Molecular Genetic Studies of Melanocortin Receptors in Morbid Obesity

The prevalence of obesity is increasing at an alarming rate in all age groups worldwide. Obesity is a serious health problem due to increased risk of morbidity and mortality. Although environmental factors play a major role in the development of obesity, the identification of rare monogenic defects in human genes have confirmed that obesity has a strong genetic component. Mutations have been identified in genes encoding proteins of the leptin-melanocortin signaling system, which has an important role in the regulation of appetite and energy balance. The present study aimed at identifying mutations and genetic variations in the melanocortin receptors 2-5 and other genes active on the same signaling pathway accounting for severe early-onset obesity in children and morbid obesity in adults. The main achievement of this thesis was the identification of melanocortin-4 receptor (MC4R) mutations in Finnish patients. Six pathogenic MC4R mutations (308delT, P299H, two S127L and two -439delGC mutations) were identified, corresponding to a prevalence of 3% in severe early-onset obesity. No obesity causing MC4R mutations were found among patients with adult-onset morbid obesity. The MC4R 308delT deletion is predicted to result in a grossly truncated nonfunctional receptor of only 107 amino acids. The C-terminal residues, which are important in MC4R cell surface targeting, are totally absent from the mutant 308delT receptor. In vitro functional studies supported a pathogenic role for the S127L mutation since agonist induced signaling of the receptor was impaired. Cell membrane localization of the S127L receptor did not differ from that of the wild-type receptor, confirming that impaired function of the S127L receptor was due to reduced signaling properties. The P299H mutation leads to intracellular retention of the receptor. The -439delGC deletion is situated at a potential nescient helix-loop-helix 2 (NHLH2) -binding site in the MC4R promoter. It was demonstrated that the transcription factor NHLH2 binds to the consensus sequence at the -439delGC site in vitro, possibly resulting in altered promoter activity. Several genetic variants were identified in the melanocortin-3 receptor (MC3R) and pro-opiomelanocortin (POMC) genes. These polymorphisms do not explain morbid obesity, but the results indicate that some of these genetic variations may be modifying factors in obesity, resulting in subtle changes in obesity-related traits. A risk haplotype for obesity was identified in the ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1) gene through a candidate gene single nucleotide polymorphism (SNP) genotyping approach. An ENPP1 haplotype, composed of SNPs rs1800949 and rs943003, was shown to be significantly associated with morbid obesity in adults. Accordingly, the MC3R, POMC and ENPP1 genes represent examples of susceptibility genes in which genetic variants predispose to obesity. In conclusion, pathogenic mutations in the MC4R gene were shown to account for 3% of cases with severe early-onset obesity in Finland. This is in line with results from other populations demonstrating that mutations in the MC4R gene underlie 1-6% of morbid obesity worldwide. MC4R deficiency thus represents the most common monogenic defect causing human obesity reported so far. The severity of the MC4-receptor defect appears to be associated with time of onset and the degree of obesity. Classification of MC4R mutations may provide a useful tool when predicting the outcome of the disease. In addition, several other genetic variants conferring susceptibility to obesity were detected in the MC3R, MC4R, POMC and ENPP1 genes.

Structural Studies on the Second Mycobacterium smegmatis Dps: Invariant and Variable Features of Structure, Assembly and Function

A second DNA binding protein from stationary-phase cells of Mycobacterium smegmatis (MsDps2) has been identified from the bacterial genome. It was cloned, expressed and characterised and its crystal structure was determined. The core dodecameric structure of MsDps2 is the same as that of the Dps from the organism described earlier (MsDps1). However, MsDps2 possesses a long N-terminal tail instead of the C-terminal tail in MsDps1. This tail appears to be involved in DNA binding. It is also intimately involved in stabilizing the dodecamer. Partly on account of this factor, MsDps2 assembles straightway into the dodecamer, while MsDps1 does so on incubation after going through an intermediate trimeric stage. The ferroxidation centre is similar in the two proteins, while the pores leading to it exhibit some difference. The mode of sequestration of DNA in the crystalline array of molecules, as evidenced by the crystal structures, appears to be different in MsDps1 and MsDps2, highlighting the variability in the mode of Dps–DNA complexation. A sequence search led to the identification of 300 Dps molecules in bacteria with known genome sequences. Fifty bacteria contain two or more types of Dps molecules each, while 195 contain only one type. Some bacteria, notably some pathogenic ones, do not contain Dps. A sequence signature for Dps could also be derived from the analysis.

Data integration from two microarray platforms identifies bi-allelic genetic inactivation of RIC8A in a breast cancer cell line

Background: Using array comparative genomic hybridization (aCGH), a large number of deleted genomic regions have been identified in human cancers. However, subsequent efforts to identify target genes selected for inactivation in these regions have often been challenging. Methods: We integrated here genome-wide copy number data with gene expression data and non-sense mediated mRNA decay rates in breast cancer cell lines to prioritize gene candidates that are likely to be tumour suppressor genes inactivated by bi-allelic genetic events. The candidates were sequenced to identify potential mutations. Results: This integrated genomic approach led to the identification of RIC8A at 11p15 as a putative candidate target gene for the genomic deletion in the ZR-75-1 breast cancer cell line. We identified a truncating mutation in this cell line, leading to loss of expression and rapid decay of the transcript. We screened 127 breast cancers for RIC8A mutations, but did not find any pathogenic mutations. No promoter hypermethylation in these tumours was detected either. However, analysis of gene expression data from breast tumours identified a small group of aggressive tumours that displayed low levels of RIC8A transcripts. qRT-PCR analysis of 38 breast tumours showed a strong association between low RIC8A expression and the presence of TP53 mutations (P = 0.006). Conclusion: We demonstrate a data integration strategy leading to the identification of RIC8A as a gene undergoing a classical double-hit genetic inactivation in a breast cancer cell line, as well as in vivo evidence of loss of RIC8A expression in a subgroup of aggressive TP53 mutant breast cancers.

Streptomyces scabies and S. turgidiscabies as pathogens causing potato common scab, and potential methods for their control

Tieteellinen tiivistelmä Common scab is one of the most important soil-borne diseases of potato (Solanum tuberosum L.) in many potato production areas. It is caused by a number of Streptomyces species, in Finland the causal agents are Streptomyces scabies (Thaxter) Lambert & Loria and S. turgidiscabies Takeuchi. The scab-causing Streptomyces spp. are well-adapted, successful plant pathogens that survive in soil also as saprophytes. Control of these pathogens has proved to be difficult. Most of the methods used to manage potato common scab are aimed at controlling S. scabies, the most common of the scab-causing pathogens. The studies in this thesis investigated S. scabies and S. turgidiscabies as causal organisms of common scab and explored new approaches for control of common scab that would be effective against both species. S. scabies and S. turgidiscabies are known to co-occur in the same fields and in the same tuber lesions in Finland. The present study showed that both these pathogens cause similar symptoms on potato tubers, and the types of symptoms varied depending on cultivar rather than the pathogen species. Pathogenic strains of S. turgidiscabies were antagonistic to S. scabies in vitro indicating that these two species may be competing for the same ecological niche. In addition, strains of S. turgidiscabies were highly virulent in potato and they tolerated lower pH than those of S. scabies. Taken together these results suggest that S. turgidiscabies has become a major problem in potato production in Finland. The bacterial phytotoxins, thaxtomins, are produced by the scab-causing Streptomyces spp. and are essential for the induction of scab symptoms. In this study, thaxtomins were produced in vitro and four thaxtomin compounds isolated and characterized. All four thaxtomins induced similar symptoms of reduced root and shoot growth, root swelling or necrosis on micro-propagated potato seedlings. The main phytotoxin, thaxtomin A, was used as a selective agent in a bioassay in vitro to screen F1 potato progeny from a single cross. Tolerance to thaxtomin A in vitro and scab resistance in the field were correlated indicating that the in vitro bioassay could be used in the early stages of a resistance breeding program to discard scab-susceptible genotypes and elevate the overall levels of common scab resistance in potato breeding populations. The potential for biological control of S. scabies and S. turgidiscabies using a non-pathogenic Streptomyces strain (346) isolated from a scab lesion and S. griseoviridis strain (K61) from a commercially available biocontrol product was studied. Both strains showed antagonistic activity against S. scabies and S. turgidiscabies in vitro and suppressed the development of common scab disease caused by S. turgidiscabies in the glasshouse. Furthermore, strain 346 reduced the incidence of S. turgidiscabies in scab lesions on potato tubers in the field. These results demonstrated for the first time the potential for biological control of S. turgidiscabies in the glasshouse and under field conditions and may be applied to enhance control of common scab in the future.

Detection,amplification and sequence homology of sodC in clinical isolates of Salmonella sp

Background & objectives: Periplasmic copper and zinc superoxide dismutase (Cu,Zn-SOD or SodC) is an important component of the antioxidant shield which protects bacteria from the phagocytic oxidative burst. Cu,Zn-SODs protect Gram-negative bacteria against oxygen damage which have also been shown to contribute to the pathogenicity of these bacterial species. We report the presence of SodC in drug resistant Salmonella sp. isolated from patients suffering from enteric fever. Further sodC was amplified, cloned into Escherichia coli and the nucleotide sequence and amino acid sequence homology were compared with the standard strain Salmonella Typhimurium 14028. Methods: Salmonella enterica serovar Typhi (S. Typhi) and Salmonellaenterica serovar Paratyphi (S. Paratyphi) were isolated and identified from blood samples of the patients. The isolates were screened for the presence of Cu, Zn-SOD by PAGE using KCN as inhibitor of Cu,Zn-SOD. The gene (sodC) was amplified by PCR, cloned and sequenced. The nucleotide and amino acid sequences of sodC were compared using CLUSTAL X.Results: SodC was detected in 35 per cent of the Salmonella isolates. Amplification of the genomic DNA of S. Typhi and S. Paratyphi with sodC specific primers resulted in 519 and 515 bp amplicons respectively. Single mutational difference at position 489 was observed between thesodC of S. Typhi and S. Paratyphi while they differed at 6 positions with the sodC of S. Typhimurium 14028. The SodC amino acid sequences of the two isolates were homologous but 3 amino acid difference was observed with that of standard strain S. Typhimurium 14028.Interpretation & conclusions: The presence of SodC in pathogenic bacteria could be a novel candidate as phylogenetic marker.

Structural studies on members of the picornavirus superfamily

The pathogenic members of the picornavirus superfamily have adverse effects on humans, their crops and their livestock. As structure is related to function, detailed structural studies on these viruses are important not only for fundamental understanding of the viral life cycle, but also for the rational design of vaccines and inhibitors for disease control. These viruses have positive sense, single-stranded RNA genomes enclosed in a protein capsid. X-ray crystallography and cryo-electron microscopy studies have revealed that the isometric members of this group have icosahedrally-symmetric capsids made up of 60 copies of each of the structural proteins. The members that infect animal cells often employ one or more cellular receptors to facilitate cell entry which in some cases is known to initiate the uncoating sequence of the genome. The nature of the interactions between individual viruses and alternative cellular receptors has rarely been probed. The capsid assembly of the members of the picornavirus superfamily is considered to be cooperative and the interactions of RNA and capsid proteins are thought to play an important role in orchestrating virus assembly. The major aims of this thesis were to solve the structures of blackcurrant reversion virus (BRV), human parechovirus 1 (HPEV1) and coxsackievirus A7 (CAV7), as well as the structure of HPEV1 complexed with two of its cellular receptors using cryo-electron microscopy, three-dimensional image reconstruction and homology modeling. Each of the selected viruses represents a taxonomic group where little or no structural data was previously available. The results enabled the detailed comparison of the new structures to those of known picornaviruses, the identification of surface-exposed epitopes potentially important for host interaction, the mapping of RNA-capsid protein interactions and the elucidation of the basis for the specificity of two different receptor molecules for the same capsid. This work will form the basis for further studies on the influence of RNA on parechovirus assembly as a potential target for drug design.

Finnish cattle as reservoir of Campylobacter spp.

The reported incidence of human campylobacteriosis in Finland is higher than in most other European countries. A high annual percentage of sporadic infections is of foreign origin, although a notable proportion of summer infections is domestically acquired. While chickens appear to be a major source of campylobacters for humans in most countries, the prevalence of campylobacters is very low in chicken slaughter batches in Finland. Data on other potential animal reservoirs of human pathogenic campylobacters in Finland are scarce. Consequently, this study aimed to investigate the status of Finnish cattle as a potential source of thermophilic Campylobacter spp. and antibiotic-resistant Campylobacter jejuni for human sporadic campylobacter infections of domestic origin. A survey of the prevalence of thermophilic Campylobacter spp. in Finnish cattle studied bovine rectal faecal samples (n=952) and carcass surface samples (n=948) from twelve Finnish slaughterhouses from January to December 2003. The total prevalence of Campylobacter spp. in faecal samples was 31.1%, and in carcass samples 3.5%. Campylobacter jejuni, the most common species, was present in 19.5% of faecal samples and in 3.1% of carcasses. In addition to thermophilic Campylobacter spp., C. hyointestinalis ssp. hyointestinalis was present in bovine samples. The prevalence of campylobacters was higher among beef cattle than among dairy cattle. Using the enrichment method, the number of positive faecal samples was 7.5 times higher than that obtained by direct plating. The predominant serotypes of faecal C. jejuni, determined by serotyping with a set of 25 commercial antisera for heat-stable antigens (Penner), were Pen2 and Pen4-complex, which covered 52% of the samples. Genotyping with pulsed-field gel electrophoresis (PFGE) using SmaI restriction yielded a high diversity of C. jejuni subtypes in cattle. Determining the minimum inhibitory concentrations of ampicillin, enrofloxacin, erythromycin, gentamicin, nalidixic acid, and oxytetracycline among bovine C. jejuni isolates using a commercial broth microdilution method yielded 9% of isolates resistant to at least one of the antimicrobials examined. No multiresistant isolates were found among the bovine C. jejuni strains. The study of the shedding patterns of Campylobacter spp. among three Finnish dairy cattle herds included the examination of fresh faecal samples and tank milk samples taken five times, as well as samples from drinking troughs taken once during the one-year study. The semiquantitative enrichment method detected C. jejuni in 169 of the 340 faecal samples, mostly at low levels. In addition, C. jejuni was present in one drinking trough sample. The prevalence between herds and sampling occasions varied widely. PFGE, using SmaI as restriction enzyme, identified only a few subtypes in each herd. In two 2 of the herds, two subtypes persisted throughout the sampling. Individual animals presented various shedding patterns during the study. Comparison of C. jejuni isolates from humans, chickens and cattle included the design of primers for four new genetic markers selected from completely sequenced C. jejuni genomes 81-176, RM1221 and NCTC 11168, and the PCR examination of domestic human isolates from southern Finland in 1996, 2002 and 2003 (n=309), chicken isolates from 2003, 2006 and 2007 (n=205), and bovine isolates from 2003 (n=131). The results revealed that bovine isolates differed significantly from human and chicken isolates. In particular, the - glutamyl transpeptidase gene was uncommon among bovine isolates. The PFGE genotyping of C. jejuni isolates, using SmaI and KpnI restriction enzymes, included a geographically representative collection of isolates from domestic sporadic human infections, chicken slaughter batches, and cattle faeces and carcasses during the seasonal peak of campylobacteriosis in the summer of 2003. The study determined that 55.4% of human isolates were indistinguishable from those of chickens and cattle. Temporal association between isolates from humans and chickens was possible in 31.4% of human infections. Approximately 19% of the human infections may have been associated with cattle. However, isolates from bovine carcasses and human cases represented different PFGE subtypes. In conclusion, this study suggests that Finnish cattle is a notable reservoir of C. jejuni, the most important Campylobacter sp. in human enteric infections. Although the concentration of these organisms in bovine faeces appeared to be low, excretion can be persistent. The genetic diversity and presence or absence of marker genes support previous suggestions of host-adapted C. jejuni strains, and may indicate variations in virulence between strains from different hosts. In addition to chickens, Finnish cattle appeared to be an important reservoir and possible source of C. jejuni in domestic sporadic human infections. However, sources of campylobacters may differ between rural and urban areas in Finland, and in general, the transmission of C. jejuni of bovine origin probably occurs via other routes than food.

Diversity of DNA methyltransferases that recognize asymmetric target sequences

DNA methyltransferases (MTases) are a group of enzymes that catalyze the methyl group transfer from S-adenosyl-L-methionine in a sequence-specific manner. Orthodox Type II DNA MTases usually recognize palindromic DNA sequences and add a methyl group to the target base (either adenine or cytosine) on both strands. However, there are a number of MTases that recognize asymmetric target sequences and differ in their subunit organization. In a bacterial cell, after each round of replication, the substrate for any MTase is hemimethylated DNA, and it therefore needs only a single methylation event to restore the fully methylated state. This is in consistent with the fact that most of the DNA MTases studied exist as monomers in solution. Multiple lines of evidence suggest that some DNA MTases function as dimers. Further, functional analysis of many restriction-modification systems showed the presence of more than one or fused MTase genes. It was proposed that presence of two MTases responsible for the recognition and methylation of asymmetric sequences would protect the nascent strands generated during DNA replication from cognate restriction endonuclease. In this review, MTases recognizing asymmetric sequences have been grouped into different subgroups based on their unique properties. Detailed characterization of these unusual MTases would help in better understanding of their specific biological roles and mechanisms of action. The rapid progress made by the genome sequencing of bacteria and archaea may accelerate the identification and study of species- and strain-specific MTases of host-adapted bacteria and their roles in pathogenic mechanisms.

The effects of abiotic and biotic factors on somatic embryogenesis and seedlings of Pinus sylvestris (L.)

Somatic embryogenesis (SE) is an asexual form of plant propagation that occurs in nature and mimics many of the events of sexual reproduction. Pinus sylvestris (L.) is an important source of timber in Northern Eurasia but it is recalcitrant to somatic embryogenesis. Several factors important for the success of the P. sylvestris embryogenic cultures have not been thoroughly investigated. In this study, we examined the effects of parental genotypes on the SE in P. sylvestris, the involvement of the gaseous plant growth regulator, ethylene in SE, and also biotic effects on somatic embryos as well as on seedlings. We tested parental effects on immature embryo initiation for different media, storage periods, and on the maturation process. Maternal effects were found to be crucial for SE in the absence of paternal effects. No maternal-paternal interaction was observed at any stage of somatic embryo production. Additionally the role of ethylene at different developmental stages of SE was investigated. Two ACC synthase genes, PsACS1 and PsACS2, were isolated and characterized. PsACS1 was expressed during the proliferation stage in all tested genotypes, whereas PsACS2 was only expressed in somatic embryos of each genotype. Ethylene production in embryos at stage 3 was significantly higher than the other stages. In a parallel study, the response of somatic embryos to fungal elicitors was investigated. Three fungi, a mutualistic ectomycorrhizal (ECM) fungus (Suillus bovinus), a weak Scots pine pathogen (Heterobasidion parviporum) and a strong pathogen (H. annosum) were used. The gene expression patterns for embryos exposed to the H. parviporum elicitor were found to be similar to that documented for S. bovinus among the tested genes. By contrast somatic embryos exposed to the H. annosum elicitor had a different pattern of regulation which was marked by a delayed response, and in some cases death of the embryos. Furthermore, interaction without direct contact between P. sylvestris seedlings and microbes (mutualistic and pathogenic fungus, cyanobacterium) were investigated. Several novel genes expressed in seedlings treated with ECM fungus were isolated which suggested that physical contact is not necessary for elicitation of host responses. The results suggest that somatic embryos and seedlings of P. sylvestris are genetically well equipped to respond to fungal elicitor/exudates and could serve as a suitable model for reproducible molecular studies in conifer tree patho- and symbiotic systems.

X-ray Crystallographic Analysis of the Complexes of Enoyl Acyl Carrier Protein Reductase of Plasmodium falciparum with Triclosan Variants to Elucidate the Importance of Different Functional Groups in Enzyme Inhibition

Triclosan, a well-known inhibitor of Enoyl Acyl Carrier Protein Reductase (ENR) from several pathogenic organisms, is a promising lead compound to design effective drugs. We have solved the X-ray crystal structures of Plasmodium falciparum ENR in complex with triclosan variants having different substituted and unsubstituted groups at different key functional locations. The structures revealed that 4 and 2' substituted compounds have more interactions with the protein, cofactor, and solvents when compared with triclosan. New water molecules were found to interact with some of these inhibitors. Substitution at the 2' position of triclosan caused the relocation of a conserved water molecule, leading to an additional hydrogen bond with the inhibitor. This observation can help in conserved water-based inhibitor design. 2' and 4' unsubstituted compounds showed a movement away from the hydrophobic pocket to compensate for the interactions made by the halogen groups of triclosan. This compound also makes additional interactions with the protein and cofactor which compensate for the lost interactions due to the unsubstitution at 2' and 4'. In cell culture, this inhibitor shows less potency, which indicates that the chlorines at 2' and 4' positions increase the ability of the inhibitor to cross multilayered membranes. This knowledge helps us to modify the different functional groups of triclosan to get more potent inhibitors. (C) 2010 IUBMB IUBMB Life, 62(6): 467-476.

Diversity of DNA methyltransferases that recognize asymmetric target sequences

DNA methyltransferases (MTases) are a group of enzymes that catalyze the methyl group transfer from S-adenosyl-L-methionine in a sequence-specific manner. Orthodox Type II DNA MTases usually recognize palindromic DNA sequences and add a methyl group to the target base (either adenine or cytosine) on both strands. However, there are a number of MTases that recognize asymmetric target sequences and differ in their subunit organization. In a bacterial cell, after each round of replication, the substrate for any MTase is hemimethylated DNA, and it therefore needs only a single methylation event to restore the fully methylated state. This is in consistent with the fact that most of the DNA MTases studied exist as monomers in solution. Multiple lines of evidence suggest that some DNA MTases function as dimers. Further, functional analysis of many restriction-modification systems showed the presence of more than one or fused MTase genes. It was proposed that presence of two MTases responsible for the recognition and methylation of asymmetric sequences would protect the nascent strands generated during DNA replication from cognate restriction endonuclease. In this review, MTases recognizing asymmetric sequences have been grouped into different subgroups based on their unique properties. Detailed characterization of these unusual MTases would help in better understanding of their specific biological roles and mechanisms of action. The rapid progress made by the genome sequencing of bacteria and archaea may accelerate the identification and study of species- and strain-specific MTases of host-adapted bacteria and their roles in pathogenic mechanisms.

Molecular characterization of sediment bacterial communities affected by fish farming

Fish farming introduces nutrients, microbes and a wide variety of chemicals such as heavy metals, antifoulants and antibiotics to the surrounding environment. Introduction of antibiotics has been linked with the increased incidence of antibiotic resistant pathogenic bacteria in the farm vicinities. In this thesis molecular methods such as quantitative PCR and DNA sequencing were applied to analyze bacterial communities in sediments from fish farms and pristine locations. Altogether four farms and four pristine sites were sampled in the Baltic Sea. Two farm and two pristine locations were sampled over a surveillance period of four years. Furthermore, a new methodology was developed as a part of the study that permits amplifying single microbial genomes and capturing them according to any genetic traits, including antibiotic resistance genes. The study revealed that several resistance genes for tetracycline were found at the sediment underneath the aquaculture farms. The copy number of these genes remained elevated even at a farm that had not used any antibiotics since year 2000, six years before this study started. Similarly, an increase in the amount of mercury resistance gene merA was observed at the aquaculture sediment. The persistence of the resistance genes in absence of any selection pressure from antibiotics or heavy metals suggests that the genes may be introduced to the sediment by the farming process. This is also supported by the diversity pattern of the merA gene between farm and pristine sediments. The bacterial community-level changes in response to fish farming were very complex and no single phylogenetic groups were found that would be typical to fish farm sediments. However, the community structures had some correlation with the exposure to fish farming. Our studies suggest that the established approaches to deal with antibiotic resistance at the aquaculture, such as antibiotic cycling, are fundamentally flawed because they cannot prevent the introduction of the resistance genes and resistant bacteria to the farm area by the farming process. Further studies are required to study the entire fish farming process to identify the sources of the resistance genes and the resistant bacteria. The results also suggest that in order to prevent major microbiological changes in the surrounding aquatic environment, the farms should not be founded in shallow water where currents do not transport sedimenting matter from the farms. Finally, the technique to amplify and select microbial genomes will potentially have a considerable impact in microbial ecology and genomics.

cAMP-regulated Protein Lysine Acetylases in Mycobacteria

Cyclic AMP synthesized by Mycobacterium tuberculosis has been shown to play a role in pathogenesis. However, the high levels of intracellularcAMP found in both pathogenic and nonpathogenic mycobacteria suggest that additional and important biological processes are regulated by characterization of novel cAMP-binding proteins in M. smegmatis and M. tuberculosis (MSMEG_5458 and Rv0998, respectively) that contain a cyclic nucleotide binding domain fused to a domain that shows similarity to the GNAT family of acetyltransferases. We detect protein lysine acetylation in mycobacteria and identify a universal stress protein (USP) as a substrate of MSMEG_5458. Acetylation of a lysine residue in USP is regulated by cAMP, and using a strain deleted for MSMEG_5458, we show that USP is indeed an in vivo substrate for MSMEG_5458. The Rv0998 protein shows a strict cAMP-dependent acetylation of USP, despite a lower affinity for cAMP than MSMEG_5458. Thus, this report not only represents the first demonstration of protein lysine acetylation in mycobacteria but also describes a unique functional interplay between a cyclic nucleotide binding domain and a protein acetyltransferase.

Real-Time PCR : A Molecular Approach to Investigate the Role of Intestinal Microbiota in the Pathophysiology of Irritable Bowel Syndrome

Irritable bowel syndrome (IBS) is a common multifactorial functional intestinal disorder, the pathogenesis of which is not completely understood. Increasing scientific evidence suggests that microbes are involved in the onset and maintenance of IBS symptoms. The microbiota of the human gastrointestinal (GI) tract constitutes a massive and complex ecosystem consisting mainly of obligate anaerobic microorganisms making the use of culture-based methods demanding and prone to misinterpretation. To overcome these drawbacks, an extensive panel of species- and group-specific assays for an accurate quantification of bacteria from fecal samples with real-time PCR was developed, optimized, and validated. As a result, the target bacteria were detectable at a minimum concentration range of approximately 10 000 bacterial genomes per gram of fecal sample, which corresponds to the sensitivity to detect 0.000001% subpopulations of the total fecal microbiota. The real-time PCR panel covering both commensal and pathogenic microorganisms was assessed to compare the intestinal microbiota of patients suffering from IBS with a healthy control group devoid of GI symptoms. Both the IBS and control groups showed considerable individual variation in gut microbiota composition. Sorting of the IBS patients according to the symptom subtypes (diarrhea, constipation, and alternating predominant type) revealed that lower amounts of Lactobacillus spp. were present in the samples of diarrhea predominant IBS patients, whereas constipation predominant IBS patients carried increased amounts of Veillonella spp. In the screening of intestinal pathogens, 17% of IBS samples tested positive for Staphylococcus aureus, whereas no positive cases were discovered among healthy controls. Furthermore, the methodology was applied to monitor the effects of a multispecies probiotic supplementation on GI microbiota of IBS sufferers. In the placebo-controlled double-blind probiotic intervention trial of IBS patients, each supplemented probiotic strain was detected in fecal samples. Intestinal microbiota remained stable during the trial, except for Bifidobacterium spp., which increased in the placebo group and decreased in the probiotic group. The combination of assays developed and applied in this thesis has an overall coverage of 300-400 known bacterial species, along with the number of yet unknown phylotypes. Hence, it provides good means for studying the intestinal microbiota, irrespective of the intestinal condition and health status. In particular, it allows screening and identification of microbes putatively associated with IBS. The alterations in the gut microbiota discovered here support the hypothesis that microbes are likely to contribute to the pathophysiology of IBS. The central question is whether the microbiota changes described represent the cause for, rather than the effect of, disturbed gut physiology. Therefore, more studies are needed to determine the role and importance of individual microbial species or groups in IBS. In addition, it is essential that the microbial alterations observed in this study will be confirmed using a larger set of IBS samples of different subtypes, preferably from various geographical locations.

Molecular and Clinical Characteristics of Pituitary Adenoma Predisposition (PAP)

Pituitary adenomas are common benign neoplasms. Although most of them are sporadic, a minority occurs in familial settings. Heterozygous germline mutations in the aryl hydrocarbon receptor interacting protein (AIP) gene were found to underlie familial pituitary adenomas, a condition designated as pituitary adenoma predisposition (PAP). PAP confers incomplete penetrance of mostly growth hormone (GH) secreting adenomas in young patients, who often lack a family history of pituitary adenomas. This thesis work aimed to clarify the molecular and clinical characteristics of PAP. Applying the multiplex ligation-dependent probe amplification assay (MLPA), we found large genomic AIP deletions to account for a subset of PAP. Therefore, MLPA could be considered in PAP suspected patients with no AIP mutations found with conventional sequencing. We generated an Aip mouse model to examine pituitary tumorigenesis in vivo. The heterozygous Aip mutation conferred complete penetrance of pituitary adenomas that were mostly GH-secreting, rendering the phenotype of the Aip mouse similar to that of PAP patients. We suggest that AIP may function as a candidate gatekeeper gene in somatotrophs. To clarify molecular mechanisms of tumorigenesis, we elucidated the expression of AIP-related molecules in human and mouse pituitary tumors. The expression of aryl hydrocarbon receptor nuclear translocator (ARNT) was reduced in mouse Aip-deficient adenomas, and similar ARNT reduction was also evident in human AIP mutation positive adenomas. This suggests that in addition to participating in the hypoxia pathway, estrogen receptor signaling and xenobiotic response pathways, ARNT may play a role in AIP-related tumorigenesis. We also studied the characteristics and the response to therapy of PAP patients and found them to have an aggressive disease phenotype with young age at onset. Therefore, improvement in treatment outcomes of PAP patients would require their efficient identification and earlier diagnosis of the pituitary adenomas. The possible role of the RET proto-oncogene in tumorigenesis of familial AIP mutation negative pituitary adenomas was evaluated, but none of the found RET germline variants were considered pathogenic. Surprisingly, RET immunohistochemistry suggested possible underexpression of RET in AIP mutation positive pituitary adenomas an observation that merits further investigation.