The overall goal of this dissertation is to elucidate the cellular and circuit mechanisms underlying flexible behavior in the prefrontal cortex. We are often faced with situations in which the appropriate behavior in one context is inappropriate in others. If these situations are familiar, we can perform the appropriate behavior without relearning how the context relates to the behavior — an important hallmark of intelligence. Neuroimaging and lesion studies have shown that this dynamic, flexible process of remapping context to behavior (task switching) is dependent on prefrontal cortex, but the precise contributions and interactions of prefrontal subdivisions are still unknown. This dissertation investigates two prefrontal areas that are thought to be involved in distinct, but complementary executive roles in task switching — the dorsolateral prefrontal cortex (dlPFC) and the anterior cingulate cortex (ACC). Using electrophysiological recordings from macaque monkeys, I show that synchronous network oscillations in the dlPFC provide a mechanism to flexibly coordinate context representations (rules) between groups of neurons during task switching. Then, I show that, wheras the ACC neurons can represent rules at the cellular level, they do not play a significant role in switching between contexts — rather they seem to be more related to errors and motivational drive. Finally, I develop a set of web-enabled interactive visualization tools designed to provide a multi-dimensional integrated view of electrophysiological datasets. Taken together, these results contribute to our understanding of task switching by investigating new mechanisms for coordination of neurons in prefrontal cortex, clarifying the roles of prefrontal subdivisions during task switching, and providing visualization tools that enhance exploration and understanding of large, complex and multi-scale electrophysiological data.

Webbserver-loggar är kraftfullt att använda för att spåra användning och förstå användares beteenden, men växer snabbt i storlek och kan vara svåra att tyda i textformat. Med hjälp av visualisering kan förståelse för data skapas. Diagram är vanligast att använda och interaktivitet är ett stort hjälpmedel för att skapa mer meningsfulla grafiska representationer. Vid skapade av diagram kan JavaScript-bibliotek användas och två välanvända och populära bibliotek är D3.js och Google Charts. På grund av en ökning av webbaserad visualisering och lättillgängligheten med analyseringsverktyg online, kommer renderingstider vid visualisering av loggdata analyseras i kombination med visualisering baserat på JavaScript-bibliotek. Ett tekniskt experiment genomförs för att ta reda på vilket bibliotek som har snabbast renderingstid. För att studien ska åstadkomma ett brett resultat utförs experiment vid både initial och interaktiv rendering av diagram med loggdata och varierande faktorer. Resultatet indikerar på att D3.js erhåller snabbast renderingstider i samtliga fallen.

Bone formation and remodeling require continuous generation of osteoprogenitors from bone marrow stromal cells (MSC), which are regulated by local growth factors and hormones with putative roles in mesenchymal proliferation and differentiation. Hepatocyte growth factor (HGF) and its receptor c-Met are widely expressed in MSC and are thought to play a key role in the interactions between cells. 1,25-dihydroxyvitamin D (1,25OHD) is the most active metabolite of vitamin D. 1,25OHD binds to its nuclear/membrane vitamin D receptor (VDR) and generates appropriate biological responses. The purpose of this study was to investigate the regulation of proliferation and differentiation by HGF in human bone marrow-derived stromal cells (hMSC). We examined the impact of HGF on hMSC cell-cycle regulation and the combination effects of HGF and 1,25OHD on hMSC osteogenic differentiation to enhance our knowledge of hMSC regulation. hMSC isolated from bone marrow were plated and grown in DMEM supplemented with 3% FBS incubated at 37C with 5% CO2 in air. HGF treatment of hMSCs reduced the rate of cell proliferation and this result was not due to apoptosis or cell senescence. Real-time RT-PCR and Western blot analysis showed increased gene and protein expression of the cell-cycle inhibitors p53, p21, and p27 after HGF treatment. These results appear to be specific because HGF did not significantly alter the gene expression level of other cell-cycle mediators such as RB, cyclin D1, CDK2, CDK4, or CDK6. Transfection of siRNA specific for cMet, the HGF receptor, eliminated the HGF anti-proliferation effect. cMet siRNA also eliminated the increase in p53, p21, and p27, further supporting a role for these cell-cycle inhibitors in HGF¡¯s regulation of hMSC. These results suggest that treatment of hMSC with HGF slows cell proliferation by increasing the expression of p53, p21, and p27. The reduced rate of cell proliferation did not appear to be due to cell differentiation, because treatment of hMSC with HGF alone did not induce cell differentiation. However, HGF in combination with a known osteogenic differentiation activator, 1,25OHD, significantly increased cell maturation/differentiation compared to 1,25D alone, as indicated by an increase in osteocalcin mRNA (a marker for osteogenic differentiation). Whereas HGF had no effect on 1,25OHD synthesis per se, HGF did induce 1,26OHD receptor (VDR) gene expression. HGF up-regulated the expression of the p63 gene, a member of the p53 family. Knocking down the p63 gene reduced the HGF effect on VDR expression and eliminated the HGF-induced up-regulation of the osteogenic differentiation markers osteopontin (OPN) and bone sialoprotein (BSP). Moreover, the ChIP assay shows that p63 was able to bind to the VDR promoter, possibly explaining the mechanism of p63-mediated VDR up-regulation. These results indicate that HGF can also induce hMSC osteogenic differentiation when combined with 1,25OHD by up-regulating 1,25OHD receptor VDR expression.

Background: The interest of vitamin D has increased in the last years. That is because there is so many possible positive effects of vitamin D and also because many individuals has vitamin D deficiency. Modern man spends much time indoors which leads to lower levels of vitamin D. People who have emigrated from a sunny climate to a Nordic climate often gets a deficiency due to a more pigmented skin which requires a larger amount of UVB to get an adequate synthesis of vitamin D. Aim: The aim with this study is to compare and evaluate how similar the instrument mini VIDAS measures 25(OH)D total against the current existing method cobas e 602. A discussion about if 25(OH)D total has a place in primary health care is included in the study. Method: The comparison was based on 39 samples. The samples was analyzed on cobas e 602 and mini VIDAS. A precision test was performed. External controls from DEQAS was also included in the study. The results have been presented with simple linear regression analysis, mean value, SD and CV. Results: The comparison between cobas e 602 and mini VIDAS gave a coefficient of determination of 81,34 %. mini VIDAS was closest to the external controls target values. Conclusion: There is no obvious conclusions about if mini VIDAS fulfills the requirement to be introduced to primary health care. The coefficient of determination of 81,34 % should be at least 95 %. However is mini VIDAS closer to the external controls target values then cobas e 602. There is factors that implies that 25(OH)D total has a place in primary health care with regards to demand, use and because many individuals has vitamin D deficiency. The instrument is also user-friendly to a primary health care laboratory. / Bakgrund: Intresset för vitamin D har ökat de senare åren. Det beror dels på att det finns många eventuella positiva effekter av vitamin D och dels på att många individer har brist på vitamin D. Nutidens människa spenderar mycket tid inomhus vilket leder till lägre nivåer av vitamin D. Personer som har utvandrat från ett soligt klimat till nordiskt klimat får ofta brist på grund av en mer pigmenterad hud som behöver större mängd UVB för att få en adekvat syntes av vitamin D. Syfte: Syftet med den här studien är att jämföra och utvärdera hur lika det patientnära instrumentet mini VIDAS mäter 25(OH)D total mot befintlig metod cobas e 602. Diskussion om analysen 25(OH)D total har en plats i primärvården ingår även i studien. Metod: Jämförelsen baserades på 39 st prover. Proven analyserades på cobas e 602 och mini VIDAS. Ett precisionsförsök gjordes. Externkontroller från DEQAS inkluderades även i studien. Resultaten har presenterats genom enkel linjär regressionsanalys, medelvärde, SD och CV. Resultat: Jämförelsen mellan cobas e 602 och mini VIDAS gav en förklaringsgrad på 81,43 %. mini VIDAS var närmst externkontrollernas målvärden. Slutsats: Det går inte att dra självklara slutsatser ifall mini VIDAS uppfyller kraven att införas i primärvården. Förklaringsgraden som är på 81,43 % bör vara minst 95 %. Däremot överensstämmer mini VIDAS med externkontrollerna bättre än cobas e 602. Det finns faktorer som tyder på att analysen 25(OH)D har en plats i primärvården med avseende på efterfrågan, användningsområde och antal individer med brist. Instrumentet är dessutom användarvänligt för ett primärvårdslaboratorium.

The CYP27B1 enzyme that synthesizes 1alpha,25-(OH) 2D, is expressed in chondrocytes, suggesting that local production of 1alpha,25-(OH)2D could play an autocrine or paracrine role in the differentiation of these cells. To test this hypothesis, we have engineered mutant mice that do not express the Cyp27b1 gene in chondrocytes. This led to increased width of the hypertrophic zone of the growth plate at E15.5, increased bone mass in neonatal long bones, and increased expression of the chondrocytic differentiation markers Indian Hedgehog and PTH/PTHrP receptor. VEGF mRNA levels were decreased, accompanied by decreased PECAM-1 immunostaining, suggesting a delay in vascularization. We have also engineered mice overexpressing a Cyp27b1 transgene in chondrocytes. The transgenic mice showed a partial mirror image phenotype compared to the tissue-specific inactivation model. These results support an autocrine/paracrine role of 1alpha,25-(OH) 2D in endochondral ossification and chondrocyte development in vivo. / The CYP24A1 enzyme is involved in the catabolic breakdown of 1alpha,25-(OH)2D but also synthesizes the 24R,25-(OH) 2D metabolite. Studies in chicken suggest a role for 24R,25-(OH) 2D in fracture repair. We induced stabilized transverse mid-diaphysial fractures of the tibia in four-month-old wild-type and Cyp24a1-deficient mice. Examination of the callus sections showed delayed hard callus formation in the homozygous mutant animals compared to wild-type littermates. RT-qPCR showed perturbed levels of type X collagen transcripts in mutant mice at 14 and 21 days post-fracture, reflecting the delayed healing. Rescue of the impaired healing by subcutaneous injection of 24R,25-(OH)2D3 normalized the histological appearance of the callus, static histomorphometric index and type X collagen mRNA expression, while 1alpha,25-(OH)2D 3 did not. These results show that Cyp24a1 deficiency delays fracture repair and strongly suggest that vitamin D metabolites hydroxylated at position 24, such as 24R,25-(OH)2D, play an important role in the mechanisms leading to normal fracture healing.

Dopamine D3 receptors (D3Rs) have been implicated in mediating behavioural sensitization to various drugs of abuse, but their role in ethanol (EtOH) sensitization has not been directly examined. Neil Richtand proposed a role for D3Rs in the modulation of sensitization by acting as an inhibitor of D1/D2 receptor-mediated behaviours, and several reports suggest D3Rs up-regulate in response to chronic drugs of abuse. In separate experiments, we examined EtOH sensitization in D3R knockout (KO) as well as in D1R and D2R KO mice. We also examined amphetamine sensitization in D3R KOs compared to wild type mice. We challenged C57Bl/6 and DBA/2 mice with a D3R agonist (PD128907) and antagonist (U99194A) to examine how acute and chronic D3R activation and inactivation may affect the induction and expression of EtOH sensitization. We investigated D1/D3R interactions in sensitized and control mice and examined whether EtOH sensitization leads to changes in D3R binding using [125I]-7-OH-PIPAT autoradiography. Results showed that D3R KOs, were resistant to EtOH but not to amphetamine sensitization. Chronic but not acute D3R blockade with U99194A inhibited the induction, whereas acute D3R activation with PD128907 attenuated the expression of EtOH sensitization. In our D1/D3R interaction study we observed that although PD128907 attenuated D1 agonist-induced hyperactivity with SKF81297, this effect was the same in sensitized and control animals, even though sensitized mice were more responsive to PD128907 than controls. This enhanced response, which suggests a functional up-regulation of D3Rs, was not accompanied by changes in D3R binding as indicated by autoradiography, and could mean that functional changes in the D3R associated with EtOH sensitization occur elsewhere than at the level of the membrane-bound receptor. Taken together, these results suggest a modulatory role for the D3R in EtOH but not amphetamine sensitization, where D3R activation attenuates the expression and D3R blockade prevents the induction of EtOH sensitization. These results are important because a better understanding of the role of the D3R in EtOH sensitization may help not only to identify some of the underlying neural mechanisms of sensitization, but also help in the identification of treatment strategies for patients that may be susceptible to alcohol abuse.

Biologically active 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) binds the vitamin D receptor (VDR) to exert its effect on target cells. VDR expression is found in a number of immune cells including professional antigen-presenting cells such as dendritic cells. It has been found that the actions of 1,25-(OH)2D3 on the immune system are mainly immunosuppressive. The cross-presentation pathway allows for exogenously derived antigens to be presented by pAPCs on MHC-I molecules to CD8+ T cells. CD8+ T cell activation results in the expansion of epitope-specific T cell populations that confer host protection. These epitopes can be organized into an immunodominance hierarchy. Previous work demonstrated that introducing LCMV-NP via the cross-priming pathway significantly alters the immunodominance hierarchy of a subsequent LCMV infection. Building upon these observations, our study assessed the effects of LCMV-NP cross priming in the presence of a single dose of 1,25-(OH)2D3. Treatment with 1,25-(OH)2D3 was found to have biological effects in our model system. In vitro pAPCs were demonstrated to up-regulate IL-10 and CYP24A1 mRNA, in addition to the transactivation of cellular VDR, as demonstrated by a relocalization to the nuclear region. Mice treated with 1,25-(OH)2D3 were found to produce up-regulated IL-10 and CYP24A1 transcripts. Expression of VDR was increased at both the transcript and protein level. Our results demonstrate that a single dose of 1,25-(OH)2D3 does not affect the cross-priming pathway in this system. Treatment with 1,25-(OH)2D3 did not influence the ability of differentiated pAPCs to phagocytose or cross-present exogenous antigen to epitope-specific CD8+ T cells. Furthermore, 1,25-(OH)2D3 did not alter cross-priming or the establishment of the LCMV immunodominance hierarchy in vivo. By confirming that 1,25-(OH)2D3 does not suppress cross-priming in our model, our study helps to expand the understanding of the immunomodulatory role of exogenous 1,25-(OH)2D3 on the outcome of virus infection. Collectively, our data supports the observation that the role of 1,25-(OH)2D3 in the immune system is not always associated with suppressive effects. / Thesis (Master, Microbiology & Immunology) -- Queen's University, 2011-08-29 14:53:18.766

Dopamine D3 receptors (D3Rs) have been implicated in mediating behavioural sensitization to various drugs of abuse, but their role in ethanol (EtOH) sensitization has not been directly examined. Neil Richtand proposed a role for D3Rs in the modulation of sensitization by acting as an inhibitor of D1/D2 receptor-mediated behaviours, and several reports suggest D3Rs up-regulate in response to chronic drugs of abuse. In separate experiments, we examined EtOH sensitization in D3R knockout (KO) as well as in D1R and D2R KO mice. We also examined amphetamine sensitization in D3R KOs compared to wild type mice. We challenged C57Bl/6 and DBA/2 mice with a D3R agonist (PD128907) and antagonist (U99194A) to examine how acute and chronic D3R activation and inactivation may affect the induction and expression of EtOH sensitization. We investigated D1/D3R interactions in sensitized and control mice and examined whether EtOH sensitization leads to changes in D3R binding using [125I]-7-OH-PIPAT autoradiography. Results showed that D3R KOs, were resistant to EtOH but not to amphetamine sensitization. Chronic but not acute D3R blockade with U99194A inhibited the induction, whereas acute D3R activation with PD128907 attenuated the expression of EtOH sensitization. In our D1/D3R interaction study we observed that although PD128907 attenuated D1 agonist-induced hyperactivity with SKF81297, this effect was the same in sensitized and control animals, even though sensitized mice were more responsive to PD128907 than controls. This enhanced response, which suggests a functional up-regulation of D3Rs, was not accompanied by changes in D3R binding as indicated by autoradiography, and could mean that functional changes in the D3R associated with EtOH sensitization occur elsewhere than at the level of the membrane-bound receptor. Taken together, these results suggest a modulatory role for the D3R in EtOH but not amphetamine sensitization, where D3R activation attenuates the expression and D3R blockade prevents the induction of EtOH sensitization. These results are important because a better understanding of the role of the D3R in EtOH sensitization may help not only to identify some of the underlying neural mechanisms of sensitization, but also help in the identification of treatment strategies for patients that may be susceptible to alcohol abuse.

7-Dehydrocholesterol (7-DHC) is the substrate of 7-dehydrocholesterol reductase (DHCR7) in the cholesterol synthesis pathway. Keratinocytes in human skin possess the enzymes necessary for cholesterol synthesis but are also responsible for vitamin D3 synthesis from 7-DHC by exposure to UVB irradiation. It has been well established that DHCR7 is regulated by the SREBP pathway in the regulation of cholesterol synthesis, but little is known about the regulation of DHCR7 by the vitamin D pathway. In this study, the regulation of DHCR7 activity by vitamin D was explored. Treatment of adult human epidermal keratinocyte (HEKa) cells with vitamin D3 resulted in a rapid decrease in DHCR7 activity which was not due to changes in the amount of enzyme present. This suppression of activity was observed only in HEKa cells, a primary cell line cultured from normal human skin, and not in an immortalized skin cell line (HaCaT cells) nor in two liver-derived hepatoma cell lines. Because vitamin D3 treatment of HEKa cells did not change the content of lanosterol nor 7-DHC, these results suggest that vitamin D3 rapidly down-regulates the entire cholesterolgenesis pathway, presumably at a very early step in the pathway. 25-Hydroxyvitamin D3, the first metabolite and circulating form of vitamin D3, had a lesser effect on DHCR7 activity, while 1,25-dihydroxyvitamin D3, the activated form of the vitamin, had no effect on DHCR7, indicating that the vitamin D receptor is not involved. The decrease in DHCR7 activity was due neither to the dephosphorylation of the enzyme, an established mechanism of inactivation, nor to direct inhibition by vitamin D3. Vitamin D3 markedly inhibited proliferation and induced differentiation of HEKa cells, suggesting a possible role for hedgehog signaling in the decrease in DHCR7 activity.

A B S T R A C T Title: Recombination of Ions in Plasma at 50 − 300 K Author: Peter Rubovič Supervisor: Prof. RNDr. Juraj Glosík, DrSc. Abstract: Main part of this doctoral thesis lies in a study of recom- bination of atomic and molecular ions in low temperature plasmatic environment with emphasis on effect of third bodies. Stationary After- glow equipped with Cavity Ring Down Spectrometer and Cryogenic Flowing Afterglow with Langmuir Probe II were used to obtain recom- bination rate coefficients. Electron assisted collisional radiative recom- bination of Ar+ ion was studied in the temperature range of 50−100 K and helium assisted collisional radiative recombination was observed too. Both H+ 3 and its isotopologue D+ 3 were studies in flowing after- glow and spectroscopically in stationary afterglow as well. Binary re- combination rate coefficients and ternary recombination rate coeffi- cients for helium assisted ternary recombination were determined in the temperature range of 50 − 250 K. These coefficients were deter- mined also for pure ortho- and para- nuclear spin configurations of H+ 3 in the temperature range of 80 − 200 K. Keywords: dissociative recombination, collisional radiative recombi- nation, H+ 3 , D+ 3 , Ar+ viii