Response of the calcifying coccolithophore Emiliania huxleyi to low pH/high pCO2: from physiology to molecular level, 2011

The emergence of ocean acidification as a significant threat to calcifying organisms in marine ecosystems creates a pressing need to understand the physiological and molecular mechanisms by which calcification is affected by environmental parameters. We report here, for the first time, changes in gene expression induced by variations in pH/pCO2 in the widespread and abundant coccolithophore Emiliania huxleyi. Batch cultures were subjected to increased partial pressure of CO2 (pCO2; i.e. decreased pH), and the changes in expression of four functional gene classes directly or indirectly related to calcification were investigated. Increased pCO2 did not affect the calcification rate and only carbonic anhydrase transcripts exhibited a significant down-regulation. Our observation that elevated pCO2 induces only limited changes in the transcription of several transporters of calcium and bicarbonate gives new significant elements to understand cellular mechanisms underlying the early response of E. huxleyi to CO2-driven ocean acidification.

Caracterización de la necrosis sistémica inducida por la interacción sinérgica entre el Virus X de la patata y potyvirus en Nicotiana benthamiana

Los virus de plantas pueden causar enfermedades severas que conllevan serias pérdidas económicas a nivel mundial. Además, en la naturaleza son comunes las infecciones simultáneas con distintos virus que conducen a la exacerbación de los síntomas de enfermedad, fenómeno al que se conoce como sinergismo viral. Una de las sintomatologías más severas causadas por los virus en plantas susceptibles es la necrosis sistémica (NS), que incluso puede conducir a la muerte del huésped. Este fenotipo ha sido comparado en ocasiones con la respuesta de resistencia de tipo HR, permitiendo establecer una serie de paralelismos entre ambos tipos de respuesta que sugieren que la NS producida en interacciones compatibles sería el resultado de una respuesta hipersensible sistémica (SHR). Sin embargo, los mecanismos moleculares implicados en el desarrollo de la NS, su relación con procesos de defensa antiviral o su relevancia biológica aún no son bien entendidos, al igual que tampoco han sido estudiados los cambios producidos en la planta a escala genómica en infecciones múltiples que muestran sinergismo en patología. En esta tesis doctoral se han empleado distintas aproximaciones de análisis de expresión génica, junto con otras técnicas genéticas y bioquímicas, en el sistema modelo de Nicotiana benthamiana para estudiar la NS producida por la infección sinérgica entre el Virus X de la patata (PVX) y diversos potyvirus. Se han comparado los cambios producidos en el huésped a nivel genómico y fisiológico entre la infección doble con PVX y el Virus Y de la patata (PVY), y las infecciones simples con PVX o PVY. Además, los cambios transcriptómicos y hormonales asociados a la infección con la quimera viral PVX/HC‐Pro, que reproduce los síntomas del sinergismo entre PVX‐potyvirus, se han comparado con aquellos producidos por otros dos tipos de muerte celular, la PCD ligada a una interacción incompatible y la PCD producida por la disfunción del proteasoma. Por último, técnicas de genética reversa han permitido conocer la implicación de factores del huésped, como las oxilipinas, en el desarrollo de la NS asociada al sinergismo entre PVXpotyvirus. Los resultados revelan que, respecto a las infecciones con solo uno de los virus, la infección doble con PVX‐PVY produce en el huésped diferencias cualitativas además de cuantitativas en el perfil transcriptómico relacionado con el metabolismo primario. Otros cambios en la expresión génica, que reflejan la activación de mecanismos de defensa, correlacionan con un fuerte estrés oxidativo en las plantas doblemente infectadas que no se detecta en las infecciones simples. Además, medidas en la acumulación de determinados miRNAs implicados en diversos procesos celulares muestran como la infección doble altera de manera diferencial tanto la acumulación de estos miRNAs como su funcionalidad, lo cual podría estar relacionado con los cambios en el transcriptoma, así como con la sintomatología de la infección. La comparación a nivel transcriptómico y hormonal entre la NS producida por PVX/HC‐Pro y la interacción incompatible del Virus del mosaico del tabaco en plantas que expresan el gen N de resistencia (SHR), muestra que la respuesta en la interacción compatible es similar a la que se produce durante la SHR, si bien se presenta de manera retardada en el tiempo. Sin embargo, los perfiles de expresión de genes de defensa y de respuesta a hormonas, así como la acumulación relativa de ácido salicílico (SA), ácido jasmonico (JA) y ácido abscísico, en la interacción compatible son más semejantes a la respuesta PCD producida por la disfunción del proteasoma que a la interacción incompatible. Estos datos sugieren una contribución de la interferencia sobre la funcionalidad del proteasoma en el incremento de la patogenicidad, observado en el sinergismo PVX‐potyvirus. Por último, los resultados obtenidos al disminuir la expresión de 9‐LOX, α‐DOX1 y COI1, relacionados con la síntesis o con la señalización de oxilipinas, y mediante la aplicación exógena de JA y SA, muestran la implicación del metabolismo de las oxilipinas en el desarrollo de la NS producida por la infección sinérgica entre PVXpotyvirus en N. benthamiana. Además, estos resultados indican que la PCD asociada a esta infección, al igual que ocurre en interacciones incompatibles, no contiene necesariamente la acumulación viral, lo cual indica que necrosis e inhibición de la multiplicación viral son procesos independientes. ABSTRACT Plant viruses cause severe diseases that lead to serious economic losses worldwide. Moreover, simultaneous infections with several viruses are common in nature leading to exacerbation of the disease symptoms. This phenomenon is known as viral synergism. Systemic necrosis (SN) is one of the most severe symptoms caused by plant viruses in susceptible plants, even leading to death of the host. This phenotype has been compared with the hypersensitive response (HR) displayed by resistant plants, and some parallelisms have been found between both responses, which suggest that SN induced by compatible interactions could be the result of a systemic hypersensitive response (SHR). However, the molecular mechanisms involved in the development of SN, its relationship with antiviral defence processes and its biological relevance are still unknown. Furthermore, the changes produced in plants by mixed infections that cause synergistic pathological effects have not been studied in a genome‐wide scale. In this doctoral thesis different approaches have been used to analyse gene expression, together with other genetic and biochemical techniques, in the model plant Nicotiana benthamiana, in order to study the SN produced by the synergistic infection of Potato virus X (PVX) with several potyviruses. Genomic and physiological changes produced in the host by double infection with PVX and Potato virus Y (PVY), and by single infection with PVX or PVY have been compared. In addition, transcriptional and hormonal changes associated with infection by the chimeric virus PVX/HC‐Pro, which produces synergistic symptoms similar to those caused by PVX‐potyvirus, have been compared with those produced by other types of cell death. These types of cell death are: PCD associated with an incompatible interaction, and PCD produced by proteasome disruption. Finally, reverse genetic techniques have revealed the involvement of host factors, such as oxylipins, in the development of SN associated with PVX‐potyvirus synergism. The results revealed that compared with single infections, double infection with PVX‐PVY produced qualitative and quantitative differences in the transcriptome profile, mainly related to primary metabolism. Other changes in gene expression, which reflected the activation of defence mechanisms, correlated with a severe oxidative stress in doubly infected plants that was undetected in single infections. Additionally, accumulation levels of several miRNAs involved in different cellular processes were measured, and the results showed that double infection not only produced the greatest variations in miRNA accumulation levels but also in miRNA functionality. These variations could be related with transcriptomic changes and the symptomatology of the infection. Transcriptome and hormone level comparisons between SN induced by PVX/HCPro and the incompatible interaction produced by Tobacco mosaic virus in plants expressing the N resistance gene (SHR), showed some similarities between both responses, even though the compatible interaction appeared retarded in time. Nevertheless, the expression profiles of both defence‐related genes and hormoneresponsive genes, as well as the relative accumulation of salicylic acid (SA), jasmonic acid (JA) and abscisic acid in the compatible interaction are more similar to the PCD response produced by proteasome disruption. These data suggest that interference with proteasome functionality contributes to the increase in pathogenicity associated with PVX‐potyvirus synergism. Finally, the results obtained by reducing the expression of 9‐LOX, α‐DOX1 and COI1, related with synthesis or signalling of oxylipins, and by applying exogenously JA and SA, revealed that oxylipin metabolism is involved in the development of SN induced by PVX‐potyvirus synergistic infections in N. benthamiana. Moreover, these results also indicated that PVX‐potyvirus associated PCD does not necessarily restrict viral accumulation, as is also the case in incompatible interactions. This indicates that both necrosis and inhibition of viral multiplication are independent processes.

Cartografía de QTL asociados a la tolerancia a estrés hídrico en frijol común (Phaseolus vulgaris L.)

Phaseolus vulgaris L. (frijol común o judía) es una leguminosa de gran demanda para la nutrición humana y un producto agrícola muy importante. Sin embargo, la producción de frijol se ve limitada por presiones ambientales como la sequía. En México, el 85% de la cosecha de frijol se produce en la temporada de primavera-verano, principalmente en las regiones del altiplano semiárido con una precipitación anual entre 250 y 400 mm. A pesar del implemento de tecnología en el campo, los factores naturales impiden al agricultor llegar a los rendimientos deseados. El Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias (INIFAP), como instituto de investigación gubernamental en México, tiene como objetivo la mejora de cultivos estratégicos, uno de ellos, P. vulgaris. Los estudios en relación a la sequía se enfocan especialmente en la selección de genotipos tolerantes, los cuales son sometidos en condiciones de estrés y monitoreando parámetros como el rendimiento y peso de semilla, además de algunos indicadores tales como índice de cosecha. El resultado de estos trabajos ha sido la obtención de variedades con mayor tolerancia a la sequía, tales como Pinto Villa y Pinto Saltillo. En los últimos años se ha avanzado notablemente en el conocimiento de las bases moleculares en las respuestas de las plantas al estrés. De acuerdo a diversos estudios se ha demostrado que las plantas bajo estrés por sequía experimentan cambios en la expresión de genes involucrados en la señalización, regulación de la transcripción y la traducción, transporte de agua y la función directa en la protección celular. También se ha observado que el déficit de agua es causado por las temperaturas extremas y la alta concentración de sales, por lo que al nivel molecular, las respuestas al estrés tienen puntos de especificidad y puntos de entrecruzamiento. La sequía puede generar estreses secundarios, tales como el nutricional, oxidativo y osmótico. Sin embargo, es necesario identificar y caracterizar muchos de los componentes involucrados en las respuestas al déficit hídrico, la caracterización de estos genes permitirá tener una mejor comprensión de los mecanismos bioquímicos y fisiológicos involucrados en la tolerancia al estrés. Actualmente, con el apoyo de la biología molecular se han identificado algunos genes que otorgan ventajas para la adaptación a ambientes desfavorables. Por lo que el objetivo del presente trabajo es identificar marcadores genéticos asociados a rasgos fenotípicos con énfasis a la tolerancia a estrés hídrico en P. vulgaris. Una vez establecidos los marcadores asociados al estrés hídrico, es factible considerar su uso para la selección asistida por marcadores en líneas o variedades de frijol de interés para los mejoradores. Se evaluaron 282 familias F3:5 derivadas de la cruza entre los cultivares Pinto Villa y Pinto Saltillo. Las familias se sembraron bajo un diseño simple de látice 17x17, el experimento se llevo acabo en el ciclo primavera-verano del 2010 y 2011, y otoñoinvierno de 2010 en el Campo Experimental Bajío del INIFAP con dos repeticiones para cada tratamiento de humedad (riego completo y sequía terminal). En todos los genotipos se realizó el fenotipado (variables fenotípicas) y el genotipado a través de marcadores moleculares. Los análisis estadísticos se basaron en el análisis de componentes principales (Eigen Analysis Selection Index Method, ESIM), la asociación entre marcadores SNP y el fenotipado (paquete SNPassoc para R) y el análisis de varianza (ANOVA). Los valores ESIM mostraron que las variables de Rendimiento, Días a floración, Días a madurez fisiológica e Índice de cosecha fueron sobresalientes en sequía terminal, por lo que se sugieren tomarse en consideración para los estudios de sequía en P. vulgaris como monitores de evaluación a la resistencia. Se identificaron nueve familias sobresalieron por sus valores ESIM (PV/PS6, 22, 131, 137, 149, 154, 201, 236 y 273), además de presentar valores superiores para el rendimiento en comparación con los parentales. Estos genotipos son candidatos interesantes para realizar estudios de identificación de loci asociados con la respuesta al estrés, y como potenciales parentales en el desarrollo de nuevas variedades de frijol. En los análisis de asociación SNPassoc se identificaron 83 SNPs significativos (p<0,0003) asociados a los rasgos fenotípicos, obteniendo un total de 222 asociaciones, de las cuales predomina el modelo genético de codominancia para las variables Días a floración, Periodo reproductivo y Biomasa total. Treinta y siete SNPs se identificaron a diferentes funciones biológicas a través del análisis de anotación funcional, de los cuales 12 SNPs (9, 18, 28, 39, 61, 69, 80, 106, 115, 128, 136 y 142) sobresalen por su asociación al fenotipado, y cuya anotación funcional indica que se encuentran en genes relacionados a la tolerancia a la sequía, tales como la actividad kinasa, actividad metabólica del almidón, carbohidratos y prolina, respuesta al estrés oxidativo, así como en los genes LEA y posibles factores de transcripción. En el caso de los análisis ANOVA, se identificaron 72 asociaciones entre los SNPs y las variables fenotípicas (F< 3,94E-04). Las 72 asociaciones corresponden a 30 SNPs y 7 variables fenotípicas, de las que predomina Peso de 100 semillas y Periodo reproductivo. Para los rasgos de Rendimiento, Índice de cosecha y Días a madurez fisiológica se presentaron asociaciones con seis SNPs (17, 34, 37, 50, 93 y 107), de los cuales, a los SNP37 y SNP107 fueron identificados a la anotación biológica de protein binding. Por otro lado, los SNP106 y SNP128 asociados al Periodo reproductivo, son genes con actividad kinasa y actividad metabólica del almidón, respectivamente. Para los marcadores tipo AFLP, se identificaron 271 asociaciones (F<2,34E-04). Las asociaciones corresponden a 86 AFLPs con todas las variables fenotípicas evaluadas, de las que predomina peso de 100 semillas, Días a floración y Periodo reproductivo. Debido a que los en los AFLPs no es posible determinar su anotación biológica, se proponen como marcadores potenciales relacionados a la resistencia a la sequía en frijol. Los AFLPs candidatos requieren más estudios tales como la secuenciación de los alelos respectivos, así como la identificación de éstas secuencias en el genoma de referencia y su anotación biológica, entre otros análisis, de esta manera podríamos establecer aquellos marcadores candidatos a la validación para la selección asistida. El presente trabajo propone tanto genotipos como marcadores genéticos, que deben ser validados para ser utilizados en el programa de mejoramiento de P. vulgaris, con el objetivo de desarrollar nuevas líneas o variedades tolerantes a la sequía. ABSTRACT Phaseolus vulgaris L. (common bean or judia) is a legume of great demand for human consumption and an important agricultural product. However, the common bean production is limited by environmental stresses, such as drought. In Mexico, 85% of the common bean crop is produced in the spring-summer season mainly in semiarid highland regions with a rainfall between 250 and 400 mm per year. In spite of the improvement of crop technology, the natural factors hamper getting an optimal yield. The National Institute for Forestry, Agriculture and Livestock (INIFAP) is a government research institute from Mexico, whose main objective is the genetic breeding of strategic crops, like P. vulgaris L. The drought tolerance studies particularly focus on the selection of bean tolerant genotypes, which are subjected to stress conditions, by means of monitoring parameters such as yield and seed weight, plus some agronomic indicators such as harvest index. The results of these works have led to obtain cultivars with higher drought tolerance such as Pinto Villa and Pinto Saltillo. Significant achievements have been recently made in understanding the molecular basis of stress plant responses. Several studies have shown that plants under drought stress present changes in gene expression related to cell signalling, transcriptional and translational regulation, water transport and cell protection. In addition, it has been observed that the extreme temperatures and high salt concentrations can cause a water deficiency so, at the molecular level, stress responses have specific and crossover points. The drought can cause secondary stresses, such as nutritional, oxidative and osmotic stress. It is required the identification of more components involved in the response to water deficit, the characterization of these genes will allow a better understanding of the biochemical and physiological mechanisms involved in stress tolerance. Currently, with the support of molecular biology techniques, some genes that confer an advantage for the crop adaptation to unfavourable environments have been identified. The objective of this study is to identify genetic markers associated with phenotypic traits with emphasis on water stress tolerance in P. vulgaris. The establishment of molecular markers linked to drought tolerance would make possible their use for marker-assisted selection in bean breeding programs. Two hundred and eighty two F3:5 families derived from a cross between the drought resistant cultivars Pinto Villa and Pinto Saltillo were evaluated. The families were sowed under a 17x17 simple lattice design. The experiment was conducted between spring-summer seasons in 2010 and 2011, and autumn-winter seasons in 2010 at the Bajio Experimental Station of INIFAP with two treatments (full irrigation and terminal drought). All families were phenotyped and genotyped using molecular markers. Statistical analysis was based on principal component analysis (Eigen Analysis Selection Index Method, ESIM), association analysis between SNP markers and phenotype (SNPassoc package R) and analysis of variance (ANOVA). The ESIM values showed that seed yield, days to flowering, days to physiological maturity and harvest index were outstanding traits in terminal drought treatment, so they could be considered as suitable parameters for drought-tolerance evaluation in P. vulgaris. Nine outstanding families for the ESIM values were identified (PV/PS6, 22, 131, 137, 149, 154, 201, 236 and 273), in addition, these families showed higher values for seed yield compared to the parental cultivars. These families are promising candidates for studies focused on the identification of loci associated to the stress response, and as potential parental cultivars for the development of new varieties of common bean. In the SNPassoc analysis, 83 SNPs were found significantly associated (p<0.0003) with phenotypic traits, obtaining a total of 222 associations, most of which involved the traits days to flowering, reproductive period and total biomass under a codominant genetic model. The functional annotation analysis showed 37 SNPs with different biological functions, 12 of them (9, 18, 28, 39, 61, 69, 80, 106, 115, 128, 136 and 142) stand out by their association to phenotype. The functional annotation suggested a connection with genes related to drought tolerance, such as kinase activity, starch, carbohydrates and proline metabolic processes, responses to oxidative stress, as well as LEA genes and putative transcription factors. In the ANOVA analysis, 72 associations between SNPs and phenotypic traits (F<3.94E- 04) were identified. All of these associations corresponded to 30 SNPs markers and seven phenotypic traits. Weight of 100 seeds and reproductive period were the traits with more associations. Seed yield, harvest index and days to physiological maturity were associated to six SNPs (17, 34, 37, 50, 93 and 107), the SNP37 and SNP107 were identified as located in protein binding genes. The SNP106 and SNP128 were associated with the reproductive period and belonged to genes with kinase activity and genes related to starch metabolic process, respectively. In the case of AFLP markers, 271 associations (F<2.34E-04) were identified. The associations involved 86 AFLPs and all phenotypic traits, being the most frequently associated weight of 100 seeds, days to flowering and reproductive period. Even though it is not possible to perform a functional annotation for AFLP markers, they are proposed as potential markers related to drought resistance in common bean. AFLPs candidates require additional studies such as the sequencing of the respective alleles, identification of these sequences in the reference genome and gene annotation, before their use in marker assisted selection. This work, although requires further validation, proposes both genotypes and genetic markers that could be used in breeding programs of P. vulgaris in order to develop new lines or cultivars with enhanced drought-tolerance.

Tissue phenotype depends on reciprocal interactions between the extracellular matrix and the structural organization of the nucleus

What determines the nuclear organization within a cell and whether this organization itself can impose cellular function within a tissue remains unknown. To explore the relationship between nuclear organization and tissue architecture and function, we used a model of human mammary epithelial cell acinar morphogenesis. When cultured within a reconstituted basement membrane (rBM), HMT-3522 cells form polarized and growth-arrested tissue-like acini with a central lumen and deposit an endogenous BM. We show that rBM-induced morphogenesis is accompanied by relocalization of the nuclear matrix proteins NuMA, splicing factor SRm160, and cell cycle regulator Rb. These proteins had distinct distribution patterns specific for proliferation, growth arrest, and acini formation, whereas the distribution of the nuclear lamina protein, lamin B, remained unchanged. NuMA relocalized to foci, which coalesced into larger assemblies as morphogenesis progressed. Perturbation of histone acetylation in the acini by trichostatin A treatment altered chromatin structure, disrupted NuMA foci, and induced cell proliferation. Moreover, treatment of transiently permeabilized acini with a NuMA antibody led to the disruption of NuMA foci, alteration of histone acetylation, activation of metalloproteases, and breakdown of the endogenous BM. These results experimentally demonstrate a dynamic interaction between the extracellular matrix, nuclear organization, and tissue phenotype. They further show that rather than passively reflecting changes in gene expression, nuclear organization itself can modulate the cellular and tissue phenotype.

Gradual Phenotypic Conversion Associated with Immortalization of Cultured Human Mammary Epithelial Cells

Examination of the process of immortal transformation in early passages of two human mammary epithelial cell (HMEC) lines suggests the involvement of an epigenetic step. These lines, 184A1 and 184B5, arose after in vitro exposure of finite lifespan 184 HMEC to a chemical carcinogen, and both are clonally derived. Although early-passage mass cultures of 184A1 and 184B5 maintained continuous slow growth, most individual cells lost proliferative ability. Uniform good growth did not occur until 20–30 passages after the lines first appeared. Early-passage cultures expressed little or no telomerase activity and telomeres continued to shorten with increasing passage. Telomerase activity was first detected when the telomeres became critically short, and activity levels gradually increased thereafter. Early-passage cultures had little or no ability to maintain growth in transforming growth factor-β (TGFβ); however, both mass cultures and clonal isolates showed a very gradual increase in the number of cells displaying progressively increased ability to maintain growth in TGFβ. A strong correlation between capacity to maintain growth in the presence of TGFβ and expression of telomerase activity was observed. We have used the term “conversion” to describe this process of gradual acquisition of increased growth capacity in the absence or presence of TGFβ and reactivation of telomerase. We speculate that the development of extremely short telomeres may result in gradual, epigenetic-based changes in gene expression. Understanding the underlying mechanisms of HMEC conversion in vitro may provide new insight into the process of carcinogenic progression in vivo and offer novel modes for therapeutic intervention.

Influences of aging and caloric restriction on the transcriptional profile of skeletal muscle from rhesus monkeys

In laboratory rodents, caloric restriction (CR) retards several age-dependent physiological and biochemical changes in skeletal muscle, including increased steady-state levels of oxidative damage to lipids, DNA, and proteins. We have previously used high-density oligonucleotide arrays to show that CR can prevent or delay most of the major age-related transcriptional alterations in the gastrocnemius muscle of C57BL/6 mice. Here we report the effects of aging and adult-onset CR on the gene expression profile of 7,070 genes in the vastus lateralis muscle from rhesus monkeys. Gene expression analysis of aged rhesus monkeys (mean age of 26 years) was compared with that of young animals (mean age of 8 years). Aging resulted in a selective up-regulation of transcripts involved in inflammation and oxidative stress, and a down-regulation of genes involved in mitochondrial electron transport and oxidative phosphorylation. Middle-aged monkeys (mean age of 20 years) subjected to CR since early adulthood (mean age of 11 years) were studied to determine the gene expression profile induced by CR. CR resulted in an up-regulation of cytoskeletal protein-encoding genes, and also a decrease in the expression of genes involved in mitochondrial bioenergetics. Surprisingly, we did not observe any evidence for an inhibitory effect of adult-onset CR on age-related changes in gene expression. These results indicate that the induction of an oxidative stress-induced transcriptional response may be a common feature of aging in skeletal muscle of rodents and primates, but the extent to which CR modifies these responses may be species-specific.

Controlling protein association and subcellular localization with a synthetic ligand that induces heterodimerization of proteins.

Extracellular growth and differentiation factors induce changes in gene expression in the nucleus by initiating a series of protein associations that alter the subcellular localization of intracellular signaling proteins. Initial events involve receptor homo- or heterodimerization and subsequent recruitment of cytosolic signaling proteins to the inner leaflet of the plasma membrane. Intermediate events involve the translocation of proteins into the nucleus. Late events involve the recruitment of transcriptional activators to the vicinity of specific genes in the nucleus, resulting in increased gene transcription. The ability to induce signals at each of these three phases of signaling pathways is illustrated by the use of a heterodimeric chemical inducer of dimerization that causes a proximal relationship between two different target proteins.

Epigenetics of lung cancer

Epigenetics is the study of heritable changes in gene expression that occur without changes in DNA sequence. It has a role in determining when and where a gene is expressed during development. Perhaps the most well known epigenetic mechanism is DNA methylation whereby cytosines at position 5 in CpG dinucleotides are methylated. Histone modification is another form of epigenetic control, which is quite complex and diverse. Histones and DNA make up the nucleosome which is the structural unit of chromatin which are involved in packaging DNA. Apart from the crucial role epigenetics plays in embryonic development, transcription, chromatin structure, X chromosome inactivation and genomic imprinting, its role in an increasing number of human diseases is more and more recognized. These diseases include cancer, and lung cancer in particular has been increasingly studied for the potential biological role of epigenetic changes with the promise of better and novel diagnostic and therapeutic tools.

MyT1 Counteracts the Neural Progenitor Program to Promote Vertebrate Neurogenesis

The generation of neurons from neural stem cells requires large-scale changes in gene expression that are controlled to a large extent by proneural transcription factors, such as Ascl1. While recent studies have characterized the differentiation genes activated by proneural factors, less is known on the mechanisms that suppress progenitor cell identity. Here, we show that Ascl1 induces the transcription factor MyT1 while promoting neuronal differentiation. We combined functional studies of MyT1 during neurogenesis with the characterization of its transcriptional program. MyT1 binding is associated with repression of gene transcription in neural progenitor cells. It promotes neuronal differentiation by counteracting the inhibitory activity of Notch signaling at multiple levels, targeting the Notch1 receptor and many of its downstream targets. These include regulators of the neural progenitor program, such as Hes1, Sox2, Id3, and Olig1. Thus, Ascl1 suppresses Notch signaling cell-autonomously via MyT1, coupling neuronal differentiation with repression of the progenitor fate.

Guided Migration and Collective Behavior: Cell Dynamics during Cancer Progression

This dissertation focuses on gaining understanding of cell migration and collective behavior through a combination of experiment, analysis, and modeling techniques. Cell migration is a ubiquitous process that plays an important role during embryonic development and wound healing as well as in diseases like cancer, which is a particular focus of this work. As cancer cells become increasingly malignant, they acquire the ability to migrate away from the primary tumor and spread throughout the body to form metastatic tumors. During this process, changes in gene expression and the surrounding tumor environment can lead to changes in cell migration characteristics. In this thesis, I analyze how cells are guided by the texture of their environment and how cells cooperate with their neighbors to move collectively. The emergent properties of collectively moving groups are a particular focus of this work as collective cell dynamics are known to change in diseases such as cancer. The internal machinery for cell migration involves polymerization of the actin cytoskeleton to create protrusions that---in coordination with retraction of the rear of the cell---lead to cell motion. This actin machinery has been previously shown to respond to the topography of the surrounding surface, leading to guided migration of amoeboid cells. Here we show that epithelial cells on nanoscale ridge structures also show changes in the morphology of their cytoskeletons; actin is found to align with the ridge structures. The migration of the cells is also guided preferentially along the ridge length. These ridge structures are on length scales similar to those found in tumor microenvironments and as such provide a system for studying the response of the cells' internal migration machinery to physiologically relevant topographical cues. In addition to sensing surface topography, individual cells can also be influenced by the pushing and pulling of neighboring cells. The emergent properties of collectively migrating cells show interesting dynamics and are relevant for cancer progression, but have been less studied than the motion of individual cells. We use Particle Image Velocimetry (PIV) to extract the motion of a collectively migrating cell sheet from time lapse images. The resulting flow fields allow us to analyze collective behavior over multiple length and time scales. To analyze the connection between individual cell properties and collective migration behavior, we compare experimental flow fields with the migration of simulated cell groups. Our collective migration metrics allow for a quantitative comparison between experimental and simulated results. This comparison shows that tissue-scale decreases in collective behavior can result from changes in individual cell activity without the need to postulate the existence of subpopulations of leader cells or global gradients. In addition to tissue-scale trends in collective behavior, the migration of cell groups includes localized dynamic features such as cell rearrangements. An individual cell may smoothly follow the motion of its neighbors (affine motion) or move in a more individualistic manner (non-affine motion). By decomposing individual motion into both affine and non-affine components, we measure cell rearrangements within a collective sheet. Finally, finite-time Lyapunov exponent (FTLE) values capture the stretching of the flow field and reflect its chaotic character. Applying collective migration analysis techniques to experimental data on both malignant and non-malignant human breast epithelial cells reveals differences in collective behavior that are not found from analyzing migration speeds alone. Non-malignant cells show increased cooperative motion on long time scales whereas malignant cells remain uncooperative as time progresses. Combining multiple analysis techniques also shows that these two cell types differ in their response to a perturbation of cell-cell adhesion through the molecule E-cadherin. Non-malignant MCF10A cells use E-cadherin for short time coordination of collective motion, yet even with decreased E-cadherin expression, the cells remain coordinated over long time scales. In contrast, the migration behavior of malignant and invasive MCF10CA1a cells, which already shows decreased collective dynamics on both time scales, is insensitive to the change in E-cadherin expression.

Guided Migration and Collective Behavior: Cell Dynamics during Cancer Progression

This dissertation focuses on gaining understanding of cell migration and collective behavior through a combination of experiment, analysis, and modeling techniques. Cell migration is a ubiquitous process that plays an important role during embryonic development and wound healing as well as in diseases like cancer, which is a particular focus of this work. As cancer cells become increasingly malignant, they acquire the ability to migrate away from the primary tumor and spread throughout the body to form metastatic tumors. During this process, changes in gene expression and the surrounding tumor environment can lead to changes in cell migration characteristics. In this thesis, I analyze how cells are guided by the texture of their environment and how cells cooperate with their neighbors to move collectively. The emergent properties of collectively moving groups are a particular focus of this work as collective cell dynamics are known to change in diseases such as cancer. The internal machinery for cell migration involves polymerization of the actin cytoskeleton to create protrusions that---in coordination with retraction of the rear of the cell---lead to cell motion. This actin machinery has been previously shown to respond to the topography of the surrounding surface, leading to guided migration of amoeboid cells. Here we show that epithelial cells on nanoscale ridge structures also show changes in the morphology of their cytoskeletons; actin is found to align with the ridge structures. The migration of the cells is also guided preferentially along the ridge length. These ridge structures are on length scales similar to those found in tumor microenvironments and as such provide a system for studying the response of the cells' internal migration machinery to physiologically relevant topographical cues. In addition to sensing surface topography, individual cells can also be influenced by the pushing and pulling of neighboring cells. The emergent properties of collectively migrating cells show interesting dynamics and are relevant for cancer progression, but have been less studied than the motion of individual cells. We use Particle Image Velocimetry (PIV) to extract the motion of a collectively migrating cell sheet from time lapse images. The resulting flow fields allow us to analyze collective behavior over multiple length and time scales. To analyze the connection between individual cell properties and collective migration behavior, we compare experimental flow fields with the migration of simulated cell groups. Our collective migration metrics allow for a quantitative comparison between experimental and simulated results. This comparison shows that tissue-scale decreases in collective behavior can result from changes in individual cell activity without the need to postulate the existence of subpopulations of leader cells or global gradients. In addition to tissue-scale trends in collective behavior, the migration of cell groups includes localized dynamic features such as cell rearrangements. An individual cell may smoothly follow the motion of its neighbors (affine motion) or move in a more individualistic manner (non-affine motion). By decomposing individual motion into both affine and non-affine components, we measure cell rearrangements within a collective sheet. Finally, finite-time Lyapunov exponent (FTLE) values capture the stretching of the flow field and reflect its chaotic character. Applying collective migration analysis techniques to experimental data on both malignant and non-malignant human breast epithelial cells reveals differences in collective behavior that are not found from analyzing migration speeds alone. Non-malignant cells show increased cooperative motion on long time scales whereas malignant cells remain uncooperative as time progresses. Combining multiple analysis techniques also shows that these two cell types differ in their response to a perturbation of cell-cell adhesion through the molecule E-cadherin. Non-malignant MCF10A cells use E-cadherin for short time coordination of collective motion, yet even with decreased E-cadherin expression, the cells remain coordinated over long time scales. In contrast, the migration behavior of malignant and invasive MCF10CA1a cells, which already shows decreased collective dynamics on both time scales, is insensitive to the change in E-cadherin expression.

Evolution of gene expression changes in newborn rats after mechanical ventilation with reversible intubation.

Mechanical ventilation (MV) is life-saving but potentially harmful for lungs of premature infants. So far, animal models dealt with the acute impact of MV on immature lungs, but less with its delayed effects. We used a newborn rodent model including non-surgical and therefore reversible intubation with moderate ventilation and hypothesized that there might be distinct gene expression patterns after a ventilation-free recovery period compared to acute effects directly after MV. Newborn rat pups were subjected to 8 hr of MV with 60% oxygen (O(2) ), 24 hr after injection of lipopolysaccharide (LPS), intended to create a low inflammatory background as often recognized in preterm infants. Animals were separated in controls (CTRL), LPS injection (LPS), or full intervention with LPS and MV with 60% O(2) (LPS + MV + O(2) ). Lungs were recovered either directly following (T:0 hr) or 48 hr after MV (T:48 hr). Histologically, signs of ventilator-induced lung injury (VILI) were observed in LPS + MV + O(2) lungs at T:0 hr, while changes appeared similar to those known from patients with chronic lung disease (CLD) with fewer albeit larger gas exchange units, at T:48 hr. At T:0 hr, LPS + MV + O(2) increased gene expression of pro-inflammatory MIP-2. In parallel anti-inflammatory IL-1Ra gene expression was increased in LPS and LPS + MV + O(2) groups. At T:48 hr, pro- and anti-inflammatory genes had returned to their basal expression. MMP-2 gene expression was decreased in LPS and LPS + MV + O(2) groups at T:0 hr, but no longer at T:48 hr. MMP-9 gene expression levels were unchanged directly after MV. However, at T:48 hr, gene and protein expression increased in LPS + MV + O(2) group. In conclusion, this study demonstrates the feasibility of delayed outcome measurements after a ventilation-free period in newborn rats and may help to further understand the time-course of molecular changes following MV. The differences obtained from the two time points could be interpreted as an initial transitory increase of inflammation and a delayed impact of the intervention on structure-related genes. Pediatr Pulmonol. 2012; 47:1204-1214. © 2012 Wiley Periodicals, Inc.

Gene expression changes in chronic inflammatory demyelinating polyneuropathy skin biopsies.

Chronic-inflammatory demyelinating polyneuropathy (CIDP) is an immune-mediated disease with no known biomarkers for diagnosing the disease or assessing its prognosis. We performed transcriptional profiling microarray analysis on skin punch biopsies from 20 CIDP patients and 17 healthy controls to identify disease-associated gene expression changes. We demonstrate changes in expression of genes involved in immune and chemokine regulation, growth and repair. We also found a combination of two upregulated genes that can be proposed as a novel biomarker of the disorder.