Agriculture, urbanization, and human activities, if not managed carefully, can expose a water body to environmental degradation, decreased water quality, and ultimately impaired benthic macroinvertebrate assemblage conditions. In streams where the benthic macroinvertebrates are impaired, the stream itself will not be meeting the water quality standards set forth in the Clean Water Act. As a result, the goal of this study was to establish relationships between benthic macroinvertebrates and their stressors so that stressor levels that would not adversely impact the benthic macroinvertebrates could be determined. Stressors such as sediment, habitat, water quality, landuse, watershed characteristics, and livestock numbers impact the benthic macroinvertebrate assemblage conditions. Since sediment is recognized as the Nation's leading pollutant and since the benthic macroinvertebrates live in the sediment on a stream bottom, this study placed emphasis upon the investigation of sediment as a primary stressor to the benthic macroinvertebrates. The specific objectives of this study were to develop relationships between the benthic macroinvertebrates and sediment and other stressors for Virginia streams, to evaluate the accuracy of the stressor/benthos relationships, and to discuss the implications of the study results for development of benthic TMDLs. A procedure to determine the relationships between stressors and benthic macroinvertebrate assemblage conditions was developed. Existing data on sediment, habitat, water quality, landuse, watershed characteristics, livestock numbers, and benthic macroinvertebrate assemblage conditions were compiled for 34 stations with 105 samples collected from the fall of 1996 to the fall of 1998. The 34 stations were located within 13 counties in Virginia (Rockbridge, Rockingham, Augusta, Frederick, Shenandoah, Page, Loudoun, Fairfax, Prince William, Fauquier, Culpeper, Rappahannock, and Madison) and in watersheds dominated by agricultural, urban, and forested landuses. Virginia currently uses the Rapid Bioassessment Protocol (RBP) method in its Biological Assessment Program. The RBP compares habitat and biological measures of the benthic macroinvertebrates to reference conditions using individual metrics. VADEQ's Biomonitoring Database, together with Ambient Water Quality Monitoring reports, GIS data layers, and VADCR's Hydrologic Unit Animal Census Database provided all of the necessary information for the stressor variables and benthic macroinvertebrate conditions. Accordingly, the stressor/benthos relationships were evaluated using statistical analyses procedures such as forward, backward, and stepwise multiple regression techniques; correlation analysis; principal component analysis; and r-square analysis. The statistical results indicated that sediment alone cannot be used to assess the benthic macroinvertebrate assemblage conditions. Other stressors such as dissolved oxygen, flow, % urban land, total suspended solids, temperature, stream velocity, substrate, hardness and alkalinity greatly impact the benthic macroinvertebrate assemblage conditions. The study results also indicate that the individual metrics within the RBP procedure are just as critical as the final RBP values in describing the benthic macroinvertebrate assemblage conditions. Upon completing the steps needed to develop stressor/benthos relationships, the validity of the relationships were verified for their application to other streams in Virginia. Validation was completed using 10 stations with 29 samples from the fall of 1996 to the fall of 1998. The 10 stations were located within 8 counties in Virginia (Bedford, Montgomery, Pulaski, Giles, Botetourt, Albemarle, Orange, and Culpeper) and in watersheds dominated by agricultural, urban, and forested landuses to correspond with the stations used to develop the stressor/benthos relationships. The implications of the relationships with regard to TMDLs were also studied using total suspended solids (TSS) loadings, turbidity levels, and embeddedness levels as the stressors of concern. The results for all benthic stations within Virginia showed that moderately impaired streams generally need to reduce the amount of embeddedness by 11, reduce turbidity levels by 5 FTU (57%), and reduce TSS values by 7 mg/L (68%) to meet a threshold value that would no longer adversely impact the benthic macroinvertebrates. Similarly, for the severely impaired stations throughout Virginia to meet threshold values, embeddedness amounts need to be reduced by 22, turbidity reduced by 57 FTU (93%), and TSS reduced by 74 mg/L (96%). This study was important since the proposed stressor/benthos relationships can provide policymakers with a useful tool to determine stressor thresholds that will not adversely impact the benthic macroinvertebrate assemblage conditions for use in developing benthic TMDLs in Virginia. The stressor/benthos relationships could also be used to determine the impact of certain activities or stressors on the benthic macroinvertebrates assemblage conditions in a given stream. / Master of Science

<p>In recent years, the pervasiveness and importance of post-transcriptional regulation has reshaped the underlying principles of the organizational logic of gene expression. RNA-binding proteins (RBPs) and non-coding RNAs are the regulatory molecules primary responsible for interaction with target mRNAs and thereby regulating post-transcriptional processes eventually influencing characteristics of the encoded protein. Many of the mRNA targets of RBPs encode functionally related proteins, which for post-trascriptional operons, resulting in coordination of macromolecular complexes or specific cellular processes. Thus, identifying RNA targets, precise binding sites, and the dynamics of these interactions will reveal how these important regulatory factors contribute to gene regulatory networks.</p><p>ELAV family of human RBPs consist of 4 members, which all have 3 RRM (RNA-recognition motif) domains the last separated by a hinge region. It predominant role is to positively regulate the stability and translation of target mRNAs through binding to ARE (AU-rich elements) in the 3' UTR (untranslated region) of protein coding transcripts. In response to certain stimuli, HuR is subject to post-translational modifications and changes subcellular localization, which impacts its regulatory capacity. In this study on a transcriptome-wide level, we interrogate the RNA targets, precise binding sites, as well as the remodeling of these interactions in response to stimuli.</p><p>We utilized two complementary methods, RIP-chip and PAR-CLIP, to identify targets of HuR and high-resolution binding sites on a transcriptome-wide scale. We discovered that HuR-mRNA interactions are not restricted to the 3' UTR and there are thousands of intronic binding sites. A significant proportion of intronic binding sites are contained in the poly-pyrimidine tract near 3' splice sites. Binding sites in the 3' UTR and intron are often approximately 30 nucleotides apart. HuR can bind to both AU-rich and U-rich sequences, the former more prevalent in 3' UTRs and the latter more prevalent at the 3' splice site.</p><p>Next we integrated the binding data with transcriptomics of HuR siRNA mediated knockdown. We found that the degree of binding is proportional to the degree of HuR-dependent stabilization. Moreover the ability to stabilize mRNA is not restricted to 3' UTR binding sites, as intronic binding sites also exhibited the binding degree correlated stabilization. We observed that the spatial pattern of HuR binding sites relative to exons influences exon usage decisions. Specifically, binding sites upstream of the exon promote exclusion, while binding sites downstream of the exon promote inclusion.</p> / Dissertation

Le complexe EJC (Exon Junction Complex) joue un rôle central dans le couplage des processus post-transcriptionnels chez les métazoaires. Ce complexe multi-protéique est assemblé sur les ARN messagers (ARNm) par la machinerie d’épissage. Organisé autour d’un complexe cœur servant de plate-forme à de nombreux facteurs, les EJCs accompagnent les ARNm dans le cytoplasme et participent à leur transport, leur traduction et leur stabilité. L’importance physiologique de l’EJC est supportée par les nombreux défauts développementaux et les maladies génétiques associées aux composants de l’EJC. Les analyses transcriptomiques révélant un assemblage hétérogène des EJCs renforcent l’hypothèse que les EJCs participent à la régulation de l’expression des gènes. Cependant, malgré une connaissance précise de la structure de ce complexe, les liens fonctionnels entre l’assemblage de l’EJC et la régulation de transcrits spécifiques dans des conditions physiologiques doivent être établis puis caractérisés.Durant cette thèse, j’ai étudié l’expression d’eIF4A3, Y14 et MLN51, trois protéines du cœur de l’EJC, dans des cultures primaires de cellules souches neurales murines (CSN). Les CSN peuvent être différenciées en cellules épendymaires multi-ciliées qui tapissent les ventricules cérébraux et ont un rôle important dans le développement du cerveau. J’ai observé par immunofluorescence dans des CSN quiescentes que les 3 protéines sont concentrées autour du centrosome à la base du cil primaire. Ces localisations reflètent la présence d’EJC assemblés comme le prouve l’étude d’un mutant d’Y14 incapable de former l’EJC. / The Exon Junction Complex (EJC) plays a central role in coupling post-transcriptional processes in metazoans. This multi-protein complex is assembled onto messengers RNAs (mRNAs) by the splicing machinery. Organized around a core complex serving as a platform for numerous factors, EJCs accompany mRNAs to the cytoplasm and is involved in mRNA transport, translation and stability. The physiological importance of the EJC is supported by observations associating defects in EJC component expression to developmental defects and human genetic disorders. Transcriptomic studies revealing the non-ubiquitous deposition of EJCs strengthened the hypothesis that EJCs could participate to gene expression regulation. However, despite a precise picture of the structure of the EJC, functional links between EJC assembly and regulation of specific transcripts under physiological conditions is yet to be established.During this thesis, I studied the expression of eIF4A3, Y14 and MLN51 three core proteins of the EJC in primary cultures of mouse neural stem cells (NSCs). NSCs can be differentiated into multiciliated ependymal cells that line all brain ventricles and have important physiological functions in brain development. We observed by immunofluorescence that in quiescent NSCs, all three proteins are concentrated in the vicinity of the centrosome at the base of the primary cilia. This localization reflects the presence of fully assembled EJCs as proved by the study of Y14 mutant that prevent EJC core mounting.

Une des caractéristiques des cellules cancéreuses réside dans la modification de leur capacité à répondre à divers stress par rapport à des cellules saines. La modulation afférente de l'expression de gènes peut se produire à deux niveaux : transcriptionnel et post-transcriptionnel. La plupart des efforts de compréhension ont été axés sur l'étude la régulation transcriptionnelle. Néanmoins, une façon efficace et rapide de modifier l'expression des gènes est la capacité de réguler le " pool d'ARNm " pré-existant en intervenant au niveau post-transcriptionnel. Aussi, les modifications de la stabilité de l'ARNm et/ou de l'efficacité de traduction dans un contexte particulier tel que le stress tumoral et faisant intervenir des interactions ARN/protéines sont de plus en plus étudiés dans le cas des cancers. Une compréhension plus approfondie de ces mécanismes permettra de mieux comprendre 1/ l'implication des protéines liant l'ARN (RBP) dans le développement tumoral et 2/ considérer le développement de thérapies ciblées contre le cancer. Nous nous sommes concentrés sur l'étude d'une RBP pertinente vis à vis du cancer, mais très peu étudiée sur cette problématique : la protéine Staufen1 (Stau1). Stau1 est un membre de la famille des protéines qui lient l'ARN double-brin. Cette RBP contribue à la régulation post-transcriptionnelle de nombreux gènes par son implication dans des mécanismes qui vont promouvoir le transport, la dé-répression de la traduction et l'induction de la dégradation d'ARN messagers (ARNm) spécifiques. Stau1 apparait également comme un régulateur spécifique de l'expression génique intervenant dans un contexte particulier de stress cellulaire, contexte familier aux tumeurs en cours de développement. Les transcrits régulés par Stau1 se classent dans un large éventail de catégories fonctionnelles et il est intéressant de noter qu'une grande proportion d'entre eux code pour des protéines impliquées dans la régulation de processus biologiques cellulaires critique dans le développement de cancers. De part son rôle de régulateur de l'expression génique et son implication dans la réponse au stress cellulaire, nous avons émis l'hypothèse que la modification de l'expression de Stau1 puisse avoir un impact à différent niveaux sur le développement et la progression tumorale. Ce projet de thèse s'est orienté sur deux axes 1) l'étude de l'expression de Stau1 en condition de stress cellulaire et 2) l'effet de la répression de Stau1 sur le développement tumoral. / One of the characteristics of cancer cells lies in the modification of their ability to adapt to various stresses compared to healthy cells. The afferent modulation of gene expression can occur at two levels: transcriptional and post-transcriptional. Most of the efforts for understanding the gene expression process are focused on transcriptional regulation study. However, a quick and effective way to modify gene expression is the ability to regulate the "mRNA pool" by intervening in post-transcriptional level. Besides, changes in mRNA stability and/or translation efficiency in a context such as cellular stress in tumors and interactions involving RNA / protein are increasingly studied in the case of cancers. A deeper knowledge of these mechanisms will allow a better understanding of 1 / the involvement of RNA binding proteins (RBP) in tumor development and the 2 / the consideration of the development of targeted cancer therapies. We focused on the study of a relevant RBP for cancer development process, but very few studies have been undertaken on this issue: the Staufen1 protein (Stau1). Stau1 is a family member of double-stranded RNA-binding proteins. This RBP contributes to the post-transcriptional regulation of many genes through its involvement in mechanisms that will promote the transport, the derepression of translation and the induction of degradation of specific RNA transcripts (mRNA). Stau1 also appears as a regulator of specific genes expressed in respond to cellular stress induced by an unfavorable tumor microenvironment. The transcripts regulated by Stau1 can be divided into a wide range of functional categories. Interestingly, a large proportion of Stau1 targets encode proteins which regulate many critical biological cell processes in cancer development. Regarding Stau1 regulatory role and its involvement in the response to cellular stress, we made assumptions that the modification of Stau1 expression could have an impact on various levels of development and tumor progression. This project will be considered from two aspects 1/ the study of Stau1 expression under cellular stress 2/ the impact of Stau1 repression on tumor development.

Após a introdução em 1996 de uma nova classe de medicamentos antirretrovirais a potência e a eficácia da TARV aumentaram a expectativa de vida dos pacientes infectados pelo HIV. Entretanto, seu uso está associado a modificações metabólicas e de composição corporal, denominadas síndrome da lipodistrofia do HIV (SLHIV). Alguns estudos tem relatado alterações no metabolismo da glicose e aumento de níveis séricos de colesterol total, LDL-c e triglicérides; ainda, pode-se observar maiores valores de circunferência abdominal e presença de hipertensão arterial sistêmica. Tais achados são também relacionados à síndrome metabólica. Podemos estão relacionar as alterações encontradas em pacientes soropositivos para o HIV com alterações da síndrome metabólica do paciente soronegativo para o HIV. Ainda, encontra-se dados com o aumento de níveis séricos de RBP-4 e correlação positiva com componentes da síndrome metabólica em diversas populações, associados à essas alterações metabólicas, aumento de marcadores de estresse oxidativo. Assim, o presente estudo avaliou se níveis séricos de RBP-4 e marcadores de estresse oxidativo estão aumentados em população HIV+ quando comparados com HIV-. Casuística e Métodos: Foram selecionados 59 pacientes divididos em 4 grupos: HIV+SM+, HIV+SM-, HIV-SM- e HIV-SM+. Foram avaliados marcadores de estresse oxidativo (CAT, Frap, MDA, GSH, 8- OHdG), RBP-4, retinol, avaliação antropométrica (peso, estatura, IMC, circunferência abdominal) , composição corporal por impedância biolétrica, exames bioquímicos de perfil lipídico e glicemia de jejum. Resultados: Os voluntários dos grupos com síndrome metabólica apresentaram maiores valores de peso, IMC, colesterol total, LDL-c e triglicérides. Níveis aumentados de RBP-4 e MDA também foram encontrados, estando estes mais pronunciados em indivíduos HIV+. Não foi encontrada diferença significativa entre marcadores estresse oxidativo GSH e 8OHdG entre os grupos. Não foi encontrada correlação entre RBP-4 e componentes da SM. Conclusão: RBP-4 e marcadores de peroxidação lipídica estão aumentados em pacientes com síndrome metabólica, estando mais pronunciados em indivíduos soropositivos para o HIV. / Introduction: After the introduction in 1996 of a new class of antiretroviral drugs the potency and efficacy of ART increased the life expectancy of HIV-infected patients. However, its use is associated with metabolic changes and body composition, called lipodystrophy syndrome of HIV (SLHIV). Some studies have reported increased incidence and prevalence of alterations in glucose metabolism and increased serum levels of total cholesterol, LDL-C and triglycerides; yet, we can observe higher waist circumference values and the presence of hypertension. These findings are also related to metabolic syndrome. We can relate the changes are found in patients seropositive for HIV with changes in metabolic syndrome patients soronegative for HIV. In some literatures we can observed increased serum levels of RBP-4 and positive correlation with components of the metabolic syndrome in different populations. Associated with these metabolic abnormalities, increased oxidative stress markers. The present study evaluated whether serum RBP-4 and oxidative stress markers are increased in HIV + population when compared to HIV. Methods: We have selected 59 patients divided into 4 groups: HIV + SM +, HIV + SM, SM-HIV and HIV-SM +. We have evaluated oxidative stress markers (CAT, Frap, MDA, GSH) DNA damage markers, RBP-4, vitamin A, vitamin E, anthropometric measurements (weight, height, BMI, waist circumference), body composition by bioelectrical impedance, biochemical tests of lipid profile and fasting glucose. Results: Volunteers from groups with metabolic syndrome have higher weight values, BMI, total cholesterol, LDL-C and triglycerides. Increased levels of RBP-4 and MDA were also found, and these are more pronounced in HIV + individuals. There was no significant differences between GSH and oxidative stress marker 8OHdG on different groups. No correlation was found between RBP-4 and some components of MS. Conclusion: RBP-4 and lipid peroxidation markers are elevated in patients with metabolic syndrome, being more pronounced in individuals soropositive for HIV.

Após a introdução em 1996 de uma nova classe de medicamentos antirretrovirais a potência e a eficácia da TARV aumentaram a expectativa de vida dos pacientes infectados pelo HIV. Entretanto, seu uso está associado a modificações metabólicas e de composição corporal, denominadas síndrome da lipodistrofia do HIV (SLHIV). Alguns estudos tem relatado alterações no metabolismo da glicose e aumento de níveis séricos de colesterol total, LDL-c e triglicérides; ainda, pode-se observar maiores valores de circunferência abdominal e presença de hipertensão arterial sistêmica. Tais achados são também relacionados à síndrome metabólica. Podemos estão relacionar as alterações encontradas em pacientes soropositivos para o HIV com alterações da síndrome metabólica do paciente soronegativo para o HIV. Ainda, encontra-se dados com o aumento de níveis séricos de RBP-4 e correlação positiva com componentes da síndrome metabólica em diversas populações, associados à essas alterações metabólicas, aumento de marcadores de estresse oxidativo. Assim, o presente estudo avaliou se níveis séricos de RBP-4 e marcadores de estresse oxidativo estão aumentados em população HIV+ quando comparados com HIV-. Casuística e Métodos: Foram selecionados 59 pacientes divididos em 4 grupos: HIV+SM+, HIV+SM-, HIV-SM- e HIV-SM+. Foram avaliados marcadores de estresse oxidativo (CAT, Frap, MDA, GSH, 8- OHdG), RBP-4, retinol, avaliação antropométrica (peso, estatura, IMC, circunferência abdominal) , composição corporal por impedância biolétrica, exames bioquímicos de perfil lipídico e glicemia de jejum. Resultados: Os voluntários dos grupos com síndrome metabólica apresentaram maiores valores de peso, IMC, colesterol total, LDL-c e triglicérides. Níveis aumentados de RBP-4 e MDA também foram encontrados, estando estes mais pronunciados em indivíduos HIV+. Não foi encontrada diferença significativa entre marcadores estresse oxidativo GSH e 8OHdG entre os grupos. Não foi encontrada correlação entre RBP-4 e componentes da SM. Conclusão: RBP-4 e marcadores de peroxidação lipídica estão aumentados em pacientes com síndrome metabólica, estando mais pronunciados em indivíduos soropositivos para o HIV. / Introduction: After the introduction in 1996 of a new class of antiretroviral drugs the potency and efficacy of ART increased the life expectancy of HIV-infected patients. However, its use is associated with metabolic changes and body composition, called lipodystrophy syndrome of HIV (SLHIV). Some studies have reported increased incidence and prevalence of alterations in glucose metabolism and increased serum levels of total cholesterol, LDL-C and triglycerides; yet, we can observe higher waist circumference values and the presence of hypertension. These findings are also related to metabolic syndrome. We can relate the changes are found in patients seropositive for HIV with changes in metabolic syndrome patients soronegative for HIV. In some literatures we can observed increased serum levels of RBP-4 and positive correlation with components of the metabolic syndrome in different populations. Associated with these metabolic abnormalities, increased oxidative stress markers. The present study evaluated whether serum RBP-4 and oxidative stress markers are increased in HIV + population when compared to HIV. Methods: We have selected 59 patients divided into 4 groups: HIV + SM +, HIV + SM, SM-HIV and HIV-SM +. We have evaluated oxidative stress markers (CAT, Frap, MDA, GSH) DNA damage markers, RBP-4, vitamin A, vitamin E, anthropometric measurements (weight, height, BMI, waist circumference), body composition by bioelectrical impedance, biochemical tests of lipid profile and fasting glucose. Results: Volunteers from groups with metabolic syndrome have higher weight values, BMI, total cholesterol, LDL-C and triglycerides. Increased levels of RBP-4 and MDA were also found, and these are more pronounced in HIV + individuals. There was no significant differences between GSH and oxidative stress marker 8OHdG on different groups. No correlation was found between RBP-4 and some components of MS. Conclusion: RBP-4 and lipid peroxidation markers are elevated in patients with metabolic syndrome, being more pronounced in individuals soropositive for HIV.

The fat-soluble vitamin A, which is chemically referred to retinol (ROH), is known to be essential for the process of vision, the immune system but also for cell differentiation and proliferation. Recently, ROH itself has been reported to be involved in adipogenesis and a ROH transport protein, the retinol-binding protein 4 (RBP4), in insulin resistance and type 2 diabetes. However, there is still considerable scientific debate about this relation. With the increasing amount of studies investigating the relation of ROH in obesity and type 2 diabetes, basic research is an essential prerequisite for interpreting these results. This thesis enhances the knowledge on this relation by reviewing ROH metabolism on extra- and intracellular level. Aim 1: In the blood stream ROH is transported in a complex with RBP4 and a second protein, transthyretin (TTR), to the target cells. The levels of RBP4 and TTR are influenced by several factors but mainly by liver and kidney function. The reason for that is that liver and the kidneys are the sites of RBP4 synthesis and catabolism, respectively. Interestingly, obesity and type 2 diabetes involve disorders of the liver and the kidneys. Therefore the aim was to investigate factors that influence RBP4 and TTR levels in relation to obesity and type 2 diabetes (Part 1). Aim 2: Once arrived in the target cell ROH is bound to cellular retinol-binding protein type I (CRBP-I) and metabolised: ROH can either be stored as retinylesters or it can be oxidised to retinoic acid (RA). By acting as a transcription factor in the nucleus RA may influence processes such as adipogenesis. Therefore vitamin A has been postulated to be involved in obesity and type 2 diabetes. CRBP-I is known to mediate the storage of ROH in the liver, but the extra-hepatic metabolism and the functions of CRBP-I are not well known. This has been investigated in Part 2 of this work. Material & Methods: RBP4 and TTR levels were investigated by ELISA in serum samples of human subjects with overweight, type 2 diabetes, kidney or liver dysfunction. Molecular alterations of the RBP4 and TTR protein structure were analysed by MALDI-TOF mass spectrometry. The functions of intracellular CRBP-I were investigated in CRBP-I knock-out mice in liver and extra-hepatic tissues by measuring ROH levels as well as the levels of its storage form, the retinylesters, using reverse phase HPLC. The postprandial uptake of ROH into tissues was analysed using labelled ROH. The mRNA levels of enzymes that metabolize ROH were examined by real-time polymerase chain reaction (RCR). Results: The previous published results showing increased RBP4 levels in type 2 diabetic patients could not be confirmed in this work. However, it could be shown that during kidney dysfunction RBP4 levels are increased and that RBP4 and TTR levels are decreased during liver dysfunction. The important new finding of this work is that increased RBP4 levels in type 2 diabetic mice were increased when kidney function was decreased. Thus an increase in RBP4 levels in type 2 diabetes may be the effect of a reduced kidney function which is common in type 2 diabetes. Interestingly, during severe kidney dysfunction the molecular structure of RBP4 and TTR was altered in a specific manner which was not the case during liver diseases and type 2 diabetes. This underlines the important function of the kidneys in RBP4 metabolism. CRBP-I has been confirmed to be responsible for the ROH storage in the liver since CRBP-I knock-out mice had decreased ROH and retinylesters (the storage form of ROH) levels in the liver. Interestingly, in the adipose tissue (the second largest ROH storage tissue in the body) ROH and retinylesters levels were higher in the CRBP-I knock-out compared to the wild-type mice. It could be shown in this work that a different ROH binding protein, cellular retinol-binding protein type III, is upregulated in CRBP-I knock-out mice. Moreover enzymes were identified which mediate very efficiently ROH esterification in the adipose tissue of the knock-out mice. In the pancreas there was a higher postprandial ROH uptake in the CRBP-I knock-out compard to wild-type mice. Even under a vitamin A deficient diet the knock-out animals had ROH and retinylesters levels which were comparable to wild-type animals. These results underline the important role of ROH for insulin secretion in the pancreas. Summing up, there is evidence that RBP4 levels are more determined by kidney function than by type 2 diabetes and that specific molecular modifications occur during kidney dysfunction. The results in adipose tissue and pancreas of CRBP-I knock-out mice support the hypothesis that ROH plays an important role in glucose and lipid metabolism. / Vitamin A gehört zur Gruppe der fettlöslichen Vitamine und wird chemisch als Retinol bezeichnet. Es ist essentiell für den Prozess des Sehvorgangs und der Zelldifferenzierung und kann daher bestimmte Entwicklungsprozesse wie die Bildung des Fettgewebes beeinflussen. Aufgrund seiner Fettlöslichkeit muss Retinol im Blut (= extrazellulär) sowie in der Zelle (= intrazellulär) an sogenannte Transport-Moleküle, die Retinol-bindenden Proteine (RBPs) gebunden werden. Die zwei bekanntesten Vertreter der RBPs sind das Retinol-bindende Protein 4 (RBP4) und das intrazelluläre Retinol-bindende Protein Typ I (CRBP-I). RBP4 transportiert Vitamin A im Blut von der Leber zur Zielzelle und zum Abbauorgan für Vitamin A, der Niere. CRBP-I ist in der Leber für die Speicherung von Vitamin A zuständig. In den letzten Jahren wurden neben der Beteiligung des Retinols an der Bildung des Fettgewebes auch Studien veröffentlicht, in denen ein Zusammenhang zwischen erhöhten RBP4-Werte im Blut und Typ-2-Diabetes gezeigt wurde. Bis heute ist der mögliche Zusammenhang zwischen RBP4, CRBP-I und Übergewicht nicht ausreichend erforscht. Im ersten Teil der Arbeit war daher das Ziel, Einflussfaktoren, die zu Veränderungen der RBP4-Werte im Blut führen können, zu untersuchen. Dazu wurden Blutproben von Personen mit Übergewicht und/oder Typ-2-Diabetes und Patienten mit Nierenfunktionsstörungen oder mit Leberfunktionsstörungen analysiert. Es konnte gezeigt werden, dass bereits geringe Nierenfunktionsstörungen zu erhöhten RBP4-Konzentrationen im Blut führten. Bei Typ-2-Diabetikern, die sehr oft an Nierenfunktionsstörungen leiden, war eine Erhöhung der RBP4-Konzentration mit einer Abnahme der Nierenfunktion verbunden. Somit lässt sich zusammenfassen, dass nicht Typ-2-Diabetes sondern vielmehr die dabei auftretenden Nierenfunktionsstörungen zu einer Erhöhung der RBP4-Werte führen. Bei Lebererkrankten konnte ein Absinken der RBP4-Werte nachgewiesen werden, was der verminderten Bildung von RBP4 in der Leber bei diesen Patienten zuzuschreiben ist. Im zweiten Teil sollte der Frage nachgegangen werden, wie Retinol intrazellulär verstoffwechselt wird. Dabei lag der Fokus auf der Erforschung der bisher nicht bekannten Funktionen von CRBP-I im Fettgewebe und der Bauchspeicheldrüse. Zur Untersuchung der Funktionen von CRBP-I wurden Mäuse gezüchtet, bei denen das Gen für CRBP-I gelöscht wurde. Da CRBP-I für die Speicherung von Vitamin A in der Leber verantwortlich ist, zeigen diese Mäuse sehr geringe Vitamin-A-Speicher in der Leber. Das gleiche zeigte sich für die Bauchspeicheldrüse, die für die Sekretion von Insulin Vitamin A benötigt: In den Mäusen ohne CRBP-I waren die Retinol-Werte drastisch gesunken. Interessanterweise zeigte sich im Fettgewebe ein gegenteiliges Bild: Die Konzentrationen an Retinol und dessen Speicher waren in den Mäusen ohne CRBP-I höher im Vergleich zu den normalen Mäusen. Mit bestimmten Nachweismethoden konnte herausgefunden werden, dass Retinol im Fettgewebe an ein anderes RBP, das CRBP-III, gebunden wird und dadurch effektiver gespeichert werden kann als durch CRBP-I.

Proteins that bind to both DNA and RNA embody the ability to perform multiple functions by a single gene product. These nucleic acid binding proteins in prokaryotes can play a vital role in many cellular processes, including replication, transcription, gene expression, recombination, and repair, to name a few. Nucleic acid binding proteins have unique functional characteristics that stem from their structural attributes that have evolved in a widely-conserved manner. In Escherichia coli (E. coli), the highly-conserved histone-like protein, HU, which predominates as a heterodimer of HUα and HUβ, has been found to bind to both dsDNA and ssDNA. Likewise, RNA-binding proteins contain various structural motifs, many of which are also conserved amongst many bacterial species like the RNA recognition motif. However, in Porphyromonas gingivalis, a periodontal pathogen, the histone-like, HU proteins and the RNA-binding protein (RBP) are not well characterized compared to their respective structures in E. coli. In our study, we sought to characterize and compare the HU proteins and RBP in order to gain a better understanding of their structure and function in the cell. Our data showed the HU proteins predominate as homo-tetramers and RBP as a monomer. We demonstrated single-stranded DNA binding with all three proteins. We found both P. gingivalis HU subunits bind non-specifically to ssDNA but show preferential binding to poly(dG) content, while binding to poly(dA) the weakest. These results show that HUα, HUβ and RBP are novel ssDNA binding proteins in P. gingivalis, indicating an expanded role and function within the cell.

Porphyromonas gingivalis, a gram-negative anaerobic bacterium, is a major etiological agent in the initiation and progression of severe forms of periodontal disease. Oral bacteria like P. gingivalis are subject to continually changing conditions as a consequence of host eating, oral hygiene patterns and subgingival temperatures. As such survival requires an adaptive response to environmental cues, but little is known about the mechanism by which P. gingivalis controls co- and post-transcriptional regulation of RNA levels and potentially protein expression. RNA-binding proteins (RBPs) are evolutionarily conserved across species and are involved in such regulatory mechanisms. However, P. gingivalis currently has no identified RBP. Recently, PG0627 has become an ideal candidate for a putative RBP due to its sequence homology to RBPs across various species. By characterizing PG0627, we can gain better insight into the function of this hypothetical protein and determine if it indeed behaves like an RNA-binding protein. A host of studies were done on a PG0627-deficient P. gingivalis mutant, V3139, in order to determine the biological role of the protein encoded by the gene. Our bioinformatics analysis indicated that PG0627 had sequence homology to several RNA recognition motifs or RBPs. Furthermore, our PG0627-deficient mutant, when compared to W83, exhibited decreased cell-associated iron content, decreased total interactions and invasions with eukaryotic cells, and decreased protease activity. Conversely, our PG0627-deficient mutant displayed slightly increased growth in the presence of nitrosative stress, and in hemin-depleted conditions. In conclusion, our results support that PG0627 is a valid candidate for an RNA-binding protein in P. gingivalis.

Plusieurs données expérimentales suggèrent que la protéine Core du virus de l’Hépatite B (HBV), en plus de ses fonctions structurales pour la formation des nucléocapsides dans le cytoplasme, pourrait avoir des fonctions régulatrices importantes dans le noyau des hépatocytes infectés. En effet, Core s’associe à l’ADNccc et aux promoteurs de certains gènes cellulaires dans le noyau des hépatocytes infectés et pourrait ainsi contrôler leur régulation transcriptionnelle. De plus, de par sa capacité à lier les ARN, elle pourrait également participer au métabolisme post-transcriptionnel de gènes viraux et/ou cellulaires. Pour caractériser ces fonctions, nous avons réalisé une analyse protéomique des facteurs cellulaires qui interagissent avec la protéine Core dans le noyau d’hépatocytes humains. Cet interactome a mis en évidence un grand nombre de protéines de liaison aux ARN (RBP), qui participent au métabolisme des ARN et en particulier aux mécanismes d’épissage. Deux interactants majeurs de Core ont été plus particulièrement étudiés, SRSF10 et RBMX, impliqués notamment dans l’épissage et la réparation de l’ADN. Une analyse fonctionnelle effectuée par une approche siRNA a montré que SRSF10 et RBMX affectent différemment le niveau des ARN viraux, vraisemblablement en agissant à des étapes différentes du cycle viral. De même, un composé ciblant l’activité de certaines RBP diminue fortement la réplication d’HBV en affectant l’accumulation des ARN viraux. Ainsi, ces résultats suggèrent que Core pourrait interagir avec certaines RBP pour contrôler le destin des ARN viraux et/ou cellulaires, une piste intéressante pour le développement de nouvelles stratégies antivirales ciblant l’hôte / Converging evidences suggest that the Hepatitis B virus (HBV) core protein, beside its well-known structural role to form nucleocapsids in the cytoplasm, could have important regulatory functions in the nucleus of infected hepatocytes. Indeed, nuclear Core was shown to associate with the cccDNA and to the promoters of some cellular genes, suggesting that Core may control viral and/or cellular gene expression. In addition, Core has the capacity to bind RNA, and may thus regulate HBV RNA metabolism. To elucidate these functions, we performed a proteomic analysis of the cellular factors interacting with nuclear Core in human hepatocytes. This interactome revealed a majority of highly interconnected RNA-binding proteins (RBPs), which participate in several steps of mRNA metabolism, including transcription, splicing and nuclear egress. We focused on two major Core-interacting factors, SRSF10 and RBMX that were previously involved in splicing and DNA repair. Functional analyses performed by a siRNA approach indicated that RBMX and SRSF10 were able to differentially regulate the levels of all viral RNAs most likely by acting at different steps of the viral life-cycle. Similarly, a small compound, affecting the activity of selected RBPs, severely impaired HBV replication by strongly reducing viral RNA accumulation. Altogether, these results strongly suggest that Core interacts with some selected RBPs to control the fate of viral and/or cellular RNAs and provide new critical information for the development of novel host-targeting antiviral agents (HTA)