Cognitive Development of Very Low Birth Weight Children from Infancy to Pre-school Age

The survival of preterm born infants has increased but the prevalence of long-term morbidities has still remained high. Preterm born children are at an increased risk for various developmental impairments including both severe neurological deficits as well as deficits in cognitive development. According to the literature the developmental outcome perspective differs between countries, centers, and eras. Definitions of preterm infant vary between studies, and the follow-up has been carried out with diverse methods making the comparison less reliable. It is essential to offer parents upto-date information about the outcome of preterm infants born in the same area. A centralized follow-up of children at risk makes it possible to monitor the consequences of changes in the treatment practices of hospitals on developmental outcome. This thesis is part of a larger regional, prospective multidisciplinary follow-up project entitled “Development and Functioning of Very Low Birth Weight Infants from Infancy to School Age” (PIeniPAinoisten RIskilasten käyttäytyminen ja toimintakyky imeväisiästä kouluikään, PIPARI). The thesis consists of four original studies that present data of very low birth weight (VLBW) infants born between 2001 and 2006, who are followed up from the neonatal period until the age of five years. The main outcome measure was cognitive development and secondary outcomes were significant neurological deficits (cerebral palsy, CP, deafness, and blindness). In Study I, the early crying and fussing behavior of preterm infants was studied using parental diaries, and the relation of crying behavior and cognitive and motor development at the age of two years was assessed. In Study II, the developmental outcome (cognitive, CP, deafness, and blindness) at the age of two years was studied in relation to demographic, antenatal, neonatal, and brain imaging data. Development was studied in relationship to a full-term born control group born in the same hospital. In Study III, the stability of cognitive development was studied in VLBW and full-term groups by comparing the outcomes at the ages of two and five years. Finally, in Study IV the precursors of reading skills (phonological processing, rapid automatized naming, and letter knowledge) were assessed for VLBW and full-term children at the age of five years. Pre-reading skills were studied in relation to demographic, antenatal, neonatal, and brain imaging data. The main findings of the thesis were that VLBW infants who fussed or cried more in the infancy were not at greater risk for problems in their cognitive development. However, crying was associated with poorer motor development. The developmental outcome of the present population was better that has been reported earlier and this improvement covered also cognitive development. However, the difference to fullterm born peers was still significant. Major brain pathology and intestinal perforation were independent significant risk factors for adverse outcome, also when several individual risk factors were controlled for. Cognitive development at the age of two years was strongly related with development at the age of five years, stressing the importance of the early assessment, and the possibility for early interventions. Finally, VLBW children had poorer pre-reading skills compared with their full-term born peers, but the IQ was an important mediator even when children with mental retardation were excluded from the analysis. The findings suggest that counseling parents about the developmental perspectives of their preterm infant should be based on data covering the same birth hospital. Neonatal brain imaging data and neonatal morbidity are important predictors for developmental outcome. The findings of the present study stress the importance of both short-term (two years) and long-term (five years) follow-ups for the individual, and for improving the quality of care.

Psychological well-being of the parents and child development, behavior, and quality of life in very low birth weight infants

The parents of premature infants, especially the mothers, are at increased risk for distress. Infants born prematurely are at risk for developmental problems. The aim of this study was to investigate whether the psychological well-being of both parents is associated with child development in very low birth weight (VLBW, ≤1500g) children. The burden of prematurity-related morbidity to the children and to the family was also assessed. A cohort of 201 VLBW infants born during 2001–2006 in the Turku University Hospital, Finland, and their parents were studied (I–IV). One study included a control group (n=166) of full-term infants (IV). The psychological well-being of the parents was evaluated by assessments of depressive symptoms, parenting stress, the sense of coherence and general family functioning. Cognitive, behavioral, and socio-emotional development, and the health-related quality of life (HRQoL) of the children were determined when the children were 2 to 8 years old. The psychological well-being of the parents was associated with the cognitive, behavioral and social development of the VLBW children. The VLBW infants with prematurity-related morbidities had a poorer HRQoL and the general functioning of the family was inferior compared to the control children and their families. 64.5% of the VLBW children survived without morbidities. Most of the VLBW children did not have significant behavior problems (93%), had normal social skills (63%), had no emotional problems (64%), and had no problems in executive functioning (62%). Only 3% of the surviving VLBW infants had significant cognitive delay. In conclusion, the depressive symptoms and stress of the parents can be risk factors for disadvantageous child development, while a strong sense of coherence can be protective. Parents of the premature children with developmental delays might also experience more depressive symptoms and stress than other parents. Prematurity-related morbidities were a burden to the VLBW child as well as to the family.

Accelerating food safety and quality control: The use of antibody engineering techniques in the detec-tion of low-molecular weight food contaminants

The increased awareness and evolved consumer habits have set more demanding standards for the quality and safety control of food products. The production of foodstuffs which fulfill these standards can be hampered by different low-molecular weight contaminants. Such compounds can consist of, for example residues of antibiotics in animal use or mycotoxins. The extremely small size of the compounds has hindered the development of analytical methods suitable for routine use, and the methods currently in use require expensive instrumentation and qualified personnel to operate them. There is a need for new, cost-efficient and simple assay concepts which can be used for field testing and are capable of processing large sample quantities rapidly. Immunoassays have been considered as the golden standard for such rapid on-site screening methods. The introduction of directed antibody engineering and in vitro display technologies has facilitated the development of novel antibody based methods for the detection of low-molecular weight food contaminants. The primary aim of this study was to generate and engineer antibodies against low-molecular weight compounds found in various foodstuffs. The three antigen groups selected as targets of antibody development cause food safety and quality defects in wide range of products: 1) fluoroquinolones: a family of synthetic broad-spectrum antibacterial drugs used to treat wide range of human and animal infections, 2) deoxynivalenol: type B trichothecene mycotoxin, a widely recognized problem for crops and animal feeds globally, and 3) skatole, or 3-methyindole is one of the two compounds responsible for boar taint, found in the meat of monogastric animals. This study describes the generation and engineering of antibodies with versatile binding properties against low-molecular weight food contaminants, and the consecutive development of immunoassays for the detection of the respective compounds.

Retentiopolymeerin optimointi täyteaineiden yhtäaikaisannostelussa

Kirjallisuusosassa perehdyttiin retentioaineisiin ja täyteaineisiin sekä retentioaineiden ja rainanmuodostusolosuhteiden vaikutukseen retentioon, vedenpoistoon ja paperin ominaisuuksiin. Tarkemmin kirjallisuusosassa keskityttiin täyteaineiden esiflokkaukseen, retentiopolymeerin adsorptioon sekä retentiopolymeerien ja täyteaineiden annostelutapoihin. Kokeellisessa osassa tutkittiin sarjaa retentiopolymeerejä, joiden varaustiheys ja moolimassa muuttuivat. Yksi polymeereistä oli kahdesta polymeeristävalmistettu suoladispersio ja yksi modifioitu kationinen PAM. Näillä polymeereillä käytiin läpi koesarjoja, joissa muutettiin täyteaineen annosteluaikaa retentiopolymeerin annosteluajan pysyessä vakiona. Lähinnä vertailtiin keskenään perinteistä annostelua, jossa täyteaine annosteltiin paljon ennen retentiopolymeeriä,ja yhtäaikaista annostelua, jossa molemmat annosteltiin yhtä aikaa lähellä perälaatikkoa. Kokeet tehtiin MBF-laitteella, jolla pystytään paperikonetta vastaaviin pulsaatiotaajuuksiin ja sillä voidaan valmistaa tasoviirakoneella valmistetunpaperin kaltaisia laboratorioarkkeja. Valmistetuista arkeista tutkittiin retentioita ja paperiteknisiä ominaisuuksia. Laboratoriokokeiden perusteella yhtäaikainen annostelu antoi paremmat täyteaineretentiot verrattaessa perinteiseen annosteluun lähes kaikissa koesarjoissa. Varsinkin lyhytketjuiset polymeerit näyttivättoimivan hyvin yhtäaikaisannostelulla, mikä saattaisi johtua siitä, että lyhyt reagointiaika sulpun kanssa on lyhytketjuisille polymeereille edullinen, sillä silloin polymeeriketjun konformaatio ei ehdi asettua liian alhaiseksi ja ketjun toimintakyky säilyy parempana. Polymeerin varaustiheyden kasvaessa riittävästi laski täyteaineretentio seuraavissa tapauksissa: SC-massa + kaoliini ja SC-massa +GCC kummallakin annostelulla sekä SC-massa + PCC A perinteisellä annostelulla. Hienopaperimassalla samaa trendiä noudatti täyteaine GCC kummallakin annostelulla, kun taas PCC H:ta käytettäessä paranivat täyteaineretentiot molemmilla annosteluilla. Retentiopolymeerin moolimassan kasvaessa riittävästi kääntyi täyteaineretentio laskuun täyteaineilla GCC ja kaoliini, kun käytettiin SC-massaa. Hienopaperimassalla GCC noudatti tätä samaa taipumusta. Sen sijaan SC-massalla PCC A:takäytettäessä täyteaineretentio puolestaan nousi hieman moolimassan kasvaessa. Näin kävi myös hienopaperimassalla, kun täyteaineena käytettiin PCC H:ta. Käytettäessä SC-massaa, perinteisellä annostelulla saatiin parempi tai yhtä hyvä valonsironta kuin yhtäaikaisella annostelulla kaikilla täyteaineilla. Tämä saattaisi johtua siitä, että yhtäaikaisannostelulla on muodostunut suurempia täyteaineflokkeja, mikä on alentanut valoa sirottavia pintoja. Täyteaineista korkeimmat valonsirontakertoimet antoi PCC A ja alhaisimmat kaoliini. PCC A:lla oli kapein partikkelikokojakauma, mikä korottaa paperin valonsirontaa. Hienopaperimassalla valonsirontakerroin ja opasiteetti suurenivat GCC-pitoisuuden kasvaessa kummallakin annostelulla, mikä voisi johtua täyteainepartikkelien antamasta paremmasta sironnasta. Yhtäaikaisella annostelulla saavutettiin huomattavasti paremmat valonsironnan arvot perinteiseen annosteluun verrattuna. PCC H-pitoisuuden kasvaessa suurenivat myös valonsirontakerroin ja opasiteetti kummallakin annostelulla. PCC H antoi korkeammat valonsirontakertoimet kuin GCC. PCC omaa suuremman valonheijastusluvun kuin GCC, minkä vuoksi se antaa paremmat valonsirontakertoimen arvot. PCC H:n partikkelikokojakauma oli myös kapeampi kuin GCC:n, mikä mahdollisti paremman valonsironnan ja opasiteetin saavuttamisen.

Alumiinirakenteiden kulumiskestävyyden parantaminen laserpintakäsittelyillä

Alumiini on pienen ominaispainonsa, hyvän korroosiokestävyytensä ja muotoiltavuutensa ansiosta runsaasti käytetty materiaali niin koneenosissa, kuin monissa muissakin rakenteissa. Alumiinin pehmeys ja huono kulumiskestävyys ovat rajoittaneet sen käyttöä. Alumiinin kovuuden ja kulumiskestävyyden parantaminen laajentaisi sen käyttömahdollisuuksia. Laserpintakäsittelymenetelmien käyttö on potentiaalinen, mutta vielä vähän tutkittu keino alumiinin kulumiskestävyyden parantamiseksi. Diplomityön teoriaosassa käsitellään materiaalin eri kulumismekanismeja, kohdemateriaali alumiinin ominaisuuksia ja tarkastellaan keinoja parantaa kulumiskestävyyttä sekä perinteisillä tekniikoilla että erityisesti lasertekniikoiden avulla. Diplomityön kokeellisessa osassa tehtiin laserpintakäsittelykokeita Nd:YAG-laserilla tavoitteena kovuuden ja kulumiskestävyyden parantaminen. Parhaat tulokset saavutettiin laserpinnanseostustekniikalla nikkelipohjaisella Inconel 625-seosaineella. Diplomityössä tehdyissä kokeissa saavutettiin yli 10-kertainen alumiinin pinnankovuus verrattuna käsittelemättömään perusaineeseen. Abrasiivinen kulumiskestävyys parantui parhaimmillaan yli kolminkertaiseksi verrattuna käsittelemättömään alumiiniin ja anodisoituihin vertailukappaleisiin. Tutkimuksessa määriteltiin myös parametrialue kokeissa käytetyille perus- ja lisäaineelle. Diplomityössä tehdyt kokeet toimivat hyvänä pohjana jatkokokeille.

Antenatal inflammation and brain pathology in preterm infants

Chorioamnionitis is known to be an important risk factor underlying preterm delivery, and it has also been suggested to associate with brain lesions and deviant neurological development in both preterm and term infants. Cytokines are believed to be the link causing the deleterious effects of inflammation to the nervous system. Their genetic regulation has also been suggested to play a role, as interleukin (IL)-6 -174 and -572 genotypes, which partly regulate IL-6 synthesis responses, have been connected with deviant neurological development in preterm infants. We evaluated the association of histological chorioamnionitis with brain lesions, regional brain volumes, and the functioning of the auditory pathway in very low birth weight/very low gestational age (VLBW/VLGA) infants. In addition, we investigated the association between IL-6 -174 and -572 genotypes and histological chorioamnionitis, neonatal infections, and brain lesions and regional brain volumes in VLBW/VLGA infants. This study is a part of a larger multidisciplinary project PIPARI (Development and Functioning of Very Low Birth Weight Infants from Infancy to School Age), in which the survivors of a 6-year cohort of VLBW/VLGA infants (n=274) are being followed until school age in Turku University Central Hospital, Finland. Placental samples were collected in the delivery room, and were analyzed for histological inflammatory findings. Blood samples from the infants were collected and DNA was genotyped for IL-6-174 and -572 polymorphisms (GG/GC/CC). Brain ultrasound examinations were performed repeatedly in the neonatal intensive care unit and at term age, and were analysed for structural brain lesions. Brain magnetic resonance imaging was performed at term age, and was analysed for regional brain volumes. In addition, diffusion tensor imaging was performed at term, and was used to analyse fractional anisotrophy and the apparent diffusion coefficient of inferior colliculus. The brainstem auditory evoked potential recordings were carried out according to the routine clinical procedure at median age of 30 days after term age. In our study, we found that histological chorioamnionitis was not an independent risk factor for brain lesions, reduced regional brain volumes or abnormal functioning of the auditory pathway in VLBW/VLGA infants. In addition, we found that IL-6 -174 GG and -572 GC genotypes were associated with a higher incidence of histological chorioamnionitis, and that -174 CC genotype associated with higher incidence of septicaemia. The analysed IL-6 genotypes were not associated with other brain lesions, but a reduced volume of basal ganglia and thalami was associated with IL-6 -174 CC and -572 GG genotypes. In conclusion, our findings suggest that histological chorioamnionitis is not an independent risk factor for the brain development of VLBW/VLGA infants, or that the risk caused by inflammation does not exceed the risks attributed to other underlying pathologies behind preterm deliveries. In addition, our findings give reason to propose that IL-6 promoter genotypes have a role in the defence against serious infections and in the brain development of VLBW/VLGA infants.

Free PAPP-A: a Novel Marker in Acute Coronary Syndrome Patients

In recent years, one important objective of cardiovascular research has been to find new markers that would improve the risk stratification and diagnosis of patients presenting with symptoms of acute coronary syndrome (ACS). Pregnancy-associated plasma protein A (PAPP-A) is a large metalloproteinase involved in insulin-like growth factor signalling. It is expressed in various tissues and seems to be involved in many physiological and pathological processes, such as folliculogenesis, bone formation, wound healing, pregnancy and atherosclerosis. The aim of this thesis was to investigate PAPP-A in ACS patients. Circulating concentrations of PAPP-A had been previously shown to be elevated in ACS. In this study it was revealed that the form of PAPP-A causing this elevation was the free noncomplexed PAPP-A. Thus, the form of PAPP-A in the circulation of ACS patients differed from the complexed PAPP-A form abundantly present in the circulation during pregnancy. A point-of-care method based on time-resolved immunofluorometric assays was developed, which enabled the rapid detection of free PAPP-A. The method was found to perform well with serum and heparin plasma samples as well as with heparinized whole blood samples. With this method the concentrations of free PAPP-A in healthy individuals were shown to be negligible. When the clinical performance of the method was evaluated with serum samples from ACS patients, it was shown that the free PAPP-A concentration in the admission sample was an independent predictor of myocardial infarction and death. Moreover, as a prognostic marker, free PAPP-A was revealed to be superior to total PAPPA, i.e. the combination of free and complexed PAPP-A, which has been measured by the other groups in this field. As heparin products are widely used as medication in ACS patients, the effect of heparin products on free PAPP-A molecule and circulating concentrations were also investigated in this study. It was shown that intravenous administration of low molecular weight or unfractionated heparin elicits a rapid release of free PAPP-A into the circulation in haemodialysis patients and patients undergoing angiography. Moreover, the interaction between PAPP-A and heparin was confirmed in gel filtration studies. Importantly, the patients included in the clinical evaluation of the free PAPP-A detection method developed had not received any heparin product medication before the admission sample and thus the results were not affected by the heparin effect. In conclusion, free PAPP-A was identified as a novel marker associated with ACS. The point-of-care methods developed enable rapid detection of this molecule which predicts adverse outcome when measured in the admission sample of ACS patients. However, the effect revealed of heparin products on circulating PAPP-A concentrations should be acknowledged when further studies are conducted related to free or total PAPP-A in ACS.

The effect of the birth hospital and the time of birth on the outcome of Finnish very preterm infants

The purpose of this study was to evaluate the effect of the birth hospital and the time of birth on mortality and the long-term outcome of Finnish very low birth weight (VLBW) or very low gestational age (VLGA) infants. This study included all Finnish VLBW/VLGA infants born at <32 gestational weeks or with a birth weight of ≤1500g, and controls born full-term and healthy. In the first part of the study, the mortality of VLBW/VLGA infants born in 2000–2003 was studied. The second part of the study consisted of a five-year follow-up of VLBW/VLGA infants born in 2001–2002. The study was performed using data from parental questionnaires and several registers. The one-year mortality rate was 11% for live-born VLBW/VLGA infants, 22% for live-born and stillborn VLBW/VLGA infants, and 0% for the controls. In live-born and in all (including stillbirths) VLBW/VLGA infants, the adjusted mortality was lower among those born in level III hospitals compared with level II hospitals. Mortality rates of live-born VLBW/VLGA infants differed according to the university hospital district where the birth hospital was located, but there were no differences in mortality between the districts when stillborn infants were included. There was a trend towards lower mortality rates in VLBW/VLGA infants born during office hours compared with those born outside office hours (night time, weekends, and public holidays). When stillborn infants were included, this difference according to the time of birth was significant. Among five-year-old VLBW/VLGA children, morbidity, use of health care resources, and problems in behaviour and development were more common in comparison with the controls. The health-related quality of life of the surviving VLBW/VLGA children was good but, statistically, it was significantly lower than among the controls. The median and the mean number of quality-adjusted life-years were 4.6 and 3.6 out of a maximum five years for all VLBW/VLGA children. For the controls, the median was 4.8 and the mean was 4.9. Morbidity rates, the use of health care resources, and the mean quality-adjusted life-years differed for VLBW/VLGA children according to the university hospital district of birth. However, the time of birth, the birth hospital level or university hospital district were not associated with the health-related quality of life, nor with behavioural and developmental scores of the survivors at the age of five years. In conclusion, the decreased mortality in level III hospitals was not gained at the expense of long-term problems. The results indicate that VLBW/VLGA deliveries should be centralized to level III hospitals and the regional differences in the treatment practices should further be clarified. A long-term follow-up on the outcome of VLBW/VLGA infants is important in order to recognize the critical periods of care and to optimise the care. In the future, quality-adjusted life-years can be used as a uniform measure for comparing the effectiveness of care between VLBW/VLGA infants and different patient groups

Probiotic interventions in infancy: Benefit and Safety Assessment of Extended Applications

Immaturity of the gut barrier system in the newborn has been seen to underlie a number of chronic diseases originating in infancy and manifesting later in life. The gut microbiota and breast milk provide the most important maturing signals for the gut-related immune system and reinforcement of the gut mucosal barrier function. Recently, the composition of the gut microbiota has been proposed to be instrumental in control of host body weight and metabolism as well as the inflammatory state characterizing overweight and obesity. On this basis, inflammatory Western lifestyle diseases, including overweight development, may represent a potential target for probiotic interventions beyond the well documented clinical applications. The purpose of the present undertaking was to study the efficacy and safety of perinatal probiotic intervention. The material comprised two ongoing, prospective, double-blind NAMI (Nutrition, Allergy, Mucosal immunology and Intestinal microbiota) probiotic interventions. In the mother-infant nutrition and probiotic study altogether 256 women were randomized at their first trimester of pregnancy into a dietary intervention and a control group. The intervention group received intensive dietary counselling provided by a nutritionist, and were further randomized at baseline, double-blind, to receive probiotics (Lactobacillus rhamnosus GG and Bifidobacterium lactis) or placebo. The intervention period extended from the first trimester of pregnancy to the end of exclusive breastfeeding. In the allergy prevention study altogether 159 women were randomized, double-blind, to receive probiotics (Lactobacillus rhamnosus GG) or placebo 4 weeks before expected delivery, the intervention extending for 6 months postnatally. Additionally, patient data on all premature infants with very low birth weight (VLBW) treated in the Department of Paediatrics, Turku University Hospital, during the years 1997 - 2008 were utilized. The perinatal probiotic intervention reduced the risk of gestational diabetes mellitus (GDM) in the mothers and perinatal dietary counselling reduced that of fetal overgrowth in GDM-affected pregnancies. Early gut microbiota modulation with probiotics modified the growth pattern of the child by restraining excessive weight gain during the first years of life. The colostrum adiponectin concentration was demonstrated to be dependent on maternal diet and nutritional status during pregnancy. It was also higher in the colostrum received by normal-weight compared to overweight children at the age of 10 years. The early perinatal probiotic intervention and the postnatal probiotic intervention in VLBW infants were shown to be safe. To conclude, the findings in this study provided clinical evidence supporting the involvement of the initial microbial and nutritional environment in metabolic programming of the child. The manipulation of early gut microbial communities with probiotics might offer an applicable strategy to impact individual energy homeostasis and thus to prevent excessive body-weight gain. The results add weight to the hypothesis that interventions aiming to prevent obesity and its metabolic consequences later in life should be initiated as early as during the perinatal period.

The Vulnerable Brain and Very Preterm Infants - Findings from the PIPARI-Study

Preterm birth is a risk for normal brain development. Brain maturation that normally happens in the uterus is in very preterm infants a developmental challenge during their stay in a neonatal intensive care unit (NICU). Typical brain injuries of preterm infants include ischemic injuries, brain haemorrhages, ventricular dilatation (VD), and reduced brain volumes. Brain injury is a serious complication of prematurity leading to possible long term consequences for the neurodevelopment of the very low birth weight (VLBW) infant, such as cerebral palsy (CP), hearing impairments, vision problems, and delay in cognitive development.There is a need for further studies to ascertain the potential risk factors and their causal relationships to brain vulnerability, growth and development in the increasing number of surviving VLBW infants. This thesis consists of four studies evaluating the definitions, causes and consequences of brain lesions in VLBW(<1500g) or very low gestationalage (VLGA) (gestational age <32 gestational weeks) infants. We showed that the redistribution of fetal blood flow is a risk factor for smaller brain volumes at term. In addition,we showed that brain lesions related to prematurity are not associated with increased spontaneous crying behaviour or circadian rhythm development in infancy. However, the preterm infants began to fuss more often and were held more than term infants at five months of age. Furthermore, we showed that VD is associated with brain lesions and smaller brain volumes. Therefore, brain magneticresonance imaging can be recommended for infants with VD. VD together with other brain pathology is a risk factor for the onset of developmental impairments in VLBW/VLGA infants at two years of age.

The spindle assembly checkpoint as a drug target - Novel small-molecule inhibitors of Aurora kinases

Cell division (mitosis) is a fundamental process in the life cycle of a cell. Equal distribution of chromosomes between the daughter cells is essential for the viability and well-being of an organism: loss of fidelity of cell division is a contributing factor in human cancer and also gives rise to miscarriages and genetic birth defects. For maintaining the proper chromosome number, a cell must carefully monitor cell division in order to detect and correct mistakes before they are translated into chromosomal imbalance. For this purpose an evolutionarily conserved mechanism termed the spindle assembly checkpoint (SAC) has evolved. The SAC comprises a complex network of proteins that relay and amplify mitosis-regulating signals created by assemblages called kinetochores (KTs). Importantly, minor defects in SAC signaling can cause loss or gain of individual chromosomes (aneuploidy) which promotes tumorigenesis while complete failure of SAC results in cell death. The latter event has raised interest in discovery of low molecular weight (LMW) compounds targeting the SAC that could be developed into new anti-cancer therapeutics. In this study, we performed a cell-based, phenotypic high-throughput screen (HTS) to identify novel LMW compounds that inhibit SAC function and result in loss of cancer cell viability. Altogether, we screened 65 000 compounds and identified eight that forced the cells prematurely out of mitosis. The flavonoids fisetin and eupatorin, as well as the synthetic compounds termed SACi2 and SACi4, were characterized in more detail utilizing versatile cell-based and biochemical assays. To identify the molecular targets of these SAC-suppressing compounds, we investigated the conditions in which SAC activity became abrogated. Eupatorin, SACi2 and SACi4 preferentially abolished the tensionsensitive arm of the SAC, whereas fisetin lowered also the SAC activity evoked by lack of attachments between microtubules (MTs) and KTs. Consistent with the abrogation of SAC in response to low tension, our data indicate that all four compounds inhibited the activity of Aurora B kinase. This essential mitotic protein is required for correction of erratic MT-KT attachments, normal SAC signaling and execution of cytokinesis. Furthermore, eupatorin, SACi2 and SACi4 also inhibited Aurora A kinase that controls the centrosome maturation and separation and formation of the mitotic spindle apparatus. In line with the established profound mitotic roles of Aurora kinases, these small compounds perturbed SAC function, caused spindle abnormalities, such as multi- and monopolarity and fragmentation of centrosomes, and resulted in polyploidy due to defects in cytokinesis. Moreover, the compounds dramatically reduced viability of cancer cells. Taken together, using a cell-based HTS we were able to identify new LMW compounds targeting the SAC. We demonstrated for the first time a novel function for flavonoids as cellular inhibitors of Aurora kinases. Collectively, our data support the concept that loss of mitotic fidelity due to a non-functional SAC can reduce the viability of cancer cells, a phenomenon that may possess therapeutic value and fuel development of new anti-cancer drugs.

Quantification and Clinical Relevance of Cystatin C

An aging population and increasing rates of diabetes mellitus contribute to a high prevalence of kidney dysfunction – approximately 10 percent of adults in developed countries have chronic kidney disease (CKD). CKD is a progressive loss of kidney function and this remains permanent. Early recognition of this condition is important for prevention or impeding severe adverse cardiac and renal outcomes. Cystatin C is a low molecular weight cysteine protease inhibitor that has emerged as a biomarker of kidney function. The special potential of plasma cystatin C in this setting is related to its independency of muscle mass, which is a remarkable limitation of the traditional marker creatinine. Cystatin C is a sensitive marker in diagnosing mild and moderate CKD, especially in small children, in the elderly and in conditions where muscle mass is affected. Cystatin C is quantified with immunoassays, mainly based on particle-enhanced nephelometry (PENIA) or turbidimetry (PETIA). The aim of this study was to develop a rapid and reliable assay for quantification of human cystatin C in plasma or serum by utilizing time-resolved fluorescence-based immunoassay methods. This was accomplished by utilizing different antibodies, including polyclonal and 7 monoclonal antibodies against cystatin C. Different assay designs were tested and the best assay was further modified to a dry-reagent double monoclonal assay run on an automated immunonalyzer. This assay was evaluated for clinical performance in estimating reduced kidney function and in predicting risk of adverse outcomes in patients with non-ST elevation acute coronary syndrome. Of the tested assay designs, heterogeneous non-competitive assay had the best performace and was chosen to be developed further. As an automated double monoclonal assay, this assay enabled a reliable measurement of clinically relevant cystatin C concentrations. It also showed a stronger concordance with the reference clearance method than the conventional PETIA method in patients with reduced kidney function. Risk of all-cause mortality and combined events, defined by death and myocardial infarction, increased with higher cystatin C and cystatin C remained an independent predictor of death and combined events after adjustment to nonbiochemical baseline factors. In conclusion, the developed dry-reagent double monoclonal assay allows rapid and reliable quantitative measurement of cystatin C. As measured with the developed assay, cystatin C is a potential predictor of adverse outcomes in cardiac patients.

The Association between Growth and Neurodevelopment in Very Preterm Infants – A Follow-up Study in the PIPARI Study

Placental insufficiency is one major cause of intrauterine growth restriction and also relates to neurodevelopment. Preterm infants with very low birth weight are at risk of postnatal growth restriction as well as neurodevelopmental impairments. However, the optimal postnatal growth for long-term neurodevelopment is still unclear. The objective of this study was thus to investigate the association between growth and neurodevelopment in very preterm infants. The study populations consisted of 83 (I), 55 (II), 36 (III) and 181 (IV) infants with very low birth weight (below 1501 grams), and very or extremely low gestational age (below 32 and 26 weeks). Foetal blood circulation in relation to two-year neurodevelopment and the association between early growth and brain maturation at term age were studied. Postnatal growth, and its association with five-year cognitive outcome, was analysed. Changes in foetal blood circulation related to placental insufficiency associated with an adverse two-year cognitive outcome. Early postnatal growth in extremely preterm infants was comparable to a similar Swedish cohort. Preterm infants with slow intrauterine growth had less mature brains at term age; rapid catch-up growth until term age did not eliminate this difference. Weight gain and head circumference growth from birth until two years of age associated positively with five-year cognitive outcome in appropriate for gestational age infants. In small for gestational age infants, head circumference growth from term age to four months (corrected age) associated positively with their five-year cognitive outcome. The association between postnatal growth and neurodevelopment was different for prenatally normally grown versus slow grown preterm infants.

To Divide or Not to Divide; MicroRNAs and Small Compounds as Modulators of Mitosis

Mitosis is under the stringent quality control of the spindle assembly checkpoint (SAC). However, in cancer cells this control can fail, leading to excessive cellular proliferation and ultimately to the formation of a tumor. Novel cancer cell selective therapies are needed to stop the uncontrolled cell proliferation and tumor growth. The aim of the research presented in this thesis was to identify microRNAs (miRNAs) that could play a role in cancer cell proliferation as well as low molecular weight (LMW) compounds that could interfere with cell division. The findings could be used to develop better cancer diagnostics and therapies in the future. First, a high-throughput screen (HTS) was performed to identify LMW compounds that possess a similar chemical interaction field as rigosertib, an anti-cancer compound undergoing clinical trials. A compound termed Centmitor-1 was discovered that phenocopied the cellular impact of rigosertib by affecting the microtubule dynamics. Next, another HTS aimed at identifying compounds that would target the Hec1 protein, which mediates the interaction between spindle microtubules and chromosomes. Perturbation of this connection should prevent cell division and induce cell death. A compound termed VTT-006 was discovered that abrogated mitosis in several cell line models and exhibited binding to Hec1 in vitro. Lastly, using a cell-based HTS two miRNAs were identified that affected cancer cell proliferation via Aurora B kinase, which is an important mitotic regulator. MiR-378a-5p was found to indirectly suppress the production of the kinase whereas let-7b showed direct binding to the 3’UTR of Aurora B mRNA and repressed its translation. The miRNA-mediated perturbation of Aurora B induced defects in mitosis leading to abnormal chromosome segregation and induction of aneuploidy. The results of this thesis provide new information on miRNA signaling in cancer, which could be utilized for diagnostic purposes. Moreover, the thesis introduces two small compounds that may benefit future drug research.

Modification of commercial poly-lactide for extrusion coated food packaging applications

Poly-L-lactide (PLLA) is a widely used sustainable and biodegradable alternative to replace synthetic non-degradable plastic materials in the packaging industry. Conversely, its processing properties are not always optimal, e.g. insufficient melt strength at higher temperatures (necessary in extrusion coating processes). This thesis reports on research to improve properties of commercial PLLA grade (3051D from NatureWorks), to satisfy and extend end-use applications, such as food packaging by blending with modified PLLA. Adjustment of the processability by chain branching of commercial poly-L-lactide initiated by peroxide was evaluated. Several well-defined branched structures with four arms (sPLLA) were synthesized using pentaerythritol as a tetra-functional initiator. Finally, several block copolymers consisting of polyethylene glycol and PLLA (i.e. PEGLA) were produced to obtain a well extruded material with improved heat sealing properties. Reactive extrusion of poly-L-lactide was carried out in the presence of 0.1, 0.3 and 0.5 wt% of various peroxides [tert-butyl-peroxybenzoate (TBPB), 2,5-dimethyl-2,5-(tert-butylperoxy)-hexane (Lupersol 101; LOL1) and benzoyl peroxide (BPO)] at 190C. The peroxide-treated PLLAs showed increased complex viscosity and storage modulus at lower frequencies, indicating the formation of branched/cross linked architectures. The material property changes were dependent on the peroxide, and the used peroxide concentration. Gel fraction analysis showed that the peroxides, afforded different gel contents, and especially 0.5 wt% peroxide, produced both an extremely high molar mass, and a cross linked structure, not perhaps well suited for e.g. further use in a blending step. The thermal behavior was somewhat unexpected as the materials prepared with 0.5 wt% peroxide showed the highest ability for crystallization and cold crystallization, despite substantial cross linking. The peroxide-modified PLLA, i.e. PLLA melt extruded with 0.3 wt% of TBPB and LOL1 and 0.5 wt% BPO was added to linear PLLA in ratios of 5, 15 and 30 wt%. All blends showed increased zero shear viscosity, elastic nature (storage modulus) and shear sensitivity. All blends remained amorphous, though the ability of annealing was improved slightly. Extrusion coating on paperboard was conducted with PLLA, and peroxide-modified PLLA blends (90:10). All blends were processable, but only PLLA with 0.3 wt% of LOL1 afforded a smooth high quality surface with improved line speed. Adhesion levels between fiber and plastic, as well as heat seal performance were marginally reduced compared with pure 3051D. The water vapor transmission measurements (WVTR) of the blends containing LOL1 showed acceptable levels, only slightly lower than for comparable PLLA 3051D. A series of four-arm star-shaped poly-L-lactide (sPLLA) with different branch length was synthesized by ring opening polymerization (ROP) of L-lactide using pentaerythritol as initiator and stannous octoate as catalyst. The star-shaped polymers were further blended with its linear resin and studied for their melt flow and thermal properties. Blends containing 30 wt% of sPLLA with low molecular weight (30 wt%; Mwtotal: 2500 g mol-1 and 15000 g mol-1) showed lower zero shear viscosity and significantly increased shear thinning, while at the same time slightly increased crystallization of the blend. However, the amount of crystallization increased significantly with the higher molecular weight sPLLA, therefore the star-shaped structure may play a role as nucleating agent. PLLA-polyethylene glycol–PLLA triblock copolymers (PEGLA) with different PLLA block length were synthesized and their applicability as blends with linear PLLA (3051D NatureWorks) was investigated with the intention of improving heat-seal and adhesion properties of extrusion-coated paperboard. PLLA-PEG-PLLA was obtained by ring opening polymerization (ROP) of L-lactide using PEG (molecular weight 6000 g mol-1) as an initiator, and stannous octoate as catalyst. The structures of the PEGLAs were characterized by proton nuclear magnetic resonance spectroscopy (1H-NMR). The melt flow and thermal properties of all PEGLAs and their blends were evaluated using dynamic rheology, and differential scanning calorimeter (DSC). All blends containing 30 wt% of PEGLAs showed slightly higher zero shear viscosity, higher shear thinning and increased melt elasticity (based on tan delta). Nevertheless, no significant changes in thermal properties were distinguished. High molecular weight PEGLAs were used in extrusion coating line with 3051D without problems.