Molecular mechanisms by which p53/LSD1 and ERalpha/Trim24 complexes mediate gene regulation

In this dissertation, I identify two molecular mechanisms by which transcription factors cooperate with their co-regulators to mediate gene regulation. In the first part, I demonstrate that p53 directly recruits LSD1, a histone demethylase, to AFP chromatin to demethylate methylated H3K4 and actively mediate transcription repression. Loss of p53 and LSD1 interaction at chromatin leads to derepression of AFP in hepatic cells. In the second part, I reveal that Trim24 functions as an important co-activator in ERα-mediated gene activation in response to estrogen stimulation. Trim24 is recruited by ligand-bound ERα to chromatin and stabilizes ERα-chromatin interactions by binding to histone H3 via its PHD finger, which preferentially recognizes unmethylated H3K4. ^

Mechanism of methylation gene reactivation in human cancer cells

Epigenetic silencing of tumor suppressor genes by DNA hypermethylation at promoter regions is a common event in carcinogenesis and tumor progression. Abrogation of methylation and reversal of epigenetic silencing is a very potent way in cancer treatment. However, the reactivation mechanisms are poorly understood. In this study, we first developed a cell line model system named YB5, derived from SW48 cancer cell line, which bears one copy of stably integrated EGFP gene on Chromosome 1p31.1 region. The GFP gene expression is transcriptionally silenced due to the hypermethylated promoter CMV. However, the GFP expression can be restored using demethylating agent 5-aza-2' deoxycytidine (DAC), and detected by FACS and fluorescent microscopy. Using this system, we observed the heterogeneous reactivation induced by DAC treatment. After flow sorting, GFP negative cells exhibited similar level of incomplete demethylation compared to GFP positive cells on repetitive LINE1 element, tumor suppressor genes such as P16, CDH13, and RASSF1a, and CMV promoter as well. However, the local chromatin of CMV-GFP locus altered to an open structure marked by high H3 lysine 9 acetylation and low H3 lysine 27 tri-methylation in GFP positive cells, while the GFP negative cells retained mostly the original repressive marks. Thus, we concluded that DAC induced DNA hypomethylation alone does not directly determine the level of re-expression, and the resetting of the local chromatin structure under hypomethylation environment is required for gene reactivation. Besides, a lentivirus vector-based shRNA screening was performed using the YB5 system. Although it is the rare chance that vector lands in the neighboring region of GFP, we found that the exogenous vector DNA inserted into the upstream region of GFP gene locus led to the promoter demethylation and reactivated the silenced GFP gene. Thus, epigenetic state can be affected by changing of the adjacent nucleic acid sequences. Further, this hypermethylation silenced system was utilized for epigenetic drug screening. We have found that DAC combined with carboplatin would enhance the GFP% yield and increase expression of other tumor suppressor genes than DAC alone, and this synergistic effect may be related to DNA repair process. In summary, these studies reveal that reversing of methylation silencing requires coordinated alterations of DNA methylation, chromatin structure, and local microenvironment. ^

THE EFFECT OF PERSON-ENVIRONMENT FIT ON THE PSYCHOLOGICAL WELL-BEING OF PSYCHIATRIC AIDES WORKING IN A STATE HOSPITAL (COPING RESOURCES, SOCIAL SUPPORT, OCCUPATIONAL STRESS, LOCUS-OF-CONTROL)

This study addressed two purposes: (1) to determine the effect of person-environment fit on the psychological well-being of psychiatric aides and (2) to determine what role the coping resources of social support and control have on the above relationship. Two hundred and ten psychiatric aides working in a state hospital in Texas responded to a questionnaire pertaining to these issues.^ Person-environment fit, as a measure of occupational stress, was assessed through a modified version of the Work Environment Scale (WES). The WES subscales used in this study were: involvement, autonomy, job pressure, job clarity, and physical comfort. Psychological well-being was measured with the General Well-Being Schedule which was developed by the National Center for Health Statistics. Co-worker and supervisor support were measured through the WES and finally, control was assessed through Rotter's Locus of Control Scale.^ The results of this study were as follows: (1) all person-environment (p-e) dimensions appeared to have linear relationships with psychological well-being; (2) the p-e fit - well-being relationship did not appear to be confounded by demographic factors; (3) all p-e fit dimensions were significantly related to well-being except for autonomy; (4) p-e fit was more strongly related to well-being than the environmental measure alone; (5) supervisor support and non-work related support were found to have additive effects on the relationship between p-e fit and well-being, however no interaction or buffering effects were observed; (6) locus of control was found to have additive effects in the prediction of well-being and showed interactive effects with work pressure, involvement and physical comfort; and (7) the testing of the overall study model which included many of the components mentioned above yielded an R('2) = .27.^ Implications of these findings are discussed, future research suggested and applications proposed. ^

DETERMINING THE GENOTYPE-PHENOTYPE CONNECTION IN SYNTHETIC INDUCIBLE GENE EXPRESSION SYSTEMS

Introduction Gene expression is an important process whereby the genotype controls an individual cell’s phenotype. However, even genetically identical cells display a variety of phenotypes, which may be attributed to differences in their environment. Yet, even after controlling for these two factors, individual phenotypes still diverge due to noisy gene expression. Synthetic gene expression systems allow investigators to isolate, control, and measure the effects of noise on cell phenotypes. I used mathematical and computational methods to design, study, and predict the behavior of synthetic gene expression systems in S. cerevisiae, which were affected by noise. Methods I created probabilistic biochemical reaction models from known behaviors of the tetR and rtTA genes, gene products, and their gene architectures. I then simplified these models to account for essential behaviors of gene expression systems. Finally, I used these models to predict behaviors of modified gene expression systems, which were experimentally verified. Results Cell growth, which is often ignored when formulating chemical kinetics models, was essential for understanding gene expression behavior. Models incorporating growth effects were used to explain unexpected reductions in gene expression noise, design a set of gene expression systems with “linear” dose-responses, and quantify the speed with which cells explored their fitness landscapes due to noisy gene expression. Conclusions Models incorporating noisy gene expression and cell division were necessary to design, understand, and predict the behaviors of synthetic gene expression systems. The methods and models developed here will allow investigators to more efficiently design new gene expression systems, and infer gene expression properties of TetR based systems.

Inhibitor of cytochrome c messenger RNA -protein interaction in stimulated rat skeletal muscle

The purpose of this work was to examine the possible mechanisms for the regulation of cytochrome c gene expression in response to increased contractile activity in rat skeletal muscle. The working hypothesis was that increased contractile activity enhances cytochrome c gene expression through a cis-element. A 110% increase in cytochrome c mRNA concentration was observed in tibialis anterior (TA) muscle after 9 days of chronic stimulation. Similar difference (120%) exists between soleus (SO) muscle of higher contractile activity and white vastus lateralis (WV) muscle of lower contractile activity. These results suggest that the endogenous cytochrome c gene expression is regulated by contractile activity. Cytochrome c-reporter genes were injected into skeletal muscles to identify the cis-element that is responsible for the regulation. Although the data was inconclusive, part of it suggested the importance of the 3$\sp\prime$-untranslated region (3$\sp\prime$-UTR) in mediating the response to increased contractile activity.^ RNA gel mobility shift (GMSA) and ultraviolet (UV) cross-linking assays revealed specific RNA-protein interaction in a 50-nucleotide region of the 3$\sp\prime$-UTR in unstimulated TA muscle. Computer analysis predicted a stem-loop structure of 17 nucleotides, which provides a structural basis for RNA-protein interaction. These 17 nucleotides are 100% conserved among rat, mouse and human cytochrome c genes and their 13 pseudogenes, suggesting a functional role for this region. The RNA-protein interaction was significantly less in highly active SO muscle than in inactive WV muscle and was dramatically decreased in stimulated TA muscle due to a protein inhibitor(s) associated with ribosome. It is possible that cytochrome c mRNAs undergoing translation are subject to a compartmentalized regulatory influence.^ The conclusion from these results is that increases in contractile activity induce or activate a protein inhibitor(s) associated with ribosome in rat skeletal muscle. The inhibitor decreases RNA-protein interaction in the 3$\sp\prime$-UTR of cytochrome c mRNA, which may result in increased mRNA stability and/or translation. ^

Signaling pathways in the transcriptional and post-translational regulation of the human glutathione S -transferase P1 gene

The human glutathione S-transferase P1 (GSTP1) protein is an endogenous inhibitor of c-jun N-terminal kinases (JNKs) and an important phase II detoxification enzyme. ^ Recent identification of a cAMP response element (CRE) in the 5 ′-region of the human GSTP1 gene and several putative phosphorylation sites for the Ser/Thr protein kinases, including, cAMP-dependent protein kinases (PKAs), protein kinases C (PKCs), and JNKs in the GSTP1 protein raised the possibility that signaling pathways may play an important role in the transcriptional and post-translational regulation of GSTP1 gene. This study examined (a) whether the signaling pathway mediated by CAMP, via the GSTP1 CRE, is involved in the transcriptional regulation of the GSTP1 gene, (b) whether signaling pathways mediated by the Ser/Thr protein kinases (PKAs, PKCs, and JNKs) induce post-translational modification, viz. phosphorylation of the GSTP1 protein, and (c) whether such phosphorylation of the GSTP1 protein alters its functions in metabolism and in JNK signaling. ^ The first major finding in this study is the establishment of the human GSTP1 gene as a novel CAMP responsive gene in which transcription is activated via an interaction between PKA activated CRE binding protein-1 (CREB-1) and the CRE in the 5′-regulatory region. ^ The second major finding in this study is the observation that the GSTP1 protein undergoes phosphorylation and functionally activated by second messenger-activated protein kinases, PKA and PKC, in tumor cells with activated signaling pathways. Following phosphorylation by PKA or PKC, the catalytic activity of the GSTP1 protein was significantly enhanced, as indicated by a decrease in its Km (2- to 3.6-fold) and an increase in Kcat/ Km (1.6- to 2.5-fold) for glutathione. Given the frequent over-expression of GSTP1 and the aberrant PKA/PKC signaling cascade observed in tumors, these findings suggest that phosphorylation of GSTP1 may contribute to the malignant progression and drug-resistant phenotype of these tumors. ^ The third major finding in this study is that the GSTP1 protein, an inhibitor of JNKs, undergoes significant phosphorylation in tumor cells with activated JNK signaling pathway and in those under oxidative stress. Following phosphorylation by JNK, the ability of GSTP1 to inhibit JNK downstream function, i.e. c-jun phosphorylation, was significantly enhanced, suggesting a feedback mechanism of regulation of JNK-mediated cellular signaling. (Abstract shortened by UMI.) ^

Genetic integration of a nuclear -encoded mitochondrial gene with cardiac function

Cardiovascular disease (CVD) is the leading cause of death in the United States. One manifestation of CVD known to increase mortality is an enlarged, or hypertrophic heart. Hypertrophic cardiomyocytes adapt to increased contractile demand at the genetic level with a re-emergence of the fetal gene program and a downregulation of fatty acid oxidation genes with concomitant increased reliance on glucose-based metabolism. To understand the transcriptional regulatory pathways that implement hypertrophic directives we analyzed the upstream promoter region of the muscle specific isoform of the nuclear-encoded mitochondrial gene, carnitine palmitoyltransferase-1β (CPT-1β) in cultured rat neonatal cardiac myocytes. This enzyme catalyzes the rate-limiting step of fatty acid entry into β-oxidation and is downregulated in cardiac hypertrophy and failure, making it an attractive model for the study of hypertrophic gene regulation and metabolic adaptations. We demonstrate that the muscle-enriched transcription factors GATA-4 and SRF synergistically activate CPT-1β; moreover, DNA binding to cognate sites and intact protein structure are required. This mechanism coordinates upregulation of energy generating processes with activation of the energy consuming contractile promoter for cardiac α-actin. We hypothesized that fatty acid or glucose responsive transcription factors may also regulate CPT-1β. Oleate weakly stimulates CPT-1β activity; in contrast, the glucose responsive Upstream Stimulatory Factors (USF) dramatically depresses the CPT-1β reporter. USF regulates CPT-1β through a novel physical interaction with the cofactor PGC-1 and abrogation of MEF2A/PGC-1 synergistic stimulation. In this way, USF can inversely regulate metabolic gene programs and may play a role in the shift of metabolic substrate preference seen in hypertrophy. Failing hearts have elevated expression of the nuclear hormone receptor COUP-TF. We report that COUP-TF significantly suppresses reporter transcription independent of DNA binding and specific interactions with GATA-4, Nkx2.5 or USF. In summary, CPT-1β transcriptional regulation integrates mitochondrial gene expression with two essential cardiac functions: contraction and metabolic substrate oxidation. ^

Gene sequences of the lichen Cetraria aculeata

Lichens are symbioses between fungi (mycobionts) and photoautotrophic green algae or cyanobacteria (photobionts). Many lichens occupy large distributional ranges covering several climatic zones. So far, little is known about the large-scale phylogeography of lichen photobionts and their role in shaping the distributional ranges of lichens. We studied south polar, temperate and north polar populations of the widely distributed fruticose lichen Cetraria aculeata. Based on the DNA sequences from three loci for each symbiont, we compared the genetic structure of mycobionts and photobionts. Phylogenetic reconstructions and Bayesian clustering methods divided the mycobiont and photobiont data sets into three groups. An AMOVA shows that the genetic variance of the photobiont is best explained by differentiation between temperate and polar regions and that of the mycobiont by an interaction of climatic and geographical factors. By partialling out the relative contribution of climate, geography and codispersal, we found that the most relevant factors shaping the genetic structure of the photobiont are climate and a history of codispersal. Mycobionts in the temperate region are consistently associated with a specific photobiont lineage. We therefore conclude that a photobiont switch in the past enabled C. aculeata to colonize temperate as well as polar habitats. Rare photobiont switches may increase the geographical range and ecological niche of lichen mycobionts by associating them with locally adapted photobionts in climatically different regions and, together with isolation by distance, may lead to genetic isolation between populations and thus drive the evolution of lichens.

Molecular fingerprinting and amplicon sequencing of the bacterial biofilm on settlement tiles deployed along natural pH gradients in Papua New Guinea

Bacterial biofilms provide cues for the settlement of marine invertebrates such as coral larvae, and are therefore important for the resilience and recovery of coral reefs. This study aimed to better understand how ocean acidification may affect the community composition and diversity of bacterial biofilms on surfaces under naturally reduced pH conditions. Settlement tiles were deployed at coral reefs in Papua New Guinea along pH gradients created by two CO2 seeps, and upper and lower tiles surfaces were sampled 5 and 13 months after deployment. Automated Ribosomal Intergenic Spacer Analysis were used to characterize more than 200 separate bacterial communities, complemented by amplicon sequencing of the bacterial 16S rRNA gene of 16 samples. The bacterial biofilm consisted predominantly of Alpha-, Gamma- and Deltaproteobacteria, as well as Cyanobacteria, Flavobacteriia and Cytophaga, whereas putative settlement-inducing taxa only accounted for a small fraction of the community. Bacterial biofilm composition was heterogeneous with approximately 25% shared operational taxonomic units between samples. Among the observed environmental parameters, pH only had a weak effect on community composition (R² ~ 1%) and did not affect community richness and evenness. In contrast, there were strong differences between upper and lower surfaces (contrasting in light exposure and grazing intensity). There also appeared to be a strong interaction between bacterial biofilm composition and the macroscopic components of the tile community. Our results suggest that on mature settlement surfaces in situ, pH does not have a strong impact on the composition of bacterial biofilms. Other abiotic and biotic factors such as light exposure and interactions with other organisms may be more important in shaping bacterial biofilms than changes in seawater pH.

Ground maritime structure interaction. Recent failures in inner gravity constructions on the Spanish Coastline

A maritime construction is usually a slender line in the ocean.It is usual to see just its narrow surface strip and not analyse the large amount of submerged material the latter is supporting.Without doubt,it is the ground to which a notable load is transmitted in an environment subjected to periodic,alternating stresses,dynamic forces which the sea's media constitute. Both an outer and inner maritime construction works in a complex fashion.A granular solid(breakwater)breathes with the incident wave flow,dissipating part of the wave energy between its gaps.The backflow tries to extract the different items from the solid block,setting a balance between effective and neutral tensions that follow Terzaghui's principle. On some occasions,fluidification of the armour layer has caused the breakwater to collapse(Sines,Portugal,February 1978).On others,siphoning or liquefaction of sand supporting monoliths(vertical breakwaters)lead them to destruction or collapse(New Barcelona Harbour Mouth,Spain,November 2001). This is why the ground-force-structure interaction is a complicated analysis with joint design tools still in an incipient state. The purpose of this article is to describe two singular failures in inner maritime constructions in Spain deriving from ground problems(Malaga,July 2004and Barcelona,January 2007).They occurred recently and the causes are the subject of reflection and analysis.

Metadata Visualization of Cultural Heritage Information within a Collaborative Environment

Cultural content on the Web is available in various domains (cultural objects, datasets, geospatial data, moving images, scholarly texts and visual resources), concerns various topics, is written in different languages, targeted to both laymen and experts, and provided by different communities (libraries, archives museums and information industry) and individuals (Figure 1). The integration of information technologies and cultural heritage content on the Web is expected to have an impact on everyday life from the point of view of institutions, communities and individuals. In particular, collaborative environment scan recreate 3D navigable worlds that can offer new insights into our cultural heritage (Chan 2007). However, the main barrier is to find and relate cultural heritage information by end-users of cultural contents, as well as by organisations and communities managing and producing them. In this paper, we explore several visualisation techniques for supporting cultural interfaces, where the role of metadata is essential for supporting the search and communication among end-users (Figure 2). A conceptual framework was developed to integrate the data, purpose, technology, impact, and form components of a collaborative environment, Our preliminary results show that collaborative environments can help with cultural heritage information sharing and communication tasks because of the way in which they provide a visual context to end-users. They can be regarded as distributed virtual reality systems that offer graphically realised, potentially infinite, digital information landscapes. Moreover, collaborative environments also provide a new way of interaction between an end-user and a cultural heritage data set. Finally, the visualisation of metadata of a dataset plays an important role in helping end-users in their search for heritage contents on the Web.

Enhancing Interaction With Smart Objects Throught Mobile Devices

Interaction with smart objects can be accomplished with different technologies, such as tangible interfaces or touch computing, among others. Some of them require the object to be especially designed to be 'smart', and some other are limited in the variety and complexity of the possible actions. This paper describes a user-smart object interaction model and prototype based on the well known event-condition-action (ECA) reasoning, which can work, to a degree, independently of the intelligence embedded into the smart object. It has been designed for mobile devices to act as mediators between users and smart objects and provides an intuitive means for personalization of object's behavior. When the user is close to an object, this one publishes its 'event & action' capabilities to the user's device. The user may accept the object's module offering, which will enable him to configure and control that object, but also its actions with respect to other elements of the environment or the virtual world. The modular ECA interaction model facilitates the integration of different types of objects in a smart space, giving the user full control of their capabilities and facilitating creative mash-uping to build customized functionalities that combine physical and virtual actions

Detecting reliable gene interactions by a hierarchy of Bayesian network classifiers

The main purpose of a gene interaction network is to map the relationships of the genes that are out of sight when a genomic study is tackled. DNA microarrays allow the measure of gene expression of thousands of genes at the same time. These data constitute the numeric seed for the induction of the gene networks. In this paper, we propose a new approach to build gene networks by means of Bayesian classifiers, variable selection and bootstrap resampling. The interactions induced by the Bayesian classifiers are based both on the expression levels and on the phenotype information of the supervised variable. Feature selection and bootstrap resampling add reliability and robustness to the overall process removing the false positive findings. The consensus among all the induced models produces a hierarchy of dependences and, thus, of variables. Biologists can define the depth level of the model hierarchy so the set of interactions and genes involved can vary from a sparse to a dense set. Experimental results show how these networks perform well on classification tasks. The biological validation matches previous biological findings and opens new hypothesis for future studies

Solving identity delegation problem in the e-government environment

At present, many countries allow citizens or entities to interact with the government outside the telematic environment through a legal representative who is granted powers of representation. However, if the interaction takes place through the Internet, only primitive mechanisms of representation are available, and these are mainly based on non-dynamic offline processes that do not enable quick and easy identity delegation. This paper proposes a system of dynamic delegation of identity between two generic entities that can solve the problem of delegated access to the telematic services provided by public authorities. The solution herein is based on the generation of a delegation token created from a proxy certificate that allows the delegating entity to delegate identity to another on the basis of a subset of its attributes as delegator, while also establishing in the delegation token itself restrictions on the services accessible to the delegated entity and the validity period of delegation. Further, the paper presents the mechanisms needed to either revoke a delegation token or to check whether a delegation token has been revoked. Implications for theory and practice and suggestions for future research are discussed.

Oscillatory genetic circuits: new designs and mathematical models

Introduction and motivation: A wide variety of organisms have developed in-ternal biomolecular clocks in order to adapt to cyclic changes of the environment. Clock operation involves genetic networks. These genetic networks have to be mod¬eled in order to understand the underlying mechanism of oscillations and to design new synthetic cellular clocks. This doctoral thesis has resulted in two contributions to the fields of genetic clocks and systems and synthetic biology, generally. The first contribution is a new genetic circuit model that exhibits an oscillatory behav¬ior through catalytic RNA molecules. The second and major contribution is a new genetic circuit model demonstrating that a repressor molecule acting on the positive feedback of a self-activating gene produces reliable oscillations. First contribution: A new model of a synthetic genetic oscillator based on a typical two-gene motif with one positive and one negative feedback loop is pre¬sented. The originality is that the repressor is a catalytic RNA molecule rather than a protein or a non-catalytic RNA molecule. This catalytic RNA is a ribozyme that acts post-transcriptionally by binding to and cleaving target mRNA molecules. This genetic clock involves just two genes, a mRNA and an activator protein, apart from the ribozyme. Parameter values that produce a circadian period in both determin¬istic and stochastic simulations have been chosen as an example of clock operation. The effects of the stochastic fluctuations are quantified by a period histogram and autocorrelation function. The conclusion is that catalytic RNA molecules can act as repressor proteins and simplify the design of genetic oscillators. Second and major contribution: It is demonstrated that a self-activating gene in conjunction with a simple negative interaction can easily produce robust matically validated. This model is comprised of two clearly distinct parts. The first is a positive feedback created by a protein that binds to the promoter of its own gene and activates the transcription. The second is a negative interaction in which a repressor molecule prevents this protein from binding to its promoter. A stochastic study shows that the system is robust to noise. A deterministic study identifies that the oscillator dynamics are mainly driven by two types of biomolecules: the protein, and the complex formed by the repressor and this protein. The main conclusion of this study is that a simple and usual negative interaction, such as degradation, se¬questration or inhibition, acting on the positive transcriptional feedback of a single gene is a sufficient condition to produce reliable oscillations. One gene is enough and the positive transcriptional feedback signal does not need to activate a second repressor gene. At the genetic level, this means that an explicit negative feedback loop is not necessary. Unlike many genetic oscillators, this model needs neither cooperative binding reactions nor the formation of protein multimers. Applications and future research directions: Recently, RNA molecules have been found to play many new catalytic roles. The first oscillatory genetic model proposed in this thesis uses ribozymes as repressor molecules. This could provide new synthetic biology design principles and a better understanding of cel¬lular clocks regulated by RNA molecules. The second genetic model proposed here involves only a repression acting on a self-activating gene and produces robust oscil¬lations. Unlike current two-gene oscillators, this model surprisingly does not require a second repressor gene. This result could help to clarify the design principles of cellular clocks and constitute a new efficient tool for engineering synthetic genetic oscillators. Possible follow-on research directions are: validate models in vivo and in vitro, research the potential of second model as a genetic memory, investigate new genetic oscillators regulated by non-coding RNAs and design a biosensor of positive feedbacks in genetic networks based on the operation of the second model Resumen Introduccion y motivacion: Una amplia variedad de organismos han desarro-llado relojes biomoleculares internos con el fin de adaptarse a los cambios ciclicos del entorno. El funcionamiento de estos relojes involucra redes geneticas. El mo delado de estas redes geneticas es esencial tanto para entender los mecanismos que producen las oscilaciones como para diseiiar nuevos circuitos sinteticos en celulas. Esta tesis doctoral ha dado lugar a dos contribuciones dentro de los campos de los circuitos geneticos en particular, y biologia de sistemas y sintetica en general. La primera contribucion es un nuevo modelo de circuito genetico que muestra un comportamiento oscilatorio usando moleculas de ARN cataliticas. La segunda y principal contribucion es un nuevo modelo de circuito genetico que demuestra que una molecula represora actuando sobre el lazo de un gen auto-activado produce oscilaciones robustas. Primera contribucion: Es un nuevo modelo de oscilador genetico sintetico basado en una tipica red genetica compuesta por dos genes con dos lazos de retroa-limentacion, uno positivo y otro negativo. La novedad de este modelo es que el represor es una molecula de ARN catalftica, en lugar de una protefna o una molecula de ARN no-catalitica. Este ARN catalitico es una ribozima que actua despues de la transcription genetica uniendose y cortando moleculas de ARN mensajero (ARNm). Este reloj genetico involucra solo dos genes, un ARNm y una proteina activadora, aparte de la ribozima. Como ejemplo de funcionamiento, se han escogido valores de los parametros que producen oscilaciones con periodo circadiano (24 horas) tanto en simulaciones deterministas como estocasticas. El efecto de las fluctuaciones es-tocasticas ha sido cuantificado mediante un histograma del periodo y la función de auto-correlacion. La conclusion es que las moleculas de ARN con propiedades cataliticas pueden jugar el misnio papel que las protemas represoras, y por lo tanto, simplificar el diseno de los osciladores geneticos. Segunda y principal contribucion: Es un nuevo modelo de oscilador genetico que demuestra que un gen auto-activado junto con una simple interaction negativa puede producir oscilaciones robustas. Este modelo ha sido estudiado y validado matematicamente. El modelo esta compuesto de dos partes bien diferenciadas. La primera parte es un lazo de retroalimentacion positiva creado por una proteina que se une al promotor de su propio gen activando la transcription. La segunda parte es una interaction negativa en la que una molecula represora evita la union de la proteina con el promotor. Un estudio estocastico muestra que el sistema es robusto al ruido. Un estudio determinista muestra que la dinamica del sistema es debida principalmente a dos tipos de biomoleculas: la proteina, y el complejo formado por el represor y esta proteina. La conclusion principal de este estudio es que una simple y usual interaction negativa, tal como una degradation, un secuestro o una inhibition, actuando sobre el lazo de retroalimentacion positiva de un solo gen es una condition suficiente para producir oscilaciones robustas. Un gen es suficiente y el lazo de retroalimentacion positiva no necesita activar a un segundo gen represor, tal y como ocurre en los relojes actuales con dos genes. Esto significa que a nivel genetico un lazo de retroalimentacion negativa no es necesario de forma explicita. Ademas, este modelo no necesita reacciones cooperativas ni la formation de multimeros proteicos, al contrario que en muchos osciladores geneticos. Aplicaciones y futuras lineas de investigacion: En los liltimos anos, se han descubierto muchas moleculas de ARN con capacidad catalitica. El primer modelo de oscilador genetico propuesto en esta tesis usa ribozimas como moleculas repre¬soras. Esto podria proporcionar nuevos principios de diseno en biologia sintetica y una mejor comprension de los relojes celulares regulados por moleculas de ARN. El segundo modelo de oscilador genetico propuesto aqui involucra solo una represion actuando sobre un gen auto-activado y produce oscilaciones robustas. Sorprendente-mente, un segundo gen represor no es necesario al contrario que en los bien conocidos osciladores con dos genes. Este resultado podria ayudar a clarificar los principios de diseno de los relojes celulares naturales y constituir una nueva y eficiente he-rramienta para crear osciladores geneticos sinteticos. Algunas de las futuras lineas de investigation abiertas tras esta tesis son: (1) la validation in vivo e in vitro de ambos modelos, (2) el estudio del potential del segundo modelo como circuito base para la construction de una memoria genetica, (3) el estudio de nuevos osciladores geneticos regulados por ARN no codificante y, por ultimo, (4) el rediseno del se¬gundo modelo de oscilador genetico para su uso como biosensor capaz de detectar genes auto-activados en redes geneticas.