Identification of host cellular requirements for positive strand RNA viruses: Hepatitis C virus and hepatitis E virus

Investigation of the host cellular requirements of two positive strand RNA viruses, by microarray, phospho-proteome array and by siRNA screening identified differential expression of host genes during hepatitis C virus viral entry. Also my studies suggest that NS4B regulates a newly identified oncogenic pathway. For hepatitis E virus, I looked for the cellular effects conferred by the capsid protein ORF2, and the results showed that ORF2 induced activation of CHOP did not lead to apoptosis which might be due to the up regulation of anti-apoptotic chaperones like Hsp72

The role of the Fur protein for maintenance of iron homeostasis in the strictly anaerobic bacterium Clostridium acetobutylicum

Iron is an indispensable micronutrient for virtually all bacterial species. On the other hand, excess of iron could lead to formation of ROS, which could be toxic for the cell. Therefore, microorganisms have established a tight control on the intracellular iron content. The milestone of the bacterial iron-dependent response is the ferric uptake regulator (Fur) protein. In silico, biochemical and in vivo studies established the role of CAC1682 as a genuine iron-responsive regulator in the solvent-producing bacterium Clostridium acetobutylicum

Novel Targets and Functions for Xenopus pitx3 during Embryonic Development

Pitx3 is a homeodomain transcription factor with an expression pattern conserved across phyla: in the dopaminergic neurons of the midbrain, in the lens during all stages of lens development and in the forming somites. In Xenopus laevis, this gene shows novel expression areas, such as the pituitary gland, the heart and the gut. Morpholino-based knockdown of pitx3 results in phenotypes characterized by small or absent lens, bent dorsal axis and randomized coiling of the heart and gut. Comparing gene expression changes in wild-type versus knockdown embryos by microarray, we generated a vast list of genes possible downstream targets for pitx3. Confirming a number of those genes as affected by the absence of pitx3 allowed for positioning pitx3 in a variety of pathways. Given that a significant number of these genes were known as major players during somitogenesis, corroborated with the bent dorsal axis phenotype initiated the further discovery for the role of pitx3 in this developmental process. To determine direct targets for pitx3 we needed a reporter assay to test the protein-promoter interaction. Since the existing assays were deemed unsatisfactory in terms of accuracy and sensitivity we developed a new technique which permits precise detection of the reporter gene in a homogenous population of cells containing both the transcription factor and the reporter. This also enables the assessment of cooperativity for the tested transcription factors. Lastly, this new technique was employed to examine the promoters of some of the microarray candidate genes and to determine new direct targets for pitx3, thus redesigning existing pathways to incorporate the new interactions

Regulating Campylobacter jejuni flagellar gene expression: transcriptional and post-transcriptional mechanisms of control

The bacterial pathogen Campylobacter jejuni is the leading cause of foodborne gastroenteritis in the developed world. C. jejuni flagella are crucial virulence determinants, but the regulation of these complex organelles within different environments is not fully understood. Moreover, regulatory RNAs are important for virulence and flagellar gene expression in many prokaryotes, but their role in C. jejuni biology is unknown. The first aim was to understand flagellar regulation in acidic conditions and what effect this has on virulence. The most acidic pH C. jejuni was able to survive was pH 3.6 and acid-shock at this pH and pH 5 increased expression of a subset of flagellar genes and increased invasion of intestinal epithelial cells. The second aim of this study was to characterise the function of two paralogous small non-coding RNAs (less than 50 nucleotides), NC1 and NC4, which were identified in the C. jejuni NCTC11168 transcriptome and are predicted to regulate flagella gene expression. NC1 and NC4 expression was dependent on the flagellar sigma factor, sigma28, and post-transcriptionally regulated expression of predicted sigma54-dependent C. jejuni flagellar gene targets in an E. coli based GFP reporter system. However, microarray and phenotypic analysis showed no clear differences in gene expression between NC1/NC4 deletion and over-expression mutants compared to the wild-type strain. The conclusions are that flagellar gene expression is regulated by acidic conditions and C. jejuni invasion of intestinal epithelial cells may be primed in response to acid. In addition, the transcription of NC1 and NC4 is linked to flagella expression and they may function to post-transcriptionally regulate sigma54-dependent flagella genes in C. jejuni. Although the biological significance of NC1 and NC4 remains unknown, this is the first study to show that non-coding RNAs are potential regulators of gene expression in Campylobacter

Genetic elements and molecular mechanisms driving the evolution of the pathogenic marine bacterium Vibrio parahaemolyticus

Vibrio parahaemolyticus is an opportunistic human pathogen that occurs naturally in a non-pathogenic form in coastal estuarine and marine environments worldwide. Following the acquisition of virulence-associated genes, V. parahaemolyticus has emerged as a significant pathogen causing seafood-borne illnesses. The mechanisms and conditions that promote the emergence of disease causing V. parahaemolyticus strains are not well understood. In addition, V. parahaemolyticus clinical strains isolated from disease-associated samples and environmental strains from sediment, water, and marine organisms have been identified with considerable diversity; however, the evolutionary relationships of disease-causing strains and environmental strains are not known. In the following research, the evolutionary relationships of V. parahaemolyticus clinical and environmental strains are examined. In addition, the contribution of genetic elements and molecular mechanisms such as deficiency of DNA repair to the evolution of V. parahaemolyticus clinical and environmental strains is shown. Molecular analysis of the evolutionary relationships of V. parahaemolyticus clinical and environmental strains demonstrated separate lineages of pathogenic and non-pathogenic strains with the exception of several environmental strains that may represent a reservoir of disease-causing strains in the environment. Sequence characterization of plasmids isolated from diverse environmental Vibrios indicated a role of plasmids in strain evolution by horizontal transfer of housekeeping genes. In addition, analysis of plasmids from V. parahaemolyticus clinical and environmental strains indicated the existence of a plasmid family distributed among V. parahaemolyticus, V. campbellii, and V. harveyi environmental strains. Sequence characterization of a plasmid of this family from a V. parahaemolyticus environmental strain indicated the contribution of these plasmids to the emergence of the clonal pandemic strains. Investigation of the role of molecular mechanisms to the evolution of V. parahaemolyticus strains showed that inactivation of the DNA repair pathway methyl-directed mismatch repair (MMR) increased the accumulation of spontaneous mutations leading to increased nucleotide diversity in select genes. The research findings in the following chapters demonstrate a considerable contribution of genetic elements and molecular mechanisms to the evolution of genetic and phenotypic diversity.Ph.D.Committee Chair: Patricia Sobecky; Committee Member: Eric Stabb; Committee Member: Jim Spain; Committee Member: Roger Wartell; Committee Member: Thomas DiChristin

Characterization of the Role of the Rpf Motif in Mycobacteriophage Tape Measure Proteins

In order to inject their DNA into the bacterial cytoplasm and establish infection, bacteriophages must ensure their genetic material successfully traverses both the bacterial membrane(s) and the layer of peptidoglycan surrounding the host cell. Phages accomplish this in a variety of ways, and some have virion-associated murein hydrolase enzymes that facilitate this process, particularly in conditions where the peptidoglycan is highly cross-linked. Phages that infect the mycobacteria must also contend with these barriers to infection, as well an impermeable layer of mycolic acids that decorates the cell surface; however, the mechanisms by which they do this are mostly unknown. In this regard, three small sequence motifs have been identified within mycobacteriophage tape measure proteins (TMPs) − extended molecules that span the tail lumen and determine its length − at least two of which have similarity to host proteins with muralytic activity. This suggests that phages may utilize regions of the TMP, which because of its location within the tail might be uniquely primed for host interaction, to facilitate localized peptidoglycan hydrolysis and DNA injection. The focus of this study is the motif found in the TMPs of mycobacteriophages Barnyard and Giles that has identity to a group of bacterial proteins known as resuscitation promoting factors (Rpfs). These Rpf proteins stimulate growth of non-growing bacteria and seem to exert their activity by cleaving inert peptidoglycan in the cell wall. Notably, the Barnyard Rpf Motif is contained within a 70 kDa C-terminal cleavage product of TMP, which appears to be cell wall- and/or membrane-associated during infection. Further, mycobacteria expressing TMP fragments containing this motif show aberrant behavior in culture and on solid media, and hybrid proteins in which the Rpf domain of the Micrococcus luteus Rpf protein is replaced with either of the phage motifs have muralytic activity in vivo. A recombineering-based method for generating mutations on lytically replicating mycobacteriophages has been developed and utilized to make multiple mutations in the Giles TMP motifs. Mutant phages infect host cells in late-stationary phase with a reduced efficiency, an observation that further supports a role for these motifs in cell wall hydrolysis during infection

Gene expression profiling of metastatic gastric cancer

Gastric cancer is one of the main cancers in the world, and is responsible for a large number of deaths each year. In Europe, the main issue is that it is only detected at latter stages, when metastases have developed. Although metastasis is the main cause of death from such tumours, the process is a very complex one and still not fully understood. In order to unravel this mechanism, microarrays have been used to study the expression pattern of a large number of genes in primary tumours and metastatic samples. These studies aim at indentifying genes that may playa role in metastasis. A number of microarray studies have already been carried out on gastric cancer to pursue knowledge. However, they have been mainly carried out in Asian populations, which are thought to present different gastric tumours than Europeans, possibly due to genetic and environmental parameters. The present thesis therefore aimed to carry out the first microarray study on gastric tumours from a European population, to identify genes that might playa role in gastric cancer metastasis, and assess whether there were any differences between Asian and European samples. In order to achieve this aim, the printing of in-house microarrays and methods to amplify and hybridise the samples first needed to be developed. This included the development of a new amplification method, the SMARTff7 protocol. Results showed that this method allowed more genes to be amplified than with an .established protocol. In addition, a microarray study published in 2003 identified a metastasis specific gene expression signature that could differentiate between metastatic and nonmetastatic primary tumours from different sites. However, this study did not include samples from gastric cancer. It was thus decided to test whether this signature could be applied to primary gastric tumours, using the new MetriGenix® platform. Although the analysis seemed to indicate that the signature did not apply to gastric tumours, technical issues meant that the results were inconclusive. On the other hand, a larger microarray analysis using the techniques developed during this project allowed the indentification of a number of genes of interest which may playa role in the metastatic spread of gastric cancer

Identificación de dianas efectoras de ANp73 asociadas a angiogénesis y linfangiogénesis. Valor diagnóstico y pronóstico en vesículas extracelulares en pacientes con cáncer colorrectal

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de Lectura: 14-10-2022El cáncer colorrectal es el tumor con mayor incidencia en la población general de nuestro país y el segundo en mortalidad. La detección de este tipo de cáncer en etapas tempranas podría mejorar la supervivencia y pronóstico del paciente, sin embargo, las técnicas de cribado poblacional actuales presentan limitaciones en este propósito. En el origen del cáncer colorrectal tiene lugar la desactivación de diversos genes clave para las células y/o la activación de oncogenes por distintos eventos genéticos y epigenéticos. En este contexto tumoral, se ha demostrado que la isoforma oncogénica ΔNp73 está implicada en el proceso de carcinogénesis y progresión tumoral, además de asociarse a peor pronóstico en los pacientes con cáncer colorrectal. En esta tesis doctoral nos hemos centrado en estudiar e identificar las posibles dianas efectoras de la isoforma ΔNp73 y en qué procesos o actividades protumorales están implicadas. Para ello hemos llevado a cabo ensayos proteómicos basados en microarrays de anticuerpos y marcaje metabólico con isótopos estables junto con ensayos funcionales in vitro de ganancia y pérdida de función. Hemos identificado en el secretoma de células tumorales de cáncer colorrectal diversas proteínas desreguladas implicadas en adhesión celular, migración e invasión, metabolismo, unión y reparación del ADN, angiogénesis y linfangiogénesis. Aquí, validamos la implicación de BDNF y EMAP-II como mediadores del papel de ΔNp73 en angiogénesis y linfangiogénesis. Además, comprobamos que la desregulación de estos dos mediadores es independiente del estatus de p53. Asimismo, hemos mostrado que BDNF es un excelente candidato a biomarcador de diagnóstico precoz dada su presencia diferencial en el plasma sanguíneo de individuos con lesiones premalignas y pacientes con cáncer colorrectal. Por otro lado, hemos explorado el papel de las isoformas ΔNp73, TAp73 y Δ133p53 como biomarcadores de diagnóstico y pronóstico en biopsia líquida. Con este fin, se ha analizado el contenido de ARN mensajero de dichas isoformas en vesículas extracelulares de individuos sanos, con lesiones premalignas y pacientes con cáncer colorrectal. Hemos observado que las isoformas TAp73 no se empaquetan en vesículas extracelulares. Además, hemos encontrado que el contenido de ΔNp73 y Δ133p53 en vesículas extracelulares del plasma sanguíneo presenta potencial como biomarcador de diagnóstico precoz y pronóstico, respectivament

Analysis of the murine granulosa cell transcriptome during luteinisation

The granulosa cells of the ovarian follicle surround the oocyte and support it during follicle development. Once exposed to the LH surge, the granulosa cells are characterised by the induction of genes necessary for cellular differentiation. The extensive morphological and functional changes which characterise luteinisation involve the regulation of gene and protein expression responsible for the cessation of proliferation and the induction of differentiation in the individual granulosa cells. The differentiating granulosa cell also functions in both an endocrine and paracrine manner mediating follicle and oocyte maturation and subsequent corpus luteum remodelling. The formation of the functional corpus luteum and secretion of progesterone is essential for the establishment of pregnancy following ovulation. Although much is known about the molecular mechanisms responsible for follicular development comparatively little study has been carried out to analyse the control of, and events which occur during, luteinisation. It is therefore pertinent to study gene expression changes to try to clarify and understand mechanisms which regulate and underpin ovarian granulosa cell luteinisation. In order to investigate the mechanisms underlying these processes we embarked on a time- and cell-specific analysis of gene expression in the granulosa cell during late follicle development and early luteinisation. Changes in gene expression during granulosa cell luteinisation were measured using serial analysis of gene expression (SAGE). Immature normal mice were treated with gonadotrophin to induce formation and luteinisation of ovarian follicles. SAGE libraries were generated from mRNA isolated from granulosa cells collected before and after induction of luteinisation. The combined libraries contained 105,224 tags representing 40,248 unique transcripts. Overall, 715 transcripts showed a significant difference in abundance between the two libraries of which 216 were significantly down-regulated by luteinisation and 499 were significantly up-regulated. Among transcripts differentially regulated, there were clear and expected changes in genes involved in modelling of the extracellular matrix, regulation of the cytoskeleton and intra and intercellular signalling. Also identified were transcripts relating to genes and cellular signalling pathways novel to the granulosa cell, including members of the E2F family of cell cycle regulators and the Notch signalling pathway as well as genes implicated in angiogenesis and cellular metabolism not previously associated with the granulosa cell. Further studies into an unmatched SAGE tag which was highly differentially expressed revealed that it represented a variable length non-coding transcript which showed a tissue- and temporal-specific expression pattern within granulosa tissue. This transcript is highly conserved across species and lies distal to the 3' end of the inhibin BA subunit. Highest levels of expression were found within the gonadotrophin-stimulated, mature antral follicle prior to the LH surge where it was the 6th most highly expressed transcript. After luteinisation there was a rapid downregulation of expression. It is suggested that this transcript may have involvement in regulating the transcription and/or translation of the inhibin BA subunit during follicle development and luteinisation. In conclusion, this thesis provides insight into some of the important mechanisms involved in the regulation of luteinisation, namely angiogenesis, differentiation, cell cycle control and the metabolic machinery within the granulosa cell. We have isolated a large number of candidate genes related to the cellular differentiation processes occurring within the granulosa cell during luteinisation. The data generated and presented here constitutes a new base for the testing of hypotheses in the field of follicle development and luteinisation