Effects of oral calcium sensitizers on experimental heart failure

Suun kautta annosteltava kalsiumherkistäjä parantaa sydämen vajaatoimintaan liittyvää pumppausvajetta kokeellisissa sydämen vajaatoimintamalleissa Huolimatta viime vuosikymmenien lääketieteellisestä kehityksestä krooninen sydämen vajaatoiminta on silti edelleen vakava, elämänlaatua voimakkaasti rajoittava sairaus. Kalsiumherkistäjät ovat uusi, sydämen pumppausvoimaa lisäävä lääkeryhmä. Levosimendaani, kotimaista alkuperää oleva kalsiumherkistäjä, on kliinisessä käytössä akuutin vajaatoiminnan hoitoon suonensisäisesti ja lyhytaikaisesti annosteltavana valmisteena. Levosimendaanilla on aktiivinen metaboliitti, OR-1896, jonka oletetaan olevan vuorokauden mittaisen levosimendaani-infuusion jälkeen havaittujen useita päiviä kestävien hyödyllisisten vaikutuksisten takana. Levosimendaanin kroonisen, suun kautta tapahtuvan annostelun vaikutuksista tieto on vähäisempää, mutta sillä näyttää olevan positiivisia vaikutuksia potilaiden raportoimana. FM Marjut Louhelainen on selvittänyt väitöskirjassaan suun kautta annosteltavan levosimendaanin ja sen pitkäkestoisen aktiivisen metaboliitin vaikutuksia kroonisen vajaatoiminnan hoidossa käyttämällä sekä hypertensiivisen sydäntaudin että 2 tyypin diabeteksen komplisoimaan sydäninfarktin kokeellisia malleja. Tutkimuksessa selvitettiin lisäksi vajaatoimintaan johtavia molekyylitason tapahtumia sydänlihaksessa. Tutkimuksessa osoitettiin, että krooninen suun kautta annosteltu hoito sekä kalsiumherkistäjä levosimendaanilla että sen aktiivisella metaboliitilla estää hypertensiiviseen sydämen vajaatoiminnan aikaasaamaa sydämen uudelleenmuovaantumista ja siihen liittyvää kuolleisuutta. Nämä vaikutukset välittyivät vähentyneen sydänlihassoluhypertrofian, solukuolleisuuden ja neurohumaraalisen aktivaation kautta. Levosimendaanin ja OR-1896:n osoitettiin myös parantavan sydämen pumppausfunktiota tyyppi 2 diabeteksen komplisoimassa sydäninfarktissa. Ei-diabeettiseen tilanteeseen verrattuna diabetekseen liittyvä infarktin jälkeinen vajaatoiminnan kehitys oli yhteydessä lisääntyneeseen tulehdukseen, fibroosiin, solukuolemaan, neurohumoraaliseen aktivaatioon ja ennenaikaiseen kudoksen vanhenemiseen. Sekä levosimendaani, että OR-1869 vähensivät tulehduksen, fibroosin ja solukuoleman merkkejä ja vaimensi neurohumoraalista aktivaatiota. OR-1896 myös vähensi solujen vanhenemiseen liittyvien merkkiaineiden ilmentymistä. Väitöskirjassa todettiin, että suun kautta annosteltuna sekä levosimendaani, että sen aktiivinen metaboliitti OR-1896, omaavat terapeuttista potentiaalia sekä hypertensiivisen sydäntaudin hoitoon että sydäninfarktin jälkeisen vajaatoiminnan estoon. FM Marjut Louhelaisen farmakologian alaan kuuluva väitöskirja Effects of oral calcium sensitizers on experimental heart failure tarkastetaan Helsingin yliopiston Lääketieteellisessä tiedekunnassa perjantaina 29.01.2010 klo 12 (Biomedicum Helsinki, luentosali 2, Haartmaninkatu 8, Helsinki). Vastaväittäjänä toimii professori Raimo Tuominen, Helsingin yliopiston Farmasian tiedekunnasta ja kustoksena professori Eero Mervaala Helsingin yliopiston Lääketieteellisestä tiedekunnasta.

Effects of dairy proteins and calcium on diet-induced obesity in mice

Diet high in dairy products is inversely associated with body mass index, risk of metabolic syndrome and prevalence of type 2 diabetes in several populations. Also a number of intervention studies support the role of increased dairy intake in the prevention and treatment of obesity. Dairy calcium has been suggested to account for the effect of dairy on body weight, but it has been repeatedly shown that the effect of dairy is superior to the effect of supplemental calcium. Dairy proteins are postulated to either enhance the effect of calcium or have an independent effect on body weight, but studies in the area are scarce. The aim of this study was to evaluate the potential of dairy proteins and calcium in the prevention and treatment of diet-induced obesity in C57Bl/6J mice. The effect of dairy proteins and calcium on the liver and adipose tissue was also investigated in order to characterise the potential mechanisms explaining the reduction of risk for metabolic syndrome and type 2 diabetes. A high-calcium diet (1.8%) in combination with dietary whey protein inhibited body weight and fat gain and accelerated body weight and fat loss in high-fat-fed C57Bl/6J mice during long-term studies of 14 to 21 weeks. α-lactalbumin, one of the major whey proteins, was the most effective whey protein fraction showing significantly accelerated weight and fat loss during energy restriction and reduced the amount of visceral fat gain during ad libitum feeding after weight loss. The microarray data suggest sensitisation of insulin signalling in the adipose tissue as a result of a calcium-rich whey protein diet. Lipidomic analysis revealed that weight loss on whey protein-based high-calcium diet was characterised by significant decreases in diabetogenic diacylglycerols and lipotoxic ceramide species. The calcium supplementation led to a small, but statistically significant decrease in fat absorption independent of the protein source of the diet. This augments, but does not fully explain the effects of the studied diets on body weight. A whey protein-containing high-calcium diet had a protective effect against a high-fat diet-induced decline of β3 adrenergic receptor expression in adipose tissue. In addition, a high-calcium diet with whey protein increased the adipose tissue leptin expression which is decreased in this obesity-prone mouse strain. These changes are likely to contribute to the inhibition of weight gain. The potential sensitisation of insulin signalling in adipose tissue together with the less lipotoxic and diabetogenic hepatic lipid profile suggest a novel mechanistic link to explain why increased dairy intake is associated with a lower prevalence of metabolic syndrome and type 2 diabetes in epidemiological studies. Taken together, the intake of a high-calcium diet with dairy proteins has a body weight lowering effect in high-fat-fed C57Bl/6J mice. High-calcium diets containing whey protein prevent weight gain and enhance weight loss, α-lactalbumin being the most effective whey protein fraction. Whey proteins and calcium have also beneficial effects on hepatic lipid profile and adipose tissue gene expression, which suggest a novel mechanistic link to explain the epidemiological findings on dairy intake and metabolic syndrome. The clinical relevance of these findings and the precise mechanisms of action remain an intriguing field of future research.

Oncolytic adenoviruses for treatment of ovarian cancer

Virotherapy, the use of oncolytic properties of viruses for eradication of tumor cells, is an attractive strategy for treating cancers resistant to traditional modalities. Adenoviruses can be genetically modified to selectively replicate in and destroy tumor cells through exploitation of molecular differences between normal and cancer cells. The lytic life cycle of adenoviruses results in oncolysis of infected cells and spreading of virus progeny to surrounding cells. In this study, we evaluated different strategies for improving safety and efficacy of oncolytic virotherapy against human ovarian adenocarcinoma. We examined the antitumor efficacy of Ad5/3-Δ24, a serotype 3 receptor-targeted pRb-p16 pathway-selective oncolytic adenovirus, in combination with conventional chemotherapeutic agents. We observed synergistic activity in ovarian cancer cells when Ad5/3-Δ24 was given with either gemcitabine or epirubicin, common second-line treatment options for ovarian cancer. Our results also indicate that gemcitabine reduces the initial rate of Ad5/3-Δ24 replication without affecting the total amount of virus produced. In an orthotopic murine model of peritoneally disseminated ovarian cancer, combining Ad5/3-Δ24 with either gemcitabine or epirubicin resulted in greater therapeutic benefit than either agent alone. Another useful approach for increasing the efficacy of oncolytic agents is to arm viruses with therapeutic transgenes such as genes encoding prodrug-converting enzymes. We constructed Ad5/3-Δ24-TK-GFP, an oncolytic adenovirus encoding the thymidine kinase (TK) green fluorescent protein (GFP) fusion protein. This novel virus replicated efficiently on ovarian cancer cells, which correlated with increased GFP expression. Delivery of prodrug ganciclovir (GCV) immediately after infection abrogated viral replication, which might have utility as a safety switch mechanism. Oncolytic potency in vitro was enhanced by GCV in one cell line, and the interaction was not dependent on scheduling of the treatments. However, in murine models of metastatic ovarian cancer, administration of GCV did not add therapeutic benefit to this highly potent oncolytic agent. Detection of tumor progression and virus replication with bioluminescence and fluorescence imaging provided insight into the in vivo kinetics of oncolysis in living mice. For optimizing protocols for upcoming clinical trials, we utilized orthotopic murine models of ovarian cancer to analyze the effect of dose and scheduling of intraperitoneally delivered Ad5/3-Δ24. Weekly administration of Ad5/3-Δ24 did not significantly enhance antitumor efficacy over a single treatment. Our results also demonstrate that even a single intraperitoneal injection of only 100 viral particles significantly increased the survival of mice compared with untreated animals. Improved knowledge of adenovirus biology has resulted in creation of more effective oncolytic agents. However, with more potent therapy regimens an increase in unwanted side-effects is also possible. Therefore, inhibiting viral replication when necessary would be beneficial. We evaluated the antiviral activity of chlorpromazine and apigenin on adenovirus replication and associated toxicity in fresh human liver samples, normal cells, and ovarian cancer cells. Further, human xenografts in mice were utilized to evaluate antitumor efficacy, viral replication, and liver toxicity. Our data suggest that these agents can reduce replication of adenoviruses, which could provide a safety switch in case of replication-associated side-effects. In conclusion, we demonstrate that Ad5/3-Δ24 is a useful oncolytic agent for treatment of ovarian cancer either alone or in combination with conventional chemotherapeutic drugs. Insertion of genes encoding prodrug-converting enzymes into the genome of Ad5/3-Δ24 might not lead to enhanced antitumor efficacy with this highly potent oncolytic virus. As a safety feature, viral activity can be inhibited with pharmacological substances. Clinical trials are however needed to confirm if these preclinical results can be translated into efficacy in humans. Promising safety data seen here, and in previous publications suggest that clinical evaluation of the agent is feasible.

Controlling transduction and replication of oncolytic adenoviruses

Despite progress in conventional cancer treatment regimes, metastatic disease essentially remains incurable and new treatment alternatives are needed. Virotherapy is a relatively novel approach in cancer treatment. It harnesses the natural ability of oncolytic viruses to kill the cells they proliferate in and to spread to neighboring cells, thereby amplifying the therapeutic effect of the initial input dose. The use of replicating, oncolytic viruses for cancer treatment necessitates introduction of various genetic modifications to the viral genome, thereby restraining replication exclusively to tumor cells and eventually obtaining selective eradication of the tumor without side effects to healthy tissue. Furthermore, various modifications can be applied to the viral capsid in hope of gaining effective transduction of target tissue. In other words, the entry of viruses into tumor tissue can be augmented by allowing the virus to utilize non-native receptors for entry. Genetic capsid modifications may also help to avoid some major hurdles in systemic delivery that ultimately lead to the rapid clearance of the virus from the blood and virus induced toxicity. In addition to genetic modifications that alter the phenotype of the virus, some pharmacologic agents may be utilized to enhance the virus entry to target site. Liver kupffer cells (KC) are responsible for the majority of viral clearance after systemic viral delivery and they play a major role in adenovirus induced acute toxicity. The therapeutic window could possibly be widened by transiently depleting KCs, allowing smaller viral input doses and diminishing KC related toxicity. The transductional efficacy of various capsid modified viruses was analyzed in vitro and in vivo in murine orthotopic breast cancer model. The effect of capsid modifications on the oncolytic efficacy, i.e. the ability of the viruses to kill cancer cells, was evaluated in vitro and in vivo in murine cancer models. We concluded that capsid modifications result in transductional enhancement, and that enhanced transduction translates into more potent oncolysis in vitro and in vivo. When KC depleting agents were used in vivo prior to viral injections, enhanced tumor transduction was seen, but this effect was not translated into enhanced antitumor activity. Transcriptional regulation of replicative oncolytic viruses is a prerequisite for virotherapy. Tumor or tissue specific promoters can be used to control the transcription of adenoviral early genes to gain cancer specific viral replication. Specific deletions in viral regions essential for virus replication in normal cells can further increase the safety by allowing viral genome replication in cancer cells featuring specific mutations. Genetically modified viruses were shown to be able to kill putative cancer stem cells that are thought to be responsible for post treatment relapses and metastasis. Further, pharmacologic intervention reduced viral replication and thereby might offer an additional safety switch in case viral replication related side effects are encountered.

Zinc, cadmium and lead resistance mechanisms in bacteria and their contribution to biosensing

In bacteria resistance to heavy metals is mainly achieved through active efflux, but also sequestration with proteins or as insoluble compounds is used. Although numerous studies have dealt with zinc, cadmium and lead resistance mechanisms in bacteria, it has still remained unclear how different transporters are integrated into an effective homeostasis/resistance network and whether specific mechanisms for lead sequestration exist. Furthermore, since metals are toxic not only to bacteria but to higher organisms as well, it is important to be able to estimate possible biological effects of heavy metals in the environment. This could be done by determining the bioavailable amount of the metals in the environment with bacterial bioreporters. That is, one can employ bacteria that respond to metal contamination by a measurable signal to assess the property of metals to cross biological membranes and to cause harmful effects in a possibly polluted environment. In this thesis a new lead resistance mechanism is described, interplay between CBA transporters and P-type ATPases in zinc and cadmium resistance is presented and finally the acquired knowledge is used to construct bacterial bioreporters for heavy metals with increased sensitivity and specificity. The new lead resistance model employs a P-type ATPase that removes Pb2+ ions from the cytoplasm and a phosphatase that produces inorganic phosphate for lead sequestration in the periplasm. This was the first study where the molecular mechanism of lead sequestration has been described. Characterization of two P-type ATPases and two CBA transporters showed that resistance mechanisms for Zn2+ and Cd2+ are somewhat different than for Pb2+ as these metals cannot be sequestered as insoluble compounds as easily. Resistance to Zn2+ was conferred merely by the CBA transporter that could export both cytoplasmic and periplasmic ions; whereas, full resistance to Cd2+ required interplay of a P-type ATPase that exported cytoplasmic ions to periplasm and a CBA transporter that further exported periplasmic ions to the outside. The knowledge on functionality of the transporters and metal-inducible promoters was exploited in bioreporter technology. A transporter-deficient bioreporter strain that lacked exporters for Zn2+/Cd2+/Pb2+ could detect up to 45-fold lower metal concentrations than its wild type counterpart due to the accumulation of metals in the cell. The broad specificity issue of bioreporters was overcome by using Zn-specific promoter as a sensor element, thus achieving Zn-specific bioreporter.

Microbial life and deposits in paper machine circuits

Väitöskirjani käsittele mikrobien ja erilaisten kemikaalien rooleja saostumien ja biofilmien muodostumisessa paperi- ja kartonkikoneilla. "Saostuma" tässä työssä tarkoittaa kiinteän aineen kertymää konepinnoille tai rajapinnoille konekierroissa, jotka on tarkoitettu massasulppujen, lietteiden, vesien tai ilman kuljetukseen. Saostumasta tulee "biofilmi" silloin kun sen oleellinen rakennekomponentti on mikrobisolut tai niiden tuotteet. Väitöstyöni työhypoteesina oli, että i. tietämys saostumien koostumuksesta, sekä ii. niiden rakenteesta, biologisista, fysikaalis-kemiallisista ja teknisistä ominaisuuksista ohjaavat tutkijaa löytämään ympäristöä säästäviä keinoja estää epätoivottujen saostumien muodostus tai purkaa jo muodostuneita saostumia. Selvittääkseni saostumien koostumista ja rakennetta käytin monia erilaisia analytiikan työkaluja, kuten elektronimikroskopiaa, konfokaali-laser mikroskopiaa (CLSM), energiadispersiivistä röntgenanalyysiä (EDX), pyrolyysi kaasukromatografiaa yhdistettynä massaspektrometriaan (Py-GCMS), joninvaihtokromatografiaa, kaasukromatografiaa ja mikrobiologisia analyysejä. Osallistuin aktiivisesti innovatiivisen, valon takaisinsirontaan perustuvan sensorin kehittämistyöhön, käytettäväksi biofilmin kasvun mittaukseen suoraan koneen vesikierroista ja säiliöistä. Työni osoitti, että monet paperinvalmistuksessa käytetyistä kemikaaleista reagoivat keskenään tuottaen orgaanisia tahmakerroksia konekiertojen teräspinnoille. Löysin myös kerrostumia, jotka valomikroskooppisessa tarkastelussa oli tulkittu mikrobeiksi, mutta jotka elektronimikroskopia paljasti alunasta syntyneiksi, alumiinihydroksidiksi joka saostui pH:ssa 6,8 kiertokuitua käyttävän koneen viiravesistä. Monet paperintekijät käyttävät vieläkin alunaa kiinnitysaineena vaikka prosessiolot ovat muuttuneet happamista neutraaleiksi. Sitä pidetään paperitekijän "aspiriinina", mutta väitöstutkimukseni osoitti sen riskit. Löysin myös orgaanisia saostumia, joiden alkuperä oli aineiden, kuten pihkan, saippuoituminen (kalsium saippuat) niin että muodostui tahmankasvua ylläpitävä alusta monilla paperi- ja kartonkikoneilla. Näin solumuodoiltaan Deinococcus geothermalista muistuttavia bakteereita kasvamassa lujasti teräskoepalojen pintaan kiinnittyneinä pesäkkeinä, kun koepaloja upotettiin paperikoneiden vesikiertoihin. Nämä deinokokkimaiset pesäkkeet voivat toimia jalustana, tarttumisalustana muiden mikrobien massoille, joka selittäisi miksi saostumat yleisesti sisältävät deinokokkeja pienenä, muttei koskaan pääasiallisena rakenneosana. Kun paperikoneiden käyttämien vesien (raakavedet, lämminvesi, biologisesti puhdistettu jätevesi) laatua tutkitaan, mittausmenetelmällä on suuri merkitys. Koepalan upotusmenetelmällä todettu biofilmikasvu ja viljelmenetelmällä mitattu bakteerisaastuneisuus korreloivat toisiinsa huonosti etenkin silloin kun likaantumisessa oli mukana rihmamaiseti kasvavia bakteereja. Huoli ympäristöstä on pakottanut paperi- ja kartonkikoneiden vesikiertojen sulkemiseen. Vesien kierrätys ja prosessivesien uudelleenkäyttö nostavat prosessilämpötilaa ja lisäävät koneella kiertävien kolloidisten ja liuenneiden aineiden määriä. Tutkin kiertovesien pitoisuuksia kolmessa eriasteisesti suljetussa tehtaassa, joiden päästöt olivat 0 m3, 0,5 m3 ja 4 m3 jätevettä tuotetonnia kohden, perustuen puhdistetun jäteveden uudelleen käyttöön. Nollapäästöisellä tehtaalla kiertovesiin kertyi paljon orgaanisesti sidottua hiiltä (> 10 g L-1), etenkin haihtuvina happoina (maito-, etikka-, propioni- ja voi-). Myös sulfaatteja, klorideja, natriumia ja kalsiumia kertyi paljon, > 1 g L-1 kutakin. Pääosa (>40%) kaikista bakteereista oli 16S rRNA geenisekvenssianalyysien tulosten perusteella sukua, joskin etäistä (< 96%) ainoastaan Enterococcus cecorum bakteerille. 4 m3 päästävältä tehtaalta löytyi lisäksi Bacillus thermoamylovorans ja Bacillus coagulans. Tehtaiden saostumat sisälsivät arkkeja suurina pitoisuuksina, ≥ 108 g-1, mutta tunnistukseen riittävää sekvenssisamanlaisuutta löytyi vain yhteen arkkisukuun, Methanothrix. Tutkimustulokset osoittivat että tehtaan vesikiertojen sulkeminen vähensi rajusti mikrobiston monimuotoisuutta, muttei estänyt liuenneen aineen ja kiintoaineen mineralisoitumista.

Effects of dietary phosphorus and calcium-to-phosphorus ratio on calcium and bone metabolism in healthy 20- to 43-year-old Finnish women

Dietary habits have changed during the past decades towards an increasing consumption of processed foods, which has notably increased not only total dietary phosphorus (P) intake, but also intake of P from phosphate additives. While the intake of calcium (Ca) in many Western countries remains below recommended levels (800 mg/d), the usual daily P intake in a typical Western diet exceeds by 2- to 3-fold the dietary guidelines (600 mg/d). The effects of high P intake in healthy humans have been investigated seldom. In this thesis healthy 20- to 43-year-old women were studied. In the first controlled study (n = 14), we examined the effects of P doses, and in a cross-sectional study (n = 147) the associations of habitual P intakes with Ca and bone metabolism. In this same cross-sectional study, we also investigated whether differences exist between dietary P originating from natural P sources and phosphate additives. The second controlled study (n = 12) investigated whether by increasing the Ca intake, the effects of a high P intake could be reduced. The associations of habitual dietary calcium-to-phosphorus ratios (Ca:P ratio) with Ca and bone metabolism were determined in a cross-sectional study design (n = 147). In the controlled study, the oral intake of P doses (495, 745, 1245 and 1995 mg/d) with a low Ca intake (250 mg/d) increased serum parathyroid hormone (S-PTH) concentration in a dose-dependent manner. In addition, the highest P dose decreased serum ionized calcium (S-iCa) concentration and bone formation and increased bone resorption. In the second controlled study with a dietary P intake of 1850 mg/d, by increasing the Ca intake from 480 mg/d to 1080 mg/d and then to 1680 mg/d, the S-PTH concentration decreased, the S-iCa concentration increased and bone resorption decreased dose-dependently. However, not even the highest Ca intake could counteract the effect of high dietary P on bone formation, as indicated by unchanged bone formation activity. In the cross-sectional studies, a higher habitual dietary P intake (>1650 mg/d) was associated with lower S-iCa and higher S-PTH concentrations. The consumption of phosphate additive-containing foods was associated with a higher S-PTH concentration. Moreover, habitual low dietary Ca:P ratios (≤0.50, molar ratio) were associated with higher S-PTH concentrations and 24-h urinary Ca excretions, suggesting that low dietary Ca:P ratios may interfere with homeostasis of Ca metabolism and increase bone resorption. In summary, excessive dietary P intake in healthy Finnish women seems to be detrimental to Ca and bone metabolism, especially when dietary Ca intake is low. The results indicate that by increasing dietary Ca intake to the recommended level, the negative effects of high P intake could be diminished, but not totally prevented. These findings imply that phosphate additives may be more harmful than natural P. Thus, reduction of an excessively high dietary P intake is also beneficial for healthy individuals.

Associations of gene polymorphisms and nutrition with calcium homeostasis and bone mineral density : Studies on skeletal nutrigenetics

Heredity explains a major part of the variation in calcium homeostasis and bone strength, and the susceptibility to osteoporosis is polygenetically regulated. Bone phenotype results from the interplay between lifestyle and genes, and several nutritional factors modulate bone health throughout life. Thus, nutrigenetics examining the genetic variation in nutrient intake and homeostatic control is an important research area in the etiology of osteoporosis. Despite continuing progress in the search for candidate genes for osteoporosis, the results thus far have been inconclusive. The main objective of this thesis was to investigate the associations of lactase, vitamin D receptor (VDR), calcium sensing receptor (CaSR) and parathyroid hormone (PTH) gene polymorphisms and lifestyle factors and their interactions with bone health in Finns at varying stages of the skeletal life span. Markers of calcium homeostasis and bone remodelling were measured from blood and urine samples. Bone strength was measured at peripheral and central bone sites. Lifestyle factors were assessed with questionnaires and interviews. Genetic lactase non-persistence (the C/C-13910 genotype) was associated with lower consumption of milk from childhood, predisposing females in particular to inadequate calcium intake. Consumption of low-lactose milk and milk products was shown to decrease the risk for inadequate calcium intake. In young adulthood, bone loss was more common in males than in females. Males with the lactase C/C-13910 genotype may be more susceptible to bone loss than males with the other lactase genotypes, although calcium intake predicts changes in bone mass more than the lactase genotype. The BsmI and FokI polymorphisms of the VDR gene were associated with bone mass in growing adolescents, but the associations weakened with age. In young adults, the A986S polymorphism of the calcium sensing receptor gene was associated with serum ionized calcium concentrations, and the BstBI polymorphism of the parathyroid gene was related to bone strength. The FokI polymorphism and sodium intake showed an interaction effect on urinary calcium excretion. A novel gene-gene interaction between the VDR FokI and PTH BstBI gene polymorphisms was found in the regulation of PTH secretion and urinary calcium excretion. Further research should be carried out with more number of Finns at varying stages of the skeletal life span and more detailed measurements of bone strength. Research should concern mechanisms by which genetic variants affect calcium homeostasis and bone strength, and the role of diet-gene and gene-gene interactions in the pathogenesis of osteoporosis.

Evaluation of methods and applications for behavioural profiling of transgenic mice

During the last 10-15 years interest in mouse behavioural analysis has evolved considerably. The driving force is development in molecular biological techniques that allow manipulation of the mouse genome by changing the expression of genes. Therefore, with some limitations it is possible to study how genes participate in regulation of physiological functions and to create models explaining genetic contribution to various pathological conditions. The first aim of our study was to establish a framework for behavioural phenotyping of genetically modified mice. We established comprehensive battery of tests for the initial screening of mutant mice. These included tests for exploratory and locomotor activity, emotional behaviour, sensory functions, and cognitive performance. Our interest was in the behavioural patterns of common background strains used for genetic manipulations in mice. Additionally we studied the behavioural effect of sex differences, test history, and individual housing. Our findings highlight the importance of careful consideration of genetic background for analysis of mutant mice. It was evident that some backgrounds may mask or modify the behavioural phenotype of mutants and thereby lead to false positive or negative findings. Moreover, there is no universal strain that is equally suitable for all tests, and using different backgrounds allows one to address possible phenotype modifying factors. We discovered that previous experience affected performance in several tasks. The most sensitive traits were the exploratory and emotional behaviour, as well as motor and nociceptive functions. Therefore, it may be essential to repeat some of the tests in naïve animals for assuring the phenotype. Social isolation for a long time period had strong effects on exploratory behaviour, but also on learning and memory. All experiments revealed significant interactions between strain and environmental factors (test history or housing condition) indicating genotype-dependent effects of environmental manipulations. Several mutant line analyses utilize this information. For example, we studied mice overexpressing as well as those lacking extracellular matrix protein heparin-binding growth-associated molecule (HB-GAM), and mice lacking N-syndecan (a receptor for HB-GAM). All mutant mice appeared to be fertile and healthy, without any apparent neurological or sensory defects. The lack of HB-GAM and N-syndecan, however, significantly reduced the learning capacity of the mice. On the other hand, overexpression of HB-GAM resulted in facilitated learning. Moreover, HB-GAM knockout mice displayed higher anxiety-like behaviour, whereas anxiety was reduced in HB-GAM overexpressing mice. Changes in hippocampal plasticity accompanied the behavioural phenotypes. We conclude that HB-GAM and N-syndecan are involved in the modulation of synaptic plasticity in hippocampus and play a role in regulation of anxiety- and learning-related behaviour.