En esta tesis doctoral se estudia el modo de vida tritrófico del hongo nematófago Pochonia chlamydosporia utilizando técnicas "multiómicas". Pochonia chlamydosporia (= Metacordyceps chlamydosporia) (Goddard) Zare y Gams es un hongo nematófago usado para el control de nematodos agalladores de la raíz (Meloidogyne spp.) (Forghani and Hajihassani, 2020), entre otros. P. chlamydosporia se distribuye por todo el mundo y tiene un modo de vida tritrófico, pudiendo también adoptar estilos de vida endófito y saprófito. El mecanismo que utiliza P. clamydosporia para infectar huevos de nematodo comprende la desacetilación de la quitina de su pared celular a quitosano para facilitar su degradación por quitosanasas (Aranda-Martinez et al., 2016). El quitosano es un biopolímero derivado de la quitina que también se encuentra en el exoesqueleto de artrópodos y crustáceos. El genoma de P. chlamydosporia codifica un elevado número de quitosanasas, gracias a las cuales es resistente a quitosano y puede utilizarlo como fuente de nutrientes (Palma-Guerrero et al., 2010). Ambos pueden combinarse para el control de plagas. En este trabajo de tesis doctoral se pretende estudiar mediante metabolómica, transcriptómica y genómica el modo de vida tritrófico de P. chlamydosporia añadiendo quitosano, para determinar los mecanismos de interacción del hongo en ese entorno. En último término, se pretende sentar las bases para desarrollar un sistema para reducir plagas y enfermedades de forma sostenible.

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<div> <p>Pancreatic ductal adenocarcinoma (PDAC) is a poorly immune responsive, treatment refractory disease, representing the fourth leading cause of cancer deaths in the United States. A lack of significant improvements in patient prognoses over the last few decades highlights the necessity for a more basic understanding of how PDAC develops and progresses. To this end, the research outlined here investigates the contributions of SOX9 and PAR1 in PDAC initiation and tumor immune evasion, respectively. </p> <p>SOX9 is a developmental transcription factor important for proper pancreas development that is restricted to only a small subset of cells in the adult organ. However, SOX9 is aberrantly expressed in precancerous lesions of the pancreas and throughout PDAC development. Using genetically engineered mouse models we demonstrated that PDAC precursor lesions cannot form in the absence of SOX9 and conversely formed at an accelerated rate when SOX9 was ectopically expressed. Surprisingly deletion of SOX9 in primary mouse PDAC cell lines had no impact on tumor growth in subcutaneous allograft experiments, indicating that although SOX9 expression is necessary for PDAC initiation, it is dispensable in many cases for tumor maintenance and growth. Research investigating the transcriptional changes induced by SOX9 prior to lesion formation is ongoing to identify additional downstream factors critical for disease initiation. </p> <p>Previous research has shown that PDAC tumors frequently display low levels of immune infiltration, which is a major limitation for the use of immune-based therapeutics and is generally an unfavorable prognostic factor. We show that in primary mouse tumor cells ablation of the thrombin receptor PAR1 caused a significant increase in the infiltration of tumor targeting CD8a⁺T cells which in turn were found to eliminate PAR1 knockout tumors. When PAR1^KO and PAR1 expressing PDAC tumor cells were co-injected into wild type mice, cells lacking PAR1 were preferentially targeted and eliminated by the immune system, indicating that PAR1 provides cell autonomous protection during an active anti-tumor adaptive immune response. Furthermore, we identified a previously underappreciated association between PAR1-mediated expression of <i>Csf2</i> and <i>Ptgs2</i>, and PDAC tumor immune evasion. Together these findings provide novel insights into the mechanisms and drivers of PDAC initiation and immune evasion.</p> </div> <br>

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Organisms modulate their response to changing environmental conditions through changes in gene expression, and extensive variations in gene expression are prevalent among individuals even within a population. This widespread plasticity and variability of gene expression is thought to play roles in adaptation and drive novel phenotypes in species. Understanding the mechanisms that contribute to such variations requires the analysis of interactions between the genome and its environment and sequence variations within the genome. This work consists of two projects investigating the plasticity and variation of gene expression during post-embryonic development in the nematode C. elegans. In the first study, I examined the response to changes in population density in developmentally arrested L1 larvae. I systematically characterized arrested L1 larvae from low to high densities using single-worm RNA-seq and uncovered that the density of resuspended L1 larvae regulates the expression of hundreds of mRNAs. Further analysis revealed that the physiological response to changes in density is rapid and signaled by a non-canonical daf-22 ascaroside independent pathway. In the second study, I investigated the evolution of gene expression within species using two genetically divergent C. elegans strains (N2 and CB4856). I carried out RNA-seq and allele-specific analysis across six different conditions and four developmental stages, and we examined gene expression divergence using the homozygous parent and F1 hybrid system. This work provides a new experimental model for studying the evolution of gene expression and a comprehensive view of gene expression variation during development in C. elegans.

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The placement of genetic information within bacterial genomes is intentionally organized, creates predictable gradients of molecular properties along the origin-terminus of replication axis. Previous studies have reported that genes located near the origin of replication generally have a higher expression level, increased dosage, and are more conserved than genes located near the terminus of replication. Additionally, substitution rates usually increases with increasing distance from the origin of replication. However, the constant reorganization of genetic information is often overlooked when considering spatial molecular trends. Here, we explore the interplay of genomic reorganization along the origin and terminus of replication axis of gene expression and substitutions in Escherichia coli, Bacillus subtilis, Streptomyces, and Sinorhizobium meliloti. Using ancestral reconstruction to account for genome reorganization, we demonstrated that the correlation between the number of substitutions and distance from the origin of replication is significant but small and inconsistent in direction. In another study, we looked at the overall expression levels of all genes from the same bacteria, and confirmed that gene expression tends to decrease when moving away from the origin of replication. We looked specifically at how inversions - one type of genomic reorganization - impact gene expression between closely related strains of E. coli. Some inversions cause significant differences in gene expression compared to non-inverted regions, however, the variation in expression does not significantly differ between inverted and non-inverted regions. This change in gene expression may be due to the expression regulation properties of two nucleoid proteins, Histone-like Nucleoid-Structuring (H-NS) and Factor for inversion stimulation (Fis), who’s binding sites had a significant positive correlation with inverted regions. In conclusion, we highlight the impact that genomic rearrangements and location have on molecular trends in bacteria, illustrating the importance of considering spatial trends in molecular evolutionary analysis, and to ensure accurate generalization of previously determined trends. Assuming that molecular trends are exclusively in one direction can be problematic. / Dissertation / Doctor of Philosophy (PhD)