the single mutant. Intriguingly, the double mutant also showed altered histone methylation but unaffected gene expression, suggesting a compensating regulatory mechanism at play. The function of SDG33 and SDG34 in immune response seems to be specific for the pathogen, as the double mutants exhibited enhanced resistance the single mutants showed no altered responses when treated with necrotrophic fungal pathogen <em>Botrytis cinerea</em>. Network analysis found the most regulatory gene by <em>B. cinerea</em> in a SDG33/SDG34 dependent manner have been implicated in biotic stress response such as <em>ERF4, TOPLESS, PUB23 </em>and<em> RCD1</em>. Comparing the immune response of double mutant against <em>P. syringae</em> and <em>B. cinerea</em>, we found that the disease related genes are only mis-regulated in the interaction of <em>B. cinerea</em> treatment not in the <em>P. syringae</em> treatment, which could be the reason of enhanced resistance to <em>B. cinerea</em> but not for <em>P. syringae</em> in the double mutants. In summary, we found the histone methyltransferases SDG33 and SDG34 has different functions in the immune response against <em>P. syringae</em> and <em>B. cinerea</em>, which might be direct or indirect relevant to the histone methylation level of the expression of downstream immune related gene.</p> <p>In addition to biotic stress, another complex trait studied in this thesis is the heterosis of nitrogen use efficiency (NUE) in Maize. NUE is another complex trait associated with multiple physiological processes including N sensing, uptake, assimilation, transport, and storage. Heterosis refers to a phenomenon where the progeny generated by crossing two different cultivars of the same species exhibit superior fitness than the inbred parents. Even though, heterosis has been exploited to improve complex traits including NUE, the underlying molecular mechanisms is not completely understood. Here, we analyzed N-responsive transcriptomes and physiological traits of a panel of six maize hybrids and their corresponding inbreds grown in the field at two different N levels. We observed diverse levels of trait heterosis that are dependent on the N conditions and organ types. We discovered dramatic pattern shift of beyond-parental-range gene expression in hybrids in response to varying N levels. We identified through integrative analyses a set of genes whose expression heterosis are quantitatively correlated to trait heterosis. These genes are involved in response to stimulus, photosynthesis, and N metabolism, and likely mediate the heterosis phenotype of N-use and growth traits in maize. In summary, our integrated analysis provided insights into the mechanistic basis of the heterosis of NUE. </p> <p>Together, applying systems and functional genomics approaches to investigate important agricultural traits could lead to a comprehensive understanding of plant complex traits to inform future engineering and breeding for better crops.</p>

Tfap2a gehört zur Familie der AP-2-Transkriptionsfaktoren. Heterozygote Mutationen von TFAP2A im Menschen führen zum Branchio-Okulo-Fazialen-Syndrom (BOFS) und sind mit Nierenanomalien assoziiert. Molekulare Mechanismen, die zu diesen BOFS-assoziierten Nierenanomalien führen, sind noch unbekannt. In diesem Projekt wurde die Expression von Mitgliedern der AP-2-Familie in neugeborenen und erwachsenen Wildtyp-Mäusen analysiert. Tfap2a wurde in der Ureterknospe und der distalen Region des S-förmigen Körpers in den Nieren neugeborener Mäuse exprimiert. Die Expression blieb in ausgereiften distalen Tubuli und Sammelrohren erhalten. Tfap2b, ein zweites Mitglied der AP-2-Familie, das in der Niere exprimiert wird und mit Zystenbildung assoziiert ist, wurde im aufsteigenden Ast der Henleschen Schleife sowie in den distalen Tubuli und dem in der Nierenrinde liegenden Sammelrohr exprimiert. Um die Rolle von Tfap2a in der Niere zu untersuchen, wurden Mäuse mit einer sammelrohrspezifischen Deletion von Tfap2a (Tfap2a-KO) erzeugt. Phänotypische und morphologische Analysen ergaben, dass Tfap2a-KO-Mäuse mäßig reduzierte Nierengewichte und eine fortschreitende Dilatation der äußeren medullären Sammelrohre aufwiesen. Einzelkern- und RNA-Sequenzierung der Nieren adulter Mäuse zeigte eine deregulierte Expression von Genen, die mit der Organisation von Aktinfilamenten, Zelladhäsion, Wnt-Signalen und anderen Signalwegen der Nierenentwicklung in Verbindung stehen. In einem isolierten Modell von kultivierten Sammelrohrzellen mit einer Deletion von Tfap2a waren ähnliche Signalwege dereguliert. Insgesamt deutet diese Studie darauf hin, dass Tfap2a für die Differenzierung des Sammelrohrepithels und die Regulierung des Durchmessers des Tubuluslumens erforderlich ist. Dies ermöglicht Einblicke in die molekularen Grundlagen der beim BOFS beobachteten Nierenfehlbildungen. / The transcriptional regulator Tfap2a is part of the AP-2 transcription factor family. Heterozygous mutations of TFAP2A in humans lead to branchio-oculo-facial syndrome (BOFS) and are associated with renal anomalies. Molecular mechanisms leading to BOFS-associated renal anomalies are still unknown. In this project, expression patterns of AP-2 family members were analyzed in newborn and adult wildtype mice. Tfap2a was expressed in the ureteric bud and distal region of the S-shaped body in kidneys of newborn mice. Expression was maintained in mature distal tubules and collecting ducts. Tfap2b, a second AP-2 family member expressed in the kidney and associated with cyst formation, was found in the ascending limb and showed overlapping expression with Tfap2a in distal tubules and the cortical collecting duct. To investigate the role of Tfap2a in the kidney, mice with a collecting duct-specific deletion of Tfap2a (Tfap2a-KO) were generated by crossing mice carrying a Cre-recombinase under the Hoxb7 promotor and mice with floxed Tfap2a alleles. Phenotypic and morphological analyses revealed that Tfap2a-KO mice displayed moderately reduced kidney weights and a progressive dilation of outer medullary collecting ducts. Single-nucleus and bulk RNA sequencing of kidneys of three months old Tfap2a-KO mice and littermate controls indicated deregulated expression of genes associated with actin filament organization, cell adhesion, Wnt signaling, and other kidney developmental pathways. Genes deregulated in Tfap2a-deficient mice included several genes previously implicated in the development of congenital anomalies of the kidney and urinary tract. In an isolated model of cultured collecting duct cells carrying a Tfap2a knockout similar pathways were deregulated. Taking together, this study indicates that Tfap2a is required for collecting duct epithelium differentiation and tubular lumen diameter regulation, providing insights into the molecular basis of renal defects observed in BOFS.

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