Pyhä Teresa ja keskiajan mystiikan teoriat

In my dissertation I have studied St Teresa (1515-1582) in the light of medieval mystical theories. I have two main levels in my research: historical and theological. On the historical level I study St Teresa s personal history in the context of her family and the Spanish society. On the theological level I study both St Teresa s mysticism and her religious experience in the light of medieval mysticism. St Teresa wrote a book called Life , which is her narrative autobiography and story about her mystical spiritual formation. She reflected herself through biblical texts interpreting them in the course of the biblical hermeneutics like allegory, typology, tropology and anagogy. In addition to that she read others life stories from her period of time, but reflected herself only slightly through the sociological point of view. She used irony as a means to gain acceptance to her authority and motive to write. Her position has been described as a double bind because of writing at the request of educated men and to the non-educated women as she herself was uneducated. She used irony as a means to achieve valuation to women, to gain negative attributes connected to them and to gain authority to teach them mystical spirituality, the Bible and prayer. In this ironic tendency she was a feminist writer. In order to understand medieval mysticism I have written in the first chapter a review of the main trends in medieval mysticism in connection with the classical emotional theories. Two medieval mystical theories show an important role in St Teresa s mysticism. One is love mysticism and the other is the three partite way of mysticism (purification, illumination and union). The classic-philosophical emotional theories play a role in both patterns. The theory of love mysticism St Teresa interpreted in the traditional way stressing the spiritual meaning of love in connexion with God and neighbors. Love is an emotion, which is bound with other emotions, but all objects of love don t strengthen spiritual love. In the three partite way of mysticism purification means to find biblical values in life and to practice meditative self-knowledge theologically interpreted. In illumination human understanding has to be illuminated by God and united to mystical knowledge from God. St Teresa considered illumination a way to learn things. Illumination has also psychological aspects like recognition of many trials and pains, which come from life on earth. Theologically interpreted in illumination one should die to oneself, let oneself be transformed and renewed by God. I have also written a review of the modern philosophical discussion on personal identity where memory and mental experiences are important creators of personal identity. St Teresa bound medieval mystical teaching together with her personal religious experience. Her personal identity is by its character based on her narrative life story where mental experiences play important role. Previous researchers have labelled St Teresa as an ecstatic person whose experiences produced ecstatic phenomena to the mysticism. These phenomena combined with visions have in one respect made of her a person who has brought physical and visionary tendencies to theology. In spite of that she also represents a modern tendency trying to give words to experiences, which at first seem to be exceptional and extreme and which are easily interpreted as one-sided either physical or sexual or unsaid. In other respect I have stressed the personality of St Teresa that was represented as both strong and weak. The strong personality for her is demonstrated by religious faith and in its practice. The weak personality was for her a natural personal identity. St Teresa saw a unifying aspect in almost all. Firstly, her mysticism was aimed towards union with God and secondly, the unifying aspects and common rules in human relations in community life were central. Union with God is based on the fact that in a soul God is living in its centre, where God is present in the Trinitarian way. The picture of God in ourselves is a mirror but to get to know God better is to recognize his/her presence in us. When the soul recognizes itself as a dwelling place of God, it knows itself as God knows him/herself. There is equality between God and the soul. To be a Christian means to participate in God in his Trinitarian being. The participation to God is a process of divinization that puts a person into transformation, change and renewal. The unitive aspect concludes also knowledge of opposites between experience of community and solitude as well as community and separateness. As a founder of monasteries St Teresa practiced theology of poverty. She renewed the monastic life founding a rule called discalced that stressed ascetic tendencies. Supporters of her work were after the difficulties in the beginning both society and churchly leaders. She wrote about the monasteries including in her description at times seriousness at times humor and irony. Her stories are said to be picaresque histories that contain stories of ordinary laymen and many unexpected occasions. She exercised a kind of Bakhtinian dialogue in her letters. St Teresa stressed the virtues like sacrifice, determination and courage in the monastic life. Most of what she taught of virtues is based on biblical spirituality but there are also psychological tendencies in her writings. The theological pedagogical advice is mixed with psychology, but she herself made no distinction between different aspects in her teaching. To understand St Teresa and her mysticism is to recognize that she mixes her personal religious experience and mysticism, which widens mysticism to religious experience in a new way, although this corresponds also the very definition of mysticism. St Teresa concentrated on mental-spiritual experiences and the aim of her mystical teaching was to produce a human mind well cured like a garden that has God as its gardener.

Novel CDNF/MANF protein family : Molecular structure, expression and neurotrophic activity

Neurotrophic factors (NTFs) are secreted proteins which promote the survival of neurons, formation and maintenance of neuronal contacts and regulate synaptic plasticity. NTFs are also potential drug candidates for the treatment of neurodegenerative diseases. Parkinson’s disease (PD) is mainly caused by the degeneration of midbrain dopaminergic neurons. Current therapies for PD do not stop the neurodegeneration or repair the affected neurons. Thus, search of novel neurotrophic factors for midbrain dopaminergic neurons, which could also be used as therapeutic proteins, is highly warranted. In the present study, we identified and characterized a novel protein named conserved dopamine neurotrophic factor (CDNF), a homologous protein to mesencephalic astrocyte-derived neurotrophic factor (MANF). Others have shown that MANF supports the survival of embryonic midbrain dopaminergic neurons in vitro, and protects cultured cells against endoplasmic reticulum (ER) stress. CDNF and MANF form a novel evolutionary conserved protein family with characteristic eight conserved cysteine residues in their primary structure. The vertebrates have CDNF and MANF encoding genes, whereas the invertebrates, including Drosophila and Caenorhabditis have a single homologous CDNF/MANF gene. In this study we show that CDNF and MANF are secreted proteins. They are widely expressed in the mammalian brain, including the midbrain and striatum, and in several non-neuronal tissues. We expressed and purified recombinant human CDNF and MANF proteins, and tested the neurotrophic activity of CDNF on midbrain dopaminergic neurons using a 6-hydroxydopamine (6-OHDA) rat model of PD. In this model, a single intrastriatal injection of CDNF protected midbrain dopaminergic neurons and striatal dopaminergic fibers from the 6-OHDA toxicity. Importantly, an intrastriatal injection of CDNF also restored the functional activity of the nigrostriatal dopaminergic system when given after the striatal 6-OHDA lesion. Thus, our study shows that CDNF is a potential novel therapeutic protein for the treatment of PD. In order to elucidate the molecular mechanisms of CDNF and MANF activity, we resolved their crystal structure. CDNF and MANF proteins have two domains; an amino (N)-terminal saposin-like domain and a presumably unfolded carboxy (C)-terminal domain. The saposin-like domain, which is formed by five α-helices and stabilized by three intradomain disulphide bridges, may bind to lipids or membranes. The C-terminal domain contains an internal cysteine bridge in a CXXC motif similar to that of thiol/disulphide oxidoreductases and isomerases, and may thus facilitate protein folding in the ER. Our studies suggest that CDNF and MANF are novel potential therapeutic proteins for the treatment of neurodegenerative diseases. Future studies will reveal the neurotrophic and cytoprotective mechanisms of CDNF and MANF in more detail.

Transcriptome and glycome profiling of human hematopoietic CD133+ and CD34+ cells

New blood cells are continuously provided by self-renewing multipotent hematopoietic stem cells (HSC). The capacity of HSCs to regenerate the hematopoietic system is utilized in the treatment of patients with hematological malignancies. HSCs can be enriched using an antibody-based recognition of CD34 or CD133 glycoproteins on the cell surface. The CD133+ and CD34+ cells may have partly different roles in hematopoiesis. Furthermore, each cell has a glycome typical for that cell type. Knowledge of HSC glycobiology can be used to design therapeutic cells with improved cell proliferation or homing properties. The present studies characterize the global gene expression profile of human cord blood-derived CD133+ and CD34+ cells, and demonstrate the differences between CD133+ and CD34+ cell populations that may have an impact in transplantation when CD133+ and CD34+ selected cells are used. In addition, these studies unravel the glycome profile of primitive hematopoietic cells and reveal the transcriptional regulation of N-glycan biosynthesis in CD133+ and CD34+ cells. The gene expression profile of CD133+ cells represents 690 differentially expressed transcripts between CD133+ cells and CD133- cells. CD34+ cells have 620 transcripts differentially expressed when compared to CD34- cells. The integrated CD133+/CD34+ cell gene expression profiles proffer novel transcripts to specify HSCs. Furthermore, the differences between the gene expression profiles of CD133+ and CD34+ cells indicate differences in the transcriptional regulation of CD133+ and CD34+ cells. CD133+ cells express a lower number of hematopoietic lineage differentiation marker genes than CD34+ cells. The expression profiles suggest a more primitive nature of CD133+ cells. Moreover, CD133+ cells have characteristic glycome that differ from the glycome of CD133- cells. High mannose-type and biantennary complex-type N-glycans are enriched in CD133+ cells. N-glycosylation-related gene expression pattern of CD133+ cells identify the key genes regulating the CD133+ cell-specific glycosylation including the overexpression of MGAT2 and underexpression of MGAT4. The putative role of MAN1C1 in the increase of unprocessed high mannose-type N-glycans in CD133+ cells is also discussed. These studies provide new information on the characteristics of HSCs. Improved understanding of HSC biology can be used to design therapeutic cells with improved cell proliferation and homing properties. As a result, HSC engineering could further their clinical use.

Functional and work disability and treatment received by patients with major depressive disorder

This study is one part of a collaborative depression research project, the Vantaa Depression Study (VDS), involving the Department of Mental and Alcohol Research of the National Public Health Institute, Helsinki, and the Department of Psychiatry of the Peijas Medical Care District (PMCD), Vantaa, Finland. The VDS includes two parts, a record-based study consisting of 803 patients, and a prospective, naturalistic cohort study of 269 patients. Both studies include secondary-level care psychiatric out- and inpatients with a new episode of major depressive disorder (MDD). Data for the record-based part of the study came from a computerised patient database incorporating all outpatient visits as well as treatment periods at the inpatient unit. We included all patients aged 20 to 59 years old who had been assigned a clinical diagnosis of depressive episode or recurrent depressive disorder according to the International Classification of Diseases, 10th edition (ICD-10) criteria and who had at least one outpatient visit or day as an inpatient in the PMCD during the study period January 1, 1996, to December 31, 1996. All those with an earlier diagnosis of schizophrenia, other non-affective psychosis, or bipolar disorder were excluded. Patients treated in the somatic departments of Peijas Hospital and those who had consulted but not received treatment from the psychiatric consultation services were excluded. The study sample comprised 290 male and 513 female patients. All their psychiatric records were reviewed and each patient completed a structured form with 57 items. The treatment provided was reviewed up to the end of the depression episode or to the end of 1997. Most (84%) of the patients received antidepressants, including a minority (11%) on treatment with clearly subtherapeutic low doses. During the treatment period the depressed patients investigated averaged only a few visits to psychiatrists (median two visits), but more to other health professionals (median seven). One-fifth of both genders were inpatients, with a mean of nearly two inpatient treatment periods during the overall treatment period investigated. The median length of a hospital stay was 2 weeks. Use of antidepressants was quite conservative: The first antidepressant had been switched to another compound in only about one-fifth (22%) of patients, and only two patients had received up to five antidepressant trials. Only 7% of those prescribed any antidepressant received two antidepressants simultaneously. None of the patients was prescribed any other augmentation medication. Refusing antidepressant treatment was the most common explanation for receiving no antidepressants. During the treatment period, 19% of those not already receiving a disability pension were granted one due to psychiatric illness. These patients were nearly nine years older than those not pensioned. They were also more severely ill, made significantly more visits to professionals and received significantly more concomitant medications (hypnotics, anxiolytics, and neuroleptics) than did those receiving no pension. In the prospective part of the VDS, 806 adult patients were screened (aged 20-59 years) in the PMCD for a possible new episode of DSM-IV MDD. Of these, 542 patients were interviewed face-to-face with the WHO Schedules for Clinical Assessment in Neuropsychiatry (SCAN), Version 2.0. Exclusion criteria were the same as in the record-based part of the VDS. Of these, 542 269 patients fulfiled the criteria of DSM-IV MDE. This study investigated factors associated with patients' functional disability, social adjustment, and work disability (being on sick-leave or being granted a disability pension). In the beginning of the treatment the most important single factor associated with overall social and functional disability was found to be severity of depression, but older age and personality disorders also significantly contributed. Total duration and severity of depression, phobic disorders, alcoholism, and personality disorders all independently contributed to poor social adjustment. Of those who were employed, almost half (43%) were on sick-leave. Besides severity and number of episodes of depression, female gender and age over 50 years strongly and independently predicted being on sick-leave. Factors influencing social and occupational disability and social adjustment among patients with MDD were studied prospectively during an 18-month follow-up period. Patients' functional disability and social adjustment were alleviated during the follow-up concurrently with recovery from depression. The current level of functioning and social adjustment of a patient with depression was predicted by severity of depression, recurrence before baseline and during follow-up, lack of full remission, and time spent depressed. Comorbid psychiatric disorders, personality traits (neuroticism), and perceived social support also had a significant influence. During the 18-month follow-up period, of the 269, 13 (5%) patients switched to bipolar disorder, and 58 (20%) dropped out. Of the 198, 186 (94%) patients were at baseline not pensioned, and they were investigated. Of them, 21 were granted a disability pension during the follow-up. Those who received a pension were significantly older, more seldom had vocational education, and were more often on sick-leave than those not pensioned, but did not differ with regard to any other sociodemographic or clinical factors. Patients with MDD received mostly adequate antidepressant treatment, but problems existed in treatment intensity and monitoring. It is challenging to find those at greatest risk for disability and to provide them adequate and efficacious treatment. This includes great challenges to the whole society to provide sufficient resources.

Novel matrix metalloproteinases in intestinal inflammation and in cancer of the gastrointestinal tract

Matrix metalloproteinases (MMPs) comprise a family of 23 zinc-dependent human endopeptidases that can degrade virtually all components of the extracellular matrix (ECM). They are classified into eight subgroups according to their structure and into six subgroups based on their substrate-specificity. MMPs have been implicated in inflammation, tissue destruction, cell migration, arthritis, vascular remodeling, angiogenesis, and tumor growth and invasion. MMPs are inhibited by their natural inhibitors, tissue inhibitors of metalloproteinases (TIMPs). Different MMPs function in the same tasks depending on the tissue or cancer subtype. I investigated the role of recently discovered MMPs, especially MMPs-19 and -26, in intestinal inflammation, in intestinal and cutaneous wound healing, and in intestinal cancer. Several MMPs and TIMPs were studied to determine their exact location at tissue level and to obtain information on possible functions of MMPs in such tissues and diseases as the healthy intestine, inflammatory bowel disease (IBD), neonatal necrotizing enterocolitis (NEC), pyoderma gangrenosum (PG), and colorectal as well as pancreatic cancers. In latent celiac disease (CD), I attempted to identify markers to predict later onset of CD in children and adolescents. The main methods used were immunohistochemistry, in situ hybridization, and Taqman RT-PCR. My results show that MMP-26 is important for re-epithelialization in intestinal and cutaneous wound healing. In colon and pancreatic cancers, MMP-26 seems to be a marker of invasive potential, although it is not itself expressed at the invasive front. MMP-21 is upregulated in pancreatic cancer and may be associated with tumor differentiation. MMPs-19 and -28 are associated with normal tissue turnover in the intestine, but they disappear in tumor progression as if they were protective markers . MMP-12 is an essential protease in intestinal inflammation and tissue destruction, as seen here in NEC and in previous CD studies. In patients with type 1 diabetes (T1D), MMPs-1, -3, and -12 were upregulated in the intestinal mucosa. Furthermore, MMP-7 was strongly elevated in NEC. In a model of aberrant wound repair, PG, MMPs-8, -9, and 10 and TNFα may promote ECM destruction, while absence of MMP-1 and MMP-26 from keratinocytes retards re-epithelialization. Based on my results, I suggest MMP-26 to be considered a putative marker for poor prognosis in pancreatic and colon cancer. However, since it functions differently in various tissues and tumor subtypes, this use cannot be generalized. Furthermore, MMP-26 is a beneficial marker for wound healing if expressed by migrating epithelial cells. MMP-12 expression in latent CD patients warrants research in a larger patient population to confirm its role as a specific marker for CD in pathologically indistinct cases. MMP-7 should be considered one of the most crucial proteases in NEC-associated tissue destruction; hence, specific inhibitors of this MMP are worth investigating. In PG, TNFα inhibitors are potential therapeutic agents, as shown already in clinical trials. In conclusion, studies of several MMPs in specific diseases and in healthy tissues are needed to elucidate their roles at the tissue level. MMPs and TIMPs are not exclusively destructive or reparative in tissues. They seem to function differently in different tissues. To identify selective MMP inhibitors, we must thoroughly understand the MMP profile (degradome) and their functions in various organs not to interfere with normal reparative functions during wound repair or beneficial host-response effects during cancer initiation and growth.

SPECT-aided diagnostics and genetic risk factors in Parkinson's disease

Hypokinesia, rigidity, tremor, and postural instability are the cardinal symptoms of Parkinson s disease (PD). Since these symptoms are not specific to PD the diagnosis may be uncertain in early PD. Etiology and pathogenesis of PD remain unclear. There is no neuroprotective therapy. Genetic findings are expected to reveal metabolic routes in PD pathogenesis and thereby eventually lead to therapeutic innovations. In this thesis, we first aimed to study the usefulness and accuracy of 123I-b-CIT SPECT in the diagnosis of PD in a consecutive clinic-based material including various movement disorders. We subsequently a genetic project to identify genetic risk factors for sporadic PD using a candidate gene approach in a case-control setting including 147 sporadic PD patients and 137 spouse controls. Dopamine transporter imaging by 123I-b-CIT SPECT could distinguish PD from essential tremor, drug-induced parkinsonism, dystonia and psychogenic parkinsonism. However, b-CIT uptake in Parkinson plus syndromes (PSP and multiple system atrophy) and dementia with Lewy bodies was not significantly different from PD. 123I-b-CIT SPECT could not reliably differentiate PD from vascular parkinsonism. 123I-b-CIT SPECT was 100% sensitive and specific in the diagnosis of PD in patients younger than 55 years but less specific in older patients, due to differential distribution of the above conditions in the younger and older age groups. 123I-b-CIT SPECT correlated with symptoms and detected bilateral nigrostriatal defect in patients whose PD was still in unilateral stage. Thus, in addition to as a differential diagnostic aid, 123I-b-CIT SPECT may be used to detect PD early, even pre-symptomatically in at-risk individuals. 123I-b-CIT SPECT was used to aid in the collection of patients to the genetic studies. In the genetic part of this thesis we found an association between PD and a polymorphic CAG-repeat in POLG1 gene encoding the catalytic subunit of mitochondrial polymerase gamma. The CAG-repeat encodes a polyglutamine tract (polyQ), the two most common lengths of which are 10Q (86-90%) and 11Q. In our Finnish material, the rarer non-10Q or non-11Q length variants (6Q-9Q, 12Q-14Q, 4R+9Q) were more frequent in patients than in spouse controls (10% vs. 3.5 %, p=0.003), or population controls (p=0.001). Therefore, we performed a replication study in 652 North American PD patients and 292 controls. Non-10/11Q alleles were more common in the US PD patients compared to the controls but the difference did not reach statistical significance (p=0.07). This larger data suggested our original definition of variant length allele might need reconsideration. Most previous studies on phenotypic effects of POLG1 polyQ have defined 10Q as the only normal allele. Non-10Q alleles were significantly more common in patients compared to the controls (17.3% vs. 12.3 %, p= 0.005). This association between non-10Q length variants and PD remained significant when compared to a larger set of 1541 literature controls (p=0.00005). In conclusion, POLG1 polyQ alleles other than 10Q may predispose to PD. We did not find association between PD and parkin or DJ-1, genes underlying autosomal recessive parkinsonism. The functional Val158Met polymorphism, which affects the catalytic effect of COMT enzyme, and another coding polymorphism in COMT were not associated with PD in our patient material. The APOE e2/3/4 polymorphism modifies risk for Alzheimer s disease and prognosis of for example brain trauma. APOE promoter and enhancer polymorphisms 219G/T and +113G/C, and APOE e3 haplotypes, have also been shown to modify the risk of Alzheimer s disease but not reported in PD. No association was found between PD and APOE e2/3/4 polymorphism, the promoter or enhancer polymorphisms, or the e3 haplotypes.

Therapeutic hypothermia after cardiac arrest : Studies on neurological and cardiological outcome and prediction of outcome in hypothermia-treated patients resuscitated from out-of-hospital cardiac arrest

The outcome of the successfully resuscitated patient is mainly determined by the extent of hypoxic-ischemic cerebral injury, and hypothermia has multiple mechanisms of action in mitigating such injury. The present study was undertaken from 1997 to 2001 in Helsinki as a part of the European multicenter study Hypothermia after cardiac arrest (HACA) to test the neuroprotective effect of therapeutic hypothermia in patients resuscitated from out-of-hospital ventricular fibrillation (VF) cardiac arrest (CA). The aim of this substudy was to examine the neurological and cardiological outcome of these patients, and especially to study and develop methods for prediction of outcome in the hypothermia-treated patients. A total of 275 patients were randomized to the HACA trial in Europe. In Helsinki, 70 patients were enrolled in the study according to the inclusion criteria. Those randomized to hypothermia were actively cooled externally to a core temperature 33 ± 1ºC for 24 hours with a cooling device. Serum markers of ischemic neuronal injury, NSE and S-100B, were sampled at 24, 36, and 48 hours after CA. Somatosensory and brain stem auditory evoked potentials (SEPs and BAEPs) were recorded 24 to 28 hours after CA; 24-hour ambulatory electrocardiography recordings were performed three times during the first two weeks and arrhythmias and heart rate variability (HRV) were analyzed from the tapes. The clinical outcome was assessed 3 and 6 months after CA. Neuropsychological examinations were performed on the conscious survivors 3 months after the CA. Quantitative electroencephalography (Q-EEG) and auditory P300 event-related potentials were studied at the same time-point. Therapeutic hypothermia of 33ºC for 24 hours led to an increased chance of good neurological outcome and survival after out-of-hospital VF CA. In the HACA study, 55% of hypothermia-treated patients and 39% of normothermia-treated patients reached a good neurological outcome (p=0.009) at 6 months after CA. Use of therapeutic hypothermia was not associated with any increase in clinically significant arrhythmias. The levels of serum NSE, but not the levels of S-100B, were lower in hypothermia- than in normothermia-treated patients. A decrease in NSE values between 24 and 48 hours was associated with good outcome at 6 months after CA. Decreasing levels of serum NSE but not of S-100B over time may indicate selective attenuation of delayed neuronal death by therapeutic hypothermia, and the time-course of serum NSE between 24 and 48 hours after CA may help in clinical decision-making. In SEP recordings bilaterally absent N20 responses predicted permanent coma with a specificity of 100% in both treatment arms. Recording of BAEPs provided no additional benefit in outcome prediction. Preserved 24- to 48-hour HRV may be a predictor of favorable outcome in CA patients treated with hypothermia. At 3 months after CA, no differences appeared in any cognitive functions between the two groups: 67% of patients in the hypothermia and 44% patients in the normothermia group were cognitively intact or had only very mild impairment. No significant differences emerged in any of the Q-EEG parameters between the two groups. The amplitude of P300 potential was significantly higher in the hypothermia-treated group. These results give further support to the use of therapeutic hypothermia in patients with sudden out-of-hospital CA.

Matrix proteinases in lung injury in the preterm infant

During inflammation, excess production and release of matrix proteinases, including matrix metalloproteinases (MMPs) and serine proteinases, may result in dysregulated extracellular proteolysis leading to development of tissue damage. Pulmonary inflammation may play an important role in the pathogenesis of lung injury in the preterm infant. The aims of this study were to evaluate involvement of MMPs and serine proteinase trypsin in acute and chronic lung injury in preterm infants and to study the role of these enzymes in acute lung injury by means of an animal model of hyperoxic lung injury. Molecular forms and levels of MMP-2, -8, and -9, and their specific inhibitor, tissue inhibitor of metalloproteinases (TIMP)-2, as well as trypsin were studied in tracheal aspirate fluid (TAF) samples collected from preterm infants with respiratory distress. Expression and distribution of trypsin-2 and proteinase-activated receptor 2 (PAR2) was examined in autopsy lung specimens from fetuses, from preterm infants with respiratory distress syndrome (RDS) or bronchopulmonary dysplasia (BPD), and from newborn infants without lung injury. We detected higher MMP-8 and trypsin-2 and lower TIMP-2 in TAF from preterm infants with more severe acute respiratory distress. Infants subsequently developing BPD had higher levels of MMP-8 and trypsin-2 early postnatally than did those who survived without this chronic lung injury. Immunohistochemically, trypsin-2 was mainly detectable in bronchial epithelium, but also in alveolar epithelium, and its expression was strongest in prolonged RDS. Since trypsin-2 is potent activator of PAR2, a G-protein coupled receptor involved in inflammation, we studied PAR2 expression in the lung. PAR2 co-localized with trypsin-2 in bronchoalveolar epithelium and its expression was significantly higher in bronchoalveolar epithelium in preterm infants with prolonged RDS than in newborn controls. In the experimental study, rats were exposed to >95% oxygen for 24, 48, and 60 hours, or room air. At 48 hours of hyperoxia, MMP-8 and trypsin levels sharply increased in bronchoalveolar lavage fluid, and expression of trypsin appeared in alveolar epithelium, and MMP-8 predominantly in macrophages. In conclusion, high pulmonary MMP-8 and trypsin-2 early postnatally are associated with severity of acute lung injury and subsequent development of BPD in preterm infants. In the injured preterm lung, trypsin-2 co-localizes with PAR2 in bronchoalveolar epithelium, suggesting that PAR2 activated by high levels of trypsin-2 is involved in lung inflammation associated with development of BPD. Marked increase in MMP-8 and trypsin early in the course of experimental hyperoxic lung injury suggests that these enzymes play a role in the pathogenesis of acute lung injury. Further exploration of the roles of trypsin and MMP-8 in lung injury may offer new targets for therapeutic intervention.

Circulating Glucocorticoid Bioactivity and Serum Glucocorticoid-Responsive Biomarkers during Steroid Therapy in Children and Adolescents with Inflammatory Bowel Disease

Objective: Glucocorticoid therapy is used worldwide to treat various inflammatory and immune conditions, including inflammatory bowel disease (IBD). In IBD, 80% of the patients obtain a positive response to the therapy; however the development of glucocorticoid-related side-effects is common. Our aim was therefore to study the possibility of optimizing glucocorticoid therapy in children and adolescents with IBD by measuring circulating glucocorticoid bioactivity (GBA) and serum glucocorticoid-responsive biomarkers in patients receiving steroid treatment for active disease. Methods: A total of sixty-nine paediatric IBD patients from the Paediatric Outpatient Clinics of the University Hospitals of Helsinki and Tampere participated in the studies. Control patients included 101 non-IBD patients and 41 disease controls in remission. In patients with active disease, blood samples were withdrawn before the glucocorticoid therapy was started, at 2-4 weeks after the initiation of the steroid and at 1-month intervals thereafter. Clinical response to glucocorticoid treatment and the development of steroid adverse events was carefully registered. GBA was analyzed with a COS-1 cell bioassay. The measured glucocorticoid therapy-responsive biomarkers included adipocyte-derived adiponectin and leptin, bone turnover-related collagen markers amino-terminal type I procollagen propeptide (PINP) and carboxyterminal telopeptide of type I collagen (ICTP) as well as insulin-like growth factor 1 (IGF-1) and sex hormone-binding globulin (SHBG), and inflammatory marker high-sensitivity C-reactive protein (hs-CRP). Results: The most promising marker for glucocorticoid sensitivity was serum adiponectin that associated with steroid therapy–related adverse events. Serum leptin indicated a similar trend. In contrast, circulating GBA rose in all subjects receiving glucocorticoid treatment but did not associate with the clinical response to steroids or with glucocorticoid therapy-related side-effects. Of notice, young patients (<10 years) showed similar GBA levels than older patients, despite receiving higher weight-adjusted doses of glucocorticoid. Markers of bone formation were lower in children with active IBD than in the control patients, probably reflecting the suppressive effect of the active inflammation. The onset of the glucocorticoid therapy further suppressed bone turnover. Inflammatory marker hs-CRP decreased readily after the initiation of the steroid, however the decrease did not associate with the clinical response to glucocorticoids. Conclusions: This is the first study to show that adipocyte-derived adiponectin associates with steroid therapy-induced side-effects. Further studies are needed, but it is possible that the adiponectin measurement could aid the recognition of glucocorticoid-sensitive patients in the future. GBA and the other markers reflecting glucocorticoid activity in different tissues changed during the treatment, however their change did not correlate with the therapeutic response to steroids or with the development of glucocorticoid-related side effects and therefore cannot guide the therapy in these patients. Studies such as as the present one that combine clinical data with newly developed biomolecular technology are needed to step-by-step build a general picture of the glucocorticoid actions in different tissues.

Theoretical modeling of ionospheric electrodynamics including induction effects

This thesis deals with theoretical modeling of the electrodynamics of auroral ionospheres. In the five research articles forming the main part of the thesis we have concentrated on two main themes: Development of new data-analysis techniques and study of inductive phenomena in the ionospheric electrodynamics. The introductory part of the thesis provides a background for these new results and places them in the wider context of ionospheric research. In this thesis we have developed a new tool (called 1D SECS) for analysing ground based magnetic measurements from a 1-dimensional magnetometer chain (usually aligned in the North-South direction) and a new method for obtaining ionospheric electric field from combined ground based magnetic measurements and estimated ionospheric electric conductance. Both these methods are based on earlier work, but contain important new features: 1D SECS respects the spherical geometry of large scale ionospheric electrojet systems and due to an innovative way of implementing boundary conditions the new method for obtaining electric fields can be applied also at local scale studies. These new calculation methods have been tested using both simulated and real data. The tests indicate that the new methods are more reliable than the previous techniques. Inductive phenomena are intimately related to temporal changes in electric currents. As the large scale ionospheric current systems change relatively slowly, in time scales of several minutes or hours, inductive effects are usually assumed to be negligible. However, during the past ten years, it has been realised that induction can play an important part in some ionospheric phenomena. In this thesis we have studied the role of inductive electric fields and currents in ionospheric electrodynamics. We have formulated the induction problem so that only ionospheric electric parameters are used in the calculations. This is in contrast to previous studies, which require knowledge of the magnetospheric-ionosphere coupling. We have applied our technique to several realistic models of typical auroral phenomena. The results indicate that inductive electric fields and currents are locally important during the most dynamical phenomena (like the westward travelling surge, WTS). In these situations induction may locally contribute up to 20-30% of the total ionospheric electric field and currents. Inductive phenomena do also change the field-aligned currents flowing between the ionosphere and magnetosphere, thus modifying the coupling between the two regions.

Fertility in Namibia : Changes in fertility levels in North-Central Namibia 1960-2001, including an assessment of the impact of HIV

The aim of this study was to estimate the development of fertility in North-Central Namibia, former Ovamboland, from 1960 to 2001. Special attention was given to the onset of fertility decline and to the impact of the HIV epidemic on fertility. An additional aim was to introduce parish registers as a source of data for fertility research in Africa. Data used consisted of parish registers from Evangelical Lutheran congregations, the 1991 and 2001 Population and Housing Censuses, the 1992 and 2000 Namibia Demographic and Health Surveys, and the HIV sentinel surveillances of 1992-2004. Both period and cohort fertility were analysed. The P/F ratio method was used when analysing census data. The impact of HIV infection on fertility was estimated indirectly by comparing the fertility histories of women who died at an age of less than 50 years with the fertility of other women. The impact of the HIV epidemic on fertility was assessed both among infected women and in the general population. Fertility in the study population began to decline in 1980. The decline was rapid during the 1980s, levelled off in the early 1990s at the end of war of independence and then continued to decline until the end of the study period. According to parish registers, total fertility was 6.4 in the 1960s and 6.5 in the 1970s, and declined to 5.1 in the 1980s and 4.2 in the 1990s. Adjustment of these total fertility rates to correspond to levels of fertility based on data from the 1991 and 2001 censuses resulted in total fertility declining from 7.6 in 1960-79 to 6.0 in 1980-89, and to 4.9 in 1990-99. The decline was associated with increased age at first marriage, declining marital fertility and increasing premarital fertility. Fertility among adolescents increased, whereas the fertility of women in all other age groups declined. During the 1980s, the war of independence contributed to declining fertility through spousal separation and delayed marriages. Contraception has been employed in the study region since the 1980s, but in the early 1990s, use of contraceptives was still so limited that fertility was higher in North-Central Namibia than in other regions of the country. In the 1990s, fertility decline was largely a result of the increased prevalence of contraception. HIV prevalence among pregnant women increased from 4% in 1992 to 25% in 2001. In 2001, total fertility among HIV-infected women (3.7) was lower than that among other women (4.8), resulting in total fertility of 4.4 among the general population in 2001. The HIV epidemic explained more than a quarter of the decline in total fertility at population level during most of the 1990s. The HIV epidemic also reduced the number of children born by reducing the number of potential mothers. In the future, HIV will have an extensive influence on both the size and age structure of the Namibian population. Although HIV influences demographic development through both fertility and mortality, the effect through changes in fertility will be smaller than the effect through mortality. In the study region, as in some other regions of southern Africa, a new type of demographic transition is under way, one in which population growth stagnates or even reverses because of the combined effects of declining fertility and increasing mortality, both of which are consequences of the HIV pandemic.

Oncolytic Adenoviruses with Radiation Therapy for Treatment of Prostate Cancer

Prostate cancer is the most common cancer in males. Although many patients with localized disease can be cured with surgery and radiotherapy, advanced disease and especially castration resistant metastatic disease remains incurable, with a median life expectancy of less than 18 months. Oncolytic adenoviruses (Ads) are a new promising treatment against cancer due to their innate capacity to kill cancer cells. Viral replication in tumor cells leads to oncolysis and production of a multiplicity of new virions that are capable of further destroying cancerous tissue. Oncolytic Ads can be modified for tumor targeted infection and replication and be armed with therapeutic transgenes to maximize the oncolytic effect. Worldwide, clinical trials with oncolytic Ads have demonstrated good safety while the antitumor efficacy remains to be improved. Importantly, the best responses have been reported when oncolytic adenoviruses have been combined with standard cancer treatments, such as chemotherapy and radiation. Further, a challenge in many virotherapy approaches has been the monitoring of virus replication in vivo. Reporter genes have been extensively used as transgenes to evaluate the biodistribution of the virus and activity of specific promoters. However, these techniques are often limited to preclinical evaluation and not amenable to human use. The aim of the thesis was to find and develop new oncolytic Ads with maximum efficacy against metastatic, castration resistant prostate cancer and study them in vitro and in vivo combined to different forms of radiation therapy. Using combination therapy, we were aiming for better antitumor efficacy with reduced side effects. Capsid modified Ads for enhanced transduction were studied. Serotype 3 targeted chimera, Ad5/3, was found to have enhanced infectivity for prostate cancer and was used for developing new viruses for the study. Correlation between Ad-encoded marker peptide secretion and simultaneous viral replication was evaluated and the effects of radiotherapy on viral replication were studied in detail. We found that the repair of double strand breaks caused by ionizing radiation was inhibited by adenoviral proteins and led to autophagic cell death. Both subcutaneous models and intrapulmonary tumor models mimicking metastatic, aggressive disease were used in vivo. Virus efficacy was evaluated by intratumoral injections. Also, intravenous administration was evaluated to study the effectiveness in metastatic disease. Oncolytic adenovirus treatment led to significant tumor growth control and increased the survival rate of the mice. These results were further improved when oncolytic Ads were combined with radiation therapy. Oncolytic Ads expressing human sodium/iodide transporter (hNIS) as a transgene were evaluated for their oncolytic potency and for the functionality of hNIS in vitro and in vivo. Monitoring of viral replication was also assessed using different imaging modalities relative to clinical use. SPECT imaging of tumor-bearing mice was evaluated and combined with simultaneous CT-scanning to obtain important anatomical information on biodistribution, also in a three-dimensional form. It was shown that hNIS-expressing adenoviruses could harbour a bi-functional transgene allowing for localization and imaging of viral replication. Targeted radiotherapy was applied by systemic radioiodide administration and resulted in iodide accumulation into Ad-infected tumor. The combination treatment showed significantly enhanced antitumor efficacy in mice bearing prostate cancer tumors. In summary, the results presented above aim to provide new treatment modalities for castration resistant prostate cancer. Molecular insights were provided for better understanding of the benefits of combined radiation therapy and oncolytic adenoviruses, which will hopefully facilitate the translation of the approach into clinical use for humans.

MECHANISMS AND EFFECTS OF MITOCHONDRIAL DNA INSTABILITY AND COPY NUMBER MANIPULATION

Defects in mitochondrial DNA (mtDNA) maintenance cause a range of human diseases, including autosomal dominant progressive external ophthalmoplegia (adPEO). This study aimed to clarify the molecular background of adPEO. We discovered that deoxynucleoside triphosphate (dNTP) metabolism plays a crucial in mtDNA maintenance and were thus prompted to search for therapeutic strategies based on the modulation of cellular dNTP pools or mtDNA copy number. Human mtDNA is a 16.6 kb circular molecule present in hundreds to thousands of copies per cell. mtDNA is compacted into nucleoprotein clusters called nucleoids. mtDNA maintenance diseases result from defects in nuclear encoded proteins that maintain the mtDNA. These syndromes typically afflict highly differentiated, post-mitotic tissues such as muscle and nerve, but virtually any organ can be affected. adPEO is a disease where mtDNA molecules with large-scale deletions accumulate in patients tissues, particularly in skeletal muscle. Mutations in five nuclear genes, encoding the proteins ANT1, Twinkle, POLG, POLG2 and OPA1, have previously been shown to cause adPEO. Here, we studied a large North American pedigree with adPEO, and identified a novel heterozygous mutation in the gene RRM2B, which encodes the p53R2 subunit of the enzyme ribonucleotide reductase (RNR). RNR is the rate-limiting enzyme in dNTP biosynthesis, and is required both for nuclear and mitochondrial DNA replication. The mutation results in the expression of a truncated form of p53R2, which is likely to compete with the wild-type allele. A change in enzyme function leads to defective mtDNA replication due to altered dNTP pools. Therefore, RRM2B is a novel adPEO disease gene. The importance of adequate dNTP pools and RNR function for mtDNA maintenance has been established in many organisms. In yeast, induction of RNR has previously been shown to increase mtDNA copy number, and to rescue the phenotype caused by mutations in the yeast mtDNA polymerase. To further study the role of RNR in mammalian mtDNA maintenance, we used mice that broadly overexpress the RNR subunits Rrm1, Rrm2 or p53R2. Active RNR is a heterotetramer consisting of two large subunits (Rrm1) and two small subunits (either Rrm2 or p53R2). We also created bitransgenic mice that overexpress Rrm1 together with either Rrm2 or p53R2. In contrast to the previous findings in yeast, bitransgenic RNR overexpression led to mtDNA depletion in mouse skeletal muscle, without mtDNA deletions or point mutations. The mtDNA depletion was associated with imbalanced dNTP pools. Furthermore, the mRNA expression levels of Rrm1 and p53R2 were found to correlate with mtDNA copy number in two independent mouse models, suggesting nuclear-mitochondrial cross talk with regard to mtDNA copy number. We conclude that tight regulation of RNR is needed to prevent harmful alterations in the dNTP pool balance, which can lead to disordered mtDNA maintenance. Increasing the copy number of wild-type mtDNA has been suggested as a strategy for treating PEO and other mitochondrial diseases. Only two proteins are known to cause a robust increase in mtDNA copy number when overexpressed in mice; the mitochondrial transcription factor A (TFAM), and the mitochondrial replicative helicase Twinkle. We studied the mechanisms by which Twinkle and TFAM elevate mtDNA levels, and showed that Twinkle specifically implements mtDNA synthesis. Furthermore, both Twinkle and TFAM were found to increase mtDNA content per nucleoid. Increased mtDNA content in mouse tissues correlated with an age-related accumulation of mtDNA deletions, depletion of mitochondrial transcripts, and progressive respiratory dysfunction. Simultaneous overexpression of Twinkle and TFAM led to a further increase in the mtDNA content of nucleoids, and aggravated the respiratory deficiency. These results suggested that high mtDNA levels have detrimental long-term effects in mice. These data have to be considered when developing and evaluating treatment strategies for elevating mtDNA copy number.