Neuronal oscillations in gamma- and alpha-frequency bands: from object representations to sensory awareness

The synchronization of neuronal activity, especially in the beta- (14-30 Hz) /gamma- (30 80 Hz) frequency bands, is thought to provide a means for the integration of anatomically distributed processing and for the formation of transient neuronal assemblies. Thus non-stimulus locked (i.e. induced) gamma-band oscillations are believed to underlie feature binding and the formation of neuronal object representations. On the other hand, the functional roles of neuronal oscillations in slower theta- (4 8 Hz) and alpha- (8 14 Hz) frequency bands remain controversial. In addition, early stimulus-locked activity has been largely ignored, as it is believed to reflect merely the physical properties of sensory stimuli. With human neuromagnetic recordings, both the functional roles of gamma- and alpha-band oscillations and the significance of early stimulus-locked activity in neuronal processing were examined in this thesis. Study I of this thesis shows that even the stimulus-locked (evoked) gamma oscillations were sensitive to high-level stimulus features for speech and non-speech sounds, suggesting that they may underlie the formation of early neuronal object representations for stimuli with a behavioural relevance. Study II shows that neuronal processing for consciously perceived and unperceived stimuli differed as early as 30 ms after stimulus onset. This study also showed that the alpha band oscillations selectively correlated with conscious perception. Study III, in turn, shows that prestimulus alpha-band oscillations influence the subsequent detection and processing of sensory stimuli. Further, in Study IV, we asked whether phase synchronization between distinct frequency bands is present in cortical circuits. This study revealed prominent task-sensitive phase synchrony between alpha and beta/gamma oscillations. Finally, the implications of Studies II, III, and IV to the broader scientific context are analysed in the last study of this thesis (V). I suggest, in this thesis that neuronal processing may be extremely fast and that the evoked response is important for cognitive processes. I also propose that alpha oscillations define the global neuronal workspace of perception, action, and consciousness and, further, that cross-frequency synchronization is required for the integration of neuronal object representations into global neuronal workspace.

TlBr raw material purification, crystal growth, annealing, detector fabrication and characterisation for gamma-ray detector applications

The research reported in this thesis dealt with single crystals of thallium bromide grown for gamma-ray detector applications. The crystals were used to fabricate room temperature gamma-ray detectors. Routinely produced TlBr detectors often are poor quality. Therefore, this study concentrated on developing the manufacturing processes for TlBr detectors and methods of characterisation that can be used for optimisation of TlBr purity and crystal quality. The processes under concern were TlBr raw material purification, crystal growth, annealing and detector fabrication. The study focused on single crystals of TlBr grown from material purified by a hydrothermal recrystallisation method. In addition, hydrothermal conditions for synthesis, recrystallisation, crystal growth and annealing of TlBr crystals were examined. The final manufacturing process presented in this thesis deals with TlBr material purified by the Bridgman method. Then, material is hydrothermally recrystallised in pure water. A travelling molten zone (TMZ) method is used for additional purification of the recrystallised product and then for the final crystal growth. Subsequent processing is similar to that described in the literature. In this thesis, literature on improving quality of TlBr material/crystal and detector performance is reviewed. Aging aspects as well as the influence of different factors (temperature, time, electrode material and so on) on detector stability are considered and examined. The results of the process development are summarised and discussed. This thesis shows the considerable improvement in the charge carrier properties of a detector due to additional purification by hydrothermal recrystallisation. As an example, a thick (4 mm) TlBr detector produced by the process was fabricated and found to operate successfully in gamma-ray detection, confirming the validity of the proposed purification and technological steps. However, for the complete improvement of detector performance, further developments in crystal growth are required. The detector manufacturing process was optimized by characterisation of material and crystals using methods such as X-ray diffraction (XRD), polarisation microscopy, high-resolution inductively coupled plasma mass (HR-ICPM), Fourier transform infrared (FTIR), ultraviolet and visual (UV-Vis) spectroscopy, field emission scanning electron microscope (FESEM) and energy-dispersive X-ray spectroscopy (EDS), current-voltage (I-V) and capacity voltage (CV) characterisation, and photoconductivity, as well direct detector examination.

Microbe-induced Innate Immune Responses in Human Dendritic Cells

Two types of antigen-presenting cells (APCs), macrophages and dendritic cells (DCs), function at the interface of innate and adaptive immunity. Through recognition of conserved microbial patterns, they are able to detect the invading pathogens. This leads to activation of signal transduction pathways that in turn induce gene expression of various molecules required for immune responses and eventually pathogen clearance. Cytokines are among the genes induced upon detection of microbes. They play an important role in regulating host immune responses during microbial infection. Chemotactic cytokines, chemokines, are involved in migratory events of immune cells. Cytokines also promote the differentiation of distinct T cell responses. Because of the multiple roles of cytokines in the immune system, the cytokine network needs to be tightly regulated. In this work, the induction of innate immune responses was studied using human primary macrophages or DCs as cell models. Salmonella enterica serovar Typhimurium served as a model for an intracellular bacterium, whereas Sendai virus was used in virus experiments. The starting point of this study was that DCs of mouse origin had recently been characterized as host cells for Salmonella. However, only little was known about the immune responses initiated in Salmonella-infected human DCs. Thus, cellular responses of macrophages and DCs, in particular the pattern of cytokine production, to Salmonella infection were compared. Salmonella-induced macrophages and DCs were found to produce multiple cytokines including interferon (IFN) -gamma, which is conventionally produced by T and natural killer (NK) cells. Both macrophages and DCs also promoted the intracellular survival of the bacterium. Phenotypic maturation of DCs as characterized by upregulation of costimulatory and human leukocyte antigen (HLA) molecules, and production of CCL19 chemokine, were also detected upon infection with Salmonella. Another focus of this PhD work was to unravel the regulatory events controlling the expression of cytokine genes encoding for CCL19 and type III IFNs, which are central to DC biology. We found that the promoters of CCL19 and type III IFNs contain similar regulatory elements that bind nuclear factor kappaB (NF-kappaB) and interferon regulatory factors (IRFs), which could mediate transcriptional activation of the genes. The regulation of type III IFNs in virus infection resembled that of type I IFNs a cytokine class traditionally regarded as antiviral. The induction of type I and type III IFNs was also observed in response to bacterial infection. Taken together, this work identifies new details about the interaction of Salmonella with its phagocytic host cells of human origin. In addition, studies provide information on the regulatory events controlling the expression of CCL19 and the most recently identified IFN family genes, type III IFN genes.

Mutations of mitochondrial DNA polymerase gamma: an important cause of neurological disorders

Thesis focuses on mutations of POLG1 gene encoding catalytic subunit polγ-α of mitochondrial DNA polymerase gamma holoenzyme (polG) and the association of mutations with different clinical phenotypes. In addition, particular defective mutant variants of the protein were characterized biochemically in vitro. PolG-holoenzyme is the sole DNA polymerase found in mitochondria. It is involved in replication and repair of the mitochondrial genome, mtDNA. Holoenzyme also includes the accessory subunit polγ-β, which is required for the enhanced processivity of polγ-α. Defective polγ-α causes accumulation of secondary mutations on mtDNA, which leads to a defective oxidative phosphorylation system. The clinical consequences of such mutations are variable, affecting nervous system, skeletal muscles, liver and other post-mitotic tissues. The aims of the studies included: 1) Determination of the role of POLG1 mutations in neurological syndromes with features of mitochondrial dysfunction and an unknown molecular cause. 2) Development and set up of diagnostic tests for routine clinical purposes. 3) Biochemical characterization of the functional consequences of the identified polγ-α variants. Studies describe new neurological phenotypes in addition to PEO caused by POLG1 mutations, including parkinsonism, premature amenorrhea, ataxia and Parkinson s disease (PD). POLG1 mutations and polymorphisms are both common and/or potential genetic risk factors at least among the Finnish population. The major findings and applications reported here are: 1) POLG1 mutations cause parkinsonism and premature menopause in PEO families in either a recessive or a dominant manner. 2) A common recessive POLG1 mutations (A467T and W748S) in the homozygous state causes severe adult or juvenile-onset ataxia without muscular symptoms or histological or mtDNA abnormalities in muscles. 3) A common recessive pathogenic change A467T can also cause a mild dominant disease in heterozygote carriers. 4) The A467T variant shows reduced polymerase activity due to defective template binding. 5) Rare polyglutamine tract length variants of POLG1 are significantly enriched in Finnish idiopathic Parkinson s disease patients. 6) Dominant mutations are clearly restricted to the highly conserved polymerase domain motifs, whereas recessive ones are more evenly distributed along the protein. The present results highlight and confirm the new role of mitochondria in parkinsonism/Parkinson s disease and describe a new mitochondrial ataxia. Based on these results, a POLG1 diagnostic routine has been set up in Helsinki University Central Hospital (HUSLAB).

IL-10 in Human Newborns : Ontogeny of IL-10 secretion and relation to the secretion of IFN-g, immunoglobulin M, G, and A, and mononuclear cell composition in newborns

The purpose of this work was to elucidate the ontogeny of interleukin-10 (IL-10) secretion from newborn mononuclear cells (MCs), and to examine its relation to the secretion of interferon-g (IFN-g) and immunoglobulins (Igs). The initial hypothesis was that the decreased immunoglobulin (Ig) synthesis of newborn babies was the result of immature cytokine synthesis regulation, which would lead to excessive IL-10 production, leading in turn to suppressed IFN-g secretion. Altogether 57 full-term newborns and 34 adult volunteers were enrolled. Additionally, surface marker compositions of 29 premature babies were included. Enzyme-linked immunoassays were used to determine the amount of secreted IL-10, IFN-g, and Igs, and the surface marker composition of MC were analyzed with a FACScan flow cytometer. The three most important findings were: 1. Cord blood MC, including CD5+ B cells, are able to secrete IL-10. However, when compared with adults, the secretion of IL-10 was decreased. This indicates that reasons other than excessive IL-10 secretion are responsible of reduced IFN-g secretion in newborns. 2. As illustrated by the IL-10 and IFN-g secretion pattern, newborn cytokine profile was skewed towards the Th2 type. However, approximately 25% of newborns had an adult like cytokine profile with both good IL10 and IFN-g secretion, demonstrating that fullterm newborns are not an immunologically homogenous group at the time of birth. 3. There were significant differences in the surface marker composition of MCs between individual neonates. While gestational age correlated with the proportion of some MC types, it is evident that there are many other maternal and fetal factors that influence the maturity and nature of lymphocyte subpopulations in individual neonates. In conclusion, the reduced ability of neonates to secrete Ig and IFN-g is not a consequence of high IL-10 secretion. However, individual newborns differ significantly in their ability to secrete cytokines as well as Igs.

Observation of exclusive charmonium production and gamma+gamma to mu+mu- in p+pbar collisions at sqrt{s} = 1.96 TeV

We have observed the reactions p+pbar --> p+X+pbar, with X being a centrally produced J/psi, psi(2S) or chi_c0, and gamma+gamma --> mu+mu-, in proton- antiproton collisions at sqrt{s} = 1.96 TeV using the Run II Collider Detector at Fermilab. The event signature requires two oppositely charged muons, each with pseudorapidity |eta| mu+mu-. Events with a J/psi and an associated photon candidate are consistent with exclusive chi_c0 production through double pomeron exchange. The exclusive vector meson production is as expected for elastic photo- production, gamma+p --> J/psi(psi(2S)) + p, which is observed here for the first time in hadron-hadron collisions. The cross sections ds/dy(y=0) for p + pbar --> p + X + pbar with X = J/psi, psi(2S) orchi_c0 are 3.92+/-0.62 nb, 0.53+/-0.14 nb, and 75+/-14 nb respectively. The cross section for the continuum, with |eta(mu+/-)|

Observation of exclusive charmonium production and gamma+gamma to mu+mu- in p+pbar collisions at sqrt{s} = 1.96 TeV

We have observed the reactions p+pbar --> p+X+pbar, with X being a centrally produced J/psi, psi(2S) or chi_c0, and gamma+gamma --> mu+mu-, in proton- antiproton collisions at sqrt{s} = 1.96 TeV using the Run II Collider Detector at Fermilab. The event signature requires two oppositely charged muons, each with pseudorapidity |eta| mu+mu-. Events with a J/psi and an associated photon candidate are consistent with exclusive chi_c0 production through double pomeron exchange. The exclusive vector meson production is as expected for elastic photo- production, gamma+p --> J/psi(psi(2S)) + p, which is observed here for the first time in hadron-hadron collisions. The cross sections ds/dy(y=0) for p + pbar --> p + X + pbar with X = J/psi, psi(2S) orchi_c0 are 3.92+/-0.62 nb, 0.53+/-0.14 nb, and 75+/-14 nb respectively. The cross section for the continuum, with |eta(mu+/-)|

Transcriptional analysis of persistent Chlamydia pneumoniae infection in vitro

Chlamydia pneumoniae can cause acute respiratory infections including pneumonia. Repeated and persistent Chlamydia infections occur and persistent C. pneumoniae infection may have a role in the pathogenesis of atherosclerosis and coronary heart disease and may also contribute to the development of chronic inflammatory lung diseases like chronic obstructive pulmonary disease (COPD) and asthma. In this thesis in vitro models for persistent C. pneumonia infection were established in epithelial and monocyte/macrophage cell lines. Expression of host cell genes in the persistent C. pneumoniae infection model of epithelial cells was studied by microarray and RT-PCR. In the monocyte/macrophage infection model expression of selected C. pneumoniae genes were studied by RT-PCR and immunofluorescence microscopy. Chlamydia is able to modulate host cell gene expression and apoptosis of host cells, which may assist Chlamydia to evade the host cells' immune responses. This, in turn, may lead to extended survival of the organism inside epithelial cells and promote the development of persistent infection. To simulate persistent C. pneumoniae infection in vivo, we set up a persistent infection model exposing the HL cell cultures to IFN-gamma. When HL cell cultures were treated with moderate concentration of IFN-gamma, the replication of C. pneumoniae DNA was unaffected while differentiation into infectious elementary bodies (EB) was strongly inhibited. By transmission electron microscopy small atypical inclusions were identified in IFN-gamma treated cultures. No second cycle of infection was observed in cells exposed to IFN-gamma , whereas C. pneumoniae was able to undergo a second cycle of infection in unexposed HL cells. Although monocytic cells can naturally restrict chlamydial growth, IFN-gamma further reduced production of infectious C. pneumoniae in Mono Mac 6 cells. Under both studied conditions no second cycle of infection could be detected in monocytic cell line suggesting persistent infection in these cells. As a step toward understanding the role of host genes in the development and pathogenesis of persistent C. pneumoniae infection, modulation of host cell gene expression during IFN-gamma induced persistent infection was examined and compared to that seen during active C. pneumoniae infection or IFN-gamma treatment. Total RNA was collected at 6 to 150 h after infection of an epithelial cell line (HL) and analyzed by a cDNA array (available at that time) representing approximately 4000 human transcripts. In initial analysis 250 of the 4000 genes were identified as differentially expressed upon active and persistent chlamydial infection and IFN-gamma treatment. In persistent infection more potent up-regulation of many genes was observed in IFN-gamma induced persistent infection than in active infection or in IFN-gamma treated cell cultures. Also sustained up-regulation was observed for some genes. In addition, we could identify nine host cell genes whose transcription was specifically altered during the IFN-gamma induced persistent C. pneumoniae infection. Strongest up-regulation in persistent infection in relation to controls was identified for insulin like growth factor binding protein 6, interferon-stimulated protein 15 kDa, cyclin D1 and interleukin 7 receptor. These results suggest that during persistent infection, C. pneumoniae reprograms the host transcriptional machinery regulating a variety of cellular processes including adhesion, cell cycle regulation, growth and inflammatory response, all of which may play important roles in the pathogenesis of persistent C. pneumoniae infection. C. pneumoniae DNA can be detected in peripheral blood mononuclear cells indicating that the bacterium can also infect monocytic cells in vivo and thereby monocytes can assist the spread of infection from the lungs to other anatomical sites. Persistent infection established at these sites could promote inflammation and enhance pathology. Thus, the mononuclear cells are in a strategic position in the development of persistent infection. To investigate the intracellular replication and fate of C. pneumoniae in mononuclear cells we analyzed the transcription of 11 C. pneumoniae genes in Mono Mac 6 cells during infection by real time RT-PCR. Our results suggest that the transcriptional profile of the studied genes in monocytes is different from that seen in epithelial cells and that IFN-gamma has a less significant effect on C. pneumoniae transcription in monocytes. Furthermore, our study shows that type III secretion system (T3SS) related genes are transcribed and that Chlamydia possesses a functional T3SS during infection in monocytes. Since C. pneumoniae infection in monocytes has been implicated to have reduced antibiotic susceptibility, this creates opportunities for novel therapeutics targeting T3SS in the management of chlamydial infection in monocytes.

Hantavirus glycoprotein GN in virion assembly and regulation of interferon response

Hantaviruses have a tri-segmented negative-stranded RNA genome. The S segment encodes the nucleocapsid protein (N), M segment two glycoproteins, Gn and Gc, and the L segment the RNA polymerase. Gn and Gc are co-translationally cleaved from a precursor and targeted to the cis-Golgi compartment. The Gn glycoprotein consists of an external domain, a transmembrane domain and a C-terminal cytoplasmic domain. In addition, the S segment of some hantaviruses, including Tula and Puumala virus, have an open reading frame (ORF) encoding a nonstructural potein NSs that can function as a weak interferon antagonist. The mechanisms of hantavirus-induced pathogenesis are not fully understood but it is known that both hemorrhagic fever with renal syndrome (HFRS) and hantavirus (cardio) pulmonary syndrome (HCPS) share various features such as increased capillary permeability, thrombocytopenia and upregulation of TNF-. Several hantaviruses have been reported to induce programmed cell death (apoptosis), such as TULV-infected Vero E6 cells which is known to be defective in interferon signaling. Recently reports describing properties of the hantavirus Gn cytoplasmic tail (Gn-CT) have appeared. The Gn-CT of hantaviruses contains animmunoreceptor tyrosine-based activation motif (ITAM) which directs receptor signaling in immune and endothelial cells; and contain highly conserved classical zinc finger domains which may have a role in the interaction with N protein. More functions of Gn protein have been discovered, but much still remains unknown. Our aim was to study the functions of Gn protein from several aspects: synthesis, degradation and interaction with N protein. Gn protein was reported to inhibit interferon induction and amplication. For this reason, we also carried out projects studying the mechanisms of IFN induction and evasion by hantavirus. We first showed degradation and aggresome formation of the Gn-CT of the apathogenic TULV. It was reported earlier that the degradation of Gn-CT is related to the pathogenicity of hantavirus. We found that the Gn-CT of the apathogenic hantaviruses (TULV, Prospect Hill virus) was degraded through the ubiquitin-proteasome pathway, and TULV Gn-CT formed aggresomes upon treatment with proteasomal inhibitor. Thus the results suggest that degradation and aggregation of the Gn-CT may be a general property of most hantaviruses, unrelated to pathogenicity. Second, we investigated the interaction of TULV N protein and the TULV Gn-CT. The Gn protein is located on the Golgi membrane and its interaction with N protein has been thought to determine the cargo of the hantaviral ribonucleoprotein which is an important step in virus assembly, but direct evidence has not been reported. We found that TULV Gn-CT fused with GST tag expressed in bacteria can pull-down the N protein expressed in mammalian cells; a mutagenesis assay was carried out, in which we found that the zinc finger motif in Gn-CT and RNA-binding motif in N protein are indispensable for the interaction. For the study of mechanisms of IFN induction and evasion by Old World hantavirus, we found that Old World hantaviruses do not produce detectable amounts of dsRNA in infected cells and the 5 -termini of their genomic RNAs are monophosphorylated. DsRNA and tri-phosphorylated RNA are considered to be critical activators of innate immnity response by interacting with PRRs (pattern recognition receptors). We examined systematically the 5´-termini of hantavirus genomic RNAs and the dsRNA production by different species of hantaviruses. We found that no detectable dsRNA was produced in cells infected by the two groups of the old world hantaviruses: Seoul, Dobrava, Saaremaa, Puumala and Tula. We also found that the genomic RNAs of these Old World hantaviruses carry 5´-monophosphate and are unable to trigger interferon induction. The antiviral response is mainly mediated by alpha/beta interferon. Recently the glycoproteins of the pathogenic hantaviruses Sin Nombre and New York-1 viruses were reported to regulate cellular interferon. We found that Gn-CT can inhibit the induction of IFN activation through Toll-like receptor (TLR) and retinoic acid-inducible gene I-like RNA helicases (RLH) pathway and that the inhibition target lies at the level of TANK-binding kinase 1 (TBK-1)/ IKK epislon complex and myeloid differentiation primary response gene (88) (MyD88) / interferon regulatory factor 7 (IRF-7) complex.

NK-and T-cell function in chronic myeloid leukemia patients treated with interferon-? monotherapy

Chronic myeloid leukemia (CML) is one of the most studied human malignancies. It is caused by an autonomously active tyrosine kinase BCR-ABL, which is a result from a translocation between chromosomes 9 and 22 in the hematopoietic stem cell. As an outcome, a Philadelphia (Ph) chromosome is formed. BCR-ABL causes disturbed cell proliferation among other things. Although targeted tyrosine kinase inhibitor therapy has been developed in the beginning of the millenium and the survival rate has increased significantly, it is still not known why some patients benefit more from the treatment than others. Furthermore, the therapy is not considered to be curative. Before the era of tyrosine kinase inhibitors, the first-line treatment for CML was interferon-? (IFN-?). However, only a small proportion of patients benefitted from the treatment. Of these patients, a few were able to discontinue the treatment without renewal of the disease. The mechanism of IFN-? is not completely understood, but it is believed that differences in the immune system can be one of the reasons why some patients have better therapy response. Kreutzman, Rohon et al. have recently discovered that patients who have been able to stop IFN-? treatment have an increased number of NK- and T-cells. They also have a unique clonal T-cell population and more cytotoxic CD8+ T-cells and less CD4+ T-cells. The aim of this master’s thesis was to study the function of T- and NK-cells in IFN-? treated patients. Although it was shown earlier that IFN-? treated patients have increased NK-cell count, the function of these cells was unknown. Therefore, we have now investigated the killing potential of patients’ NK-cells, their activation status and cell surface antigen expression. In addition, we have also studied the activation status of patients’ T-cells and their cytotoxic properties. We observed that NK-cells from patients treated with IFN-? are unable to kill leukemic cells (K562) than NK-cells from healthy controls. In addition, patients on IFN-? treatment have more active T-cells and their NK-cells have an undifferentiated immunoregulatory phenotype. Patients that have been able to stop the treatment have anergic T-and NK-cells. As a conclusion our results suggest that IFN-? therapy induces increased NK-cell count, NK-cell immunoregulatory functions and more active T-cells. After stopping IFN-? therapy, NK- and T-cells from CML patients restore anergy typical for CML.