Abstract Gastric erosions are superficial mucosal breaks. With the exception of bleeding, they are considered harmless, but their aetiology, histopathology and long-term course have remained unknown and even the evolution of gastritis in patients with gastric erosions is unclear. The present study aimed to solve clinical significance and pathology of gastric erosions in a long-term follow-up study. Initially, 117 patients and 117 controls were studied in 1974–1981, and a follow-up study was performed in 1996. We evaluated the presence of Helicobacter pylori and Herpes simplex virus (HSV) infections, use of NSAIDs and alcohol, smoking, and assessed features of gastric histopathology. For follow-up, 52 patients and 66 controls were available. In the follow-up visit, 39% patients still had gastric erosions while 11% of the controls had developed erosions (p = 0.001). In H. pylori-positive subjects, peptic ulcer or a scar was more common in patients (17%) than in controls (4%, p = 0.006), but otherwise no increased morbidity or mortality was seen. High antibody titres against HSV predicted the persistence of erosions (p = 0.000), but H. pylori infection, use of NSAIDs, alcohol or smoking were not associated. Initially, inflammation was more active in the region of erosions than elsewhere in the antral mucosa, and more active inflammation in the erosion was associated with HSV seropositivity, H. pylori infection and the recent use of NSAIDs. Initially, H. pylori-positive subjects with chronic or recurrent erosions had higher scores of neutrophils compared to those with non-chronic/non-recurrent erosions. In H. pylori-positive subjects, body gastritis was initially less active in the patient group. With time, antral gastritis worsened only in the patient group. In H. pylori-negative subjects, there was no evolution of gastritis. These results show that a significant proportion of gastric erosions are chronic/recurrent but mostly without serious complications. However, H. pylori-positive patients have a significant risk to develop a peptic ulcer. A significant proportion of chronic gastric erosions is related to HSV infection. Focally enhanced inflammation modified by HSV or NSAID may be important in the pathogenesis of gastric antral erosions. Active inflammation in the erosions seems to predict their chronicity/recurrency. Patients with erosions share the characteristics of gastritis of the duodenal ulcer phenotype. / Tiivistelmä Eroosiot ovat mahalaukun pinnallisia limakalvovaurioita. Niitä pidetään vaarattomina lukuun ottamatta niihin liittyvää verenvuototaipumusta. Niiden etiologiaa, histopatologiaa ja taudinkulkua ei tunneta. Ei myöskään tiedetä eroosiopotilaiden mahan limakalvon tulehduksen kulkua. Tämän tutkimuksen tavoitteena oli selvittää mahalaukun eroosioiden kliininen merkitys ja patologia pitkäkestoisena seurantatutkimuksena. Alkujaan 117 potilasta ja 117 kontrollihenkilöä tutkittiin vuosina 1974–1981, ja seurantatutkimus tehtiin vuonna 1996. Selvitimme helikobakteerin ja Herpes simplex -viruksen (HSV) aiheuttamien infektioiden, tulehduskipulääkkeiden (NSAID) ja alkoholin käytön, sekä tupakoinnin esiintymistä. Lisäksi tutkimme histopatologisesti mahalaukun limakalvoa. Lopulta oli 52 potilaan ja 66 kontrollihenkilön aineisto käytettävissä. Seurantakäynnillä 39 prosentilla potilaista oli yhä mahalaukun eroosioita, kun taas kontrolliryhmästä vain 11 prosentilla oli kehittynyt eroosioita. Helikobakteeri -infektoituneilla maha- tai pohjukaissuolen haava/arpi oli yleisempää eroosioryhmässä (17 %) kuin kontrolleilla (4 %), mutta muuten ei esiintynyt lisääntynyttä sairastuvuutta tai kuolleisuutta. Tulehdus oli aktiivisempaa eroosioissa kuin viereisellä limakalvolla, ja tämä tulehdus liittyi korkeisiin HSV-vasta-ainetasoihin, helikobakteeri-infektioon ja NSAID:n käyttöön. Korkeat HSV-vasta-ainetasot ennustivat eroosioiden pysyvyyttä. Ensimmäisellä käynnillä aktiivinen tulehdus eroosioissa oli voimakkaampaa niillä helikobakteeri-infektoituneilla, joilla eroosiot olivat pysyviä kuin niillä, joilla eroosiot eivät uusineet. Helikobakteeri-infektoituneilla eroosiopotilailla mahalaukun runko-osan limakalvon tulehdus oli aluksi vähemmän aktiivista kuin vastaavilla kontrolliryhmän henkilöillä, mutta ajan myötä mahalaukun corpusosan limakalvon tulehdus voimistui vain eroosioryhmällä. Limakalvotulehdus ei edennyt helikobakteeri-infektoitumattomilla henkilöillä. Tulokset osoittavat, että merkittävä osa mahalaukun eroosioista on kroonisia/toistuvia, mutta enimmäkseen ilman vakavia komplikaatioita. Kuitenkin helikobakteeri-infektoituneilla eroosiopotilailla on merkittävä riski saada maha- tai pohjakaissuolen haava. HSV- infektio liittyy merkittävään osaan kroonisia mahalaukun eroosioita. Paikallisella tulehdusaktiivisuudella, jota HSV ja NSAID:n käyttö muokkaavat, saattaa olla tärkeä rooli eroosioiden synnyssä ja niiden kroonistumisessa. Eroosiopotilailla on samanlainen mahalaukun limakalvon tulehduksen jakauma kuin pohjakaissuolihaavaa sairastavilla.

Transcription initiation is an important step in the process of gene regulation in prokaryotes. Promoters are stretches of DNA sequence that are present in the upstream region of transcription start sites (TSSs), where RNA polymerase and other transcription factors bind to initiate transcription. Recent advancement in sequencing technologies has resulted in huge amount of raw data in the form of whole genome sequences. This sequence data has to be annotated, in order to identify coding, non-coding and regulatory regions. Computational tools are useful for a quick and fairly reliable annotation of many genome sequences. Promoter prediction is an important step in genome annotation process which is needed, not only for the validation of predicted genes, but also for the identification of novel genes, especially those coding for non-coding RNA, which are missed by gene prediction programs. DNA sequence dependent structural properties such as DNA duplex stability, bendability and intrinsic curvature have been found to be associated with promoter regions in all domains of life. The work presented in this thesis focuses on the analysis of these structural features in the promoter regions of published prokaryotic transcriptome data. Furthermore, promoters were predicted using these structural features and their role in gene expression were studied. The organization of thesis is as follows. An overview of transcription machinery of prokaryotes, promoter architecture, available promoter prediction programs and sequence dependent structural features is presented in chapter 1. Chapter 2 describes the datasets and methods used in entire study. Structural features of promoters associated with primary and operon TSSs of H.pylori26695 genes and their orthologs (chapter 3) Promoter regions in genomic sequences from all domains of life show similar trends in their structural properties such as stability, bendability, curvature. This chapter dis-cuss the DNA duplex stability and bendability of various classes of promoter regions (based on the identification of different classes of transcription start sites, viz. primary, secondary, internal, operon TSSs etc, in transcriptome study) of Helicobacter pylori 26695 strain. It is found that the primary TSS and operon associated TSS promoters show significantly strong structural features in their promoter regions. DNA free energy based promoter prediction tool PromPredict has been used to annotate promoters of different classes and very high recall values (80%) are obtained for primary TSS. Orthologous genes from 10 different strains of H. pylori show conservation of structural properties in promoter regions as well as coding regions. PromPredict annotates promoters of orthologous genes with very high recall and precision values. DNA duplex stability of promoter region is conserved in the orthologous genes in 10 different strains of Helicobacter pylori genome. Sequence dependent structural features of promoters in prokaryotic transcriptome (chapter 4) Next-generation sequencing studies have revealed that a wide range of transcripts such as primary, internal, antisense and non-coding RNA, are present in the prokaryotic transcriptome and a large fraction of them are functionally involved in various regulatory activities. Identification of promoters associated with different transcripts is important for characterization of transcriptome. The current chapter discusses DNA sequence dependent structural properties like stability, bendability and curvature in the promoter region of six different prokaryotic transcriptomes (Helicobacter pylori, Anabaena, Synechocystis, Escherichia coli, Salmonella and Klebsiella). Using these structural features, promoters associated with different category of transcripts were predicted, which constitute an integral part of the transcriptome. Promoter annotation using structural features is fairly accurate and reliable as compared to motif-based approach since different category of transcripts show poor sequence conservation in the promoter region. Most importantly, it is universal in nature unlike sequence-based approach that is generally organism specific. Role of sequence dependent structural properties in gene expression in prokaryotes (chapter 5) DNA duplex stability, bendability and intrinsic curvature play crucial roles in the process of transcription initiation. Hence, in order to understand the relationship be-tween these structural features and gene expression, the relative differences in stability, bendability and curvature in the promoter regions of high and low expressed genes were studied. It is found that these features are relatively accentuated in the promoter regions associated with high gene expression as compared to low gene expression. Promoter regions associated with high gene expression are annotated more reliably using DNA structural features, compared to those for low gene expression. Sequence dependent structural properties in the promoter region of essential and non-essential genes of the prokaryotes (chapter 6) Essential genes are the minimal possible set of genes required for the survival of organism. These sets of genes can be identified by experiments such as single gene deletion and transposon mediated inactivation. Here, the analysis of DNA duplex stability and bendability in the promoter regions of essential and nonessential genes of prokaryotes is reported. It is found that the average free energy and bendability pro-files are distinct in the promoters regions of essential and nonessential genes. Whole genome promoter predictions using in-house program, PromPredict, for essential and nonessential genes has also been carried out. Chapter 7 present the summary and conclusion of the entire thesis work followed by future perspectives in the field. Optimization of PromPredict algorithm and updating PromBase with newly sequenced genomes (Appendix A) PromPredict is an in-house program, which is based on the relative stability of the DNA in flanking regions. It was found to perform well in predicting promoters across all organisms. In previous studies, it was observed that for organisms having low genomic GC content (<35%), promoter prediction resulted in low precision values, which indicates higher false positive rate. Threshold values of PromPredict algorithm were re-vised in order to optimize the algorithm with low false positive rate. PromBase is a comparative genomics database of microbial genomes. It stores different genomic and structural properties of the microbial genomes. It also displays the predictions obtained from PromPredict in a graphical as well as tabular format. Newly sequenced genomes were downloaded from NCBI and processed using in-house programs and added to the mysql database (back end of the PromBase). Stability profiles for predictions were also added for the RNA coding genes, earlier only profiles for protein coding genes were displayed. Comparative genomics of asymmetric gene orientation in prokaryotes (Appendix B) Transcription proceeds in 5’ to 3’ direction on the template strand, hence it provides directionality. Prokaryotic genomes show asymmetry in gene orientation on leading and lagging strands. The different phyla of prokaryotes were analyzed in terms of asymmetry in gene orientation. It is found that organisms belonging to a particular phyla known as “Firmicutes”, show high asymmetry in gene orientation, which are known to have different DNA polymerase systems for replication.

Abstract Helicobacter pylori is a Gram-negative bacterium, which infects the stomach of more than 50% of the population worldwide. In addition to being the most important risk factor for gastric cancer and peptic ulcers, H. pylori infection is a risk factor for several extra-digestive diseases including dyslipidemia. The consequences of having an H. pylori infection are significantly influenced by the inflammatory response of the host. The pattern recognition receptor Toll-like receptor 4 (TLR4) and the cytokine interleukin 6 (IL6) are important mediators of inflammation in H. pylori related diseases. We have analyzed a series of control subjects and patients with dyspepsia, peptic ulcers or gastric cancer for frequent genetic polymorphisms of the TLR4 and IL6 genes. The prevalence of H. pylori infection, the histologic features of gastritis and cancer and serum endocrine markers and lipid concentrations were also analyzed. Furthermore, the expression of TLR4 was analyzed in specific cell types of gastric mucosa by immunohistochemistry. The TLR4 wild type genotypes of polymorphisms +896 and +1196 were associated with an increased risk of peptic ulcers. The same genotypes also associated with higher serum gastrin levels, but not with atrophy or other features of gastritis. The TLR4 expression was seen in the gastrin and somatostatin secreting cells of gastric mucosa. These results suggest a regulatory link between TLR4 and gastrin secretion. Such a link indicates the presence of a novel effector mechanism for innate immunity in modifying the host endocrine function. The IL6 -174 polymorphism associated significantly with a risk of the diffuse type of gastric carcinoma but not with the intestinal type or its precursor conditions. Finally, we demonstrated that H. pylori infections modify HDL serum levels significantly only in IL6 -174 CC genotype patients, which suggests that the detrimental effects of H. pylori infections on HDL levels are transmitted through IL6. These results clarify the mechanisms of H. pylori related diseases and open new possibilities for research on peptic ulcer disease, gastric cancer and dyslipidemia. / Tiivistelmä Helicobacter pylori on yleinen ihmisen mahalaukussa esiintyvä Gram-negatiivinen bakteeri. Helikobakteeri on tärkein mahasyövän ja maha- ja pohjukaissuolihaavan riskitekijä ja se on myös muun muassa rasva-aineenvaihdunnan häiriöiden riskitekijä. Ihmisen tulehdusvaste vaikuttaa merkittävästi helikobakteeri-infektion seurauksiin. Tollin kaltainen reseptori 4 (TLR4), joka on hahmontunnistusreseptori ja tulehduksenvälittäjäaine interleukiini 6 (IL6) ovat tärkeitä ihmisen tulehdusvasteeseen osallistuvia proteiineja. Olemme tutkineet dyspepsiaa, maha- ja pohjukaissuolihaavaa ja mahasyöpää sairastavilta potilailta sekä kontrollihenkilöiltä TLR4:n ja IL6:n geenien yleisiä emäsjärjestyksen polymorfioita. Tutkimme myös helikobakteeri-infektion yleisyyttä ja histologisia piirteitä, mahasyövän histologisia piirteitä ja seerumin merkkiaineita ja lipidipitoisuuksia. Lisäksi tutkimme TLR4:n ilmenemistä mahan limakalvolla immunohistokemiallisesti. TLR4:n polymorfismien +896 ja +1196 villin tyypin genotyypit liittyivät kohonneeseen maha- ja pohjukaissuolihaavan riskiin. Samat genotyypit liittyivät myös korkeampiin gastriinitasoihin. TLR4:ä esiintyi mahalaukun limakalvolla gastriinia tai somatostatiinia ilmentävissä soluissa. Täten TLR4:n ja maha- pohjukaissuolihaavariskin yhteys näyttää välittyvän gastriinin erityksen kautta, mikä viittaa uuteen säätely-yhteyteen luontaisen immuniteetin ja mahalaukun umpieritysjärjestelmän välillä. IL6 -174 -polymorfismi yhdistyi diffuusin tyypin mahakarsinooman riskiin mutta ei intestinaalisen tyypin karsinooman riskiin. Helikobakteeri-infektio yhdistyi pienentyneisiin HDL-kolesterolipitoisuuksiin vain potilailla, joilla oli IL6 -174 CC genotyyppi, mikä viittaa helikobakteerin kolesterolitasoille haitallisen vaikutuksen välittyvän IL6:n kautta. Nämä tulokset antavat lisätietoa helikobakteerin aiheuttamien sairauksien mekanismeista ja avaavat uusia tutkimuspolkuja myös mahahaavan, mahasyövän ja rasva-aineenvaihdunnan häiriöiden kliiniseen tutkimukseen.

DNA repair processes are crucial for mutation avoidance and the maintenance of genetic integrity in all organisms. Organisms rely on repair processes to combat genotoxic stress imposed by hostile host environment, and sometimes by therapeutic agents. Most pathogens rapidly generate genetic variability to acquire increased virulence and evade host immune response. Therefore, there needs to exist a fine balance between mutation avoidance and fixation, which is perhaps regulated by repair processes. Haemophilus influenzae and Helicobacter pylori contribute significantly to morbidity and mortality caused by bacteria worldwide. H. influenzae is an obligate commensal of upper respiratory tract with the potential to cause a variety of diseases in humans like meningitis and respiratory infections. H. pylori, which inhabits the human stomach, is associated with gastric and duodenal ulcers and cancerous gastric lesions. One of the striking differences between these two genetically diverse bacterial species is the absence of recognized DNA mismatch repair (MMR) pathway homologs in H. pylori. MMR is a highly conserved post-replicative process, which corrects base pairing mismatches and small loops arising during DNA replication and recombination due to misincorporated nucleotides, insertions, and deletions. Defective MMR results in increased mutation frequency that can alter the pathogenic potential and antibiotic resistance of pathogens. MMR has been extensively studied in Escherichia coli, and requires an orchestrated function of different proteins like MutS, MutL, MutH, UvrD, SSB, RecJ, ExoVII, ExoI, ExoX, beta-clamp, DNA polymerase III and DNA ligase. A growing body of evidence suggests that bacteria other than the well-characterized E. coli paradigm differ in basic DNA repair machinery. MMR proteins involved in mismatch recognition and strand discrimination like MutS, MutL and MutH from H. influenzae have been characterized, but other downstream repair genes like UvrD helicase and exonucleases like RecJ have not been studied functionally in detail. H. pylori harbors a UvrD homolog, which shares limited homology with other UvrD proteins (29% identity with E. coli UvrD and 31 % with H. influenzae UvrD) and its cellular functions are not clear. Moreover, it is not well-understood how the activities of UvrD and RecJ proteins are regulated within these pathogens. It was, therefore, envisaged that biochemical characterization of UvrD and RecJ would lead to a better understanding of the mechanistic aspects of repair processes within these pathogens. The following sections summarize the results presented in this investigation. Functional characterization of UvrD from H. influenzae UvrD or DNA helicase II is a member of superfamily I of DNA helicases with well-documented roles in nucleotide excision repair (NER) and MMR, in addition to roles in replication and recombination. The 727-amino acid H. influenzae Rd KW20 UvrD (HiUvrD) protein was purified as an N-terminal (His)6-tagged protein to near homogeneity, and its authenticity was confirmed by peptide mass fingerprint analysis. HiUvrD displayed robust binding with single-stranded (ss) DNA as compared to double-stranded (ds) DNA. HiUvrD was found exhibit ~ 1000-fold higher affinity for ssDNA as compared to dsDNA as determined by surface plasmon resonance (SPR). In addition, to gain insights into the role of HiUvrD in replication, repair, recombination and transcription, the ability of HiUvrD to bind different DNA structures resembling intermediates of these processes was investigated using electrophoretic mobility shift assays. HiUvrD exhibited relatively high affinities for a number of branched DNA substrates and the order of affinity observed was; splayed-duplex ≥3’-flap ≥ ssDNA > 3’-overhang > four-way junction > three-way junction > nicked duplex > looped duplex ≥ duplex. Concurrent with its high affinity for ssDNA, HiUvrD exhibited a robust ssDNA-specific and Mg2+ - dependent ATPase activity. HiUvrD was able to unwind different DNA structures with varying efficiencies (3’ flap ≥ 3’-overhang > three-way junction > splayed-duplex > four-way junction > nicked > loop = duplex >>> 5’-overhang) and with a 3’-5’ polarity, which underpins its role in replication fork reversal, recombination and different DNA repair pathways. Multiple sequence alignment of HiUvrD with other helicases showed the presence highly conserved helicase motifs of which motif I and II are essential for ATP binding and hydrolysis. Mutation of an invariant glutamate residue (E226Q) in motif II of HiUvrD resulted in a dominant negative growth phenotype since, it was not possible to recover transformants when wild-type E. coli expression strains BL21(DE3)plysS or BL21(DE3)plysE were transformed with expression vector carrying hiuvrDE226Q. Mutation of a conserved arginine residue to alanine (R288A) in motif IV resulted in approximately 80 % reduction in ATP hydrolysis, and abrogation of helicase activity as compared to the wild-type protein. This can be attributed to ~ 70 % reduced ATP binding by HiUvrDR288A as determined by UV-crosslinking of radioactive ATP without change in affinity for ssDNA. HiUvrD was found to exist predominantly as a monomer with small amounts (~ 2-3 %) of higher oligomers like dimers and tetramers in solution. Deletion of 48 amino acid residues from distal C-terminus of HiUvrD resulted in abrogation of the oligomeric species implicating C-terminus to be involved in protein oligomerization. Interplay of UvrD with MutL and MutS in H. influenzae, and its modulation by ATP To investigate the effects of H. influenzae MutS (HiMutS) and MutL (HiMutL) on the helicase activity of HiUvrD, two different nicked DNA substrates were generated- a homoduplex and a heteroduplex DNA with a GT mismatch. HiMutL and HiMutS did not exhibit any helicase activity on either homoduplex or heteroduplex DNA, and unwinding of these substrates was observed only in presence of HiUvrD. In the presence of HiMutL the helicase activity of HiUvrD was stimulated on both homoduplex and heteroduplex nicked substrates whereas no significant modulation of HiUvrD ATPase activity in presence of HiMutL was observed. A much higher stimulation of unwinding of heteroduplex DNA was obtained, in presence of increasing concentrations of HiMutS. With increasing concentrations of HiMutL a progressive increase in HiUvrD mediated unwinding of the radiolabeled DNA strand was observed, which was ~ 15-fold higher than unwinding by HiUvrD alone. To investigate the effect of ATP in the stimulation of HiUvrD by HiMutL, two mutants of HiMutL–E29A (E29 is involved in ATP hydrolysis in E. coli UvrD), and D58A (D58 is essential for ATP binding in E. coli UvrD) were generated. HiMutLE29A retained only ~ 30 % of the wild-type ATPase activity, which was completely abolished in HiMutLD58A. Similar to wild-type protein, HiMutLE29A was able to stimulate HiUvrD helicase activity whereas HiMutLD58A failed to stimulate this activity. This indicated that ATP-bound form of MutL was essential for stimulation and perhaps interaction with UvrD. SPR analysis was carried out to validate and quantitate the direct protein-protein interaction between HiUvrD and HiMutL in absence or in presence of ATP, AMPPNP, and ADP. In the presence of ATP as well as AMPPNP, almost ~ 10,000-fold increase in the affinity between HiMutL and HiUvrD was observed but the same was not the case in presence of ADP. This clearly suggested that ATP binding rather than its hydrolysis promotes the interaction of MutL with UvrD. The effect of HiMutS on MutL-stimulated DNA unwinding by HiUvrD was determined using a heteroduplex nicked DNA with a GT mismatch. Interestingly, in the presence of HiMutS ~ 20-fold activation of DNA unwinding was observed, which is higher than the stimulation by HiMutL alone. The role of ATP-hydrolysis by MutS in regulation of UvrD helicase was studied by replacing wild-type protein with HiMutSE696A in the helicase assays. HiMutSE696A failed to hydrolyze ATP but was able to bind ATP with the same affinity as the wild-type protein and interacted with heteroduplex DNA with ~ 8-fold reduced affinity as compared to wild-type MutS. Intriguingly, increasing concentrations of HiMutSE696A failed to stimulate HiUvrD helicase activity in presence of HiMutL indicating that ATP hydrolysis by HiMutS is essential for stimulation of HiUvrD helicase activity post MutH-nicking during MMR. SSB, an essential component of all DNA metabolism pathways, possibly functions to stabilize the ssDNA tract generated by UvrD and exonucleases during MMR. ATPase and helicase activities of HiUvrD were inhibited by the cognate SSB protein. This inhibition could be overcome by increasing the concentration of HiUvrD helicases thus, pointing out the fact that SSB and UvrD perhaps compete with each other for ssDNA substrate. Noticeably, MutL and MutS proteins could alleviate the inhibition of HiUvrD by HiSSB. Functional characterization of UvrD from H. pylori In H. pylori, UvrD has been reported to limit homologous recombination and DNA-damage induced genomic recombinations but the protein has not been functionally studied. UvrD from H. pylori strain 26695 (HpUvrD) was over-expressed and purified as an N-terminal (His)6-tagged protein, and its authenticity was confirmed by peptide mass fingerprint analysis. HpUvrD exhibited high affinity for ssDNA as compared to dsDNA as determined by electrophoretic mobility shift assays and SPR. In addition, HpUvrD was able to bind a number of branched DNA structures (splayed duplex > ssDNA > 3’-flap > 3’overhang > three-way junction = four-way junction > loop >>> nicked ≥ duplex) suggesting its role in different DNA processing pathways. HpUvrD exhibited a Mg2+ - dependent ssDNA-specific ATPase activity, and a 3’-5’ helicase activity. HpUvrD was able to unwind different branched DNA structures with 3’-ssDNA regions like splayed duplex, 3’-overhang and 3’-flap. Blunt-ended duplex, duplexes with nick and loop as well as three-way and four-way junctions were unwound with less efficiency. Interestingly, the helicase activity of HpUvrD was supported by GTP and dGTP to almost the same level as ATP and dATP, which is in stark contrast to other characterized UvrD proteins. Moreover, HpUvrD was able to hydrolyze GTP albeit with ~ 1.5-fold reduced rate as compared to ATP. However, motifs associated with GTP binding and hydrolysis were not found in HpUvrD and it is possible that GTP binds in the same site as ATP. To investigate this possibility, helicase assay was done in the presence of ATP together with different concentrations of GMP-PNP, which is a non-hydrolysable analog of GTP, and did not support HpUvrD helicase activity. With increasing concentrations of GMP-PNP, a progressive inhibition of DNA unwinding by HpUvrD was observed suggesting that GMP-PNP could compete with ATP for a common binding site within HpUvrD. Replacement of a highly conserved glutamate residue with gluatamine (E206Q) in Walker B motif of HpUvrD resulted in ~17-fold reduced ATPase activity, and abrogation of helicase activity as compared to the wild-type protein. HpUvrDE206Q was able to bind ssDNA and ATP with comparable affinities as the wild-type protein suggesting the role of E206 in ATP hydrolysis. Like HiUvrD, HpUvrD was found to exist predominantly as a monomer in solution together with the presence of small amounts of higher oligomeric species. However, unlike HiUvrD, deletion of distal C-terminal 63 amino acids in HpUvD did not abrogate the oligomeric species suggesting that additional regions of the protein may be involved in protein oligomerization. The ATPase and helicase activities of HpUvrD were inhibited by the cognate SSB protein, and this inhibition could be overcome by increasing HpUvrD concentrations again suggesting that both UvrD and SSB proteins compete for ssDNA substrate. To investigate the role of UvrD in the physiology of H. pylori, a knock-out of hpuvrD was constructed in H. pylori strain 26695 by insertion of chloramphenicol cassette in its open reading frame. The mutant H. pylori strain 26695 obtained after disruption of hpuvrD was extremely slow growing under the normal microaerophilic conditions compared to the wild-type strain. Growth defect of H. pylori strain 26695ΔhpuvrD highlights the importance of UvrD in H. pylori cellular processes and in vitro fitness. Characterization of H. influenzae RecJ and its interaction with SSB Among the four exonucleases involved in MMR pathway, RecJ is the only known nuclease that degrades single-stranded DNA with 5’ to 3’ polarity. RecJ exonuclease plays additional important roles in base-excision repair, repair of stalled replication forks, and recombination. RecJ exonuclease from H. influenzae (HiRecJ) is a 575 amino acid protein, which harbors the characteristic motifs conserved among RecJ homologs. Due to limited solubility of HiRecJ, the protein was purified as a fusion protein with maltose binding protein (MBP). The purified protein exhibited a Mg2+ or Mn2+- dependent, and a highly processive 5’ to 3’ exonuclease activity, which is specific for ssDNA. MBP did not affect the exonuclease activity of HiRecJ. The processivity of HiRecJ was determined as ~ 700 nucleotides per binding event, using a ssDNA substrate labelled internally with 3H and at its 5’-terminus with 32P. Cd2+ inhibited the Mg2+ - dependent exonuclease activity of RecJ, which could not be overcome by increasing Mg2+ concentration. Site-directed mutagenesis of highly conserved residues in HiRecJ- D77A, D156A and H157A abolished the enzymatic activity. Interestingly, HiRecJD77A was found to interact with ssDNA with a 10-fold higher affinity than wild-type protein suggesting that this conserved aspartate residue may function to coordinate the binding of metal ion or DNA to hydrolysis of DNA. E. coli HU protein inhibited the HiRecJ exonuclease activity in a concentration-dependent manner possibly due to sequestration of ssDNA, thus making it unavailable for HiRecJ. During MMR, ssDNA tracts generated by UvrD helicase activity are most probably stabilized by SSB and hence, the in vivo substrate for RecJ would be SSB-ssDNA complex. The exonuclease activity of HiRecJ was stimulated approximately 3-fold by H. influenzae SSB (HiSSB) protein. HiSSB was able to stimulate HiRecJ exonuclease activity on a ssDNA substrate, which formed either a very strong secondary structure or on a homopolymeric ssDNA substrate, which did not form any secondary structure, suggesting that HiRecJ exonuclease was stimulated independent of the ability to HiSSB to melt secondary structures and stabilize ssDNA. Significantly, steady-state-kinetic analysis clearly showed that HiSSB increases the affinity of HiRecJ for ssDNA. H. influenzae SSBΔC and T4 gene 32 protein, a SSB homolog from bacteriophage T4, failed to enhance the HiRecJ exonuclease activity suggesting a specific functional interaction between HiSSB and HiRecJ mediated by C-terminus tail of HiSSB. More importantly, HiRecJ was found to directly associate with its cognate SSB. The C-terminus of HiSSB protein was found to be essential for this interaction. To delineate the regions of HiRecJ that interact with HiSSB, different truncated forms of HiRecJ were generated in which regions external to conserved motifs required for exonuclease activity were deleted. Different deletion mutants of HiRecJ- RecJ∆N34, RecJ∆C76 and the core catalytic domain (which contains amino acid residues 35-498) were purified as fusion proteins with MBP. HiSSB was found to interact with all the truncated forms of HiRecJ suggesting that its core-catalytic domain harbors a site for interaction with SSB. Taken together, the results presented in this study lead to a better understanding of the structure-function relationships of the UvrD helicase and RecJ exonuclease. Importantly, they provide insights into the interplay between various proteins in DNA MMR pathway. Characterization of repair proteins that are involved in multiple genome fidelity pathways is of fundamental importance to understand repair processes, more so in pathogenic bacteria wherein they regulate mutation rates, which can alter the fitness and virulence of the pathogens. Publication Sharma R., and Rao, D.N. (2009). Orchestration of Haemophilus influenzae RecJ exonuclease by interaction with single-stranded DNA-binding protein. J. Mol. Biol., 385, 1375-1396.

No description available.

L'opinion générale est que les ulcères sont rares pendant l'enfance, les lésions provoquées par Helicobacter pylori (H. pylori) ne se produisant que des décennies après l'acquisition de l'infection. L’infection par cette bactérie est en outre moins fréquente chez les enfants dans les pays développés par rapport aux adultes. Par ailleurs, l’usage chronique de médicaments gastro-toxiques est peu fréquent dans cette tranche d’âge. Cependant, plusieurs études ont montré qu’environ 1/10 des enfants référés pour des symptômes de dyspepsie en Europe et infectés par H. pylori présentent un ulcère gastrique ou duodénal, mais aussi que la fréquence de ces lésions chez les enfants non infectés n’est pas nulle.<p>Afin de déterminer la fréquence des ulcères gastriques et duodénaux et des érosions, nous avons commencé par réaliser une étude prospective avec la participation de 19 centres répartis dans 14 pays d'Europe. Tous les enfants référés pour une endoscopie haute ont été recrutés durant une brève période de 1 mois. Parmi les 694 enfants inclus, 56 (8,1%) avaient soit des ulcères (ulcère gastrique 17/56, 30% - ulcère duodénal 7/56, 13%) soit des érosions (érosions gastriques 21/56, 37% - érosions duodénales 9/56, 16% - érosions gastriques et duodénales 2/56, 4%). Cette étude a permis de confirmer que la fréquence des lésions augmente avec l’âge, les enfants atteints de lésions étant significativement plus âgés que les témoins. En effet, les lésions ont surtout été observées chez les enfants dans la deuxième décade de vie. Une infection par H. pylori était présente seulement chez 15 des 56 enfants (27%), un médicament gastro-toxique avait été utilisé chez 13/56 (23%), une maladie inflammatoire chronique de l’intestin était présente chez 7/56 (13%) et une polyarthrite juvénile chez 2/56 (4%, plus d'un facteur de risque présent dans la plupart des cas). Aucun facteur de risque n’a pu être démontré chez 24/56 enfants (43%), une proportion beaucoup plus élevée que celle initialement attendue.<p>Nous avons ensuite réalisé une étude cas-témoins prospective et multicentrique (12 centres participants). Tous les patients avec une lésion érosive ou ulcérée de la muqueuse gastroduodénale ont été inclus avec deux témoins appariés pour l’âge, le centre et la période. Sept cent trente-deux patients (244 cas dont 153 avec seulement des érosions et 91 avec un ou des ulcères, 488 témoins) ont été inclus. Les enfants qui avaient reçu un antibiotique, un inhibiteur de la pompe à proton ou un anti-H2 durant les 4 semaines précédant l’endoscopie ont été exclus de l’analyse statistique parce que ces médicaments influencent la détermination<p>7<p>du statut H. pylori et la gravité des lésions (42 cas et 98 témoins). Nos résultats montrent que, chez les enfants, l'infection à H. pylori est un facteur de risque pour les ulcères duodénaux et les érosions duodénales, mais pas pour les lésions gastriques. Le sexe masculin, la consommation d'AINS, les maladies rénales chroniques et le tabagisme sont d'autres facteurs de risque indépendants de lésions érosives ou d’ulcères gastroduodénaux. Cependant, aucun facteur de risque identifiable n’a été retrouvé dans une grande proportion d'enfants (97/202, 48.0%) ce qui confirme les résultats de notre première étude.<p>Chez les adultes également la proportion d’ulcères sans infection à H. pylori et sans prise d’AINS est en augmentation ces dernières années tout en restant plus faible que chez l’enfant. La fréquence des ulcères gastriques et duodénaux avec un diamètre d’au moins 5 mm a été comparée, dans notre centre et dans un centre d’endoscopie adulte situé dans la même région de Bruxelles, sur une période de deux ans. Ces données montrent que les ulcères sont moins fréquents chez les enfants que chez les adultes (20/1279 enfants avec endoscopie haute - 1,6% vs adultes 58/1010 - 5,7%, OR 0,30, 95%CI 0,10-0.86, p = 0,02) et surtout moins fréquemment associés à une infection par H. pylori (8/20 vs 40/58, OR 0,26, 95%CI 0,16- 0.78, p <0,0001).<p>Comme l’activation de la réponse immunitaire locale est inefficace pour éliminer l’infection par H. pylori et serait plutôt impliquée dans la pathogenèse des lésions de la muqueuse, nous avons comparé la réponse immunitaire muqueuse des lymphocytes T et les réponses naïves chez les enfants et chez les adultes infectés par H. pylori ainsi que chez des témoins non infectés appariés pour l’âge.<p>Dans une première étude, nous avons obtenu des biopsies de la muqueuse antrale chez 43 patients dyspeptiques (12 enfants, 31 adultes). Les concentrations de cytokines libérées dans le milieu de culture et la densité de cellules CD3+, CD25+ et CD69+ ont été évaluées par cytométrie en flux. Le nombre de cellules sécrétant de l’interféron-γ (IFN-γ), de l’interleukine-4 (IL-4) et de l’IL-10 a été mesuré par ELISPOT. Les données obtenues montrent que l’augmentation de la sécrétion d'IFN-γ et l’élévation du nombre de cellules secrétant de l’IFN-γ au niveau de la muqueuse antrale lors d’une infection par H. pylori sont plus faibles chez les enfants que chez les adultes.<p>8<p>Dans une seconde étude, nous avons comparé l’infiltrat inflammatoire de la muqueuse antrale dans différents groupes d’âge (moins de 8 ans, 8 à 17 ans, 18 à 55 ans) de patients successifs infectés par H. pylori et des témoins appariés pour l’âge. Nous avons montré une corrélation entre l'âge et la densité de neutrophiles, de cellules CD3+ et de CD8+, mais pas de cellules CD20+. Le recrutement des neutrophiles dans la muqueuse antrale est plus faible chez les enfants et apparaît corrélé avec une plus faible activation du facteur de transcription NF-kB (déterminé par immunohistochimie et par EMSA) dans cette même muqueuse. L’infiltrat inflammatoire et l’activation du NF-kB sont légèrement (mais non significativement) plus intenses en cas d’infection par une souche plus virulente (facteur de virulence cagA). Ces souches cagA+ sont retrouvées en proportion équivalente dans les différents groupes d’âge. Par contre, la charge bactérienne, mesurée par un score semi-quantitatif en histologie, n’influence pas l’intensité de l’infiltrat inflammatoire.<p>En conclusion :H. pylori reste un facteur étiologique majeur pour les ulcères et les érosions duodénales chez l’enfant, mais pas pour les lésions gastriques dans les pays à faible prévalence de l'infection et la proportion de lésions associées à une infection est plus faible que chez les adultes. Aucun facteur d’exposition connu ne peut être associé aux lésions endoscopiques dans la moitié des cas, ce qui justifiera des études ultérieures pour identifier d’autres causes exogènes ou endogènes à ces lésions.<p>La réponse immunitaire de l’hôte est impliquée dans la pathogenèse des lésions gastroduodénales associées à une infection par H. pylori. Or il a été démontré dans les travaux faisant l’objet de cette thèse que cette réponse immunitaire est plus faible chez l’enfant que chez l’adulte pour certains facteurs (cytokines Th1, immunité humorale, recrutement des polynucléaires et des lymphocytes au niveau muqueux, activation du facteur de transcription NF-κB). D’autres études confirment la plus faible réponse humorale et Th1, mais également Th17 ainsi qu’une activation plus intense des Treg. Les cytokines ou les voies de signalisation responsables de cette réponse immunitaire plus faible restent inconnues, ce qui ouvre la voie à d’autres investigations. / Doctorat en Sciences médicales / info:eu-repo/semantics/nonPublished

A novel probe was developed from genomic DNA of Helicobacter pylori ATCC type strain 43629. It hybridized with all 73 H. pylori clinical isolates tested but not with any of 183 non-H. pylori DNAs in dot blot hybridization. Typing tests revealed 41 different HaeIII-digestion patterns from 57 H. pylori strains tested. Based on the sequence of the probe, a nested PCR was developed that detected as little as 2 fg of H. pylori DNA or approximately equivalent to one cell. No PCR products were amplified from any of 21 non-H. pylori strains tested. Using this nested PCR, H. pylori DNA was detected in 33 of 45 (73%) saliva samples collected from patients with gastric H. pylori infection. These data suggest that the probe is useful for typing H. pylori and that the nested PCR is a valuable tool for detecting H. pylori DNA in saliva.

No description available.

Helicobacter pylori est une bactérie à Gram négatif qui colonise plus de 50% de la population humaine. Cette bactérie est l'un des pathogènes les plus présents dans la population et la colonisation se fait dans l'enfance et l'adolescence. H. pylori est responsable de l'apparition de maladies gastriques chez l'humain comme des ulcères gastriques, mais aussi des cancers gastriques. Plusieurs mécanismes contribuent aux maladies gastriques dont une infection chronique à long terme ainsi que des facteurs de virulence comme le système de sécrétion de type 4 (SST4). Le SST4 forme une seringue protéique utilisée par la bactérie pour injecter la protéine CagA dans les cellules humaines. Cette protéine a été la première protéine bactérienne classifiée comme une oncoprotéine par sa capacite à interférer et modifier de nombreuses fonctions et signaux métaboliques des cellules épithéliales gastriques. Afin d'éradiquer Helicobacter, une antibiothérapie est utilisée, cependant depuis les 10 dernières années plus de 50% des bactéries isolées de patients ont été identifiés comme étant porteuses de résistances contre aux moins un antibiotique de première ligne. L’utilisation de petites molécules organiques capables d'interférer avec les facteurs de virulence est une alternative intéressante à la thérapie aux antibiotiques. L'utilisation de ces molécules possède des avantages dont la faible pression de sélection de résistance parce qu’elles n’impactent pas des fonctions vitales des bactéries. Le SST4 de H. pylori est composé de nombreuses protéines essentielles qui pourraient être de potentielles cibles pour des molécules inhibitrices. Nous avons choisi la cible Cagα, une ATPase homologue à VirB11 de Agrobacterium tumefaciens. Cette protéine est essentielle pour l’injection de CagA. Précédemment, notre laboratoire a identifié une petite molécule nommée 1G2 qui était capable d’interagir avec Cagα et de diminuer l’induction de l’interleukine 8 produit par les cellules gastriques lors de l’infection par des souches de H. pylori possédant un SST4 fonctionnel. A partir d’une structure cristallographique de Cagα liée à 1G2 et nous avons créé des protéines Cagα avec des mutations aux site de liaison de 1G2. En utilisant la fluorimétrie différentielle à balayage (DSF) nous avons pu identifier les acides aminés qui contribuent à la liaison de 1G2 (K41, R73 et F39). Basé sur cette information nous avons utilisé la chimie médicinale pour créer une librairie de molécules dérivées de 1G2 dans le but d’identifier des inhibiteurs plus puissants. Après avoir éliminé les molécules ayant un effet toxique sur les cellules gastriques et H. pylori, nous avons sélectionné cinq molécules (1313, 1338, 2886, 2889 et 2902) qui inhibent la production d’IL-8 plus que 1G2 dans notre modèle d’infection cellulaire. Nous avons montré par DSF que les molécules interagissent toujours avec Cagα et 1338, 2889 et 2902 sont des inhibiteurs plus puissants de son activité d’ATPase. Avec le modèle d’infection, nous avons déterminé que les cinq molécules n’affectent par la présence de CagA dans le lysat de l’infection. Cependant, nous avons observé par microscopie électronique à balayage que le SST4 pilus n’était pas présent en présence des inhibiteurs. En plus, nous avons testé les effets de 1G2 sur des souches de H. pylori résistantes, à un ou plusieurs antibiotiques de première ligne, isolées de biopsie gastriques de patients. Comme dans le cas de la bactérie modèle de laboratoire, nous avons observé une diminution de l’induction des IL-8 lors de l’infection ainsi qu’une inhibition de la formation du SST4 pilus. Nous avons aussi identifié que le gène de la protéine Cagα d’une des bactéries résistantes à 1G2 (souche #3822) porte un remplacement de R73 à K ce qui pourrait expliquer la résistance à 1G2. Pour conclure, nous avons dans cette étude caractérisé le site de liaison de 1G2 à Cagα et nous avons identifié des molécules qui sont plus puissantes comme inhibiteurs que 1G2. / Helicobacter pylori is a Gram-negative bacterium that colonizes more than 50% of the human population. This bacterium is one of the most common pathogens in the population and colonization occurs in childhood and adolescence. H. pylori is implicated in the manifestation of gastric diseases in humans such as gastric ulcers and also gastric cancer. Several mechanisms are involved in the formation of gastric diseases including long-term chronic infection as well as virulence factors such as the type 4 secretion system (T4SS). The T4SS forms a protein syringe used by the bacteria to inject the protein CagA into mammalian cells. This protein is the first bacterial protein classified as an oncoprotein by its ability to interact with numerous metabolic functions of gastric epithelial cells. To eradicate Helicobacter, antibiotic therapy is used, but for the last 10 years more than 50% of the bacteria isolated from patients have been identified as carrying resistance against at least one first-line antibiotic. The use of small molecules capable of interfering with virulence factors is being studied as an alternative to antibiotic therapy. The use of these molecules has many advantages, and they may cause lower selection pressure for resistance than antibiotics. The H. pylori T4SS is composed of many essential proteins that could be potential targets for inhibitory molecules. We chose the target Cagα, an ATPase homologous to the model VirB11 from Agrobacterium tumefaciens. This protein is essential for the injection of CagA. Previously, our laboratory identified a small molecule coined 1G2 that interacts with Cagα and decreases the induction of interleukin-8 produced by gastric cells upon infection with H. pylori strains with functional T4SS. Based on a crystallographic study of Cagα bound to 1G2, we created Cagα proteins with mutations at the 1G2 binding site. Using differential scanning fluorimetry, we identified amino acids that contribute to 1G2 binding (K41, R73 and F39). Based on these observations, we used medicinal chemistry to create a library of molecules derived from 1G2 to create more potent inhibitors. After eliminating the molecules with a toxic effect on gastric cells and H. pylori growth, we selected five molecules with stronger effects than 1G2 on IL8 induction in our cell infection model (1313, 1338, 2886, 2889 and 2902). We observed by DSF that the molecules interact with Cagα and 1338, 2889 and 2902 are stronger inhibitors of the ATPase 8 activity than 1G2. With our infection model, we determined that the five molecules do not affect the presence of CagA. However, by scanning electron microscopy we observed that the T4SS pilus was not present. In addition to the tests on a laboratory model bacterium, we evaluated 1G2 on resistant strains of H. pylori isolated from gastric biopsy from patients. Similar to the laboratory model bacterium, 1G2 decreased IL-8 induction and inhibited T4SS pilus formation. We have also identified that strain #3822 that is resistant to 1G2 carries a R73 to K mutation in the Cagα gene, which could explain the 1G2 resistance. To conclude, we have here characterized the 1G2 binding site on Cagα and we created inhibitors that are more potent than 1G2.

Das magenbesiedelnde Bakterium Helicobacter pylori gehört zu den am weitesten verbreiteten Infektionserregern. Obwohl die Infektion meist lebenslang symptomlos verläuft, kann H. pylori bei einigen Menschen schwere Erkrankungen bis hin zum Magenkarzinom verursachen. Ziele dieser Arbeit waren Magenkarzinom-assoziierte Antigene für einen diagnostischen Test zu finden und Methoden zur Untersuchung von Spotkompositionen mittels MALDI-TOF/TOF Massenspektrometrie zu entwickeln. Im ersten Teil der Promotion wurden die Antigenerkennungsmuster von 30 Magenadenokarzinom- mit 30 Ulkus duodeni-Patienten mithilfe hochauflösender zweidimensionaler Immunoblots von H. pylori Lysat verglichen. Diese fokussierte Gegenüberstellung eignet sich gut für diese Fragestellung, da beide Erkrankungen von diesem Bakterium verursacht werden, aber nur sehr selten gemeinsam auftreten. Durch univariate statistische Analysen wurden 14 Magenkarzinom korrelierte Spots gefunden (p / The stomach-colonizing bacterium Helicobacter pylori is one of the most widespread infectious agents. Although infection mostly persists unnoticed, it may cause serious diseases like gastric carcinoma. Aims of this project were to find gastric carcinoma-associated antigens for a diagnostic test and to develop methods to analyze spot compositions using MALDI-TOF/TOF mass spectrometry. In the first part of this project antigen recognition patterns of 30 gastric carcinoma- and 30 duodenal ulcer- patients were compared using high-resolution two-dimensional immunoblots of H. pylori lysate. This focused comparison lent itself to this question because both diseases are caused by the bacterium but rarely occur conjointly. Utilizing univariate statistical tests 14 gastric carcinoma-associated spots were found (p