Mechanisms and molecular regulation of mammalian tooth replacement

In most non-mammalian vertebrates, such as fish and reptiles, teeth are replaced continuously. However, tooth replacement in most mammals, including human, takes place only once and further renewal is apparently inhibited. It is not known how tooth replacement is genetically regulated, and little is known on the physiological mechanism and evolutionary reduction of tooth replacement in mammals. In this study I have attempted to address these questions. In a rare human condition cleidocranial dysplasia, caused by a mutation in a Runt domain transcription factor Runx2, tooth replacement is continued. Runx2 mutant mice were used to investigate the molecular mechanisms of Runx2 function. Microarray analysis from dissected embryonic day 14 Runx2 mutant and wild type dental mesenchymes revealed many downstream targets of Runx2, which were validated using in situ hybridization and tissue culture methods. Wnt signaling inhibitor Dkk1 was identified as a candidate target, and in tissue culture conditions it was shown that Dkk1 is induced by FGF4 and this induction is Runx2 dependent. These experiments demonstrated a connection between Runx2, FGF and Wnt signaling in tooth development and possibly also in tooth replacement. The role of Wnt signaling in tooth replacement was further investigated by using a transgenic mouse model where Wnt signaling mediator β-catenin is continuously stabilized in dental epithelium. This stabilization led to activated Wnt signaling and to the formation of multiple enamel knots. In vitro and transplantation experiments were performed to examine the process of extra tooth formation. We showed that new teeth were continuously generated and that new teeth form from pre-existing teeth. A morphodynamic activator-inhibitor model was used to simulate enamel knot formation. By increasing the intrinsic production rate of the activator (β-catenin), the multiple enamel knot phenotype was reproduced by computer simulations. It was thus concluded that β-catenin acts as an upstream activator of enamel knots, closely linking Wnt signaling to the regulation of tooth renewal. As mice do not normally replace teeth, we used other model animals to investigate the physiological and genetic mechanisms of tooth replacement. Sorex araneus, the common shrew was earlier reported to have non-functional tooth replacement in all antemolar tooth positions. We showed by histological and gene expression studies that there is tooth replacement only in one position, the premolar 4 and that the deciduous tooth is diminished in size and disappears during embryogenesis without becoming functional. The growth rates of deciduous and permanent premolar 4 were measured and it was shown by competence inference that the early initiation of the replacement tooth in relation to the developmental stage of the deciduous tooth led to the inhibition of deciduous tooth morphogenesis. It was concluded that the evolutionary loss of deciduous teeth may involve the early activation of replacement teeth, which in turn suppress their predecessors. Mustela putorius furo, the ferret, has a dentition that resembles that of the human as ferrets have teeth that belong to all four tooth families, and all the antemolar teeth are replaced once. To investigate the replacement mechanism, histological serial sections from different embryonic stages were analyzed. It was noticed that tooth replacement is a process which involves the growth and detachment of the dental lamina from the lingual cervical loop of the deciduous tooth. Detachment of the deciduous tooth leads to a free successional dental lamina, which grows deeper into the mesenchyme, and later buds the replacement tooth. A careful 3D analysis of serial histological sections was performed and it was shown that replacement teeth are initiated from the successional dental lamina and not from the epithelium of the deciduous tooth. The molecular regulation of tooth replacement was studied and it was shown by examination of expression patterns of candidate regulatory genes that BMP/Wnt inhibitor Sostdc1 was strongly expressed in the buccal aspect of the dental lamina, and in the intersection between the detaching deciduous tooth and the successional dental lamina, suggesting a role for Sostdc1 in the process of detachment. Shh was expressed in the enamel knot and in the inner enamel epithelium in both generations of teeth supporting the view that the morphogenesis of both generations of teeth is regulated by similar mechanisms. In summary, histological and molecular studies on different model animals and transgenic mouse models were used to investigate tooth replacement. This thesis work has significantly contributed to the knowledge on the physiological mechanisms and molecular regulation of tooth replacement and its evolutionary suppression in mammals.

Outer Membrane Protease/Adhesin PgtE of Salmonella enterica: Role in Salmonella-Host Interaction

Salmonella enterica serovar Typhimurium is a common cause of gastroenteritis in humans and, occasionally, also causes systemic infection. During systemic infection an important characteristic of Salmonella is its ability to survive and replicate within macrophages. The outer membrane protease PgtE of S. enterica is a member of the omptin family of outer membrane aspartate proteases, which are beta-barrel proteins with five surface-exposed loops. The main goals of this study were to characterize biological substrates and pathogenesis-associated functions of PgtE and to determine the conditions where PgtE is fully active. In this study we found that PgtE requires rough lipopolysaccharide (LPS) to be functional but is sterically inhibited by the long O-antigen side chain in smooth LPS. Salmonella isolates normally are smooth with a long oligosaccharide O-antigen, and PgtE remains functionally cryptic in wild-type Salmonella cultivated in vitro. Interestingly, our results showed that due to increased expression of PgtE and to reduced length of the LPS O-antigen chains, the wild-type Salmonella expresses highly functional PgtE when isolated from mouse macrophage-like J774A.1 cells. Salmonella is thought to be continuously released from macrophages to infect new ones, and our results suggest that PgtE is functional during these transient extracellular growth phases. Six novel host protein substrates were identified for PgtE in this work. PgtE was previously known to activate human plasminogen (Plg) to plasmin, a broad-spectrum serine protease, and in this study PgtE was shown to interfere with the Plg system by inactivating the main inhibitor of plasmin, alpha2-antiplasmin. PgtE also interferes with another important proteolytic system of mammals by activating pro-matrix metalloproteinase-9 to an active gelatinase. PgtE also directly degrades gelatin, a component of extracellular matrices. PgtE also increases bacterial resistance against complement-mediated killing in human serum and enhances survival of Salmonella within murine macrophages as well as in the liver and spleen of intraperitoneally infected mice. Taken together, the results in this study suggest that PgtE is a virulence factor of Salmonella that has adapted to interfere with host proteolytic systems and to modify extracellular matrix; these features likely assist the migration of Salmonella during systemic salmonellosis.

Functional characterization of MutL homologue mismatch repair proteins and their variants

Mismatch repair (MMR) mechanisms repair DNA damage occurring during replication and recombination. To date, five human MMR genes, MSH2, MHS6, MSH3, MLH1 and PMS2 are known to be involved in the MMR function. Human MMR proteins form 3 different heterodimers: MutSα (MSH2 and MSH6) and MutSβ (MSH2 and MSH3), which are needed for mismatch recognition and binding, and MutLα (MLH1 and PMS2), which is needed for mediating interactions between MutS homologues and other MMR proteins. The other two MutL homologues, MLH3 and PMS1, have been shown to heterodimerize with MLH1. However, the heterodimers MutLγ (MLH1and MLH3) and MutLβ (MLH1 and PMS1) are able to correct mismatches only with low or no efficiency, respectively. A deficient MMR mechanism is associated with the hereditary colorectal cancer syndrome called hereditary nonpolyposis colorectal cancer (HNPCC) or Lynch syndrome. HNPCC is the most common hereditary colorectal cancer syndrome and accounts for 2-5% of all colorectal cancer cases. HNPCC-associated mutations have been found in 5 MMR genes: MLH1, MSH2, MSH6, PMS2 and MLH3. Most of the mutations have been found in MLH1 and MSH2 (~90%) and are associated with typical HNPCC, while mutations in MSH6, PMS2 and MLH3 are mainly linked to putative HNPCC families lacking the characteristics of the syndrome. More data of MLH3 mutations are needed to assess the significance of its mutations in HNPCC. In this study, were functionally characterized 51 nontruncating mutations in the MLH1, MLH3 and MSH2 genes to address their pathogenic significance and mechanism of pathogenicity. Of the 36 MLH1 mutations, 22 were deficient in more than one assay, 2 variants were impaired only in one assay, and 12 variants behaved like the wild type protein, whereas all seven MLH3 mutants functioned like the wild type protein in the assays. To further clarify the role and relevance of MLH3 in MMR, we analyzed the subcellular localization of the native MutL homologue proteins. Our immunofluorescence analyses indicated that when all the three MutL homologues are natively expressed in human cells, endogenous MLH1 and PMS2 localize in the nucleus, whereas MLH3 stays in the cytoplasm. The coexpression of MLH3 with MLH1 results in its partial nuclear localization. Only one MSH2 mutation was pathogenic in the in vitro MMR assay. Our study on MLH1 mutations could clearly distinguish nontruncating alterations with severe functional defects from those not or only slightly impaired in protein function. However, our study on MLH3 mutations suggest that MLH3 mutations per se are not sufficient to trigger MMR deficiency and the continuous nuclear localization of MLH1 and PMS2 suggest that MutLα has a major activity in MMR in vivo. Together with our functional assays, this confirms that MutLγ is a less efficient MMR complex than MutLα.

Genotype-Phenotype Relationships in Long QT Syndrome: Role of Mental Stress, Adrenergic Activity and a Common KCNH2 Polymorphism

Long QT syndrome is a congenital or acquired arrhythmic disorder which manifests as a prolonged QT-interval on the electrocardiogram and as a tendency to develop ventricular arrhythmias which can lead to sudden death. Arrhythmias often occur during intense exercise and/or emotional stress. The two most common subtypes of LQTS are LQT1, caused by mutations in the KCNQ1 gene and LQT2, caused by mutations in the KCNH2 gene. LQT1 and LQT2 patients exhibit arrhythmias in different types of situations: in LQT1 the trigger is usually vigorous exercise whereas in LQT2 arrhythmia results from the patient being startled from rest. It is not clear why trigger factors and clinical outcome differ from each other in the different LQTS subtypes. It is possible that stress hormones such as catecholamines may show different effects depending on the exact nature of the genetic defect, or sensitivity to catecholamines varies from subject to subject. Furthermore, it is possible that subtle genetic variants of putative modifier genes, including those coding for ion channels and hormone receptors, play a role as determinants of individual sensitivity to life-threatening arrhythmias. The present study was designed to identify some of these risk modifiers. It was found that LQT1 and LQT2 patients show an abnormal QT-adaptation to both mental and physical stress. Furthermore, as studied with epinephrine infusion experiments while the heart was paced and action potentials were measured from the right ventricular septum, LQT1 patients showed repolarization abnormalities which were related to their propensity to develop arrhythmia during intense, prolonged sympathetic tone, such as exercise. In LQT2 patients, this repolarization abnormality was noted already at rest corresponding to their arrhythmic episodes as a result of intense, sudden surges in adrenergic tone, such as fright or rage. A common KCNH2 polymorphism was found to affect KCNH2 channel function as demonstrated by in vitro experiments utilizing mammalian cells transfected with the KCNH2 potassium channel as well as QT-dynamics in vivo. Finally, the present study identified a common β-1-adrenergic receptor genotype that is related a shorter QT-interval in LQT1 patients. Also, it was discovered that compound homozygosity for two common β-adrenergic polymorphisms was related to the occurrence of symptoms in the LQT1 type of long QT syndrome. The studies demonstrate important genotype-phenotype differences between different LQTS subtypes and suggest that common modifier gene polymorphisms may affect cardiac repolarization in LQTS. It will be important in the future to prospectively study whether variant gene polymorphisms will assist in clinical risk profiling of LQTS patients.

ATM, RAD50 and p53 in breast cancer

Breast cancer is the most commonly occurring cancer among women, and its incidence is increasing worldwide. Positive family history is a well established risk factor for breast cancer, and it is suggested that the proportion of breast cancer that can be attributed to genetic factors may be as high as 30%. However, all the currently known breast cancer susceptibility genes are estimated to account for 20-30% of familial breast cancer, and only 5% of the total breast cancer incidence. It is thus likely that there are still other breast cancer susceptibility genes to be found. Cellular responses to DNA damage are crucial for maintaining genomic integrity and preventing the development of cancer. The genes operating in DNA damage response signaling network are thus good candidates for breast cancer susceptibility genes. The aim of this study was to evaluate the role of three DNA damage response associated genes, ATM, RAD50, and p53, in breast cancer. ATM, a gene causative for ataxia telangiectasia (A-T), has long been a strong candidate for a breast cancer susceptibility gene because of its function as a key DNA damage signal transducer. We analyzed the prevalence of known Finnish A-T related ATM mutations in large series of familial and unselected breast cancer cases from different geographical regions in Finland. Of the seven A-T related mutations, two were observed in the studied familial breast cancer patients. Additionally, a third mutation previously associated with breast cancer susceptibility was also detected. These founder mutations may be responsible for excess familial breast cancer regionally in Northern and Central Finland, but in Southern Finland our results suggest only a minor effect, if any, of any ATM genetic variants on familial breast cancer. We also screened the entire coding region of the ATM gene in 47 familial breast cancer patients from Southern Finland, and evaluated the identified variants in additional cases and controls. All the identified variants were too rare to significantly contribute to breast cancer susceptibility. However, the role of ATM in cancer development and progression was supported by the results of the immunohistochemical studies of ATM expression, as reduced ATM expression in breast carcinomas was found to correlate with tumor differentiation and hormone receptor status. Aberrant ATM expression was also a feature shared by the BRCA1/2 and the difficult-to-treat ER/PR/ERBB2-triple-negative breast carcinomas. From the clinical point of view, identification of phenotypic and genetic similarities between the BRCA1/2 and the triple-negative breast tumors could have an implication in designing novel targeted therapies to which both of these classes of breast cancer might be exceptionally sensitive. Mutations of another plausible breast cancer susceptibility gene, RAD50, were found to be very rare, and RAD50 can only be making a minor contribution to familial breast cancer predisposition in UK and Southern Finland. The Finnish founder mutation RAD50 687delT seems to be a null allele and may carry a small increased risk of breast cancer. RAD50 is not acting as a classical tumor suppressor gene, but it is possible that RAD50 haploinsufficiency is contributing to cancer. In addition to relatively rare breast cancer susceptibility alleles, common polymorphisms may also be associated with increased breast cancer risk. Furthermore, these polymorphisms may have an impact on the progression and outcome of the disease. Our results suggest no effect of the common p53 R72P polymorphism on familial breast cancer risk or breast cancer risk in the population, but R72P seems to be associated with histopathologic features of the tumors and survival of the patients; 72P homozygous genotype was an independent prognostic factor among the unselected breast cancer patients, with a two-fold increased risk of death. These results present important novel findings also with clinical significance, as codon 72 genotype could be a useful additional prognostic marker in breast cancer, especially among the subgroup of patients with wild-type p53 in their tumors.

Common and rare variants of the renin-angiotensin system and their relation to antihypertensive drug responses

Most of the diseases affecting public health, like hypertension, are multifactorial by etiology. Hypertension is influenced by genetic, life style and environmental factors. Estimation of the influence of genes to the risk of essential hypertension varies from 30 to 50%. It is plausible that in most of the cases susceptibility to hypertension is determined by the action of more than one gene. Although the exact molecular mechanism underlying essential hypertension remains obscure, several monogenic forms of hypertension have been identified. Since common genetic variations may predict, not only to susceptibility to hypertension, but also response to antihypertensive drug therapy, pharmacogenetic approaches may provide useful markers in finding relations between candidate genes and phenotypes of hypertension. The aim of this study was to identify genetic mutations and polymorphisms contributing to human hypertension, and examine their relationships to intermediate phenotypes of hypertension, such as blood pressure (BP) responses to antihypertensive drugs or biochemical laboratory values. Two groups of patients were investigated in the present study. The first group was collected from the database of patients investigated in the Hypertension Outpatient Ward, Helsinki University Central Hospital, and consisted of 399 subjects considered to have essential hypertension. Frequncies of the mutant or variant alleles were compared with those in two reference groups, healthy blood donors (n = 301) and normotensive males (n = 175). The second group of subjects with hypertension was collected prospectively. The study subjects (n=313) underwent a protocol lasting eight months, including four one-month drug treatment periods with antihypertensive medications (thiazide diuretic, β-blocker, calcium channel antagonist, and an angiotensin II receptor antagonist). BP responses and laboratory values were related to polymorphims of several candidate genes of the renin-angiotensin system (RAS). In addition, two patients with typical features of Liddle’s syndrome were screened for mutations in kidney epithelial sodium channel (ENaC) subunits. Two novel mutations causing Liddle’s syndrome were identified. The first mutation identified located in the beta-subunit of ENaC and the second mutation found located in the gamma-subunit, constituting the first identified Liddle mutation locating in the extracellular domain. This mutation showed 2-fold increase in channel activity in vitro. Three gene variants, of which two are novel, were identified in ENaC subunits. The prevalence of the variants was three times higher in hypertensive patients (9%) than in reference groups (3%). The variant carriers had increased daily urinary potassium excretion rate in relation to their renin levels compared with controls suggesting increased ENaC activity, although in vitro they did not show increased channel activity. Of the common polymorphisms of the RAS studied, angiotensin II receptor type I (AGTR1) 1166 A/C polymorphism was associated with modest changes in RAS activity. Thus, patients homozygous for the C allele tended to have increased aldosterone and decreased renin levels. In vitro functional studies using transfected HEK293 cells provided additional evidence that the AGTR1 1166 C allele may be associated with increased expression of the AGTR1. Common polymorphisms of the alpha-adducin and the RAS genes did not significantly predict BP responses to one-month monotherapies with hydroclorothiazide, bisoprolol, amlodipin, or losartan. In conclusion, two novel mutations of ENaC subunits causing Liddle’s syndrome were identified. In addition, three common ENaC polymorphisms were shown to be associated with occurrence of essential hypertension, but their exact functional and clinical consequences remain to be explored. The AGTR1 1166 C allele may modify the endocrine phenotype of hypertensive patients, when present in homozygous form. Certain widely studied polymorphisms of the ACE, angiotensinogen, AGTR1 and alpha-adducin genes did not significantly affect responses to a thiazide, β-blocker, calcium channel antagonist, and angiotensin II receptor antagonist.

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.

Probiotics in the prevention of clinical manifestations of common infectious diseases in children and in the elderly

Infectious diseases put an enormous burden on both children and the elderly in the forms of respiratory, gastrointestinal and oral infections. There is evidence suggesting that specific probiotics may be antagonistic to pathogens and may enhance the immune system, but the clinical evidence is still too sparce to make general conclusions on the disease-preventive effects of probiotics. This thesis, consisting of four independent, double-blind, placebo-controlled clinical trials, investigated whether Lactobacillus GG (LGG) or a specific probiotic combination containing LGG would reduce the risk of common infections or the prevalence of pathogens in healthy and infection-prone children and in independent and institutionalised elderly people. In healthy day-care children, the 7-month consumption of probiotic milk containing Lactobacillus GG appeared to postpone the first acute respiratory infection (ARI) by one week (p=0.03, adjusted p=0.16), and to reduce complicated infections (39% vs. 47%, p<0.05, adjusted p=0.13), as well as the need for antibiotic treatment (44% vs. 54%, p=0.03, adjusted p=0.08) and day-care absences (4.9 vs. 5.8 days, p=0.03, adjusted p=0.09) compared to the placebo milk. In infection-prone children, the 6-month consumption of a combination of four probiotic bacteria (LGG, L. rhamnosus LC705, Propionibacterium freudenreichii JS, Bifidobacterium breve 99) taken in capsules appeared to reduce recurrent ARIs (72% vs. 82%, p<0.05; adjusted p=0.06), and the effect was particularly noticeable in a subgroup of children with allergic diseases (12% vs. 33%, p=0.03), although no effect on the presence of nasopharyngeal rhinovirus or enterovirus was seen. The 5-month consumption of the same probiotic combination did not show any beneficial effects on the respiratory infections in frail, institutionalised elderly subjects. In healthy children receiving Lactobacillus GG, the reduction in complications resulted in a marginal reduction in the occurrence of acute otitis media (AOM) (31% vs. 39%, p=0.08; adjusted p=0.19), and the postponement of the first AOM episode by 12 days (p=0.04; adjusted p=0.09). However, in otitis-prone children, a probiotic combination did not reduce the occurrence of AOM or the total prevalence of common AOM pathogens (Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis), except in the case of children with allergic diseases, in whom probiotics reduced recurrent AOM episodes (0% vs. 14%, p=0.03). In addition, interaction between probiotics and bacterial carriage was seen: probiot-ics reduced AOM in children who did not carry any bacterial pathogens (63% vs. 83%), but the effect was the reverse in children carrying bacteria in the nasopharynx (74% vs 62%) (p<0.05). Long-term probiotic treatment, either LGG given in milk to healthy children for 7 months or a combination of probiotics given in capsules to institutionalised elderly subjects for 5 months, did not reduce the occurrence of acute diarrhoea. However, when the probiotic combination (LGG, L. rhamnosus LC705, Propionibacterium JS) was given in cheese to independent elderly subjects for 4 months, the oral carriage of high Candida counts was reduced in the probiotic group vs. the placebo group (21% vs. 34%, p=0.01, adjusted p=0.004). The risk of hyposalivation was also reduced in the probiotic group (p=0.05). In conclusion, probiotics appear to slightly alleviate the severity of infections by postponing their appearance, by reducing complications and the need for antimicrobial treatments. In addition, they appear to prevent recurrent infections in certain subgroups of children, such as in infection-prone children with allergic diseases. Alleviating ARI by probiotics may lead to a marginal reduction in the occurrence of AOM in healthy children but not in infection-prone children with disturbed nasopharyngeal microbiota. On the basis of these results it could be supposed that Lactobacillus GG or a specific combination containing LGG are effective against viral but not against bacterial otitis, and the mechanism is probably mediated through the stimulation of the immune system. A specific probiotic combination does not reduce respiratory infections in frail elderly subjects. Acute diarrhoea, either in children or in the elderly, is not prevented by the continuous, long-term consumption of probiotics, but the consumption of a specific probiotic combination in a food matrix is beneficial to the oral health of the elderly, through the reduction of the carriage of Candida.

Sampled longest common prefix array

When augmented with the longest common prefix (LCP) array and some other structures, the suffix array can solve many string processing problems in optimal time and space. A compressed representation of the LCP array is also one of the main building blocks in many compressed suffix tree proposals. In this paper, we describe a new compressed LCP representation: the sampled LCP array. We show that when used with a compressed suffix array (CSA), the sampled LCP array often offers better time/space trade-offs than the existing alternatives. We also show how to construct the compressed representations of the LCP array directly from a CSA

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.

Children’s Relationship to Plants among Primary School Children in Finland

There is considerable evidence that children in modern society are losing
their contact with nature and, more precisely, with green plants. Is this also the case in Finland, a northern country famous for its forests and wild nature? This study examines the relationship of 9- to 10-year-old Finnish schoolchildren with the green environment and plants. The data were gathered by a questionnaire
comprising structured and open-ended questions. The focus of the research was on two comparisons: first, on the nature and child relationship in rural and urban neighborhoods and, second, among boys and girls. Participants in the study amounted to a total of 76 children, 42 in the Helsinki suburb area and 34 in Paltamo. The results suggested that the children in rural surroundings had closer contact with nature than their urban associates. For example, the children of Paltamo reported to know the trees better, and considered human beings to be part of nature more often (100% vs. 76% of the pupils in Helsinki, P = 0,003). Similarly, the results showed that girls in the study (N = 48) were more interested in plants than boys (N = 28). For the girls, the beauty and joy of plants was important, whereas the boys appreciated plants as the source of life. After the pre-questioning, the children of Helsinki participated in an in-class horticultural intervention and 10 days later, answered a similar questionnaire again. The results of the open-ended questions revealed that equally to children in other Western countries, Finnish children may also be in danger of losing their direct contact with the natural environment. It was common to pass free time in organized sports such as hockey or football (boys), or by just walking and talking with friends (girls). Rural children told that they still built huts, pick berries, and climb trees, whereas urban children played in parks and city groves. The results suggest that it is essential to research
further the children’s own experiences if we are to understand, and subsequently, to enhance, the crucial role of the green environment in their lives. Horticultural interventions can be effective starting points to add to children’s knowledge, affection, and interest toward greenery, but it is highly recommended that they take place outdoors rather than indoors.

Children’s Relationship to Plants among Primary School Children in Finland

There is considerable evidence that children in modern society are losing
their contact with nature and, more precisely, with green plants. Is this also the case in Finland, a northern country famous for its forests and wild nature? This study examines the relationship of 9- to 10-year-old Finnish schoolchildren with the green environment and plants. The data were gathered by a questionnaire
comprising structured and open-ended questions. The focus of the research was on two comparisons: first, on the nature and child relationship in rural and urban neighborhoods and, second, among boys and girls. Participants in the study amounted to a total of 76 children, 42 in the Helsinki suburb area and 34 in Paltamo. The results suggested that the children in rural surroundings had closer contact with nature than their urban associates. For example, the children of Paltamo reported to know the trees better, and considered human beings to be part of nature more often (100% vs. 76% of the pupils in Helsinki, P = 0,003). Similarly, the results showed that girls in the study (N = 48) were more interested in plants than boys (N = 28). For the girls, the beauty and joy of plants was important, whereas the boys appreciated plants as the source of life. After the pre-questioning, the children of Helsinki participated in an in-class horticultural intervention and 10 days later, answered a similar questionnaire again. The results of the open-ended questions revealed that equally to children in other Western countries, Finnish children may also be in danger of losing their direct contact with the natural environment. It was common to pass free time in organized sports such as hockey or football (boys), or by just walking and talking with friends (girls). Rural children told that they still built huts, pick berries, and climb trees, whereas urban children played in parks and city groves. The results suggest that it is essential to research
further the children’s own experiences if we are to understand, and subsequently, to enhance, the crucial role of the green environment in their lives. Horticultural interventions can be effective starting points to add to children’s knowledge, affection, and interest toward greenery, but it is highly recommended that they take place outdoors rather than indoors.

Children’s Relationship to Plants among Primary School Children in Finland

There is considerable evidence that children in modern society are losing
their contact with nature and, more precisely, with green plants. Is this also the case in Finland, a northern country famous for its forests and wild nature? This study examines the relationship of 9- to 10-year-old Finnish schoolchildren with the green environment and plants. The data were gathered by a questionnaire
comprising structured and open-ended questions. The focus of the research was on two comparisons: first, on the nature and child relationship in rural and urban neighborhoods and, second, among boys and girls. Participants in the study amounted to a total of 76 children, 42 in the Helsinki suburb area and 34 in Paltamo. The results suggested that the children in rural surroundings had closer contact with nature than their urban associates. For example, the children of Paltamo reported to know the trees better, and considered human beings to be part of nature more often (100% vs. 76% of the pupils in Helsinki, P = 0,003). Similarly, the results showed that girls in the study (N = 48) were more interested in plants than boys (N = 28). For the girls, the beauty and joy of plants was important, whereas the boys appreciated plants as the source of life. After the pre-questioning, the children of Helsinki participated in an in-class horticultural intervention and 10 days later, answered a similar questionnaire again. The results of the open-ended questions revealed that equally to children in other Western countries, Finnish children may also be in danger of losing their direct contact with the natural environment. It was common to pass free time in organized sports such as hockey or football (boys), or by just walking and talking with friends (girls). Rural children told that they still built huts, pick berries, and climb trees, whereas urban children played in parks and city groves. The results suggest that it is essential to research
further the children’s own experiences if we are to understand, and subsequently, to enhance, the crucial role of the green environment in their lives. Horticultural interventions can be effective starting points to add to children’s knowledge, affection, and interest toward greenery, but it is highly recommended that they take place outdoors rather than indoors.

Content of zinc, iron and their absorption inhibitors in Nicaraguan common beans (Phaseolus vulgaris L.).

Protein-energy malnutrition and mineral deficiencies are two of the three forms of nutritional deficiencies that affect most developing countries due to inadequate access to food and diets based on a sole crop. Common bean (Phaseolus vulgaris L.) is the staple crop of Nicaragua and it has the potential to improve the nutritional status of the poorest group of the nation. Its high content of both protein and nonhaem iron provides many nutrients, but inhibitors also may prevent absorption of iron and zinc by the human consumer. A proper production chain must be followed to ensure the best grain quality for the consumer. To achieve food security, both production and high nutritional content must be maintained. Four nationally important accessions of common bean, with different harvesting dates, were selected to be submitted to two treatments: to evaluate the impact of storage conditions on the end quality of the grain. The duration of the study was six months with sampling every six weeks, and the two treatments were controlled one stored at 40°C and 75 RH %, and the other was stored in in-situ conditions. Proximate and mineral composition was evaluated as well as tannin, phytate and bioavailability. Significant differences among different accessions were found, being the most significant in protein, Fe and Zn content, tannins and phytate. Protein values ranged from 21-23%. Iron content was 61-81 mg/kg but only 3-4% was bioavailable. Zinc content was 21-25 mg/kg and 10-12% was bioavailable. The concentration of phytate ranged from 8.6-9.6 mg/g while tannin values ranged within 37.7-43.8 mg/g. Storage at high temperatures was demonstrated to have an impact on certain nutritional compounds and proved detrimental to final grain quality. Soluble sugar content and tannin content decreased after six months in both storage conditions, IDF decreased in the in-situ and SDF in the stress. The iron content and bioavailability in INTA Biofortificado were not as outstanding as expected, so experiments should be conducted to compare its iron uptake and delivery with other cultivars.