Properly representing the principle physical interactions of complex biological systems is paramount for building powerful, yet simple models. As an in depth look into different biological systems at different scales, multiple models are presented. At the molecular scale, an analytical solution to the (linearized) Poisson-Boltzmann equation for the electrostatic potential of any size biomolecule is derived using spherical geometry. The solution is tested both on an ideal sphere relative to an exact solution and on a multitude of biomolecules relative to a numerical solution. In all cases, the bulk of the error is within thermal noise. The computational power of the solution is demonstrated by finding the electrostatic potential at the surface of a viral capsid that is nearly half a million atoms in size. Next, a model of the nucleosome using simplified geometry is presented. This system is a complex of protein and DNA and acts as the first level of DNA compaction inside the nucleus of eukaryotes. The analytical model reveals a mechanism for controlling the stability of the nucleosome via changes to the total charge of the protein globular core. The analytical model is verified by a computational study on the stability change when the charge of individual residues is altered. Finally, a multiple model approach is taken to study bacteria that are capable of different responses depending on the size of their surrounding colony. The first model is capable of determining how the system propagates the information about the colony size to those specific genes that control the concentration of a master regulatory protein. A second model is used to analyze the direct RNA interference mechanism the cell employs to tune the available gene transcripts of the master regulatory protein, i.e. small RNA - messenger RNA regulation. This model provides a possible explanation for puzzling experimentally measured phenotypic responses. / Ph. D.

Chez la majorité des eucaryotes, les petits ARN (miRNA et siRNA) jouent des rôles essentiels au cours du développement, dans les réponses adaptatives aux stress, et dans la maintenance de la stabilité génétique. Les plantes codent quatre enzymes RNaseIII de type DICER-LIKE (DCL). DCL1, produit les miRNAs, tandis que DCL2, DCL3 et DCL4 produisent des siRNAs des tailles diverses. Les plantes codent également des enzymes appelées RNASE-THREE-LIKE (RTL) auxquelles il manque certains domaines spécifiques aux DCLs, et dont la fonction est largement inconnue.Des plantes sur-exprimant RTL1 montrent des défauts morphologiques, et n'accumulent pas les siRNAs produits par DCL2, DCL3 ou DCL4, indiquant que RTL1 est un suppresseur général des voies de siRNA chez les plantes. L’activité de RTL1 nécessite un domaine RNaseIII fonctionnel. RTL1 ne s'exprime naturellement que faiblement dans les racines, mais l'infection virale induite fortement son expression dans les feuilles, ce qui suggère que l’induction de RTL1 est une stratégie générale utilisée par les virus pour contrer la défense antivirale basée sur siRNAs. En accord avec cette hypothèse, les plantes transgéniques sur-exprimant RTL1 sont plus sensibles à l'infection par le TYMV que des plantes de type sauvage, probablement parce que RTL1 empêche la production des siRNAs dirigés contre les RNA viraux. Cependant, les plantes transgéniques sur-exprimant RTL1 ne sont pas plus sensibles à l'infection par le TCV, TVCV ou le CMV, qui codent les suppresseurs de RNA silencing (VSR) plus puissants que le TYMV. En effet, le VSR de TCV inhibe l'activité de RTL1, suggérant que l'induction de l’expression de RTL1 par les virus et l’amortissement de l’activité de RTL1 par leurs VSRs est une double stratégie permettant d’établir une infection avec succès. Des plantes sur-exprimant RTL2 ou des mutants rtl2 ne montrent aucun défaut morphologique, et ne montrent pas de changement majeur du répertoire des petits ARNs endogènes. Toutefois, la sur-expression de RTL2 augmente l’accumulation des petits ARNs exogènes dans des essais d’expression transitoire, et cette activité nécessite un domaine RNaseIII fonctionnel. Il est donc possible que RTL2 clive certains substrats pour faciliter l’action des enzymes DCL. / Small RNAs, including miRNA and siRNA, play essential regulatory roles in genome stability, development and stress responses in most eukaryotes. Plants encode four DICER-LIKE (DCL) RNaseIII enzymes. DCL1 produces miRNAs, while DCL2, DCL3 and DCL4 produce diverse size classes of siRNA. Plants also encode RNASE THREE-LIKE (RTL) enzymes that lack DCL-specific domains and whose function is largely unknown. Arabidopsis plants over-expressing RTL1 exhibit morphological defects and lack all types of small RNAs produced by DCL2, DCL3 and DCL4, indicating that RTL1 is a general suppressor of plant siRNA pathways. RTL1 activity requires a functional RNaseIII domain. RTL1 is naturally expressed only weakly in roots, but virus infection strongly induces its expression in leaves, suggesting that RTL1 induction is a general strategy used by viruses to counteract the siRNA-based plant antiviral defense. Accordingly, transgenic plants over-expressing RTL1 are more sensitive to TYMV infection than wild-type plants, likely because RTL1 prevents the production of antiviral siRNAs. However, TCV, TVCV and CMV, which encode stronger suppressors of RNA silencing (VSR) than TYMV, are insensitive to RTL1 over-expression. Indeed, TCV VSR inhibits RTL1 activity, suggesting that inducing RTL1 expression and dampening RTL1 activity is a dual strategy used by viruses to establish a successful infection. Plants over-expressing RTL2 and rtl2 mutants do not exhibit morphological defects and do not show major changes in the endogenous small RNA repertoire. However, RTL2 over-expression enhances the accumulation of exogenous siRNAs in transient assays, and this activity requires a functional RNaseIII domain. Therefore, it is possible that plant RTL2 processes certain substrates to facilitate the action of DCL enzymes.

Staphylococcus aureus un pathogène opportuniste de l’homme responsable de nombreuses maladies. Son pouvoir pathogène est dû à l’expression de nombreux facteurs de virulence, aussi qu’à sa capacité de s’adapter à son environnement. En pénétrant dans nos tissus S. aureus doit, pour survivre, faire face aux changements environnementaux et à la disponibilité des nutriments. L’expression des gènes impliqués dans ces réponses adaptatives, est soumise à une régulation fine, apportée par les systèmes à deux composants, les facteurs de transcription et les sARN (small ARN). Dans cette étude, j’ai identifié les fonctions d’un sARN, appelé RsaI, qui est réprimé en présence de glucose extérieur. RsaI, réprime la traduction, de plusieurs ARNm impliqués dans le métabolisme carboné et également de IcaR, impliqué dans la synthèse de biofilms. RsaI participe à l’inhibition de plusieurs enzymes de la voie de synthèse des pentoses phosphates et interagit également avec d’autre sARN. Cet ARN multifonctionnel est un véritable senseur du taux de glucose extérieur engendrant ainsi un switch métabolique, nécessaire à la réponse adaptative de S. aureus en conditions infectieuses. / Staphylococcus aureus is a human opportunist pathogenic bacterium capable to colonize different host tissues and organs and therefore generates multiple infectious conditions. Its pathogenic power is due to the expression of multiple virulence factors, and by it’s ability to adapt to the environment. Once entered in human tissues, S. aureus must face environmental changes, as the availability of nutriments to survive. Gene expressions implicated in these adaptive responses are submitted to a fine regulation, carried by two component systems, transcriptional factors, and sRNA (small RNA). In this study, I have identified the functions of a sRNA, called RsaI, which is repressed when the external concentration of glucose is at high levels. RsaI represses the translation of multiple mRNA implicated in the carbon metabolism, including a major glucose transporter, and IcaR, implicated in the biofilms synthesis. Furthermore, RsaI interacts with other sRNA. This multifunctional RNA is a real sensor of the external glucose levels, generating a metabolic switch that is necessary to ensure S. aureus adaptive response in infectious conditions.

Biofilms are a crucial adaptation for bacterial survival against stresses from external environments. Biofilms are adherent colonies of sessile bacteria embedded within a self-produced matrix. Bacterial control over formation, maintenance, and response to external stresses are strictly regulated. However, complexities of intracellular signaling for biofilm regulation are still not fully understood. In this thesis, I report on two distinct regulatory systems important for biofilm formation in the opportunistic pathogen Pseudomonas aeruginosa. The first regulatory system I report on is the two-component system TctD-TctE. This system is involved in regulating the uptake of tricarboxylic acids such as citric acid and is involved in biofilm-specific susceptibility to aminoglycoside antibiotics. Here I describe work I performed characterizing the involvement of TctD-TctE in biofilm development when citric acid is present as a carbon source in nutrient media. In further characterizing a previously observed aminoglycoside susceptibility, I found that a strain with a deletion of TctD-TctE (ΔtctED) has a heightened accumulation of tobramycin in its biofilms when grown in the presence of citric acid. In ΔtctED, I determined that there was an inhibition of overall cell growth when citric acid was present in nutrient media. Additionally, in the presence of citric acid, ΔtctED displayed high levels of biofilm formation. This contrasted with normal biofilm development observed in the PA14 wild type strain where biofilm mass was reduced in the presence citric acid. The second project of this thesis reports on a novel regulatory small RNA, the Small RNA Regulator of Biofilms (SrbA). SrbA was found to be unique to P. aeruginosa and displayed no homology with any other sequenced bacterial species. I found that loss of SrbA resulted in a significant reduction in biofilm mass. Subsequently, loss of SrbA also leads to attenuation of P. aeruginosa pathogenicity in Caenorhabditis elegans nematodes. Bacterial biofilms possess specific regulatory programs that are still just being appreciated for their complexity. This thesis work adds to our understanding of biofilm regulation by studying roles of the two-component system TctD-TctE and the small RNA SrbA in P. aeruginosa.

<p>The Escherichia coli small RNA (sRNA) MicA was identified recently in a genomewide search for sRNAs. It is encoded between the genes <i>gshA</i> and <i>luxS</i> in E. coli and its close relatives. The function of sRNAs in bacteria is generally believed to be in maintenance of homeostasis via stress-induced modulation of gene expression. Our studies on MicA have been aimed at attributing function(s) to this molecule.</p><p>We carried out high throughput assays aimed at identifying genes that are differentially regulated upon knocking out or overexpressing MicA. Among the protein candidates identified was the outer membrane protein, OmpA. Subsequent analysis allowed us to show this regulation to be antisense in nature with MicA binding within the translation initiation region of <i>ompA</i> mRNA. Furthermore, blocking the ribosome from loading caused a translational decoupling that instigates degradation of the mRNA. The regulation was apparent in early stationary phase and seen to be dependent on the RNA chaperone Hfq. </p><p>We went on to characterize the regulation of MicA, looking at its own transcription. Testing various stress conditions, we were able to identify putative promoter elements that we confirmed using transcriptional fusions. The results showed MicA to be dependent on the extracytoplasmic function ECF sigma E (σ^E) and could not detect MicA in mutants deleted for this factor.</p><p>Lastly, we identified an additional target for MicA being the adjacently encoded <i>luxS</i> mRNA. The LuxS protein is essential for the synthesis of the quorum sensing AI-2 molecule. Transcription of the <i>luxS </i>mRNA is commences within the <i>gshA</i> gene, on the other side of MicA coding region. We were able to show that MicA interacts with <i>luxS </i>mRNA and is recognized by RNase III which processes this complex leading to a shorter <i>luxS</i> mRNA isoform. The significance of this processing event is as yet undetermined. Our data elucidated a new promoter driving transcription of <i>luxS,</i> and we demonstrated this promoter to be stationary phase responsive.</p><p>In summary, the work presented here characterizes the sRNA MicA as a dual regulatory sRNA molecule, moonlighting between its cis-encoded target and its trans-encoded target. .</p>

The Escherichia coli small RNA (sRNA) MicA was identified recently in a genomewide search for sRNAs. It is encoded between the genes gshA and luxS in E. coli and its close relatives. The function of sRNAs in bacteria is generally believed to be in maintenance of homeostasis via stress-induced modulation of gene expression. Our studies on MicA have been aimed at attributing function(s) to this molecule. We carried out high throughput assays aimed at identifying genes that are differentially regulated upon knocking out or overexpressing MicA. Among the protein candidates identified was the outer membrane protein, OmpA. Subsequent analysis allowed us to show this regulation to be antisense in nature with MicA binding within the translation initiation region of ompA mRNA. Furthermore, blocking the ribosome from loading caused a translational decoupling that instigates degradation of the mRNA. The regulation was apparent in early stationary phase and seen to be dependent on the RNA chaperone Hfq. We went on to characterize the regulation of MicA, looking at its own transcription. Testing various stress conditions, we were able to identify putative promoter elements that we confirmed using transcriptional fusions. The results showed MicA to be dependent on the extracytoplasmic function ECF sigma E (σE) and could not detect MicA in mutants deleted for this factor. Lastly, we identified an additional target for MicA being the adjacently encoded luxS mRNA. The LuxS protein is essential for the synthesis of the quorum sensing AI-2 molecule. Transcription of the luxS mRNA is commences within the gshA gene, on the other side of MicA coding region. We were able to show that MicA interacts with luxS mRNA and is recognized by RNase III which processes this complex leading to a shorter luxS mRNA isoform. The significance of this processing event is as yet undetermined. Our data elucidated a new promoter driving transcription of luxS, and we demonstrated this promoter to be stationary phase responsive. In summary, the work presented here characterizes the sRNA MicA as a dual regulatory sRNA molecule, moonlighting between its cis-encoded target and its trans-encoded target. .

The CsgD protein is the master regulator of a pathway leading to the formation of curli, in essence regulating the switch between a motile and a sessile lifestyle for bacteria. The 5’-UTR region of the csgD mRNA is a hotspot for multiple regulatory small RNAs (sRNA) involved in a complex regulatory network. Even though it is previously known how the interaction takes place it is unknown how sRNA binding affects the translational activity. In order to suggest a mode of regulation a mutational assay was performed by making changes in the csgD 5’-UTR and investigate what the translational effects were. Mutations in different regions are shown to affect the translation levels in various ways.

<p>PIWI proteins belong to an evolutionary conserved protein family as the sister sub-family of ARGONAUTE (AGO) proteins. While AGO proteins are functionally well-characterized and shown to mediate small-RNA guided gene regulation, the function of PIWI proteins remain elusive. Here we pursued functional characterization of PIWI proteins by studying MILI and MIWI, two PIWI proteins in the mouse.</p><p>We first show that both MIWI and MILI co-immunoprecipitate with a novel class of non-coding small RNAs from the post-natal mouse testis extract, which are named Piwi-interacting RNAs (piRNAs). Our cloning efforts identified thousands of different piRNA sequences, mostly derived from intergenic regions. Interestingly, both MILI and MIWI piRNAs correspond to the same regions on the genome and differ primarily in length. We propose piRNAs in the adult testis are produced by the processing of long, single stranded RNA precursors, based on the observation that piRNAs originate in clusters from a number of sites on the genome in a head-to-tail homology. In support, we bioinformatically predicted putative promoters, and yeast one hybrid analysis on two such regions found out that they interact with Krueppel C2H2 type zinc finger transcription factors. We did not observe the features of the "ping-pong" mechanism in their biogenesis: Both MILI and MIWI piRNAs are biased for 5` Uracil without an Adenine bias on the 10th nucleotide position, and do not significantly consist of sequences complementary to each other along their first 10nt. Moreover, MILI piRNAs are not down-regulated in Miwi-/- testis. These results indicate that the post-natal testicular piRNAs are produced independent of the ping-pong mechanism. </p><p>Although piRNAs are highly complex, PAGE and in situ analyses showed that piRNAs are germ cell-specific with predominant expression in spermatocytes and round spermatids, suggestive of a meiotic function. Correspondingly, we found that Miwi-/-; Mili-/- mice undergo only male infertility with terminal spermatogenic arrest during meiosis. piRNAs show a nucleo-cytoplasmic distribution, with enrichment in the chromatoid and dense bodies, two male germ cell-specific structures. The dense body has been implicated in synapsis and in the heterochromatinization of the sex chromosomes during male meiosis, a process known as meiotic sex chromosome inactivation (MSCI). Our histological analysis on Miwi-/-; Mili-/- testes showed that, while the overall synapsis is not affected, the sex chromosomes retain the euchromatin marker acetyl-H4K16 and lacks the heterochromatin marker H3K9-dimethyl. These observations indicate that murine PIWI proteins are necessary for MSCI. Moreover, we identified piRNA production from the X chromosome before MSCI, and propose PIWI proteins utilize piRNAs to target and silence unpaired chromosomal regions during meiosis.</p> / Dissertation

<p>Small noncoding RNAs (ncRNAs) are key regulators of plant development through modulation of the processing, stability and translation of larger RNAs. I generated small RNA datasets comprising over 200 million aligned Illumina sequence reads covering all major cell types of the root as well as four distinct developmental zones. These data were analyzed for three major types of small RNAs, namely microRNAs (miRNAs), repeat associated small interfering RNAs (ra-siRNAs), and trans-acting siRNAs (ta-siRNAs). 133 of the 243 known miRNAs were found to be expressed in the root, and most showed tissue- or zone-specific expression patterns. My collaborators and I identified 70 new high-confidence miRNAs, and knockdown of three of the newly identified miRNAs resulted in altered root growth phenotypes. Ra-siRNAs specify methylation by the RNA directed DNA methylation (RdRM) pathway, requiring the generation of additional methylation datasets. Preliminary analysis shows cell-type specific methylation patterns that correlate with small RNA and mRNA expression. Analysis of ta-siRNAs revealed new ta-siRNA generating loci, and a novel triggering miRNA for TAS1 loci. In summary, our study demonstrates the power of isolating individual cell types and developmental zones in combination with deep sequencing and computational analyses to obtain detailed profiles of ncRNAs, as well as to significantly extend the compendium of known functional RNAs.</p> / Dissertation

Heidelberg, Univ., Diss., 2008.