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  • Entner, Doris (Helsingin yliopisto, 2013)
    In many fields of science, researchers are keen to learn causal connections among quantities of interest. For instance, in medical studies doctors want to infer the effect of a new drug on the recovery from a particular disease, or economists may be interested in the effect of education on income. The preferred approach to causal inference is to carry out controlled experiments. However, such experiments are not always possible due to ethical, financial or technical restrictions. An important problem is thus the development of methods to infer cause-effect relationships from passive observational data. While this is a rather old problem, in the late 1980s research on this issue gained significant momentum, and much attention has been devoted to this problem ever since. One rather recently introduced framework for causal discovery is given by linear non-Gaussian acyclic models (LiNGAM). In this thesis, we apply and extend this model in several directions, also considering extensions to non-parametric acyclic models. We address the problem of causal structure learning from time series data, and apply a recently developed method using the LiNGAM approach to two economic time series data sets. As an extension of this algorithm, in order to allow for non-linear relationships and latent variables in time series models, we adapt the well-known Fast Causal Inference (FCI) algorithm to such models. We are also concerned with non-temporal data, generalizing the LiNGAM model in several ways: We introduce an algorithm to learn the causal structure among multidimensional variables, and provide a method to find pairwise causal relationships in LiNGAM models with latent variables. Finally, we address the problem of inferring the causal effect of one given variable on another in the presence of latent variables. We first suggest an algorithm in the setting of LiNGAM models, and then introduce a procedure for models without parametric restrictions. Overall, this work provides practitioners with a set of new tools for discovering causal information from passive observational data in a variety of settings.
  • Salo, Pauli (Helsingin yliopisto, 2003)
  • Vitikainen, Emma (Helsingin yliopisto, 2010)
    Human actions cause destruction and fragmentation of natural habitats, predisposing populations to loss of genetic diversity and inbreeding, which may further decrease their fitness and survival. Understanding these processes is a main concern in conservation genetics. Yet data from natural populations is scarce, particularly on invertebrates, owing to difficulties in measuring both fitness and inbreeding in the wild. Ants are social insects, and a prime example of an ecologically important group for which the effects of inbreeding remain largely unstudied. Social insects serve key roles in all terrestrial ecosystems, and the division of labor between the females in the colonies queens reproduce, workers tend to the developing brood probably is central to their ecological success. Sociality also has important implications for the effects of inbreeding. Despite their relative abundance, the effective population sizes of social insects tend to be small, owing to the low numbers of reproductive individuals relative to the numbers of sterile workers. This may subject social insects to loss of genetic diversity and subsequent inbreeding depression. Moreover, both the workers and queens can be inbred, with different and possibly multiplicative consequences. The aim of this study was to investigate causes and consequences of inbreeding in a natural population of ants. I used a combination of long-term field and genetic data from colonies of the narrow-headed ant Formica exsecta to examine dispersal, mating behavior and the occurrence of inbreeding, and its consequences on individual and colony traits. Mating in this species takes place in nuptial flights that have been assumed to be population-wide and panmictic. My results, however, show that dispersal is local, with queens establishing new colonies as close as 60 meters from their natal colony. Even though actual sib-mating was rare, individuals from different but related colonies pair, which causes the population to be inbred. Furthermore, multiple mates of queens were related to each other, which also indicates localized mating flights. Hence, known mechanisms of inbreeding avoidance, dispersal and multiple mating, were not effective in this population, as neither reduced inbreeding level of the future colony. Inbreeding had negative consequences both at the individual and colony level. A queen that has mated with a related male produces inbred workers, which impairs the colony s reproductive success. The inbred colonies were less productive and, specifically, produced fewer new queens, possibly owing to effects of inbreeding on the caste determination of female larvae. A striking finding was that males raised in colonies with inbred workers were smaller, which reflects an effect of the social environment as males, being haploid, cannot be inbred themselves. The queens produced in the inbred colonies, in contrast, were not smaller, but their immune response was up-regulated. Inbreeding had no effect on queen dispersal, but inbred queens had a lower probability of successfully founding a new colony. Ultimately, queens that survived through the colony founding phase had a shorter lifespan. This supports the idea that inbreeding imposes a genetic stress, leading to inbreeding depression on both the queen and the colony level. My results show that inbreeding can have profound consequences on insects in the wild, and that in social species the effects of inbreeding may be multiplicative and mediated through the diversity of the social environment, as well as the genetic makeup of the individuals themselves. This emphasizes the need to take into account all levels of organization when assessing the effects of genetic diversity in social animals.
  • Pitala, Natalia (Helsingin yliopisto, 2007)
    Defence against pathogens is a vital need of all living organisms that has led to the evolution of complex immune mechanisms. However, although immunocompetence the ability to resist pathogens and control infection has in recent decades become a focus for research in evolutionary ecology, the variation in immune function observed in natural populations is relatively little understood. This thesis examines sources of this variation (environmental, genetic and maternal effects) during the nestling stage and its fitness consequences in wild populations of passerines: the blue tit (Cyanistes caeruleus) and the collared flycatcher (Ficedula albicollis). A developing organism may face a dilemma as to whether to allocate limited resources to growth or to immune defences. The optimal level of investment in immunity is shaped inherently by specific requirements of the environment. If the probability of contracting infection is low, maintaining high growth rates even at the expense of immune function may be advantageous for nestlings, as body mass is usually a good predictor of post-fledging survival. In experiments with blue tits and haematophagous hen fleas (Ceratophyllus gallinae) using two methods, methionine supplementation (to manipulate nestlings resource allocation to cellular immune function) and food supplementation (to increase resource availability), I confirmed that there is a trade-off between growth and immunity and that the abundance of ectoparasites is an environmental factor affecting allocation of resources to immune function. A cross-fostering experiment also revealed that environmental heterogeneity in terms of abundance of ectoparasites may contribute to maintaining additive genetic variation in immunity and other traits. Animal model analysis of extensive data collected from the population of collared flycatchers on Gotland (Sweden) allowed examination of the narrow-sense heritability of PHA-response the most commonly used index of cellular immunocompetence in avian studies. PHA-response is not heritable in this population, but is subject to a non-heritable origin (presumably maternal) effect. However, experimental manipulation of yolk androgen levels indicates that the mechanism of the maternal effect in PHA-response is not in ovo deposition of androgens. The relationship between PHA-response and recruitment was studied for over 1300 collared flycatcher nestlings. Multivariate selection analysis shows that it is body mass, not PHA-response, that is under direct selection. PHA-response appears to be related to recruitment because of its positive relationship with body mass. These results imply that either PHA-response fails to capture the immune mechanisms that are relevant for defence against pathogens encountered by fledglings or that the selection pressure from parasites is not as strong as commonly assumed.
  • Koivuniemi, Riitta (Helsingin yliopisto, 2009)
    Rheumatoid arthritis (RA) patients have premature mortality. Contrary to the general population, mortality in RA has not declined over time. This study aimed to evaluate determinants of mortality in RA by examining causes of death (CoDs) over time, accuracy of CoD diagnoses, and contribution of RA medication to CoDs. This study further evaluated detection rate of reactive systemic amyloid A amyloidosis, which is an important contributor to RA mortality. CoDs were examined in 960 RA patients between 1971 and 1991 (Study population A) and in 369 RA patients autopsied from 1952 to 1991, with non-RA patients serving as the reference cases (Study population B). In Study population B, CoDs by the clinician before autopsy were compared to those by the pathologist at autopsy to study accuracy of CoD diagnoses. In Study population B, autopsy tissue samples were re-examined systematically for amyloidosis (90% of patients) and clinical data for RA patients was studied from 1973. RA patients died most frequently of cardiovascular diseases (CVDs), infections, and RA. RA deaths declined over time. Coronary deaths showed no major change in Study population A, but, in Study population B, coronary deaths in RA patients increased from 1952 to 1991, while non-RA cases had a decrease in coronary deaths starting in the 1970s. Between CoD diagnoses by the clinician and those by the pathologist, RA patients had lower agreement than non-RA cases regarding cardiovascular (Kappa reliability measure: 0.31 vs. 0.51) and coronary deaths (0.33 vs. 0.46). Use of disease modifying anti-rheumatic drugs was not associated with any CoD. In RA patients, re-examination of autopsy tissue samples doubled the prevalence of amyloid compared with the original autopsy: from 18% to 30%. In the amyloid-positive RA patients, amyloidosis was diagnosed before autopsy in only 37%; and they had higher inflammatory levels and longer duration of RA than amyloid-negative RA patients. Of the RA patients with amyloid, only half had renal failure or proteinuria during lifetime. In RA, most important determinants of mortality were CVDs, RA, and infections. In RA patients, RA deaths decreased over time, but this was not true for coronary deaths. Coronary death being less accurately diagnosed in RA may indicate that coronary heart disease (CHD) often goes unrecognized during lifetime. Thus, active search for CHD and its effective treatment is important to reduce cardiovascular mortality. Reactive amyloidosis may often go undetected. In RA patients with proteinuria or renal failure, as well as with active and long-lasting RA, a systematic search for amyloid is important to enable early diagnosis and early enhancement of therapy. This is essential to prevent clinical manifestations of amyloidosis such as renal failure, which has a poor prognosis.
  • Metsälä, Markus (Helsingin yliopisto, 2006)
    This thesis contains five experimental spectroscopic studies that probe the vibration-rotation energy level structure of acetylene and some of its isotopologues. The emphasis is on the development of laser spectroscopic methods for high-resolution molecular spectroscopy. Three of the experiments use cavity ringdown spectroscopy. One is a standard setup that employs a non-frequency stabilised continuous wave laser as a source. In the other two experiments, the same laser is actively frequency stabilised to the ringdown cavity. This development allows for increased repetition rate of the experimental signal and thus the spectroscopic sensitivity of the method is improved. These setups are applied to the recording of several vibration-rotation overtone bands of both H(12)C(12)CH and H(13)C(13)CH. An intra-cavity laser absorption spectroscopy setup that uses a commercial continuous wave ring laser and a Fourier transform interferometer is presented. The configuration of the laser is found to be sub-optimal for high-sensitivity work but the spectroscopic results are good and show the viability of this type of approach. Several ro-vibrational bands of carbon-13 substituted acetylenes are recorded and analysed. Compared with earlier work, the signal-to-noise ratio of a laser-induced dispersed infrared fluorescence experiment is enhanced by more than one order of magnitude by exploiting the geometric characteristics of the setup. The higher sensitivity of the spectrometer leads to the observation of two new symmetric vibrational states of H(12)C(12)CH. The precision of the spectroscopic parameters of some previously published symmetric states is also improved. An interesting collisional energy transfer process is observed for the excited vibrational states and this phenomenon is explained by a simple step-down model.
  • Väärikkälä, Sofia (2007)
    Clostridium botulinum on anaerobinen itiöitä muodostava bakteeri, jonka tuottama neurotoksiini aiheuttaa ihmisille ja eläimille hengenvaarallisen botulismin. Tavallisimmin neurotoksiinia saadaan ruoan mukana, sillä raa’at elintarvikkeet kontaminoituvat luonnossa esiintyvillä itiöillä. C. botulinum kannat jaetaan neljään fenotyyppisesti erilaiseen ryhmään (I-IV). C. botulinum muodostaa elintarviketurvallisuusriskin, sillä ihmisille myrkytyksen aiheuttavien ryhmän I kantojen itiömuodot kestävät erittäin korkeita lämpötiloja ja ryhmän II kannat pystyvät lisääntymään jääkaappilämpötiloissa. Selvittämällä C. botulinumin kuuman- ja kylmänsietoon liittyviä mekanismeja elintarviketurvallisuus-riskejä voitaisiin hallita paremmin. Aistikinaasien ja vastesäätelijöiden väliseen fosforinsiirtoon perustuvien kaksikomponenttijärjestelmien tiedetään olevan bakteerien eniten käyttämä mekanismi ympäristön muutoksiin sopeutumisessa, mutta niiden merkitys C. botulinumilla on epäselvä. Tutkimuksen tarkoituksena oli selvittää, mitä merkitystä kaksikomponenttijärjestelmään kuuluvalla aistikinaasia koodaavalla CBO0366-geenillä on C. botulinuminin fysiologiassa sekä kylmän- ja kuumansiedossa. Tutkimuksessa käytettiin ClosTron-menetelmää tutkittavan.C. botulinumin ATCC 3502 -kannan CBO0366-geenin inaktivoimiseksi. Menetelmä perustuu pMTL007-plasmidin liikkuvan ryhmän II intronin kohdespesifisyyden muuttamiseen Splicing by Overlap Extension PCR:ää (SOE-PCR) käyttäen siten, että introni saatiin kiinnittymään spesifisesti C. botulinumin CBO0366-geeniin. Intronin integroituminen genomiin aiheutti kyseisen kohdan geenin lukukehyksen häiriintymisen. Mutatoidun kannan fysiologiaa ja kylmän- ja kuumansietoa tutkittiin käyttämällä ATCC 3502 -kannan villityyppiä kontrollina. Mutatoidun kannan pesäkemorfologia veri- ja munankeltuaislevyllä, metabolinen aktiivisuus 37 °C:ssa ja kasvukäyrät 37 °C:ssa sekä 45 °C:ssa olivat lähes identtiset villityypin kanssa. Mutatoitu kanta kasvoi kuitenkin selvästi villityypin kantaa hitaammin 15 °C:ssa ja 20 °C:ssa. CBO0366-geenillä on siis keskeinen merkitys C. botulinumin ATCC 3502 –kannan kylmänsietokyvyssä. Tulosten perusteella voidaan olettaa, että CBO0366-geenin koodaama aistikinaasi on osa kaksikomponenttijärjestelmää, joka on osallisena alhaisempiin lämpötiloihin sopeutumiseen tarvittavien vasteiden aikaansaamisessa. Geenin inaktivointi todennäköisesti estää kylmästä kertovan signaalin kuljettamisen solun sisälle ja edelleen spesifisen vasteen syntymisen. Siten bakteerin sopeutuminen kylmään heikkenee. Koska mutatoitu kanta kuitenkin kasvoi 15 °C:ssa, on oletettavaa, että geeni ei ole elintärkeä selviytymiseen viileämmässä.
  • Voutilainen, Merja (Helsingin yliopisto, 2010)
    Parkinson s disease (PD) is a neurodegenerative disorder associated with a progressive loss of dopaminergic neurons of the substantia nigra (SN). Current therapies of PD do not stop the progression of the disease and the efficacy of these treatments wanes over time. Neurotrophic factors are naturally occurring proteins promoting the survival and differentiation of neurons and the maintenance of neuronal contacts. Neurotrophic factors are attractive candidates for neuroprotective or even neurorestorative treatment of PD. Thus, searching for and characterizing trophic factors are highly important approaches to degenerative diseases. CDNF (cerebral dopamine neurotrophic factor) and MANF (mesencephalic astrocyte-derived neurotrophic factor) are secreted proteins that constitute a novel, evolutionarily conserved neurotrophic factor family expressed in vertebrates and invertebrates. The present study investigated the neuroprotective and restorative effects of human CDNF and MANF in rats with unilateral partial lesion of dopamine neurons by 6-hydroxydopamine (6-OHDA) using both behavioral (amphetamine-induced rotation) and immunohistochemical analyses. We also investigated the distribution and transportation profiles of intrastriatally injected CDNF and MANF in rats. Intrastriatal CDNF and MANF protected nigrostriatal dopaminergic neurons when administered six hours before or four weeks after the neurotoxin 6-OHDA. More importantly, the function of the lesioned nigrostriatal dopaminergic system was partially restored even when the neurotrophic factors were administered four weeks after 6-OHDA. A 14-day continuous infusion of CDNF but not of MANF restored the function of the midbrain neural circuits controlling movement when initiated two weeks after unilateral injection of 6-OHDA. Continuous infusion of CDNF also protected dopaminergic TH-positive cell bodies from toxin-induced degeneration in the substantia nigra pars compacta (SNpc) and fibers in the striatum. When injected into the striatum, CDNF and GDNF had similar transportation profiles from the striatum to the SNpc; thus CDNF may act via the same nerve tracts as GDNF. Intrastriatal MANF was transported to cortical areas which may reflect a mechanism of neurorestorative action that is different from that of CDNF and GDNF. CDNF and MANF were also shown to distribute more readily than GDNF. In conclusion, CDNF and MANF are potential therapeutic proteins for the treatment of PD.
  • Bäck, Susanne (Helsingin yliopisto, 2014)
    Neurodegenerative diseases are characterized by progressive loss of distinct neuronal populations. In Parkinson s disease (PD) the most prominent cell loss is seen in the dopamine (DA) neuron population in the substantia nigra pars compacta (SNpc). The resulting decrease in striatal DA levels causes dysregulation of neuronal circuits controlling movement and leads to motor symptoms typical to the disease. As for other neurodegenerative diseases, there are no available treatments that would interfere with the degenerative process in PD. The purpose of this work was therefore to test the therapeutic potential of long-term delivery of the neurotrophic factor (NTF) cerebral dopamine neurotrophic factor (CDNF) in the rat partial 6-hydroxydopamine (6-OHDA) lesion model of PD. When injected unilaterally in the striatum, 6-OHDA causes progressive dose-dependent loss of DA neurons in the SNpc accompanied by asymmetrical motor impairment. The 6-OHDA model used in our NTF studies (2x10 µg 6-OHDA) showed a stable lesion progression with a cell loss at two weeks post-lesion corresponding to that seen in PD at symptom onset. In the 6-OHDA model, the DAergic system is traditionally evaluated using immunodetection methods or measurements of tissue neurotransmitter levels. Imaging methods, such as single-photon emission computed tomography (SPECT), allows in vivo detection of neuronal circuits, and together with the DA transporter (DAT) radioligand 2β-carbomethoxy-3β-(4-[123I]iodophenyl)tropane ([123I]β-CIT), SPECT/CT provided reliable estimations of the DA cell degeneration showing high correlation to immunohistochemical findings. The method is sensitive and selective and provides substantial benefits in pre-clinical research allowing longitudinal studies in living animals. The neuroprotective effect of CDNF was studied by applying the NTF intrastriatally as two-week protein infusion with osmotic pumps, or as gene therapy with a recombinant adeno-associated viral vector in 6-OHDA-lesioned rats. Both CDNF delivery methods normalized the amphetamine-induced rotational asymmetry and provided partial protection of the tyrosine hydroxylase (TH) reactive DAergic cells in the SNpc and DA fibers in the striatum. As for GDNF, there were indications of retrograde transport of CDNF, but contrary to what has been reported for GDNF, CDNF did not affect the intact rat DAergic system. In addition, there were differences between the treatments in the capacity to induce sprouting of TH-reactive fibers. Our results confirm that CDNF can be considered as a potential therapy in PD, and that the neuroprotective mechanism of CDNF differs from that of GDNF.
  • Tammiruusu, Anne (Helsingin yliopisto, 2005)
  • Nybond, Susanna (Helsingin yliopisto, 2015)
    Due to the emergence of multidrug resistant bacteria, bacterial infections are still a major healthcare problem. Many factors have led to a discovery void of new antibacterial agents, rendering the current antibiotic pipeline inadequate for future medical needs. For example, the outcomes from pure biochemical high-throughput screening (HTS) assays have, in many cases, not led to successful clinical compounds. Therefore cell-based assays might be a better choice for primary screening. However, the antibacterial cell-based assays in the current use often require long incubation times and they are not always amenable for miniaturization and automation for HTS. In this work, two screening assays based on recombinant bioluminescent E. coli strains were optimized and implemented in the screening of chemical libraries and natural products in antibacterial drug discovery. One of the recombinant bacterial strains was a strain which is sensitive towards transcriptional and translational inhibitors. The assay based on this strain was successfully miniaturized into 384-format using automatized liquid handling and was validated with a proof-of-concept library containing known drugs. This provided a means to perform a larger scale high throughput screen of a compound library. Based on the HTS hit structures, a ligand-based in silico screening of a virtual chemical library was employed for hit enrichment. The most active hits and the in silico selected compounds were further investigated in more detail. Natural products have been an important source in drug discovery, especially in the discovery of antibiotics in the current use. However, matrix effects such as colour or turbidity of natural product extracts can potentially cause interference in conventional absorbance based microbial growth inhibition assays. Also, conventional antibacterial assays are usually not sensitive enough for detecting very small concentrations in fractionated natural product extracts. The feasibility of bioreporter -based assays in antimicrobial screening of natural products was demonstrated by screening an in-house natural product library. One of the assays was also implemented for investigating the antibacterial properties of an extract from a fungal culture filtrate, which demonstrated the sensitivity of the assay for identification of active components from fractionated samples. In conclusion, sensitive and reproducible bioassays amenable for further miniaturization and automation were developed for antibacterial drug discovery. Compared to conventional antimicrobial testing, the bioreporter-based methods offer important improvements such as simultaneous data acquirement on antimicrobial activity, first indication of mode of action and significant reduction of assay time to 2-4 h compared to 24 h in standard susceptibility assays. The developed bioluminescent assays led to the improvement of compound throughput in antimicrobial screening: from hundreds of samples (natural product extracts and fractions) in manually performed assays in 96-well plates, to thousands of test compounds (synthetic compound libraries) in 384-well format using automated liquid handling.
  • Kontturi, Leena-Stiina (Helsingin yliopisto, 2014)
    Cell therapy is defined as cell transplantation into the patient to treat a certain disease state. Therapies utilizing cells can be divided into two main categories, (1) tissue regeneration or engineering and (2) drug delivery. In tissue engineering, the transplanted cells are used to regenerate the functions of a diseased tissue. In drug delivery, the transplanted cells are used as biological factories that produce therapeutic molecules inside the body. For successful cell therapy applications, cells usually must be combined with biomaterials and bioactive factors to mimic the growth environment in vivo. The properties of these scaffolds are important for outcomes of the treatments, because the local environment determines the functionality of the cells. Thus, research on cell-biomaterial interactions is essential for the progress of cell based therapies. Hydrogels are promising cell therapy materials, because their structure resembles the natural tissue environment; they consist of long polymer chains with high water content and elastic properties, thereby enabling cellular functionality. The aim of this study was to investigate hydrogels for cell therapy applications. Firstly, we encapsulated human retinal pigment epithelial cell line (ARPE-19) genetically engineered to secrete an anti-angiogenic protein (1) into alginate-poly-L-lysine-alginate (APA) microcapsules and (2) into a composite hydrogel of cross-linked collagen and interpenetrating hyaluronic acid (HA). A custom-made cell encapsulation device was designed, built and optimized, and pharmacokinetic/pharmacodynamic (PK/PD) model was developed to investigate the intravitreal drug delivery of the anti-angiogenic protein by the encapsulated cells. Secondly, chondrocytes were encapsulated into the cross-linked collagen/HA hydrogel supplemented with transforming growth factor β1 (TGFβ1). Using the cell encapsulation device, cell microcapsules of symmetrical shape and narrow size distribution were produced. The encapsulated ARPE-19 cells remained viable and functional for at least five months. The cross-linked collagen-HA hydrogel was shown to be a suitable encapsulation matrix for ARPE-19 cells; the cells maintained viability and secreted the anti-angiogenic protein at a constant rate for at least 50 days. Moreover, the hydrogel composition could be modified to adjust the properties of the gel structure without compromising cell viability. This approach is suggested to have potential in the treatment of retinal neovascularization. The developed PK/PD model could be used to predict drug levels and therapeutic responses after intravitreal anti-angiogenic drug delivery. The simulations may augment the design of in vivo experiments. The collagen/HA matrix with TGFβ1 was suitable for chondrocyte encapsulation. The hydrogel supported viability and phenotypic cell stability. This hydrogel is strong, stable and biodegradable, and it can be delivered non-invasively as injection. Overall, it is potentially a useful delivery vehicle of chondrocytes for cartilage tissue engineering. In conclusion, ARPE-19 cells maintain viability in different hydrogels for prolonged periods and secrete the therapeutic transgene product constantly, supporting the suitability of ARPE-19 cells for cell therapy. The cross-linked collagen/HA hydrogel appears to be a potential matrix for cell therapy. It is an injectable system that supports functionality of cells, and it is applicable in drug delivery and tissue engineering.
  • Raatikainen-Ahokas, Anne (Helsingin yliopisto, 2014)
    Embryonic cells undergo sequential specification processes to generate multiple cell types of mature organs. Some cells retain pluripotency. They serve as stem or progenitor cells, and provide both new stem cells (self-renewal) and offspring for differentiation. The fate of some cells is to die by programmed cell death. In this thesis, the cell fates in nephrogenesis and spermatogenesis were studied. During kidney organogenesis, an outgrowth of the Wolffian duct, the ureteric bud, induces condensation of the metanephric mesenchyme into a cap condensate, the progenitor cell population that forms the epithelium of all future nephrons. The cap condensate is surrounded by stromal cells. The developmental fates of these cells that also surround the ureter and nascent nephrons, i.e. the kidney stroma, are poorly understood. Bone morphogenetic protein 4 (BMP4) inhibited the outgrowth of the ureteric bud from the Wolffian duct in organ culture. It also had an inhibitory effect on subsequent ureteric branching. The branching defect primarily reflected the effect of BMP4 on the mesenchymal components of the kidney. BMP4 promotes the recruitment of mesenchymal cells around the ureter and their differentiation into smooth muscle. This periureteric cell population likely has a regulatory function in subsequent ureteric growth and differentiation. The exogenous BMP4 also disrupted the cap condensates in kidney explants and large amounts of mesenchymal cells underwent apoptosis. BMP4 maintained the isolated metanephric mesenchymes while suppressing the nephrogenic potential, suggesting that BMP4 acts as a survival/differentiation factor for the stromal progenitors. The stromal cells are apparently essential for the formation and maintenance of the cap condensate. In some organs, such as the testis, the maintenance of stem cells throughout the life span is essential to the normal function, e.g. the formation of sperm cells. Spermatogonia with stem cell activity (SSCs) are among the undifferentiated spermatogonia located at the basement membrane of the seminiferous tubule. Daughters of SSCs both replenish the stem cell pool and enter the differentiation pathway into spermatozoa. Glial cell line-derived neurotrophic factor (GDNF), essential for ureteric branching morphogenesis, is also crucial to the self-renewal of the SSCs. Haploinsufficiency of the Gdnf gene in Gdnf+/- mice caused segmental exhaustion of stem cells, resulting in germ cell loss in old mice. In mice overexpressing GDNF in the testis, spermatogenesis was arrested and large clusters of spermatogonia accumulated in prepubertal animals. Thus, high GDNF concentration promotes the propagation of undifferentiated spermatogonia, whereas low GDNF levels allow SCCs to differentiate in excess and make them prone to depletion. In conclusion, signalling molecules, such as BMP4 and GDNF, affect the cell fates both in nephrogenesis and spermatogenesis by maintaining the precursor cells and promoting their differentiation.
  • Vellonen, Kati-Sisko (2010)
    Drug discovery and development from its very onset up to market approval is a long process lasting 10-15 years. New research tools are needed to accelerate and rationalize this process. Ocular drug research still relies heavily on animal testing with rabbits and other rodents. Computational methods and cell culture models are promising tools for early pharmacokinetic studies and may partly replace the animals in pharmacokinetic and toxicological studies. Computational methods are initially based on experimental data, but thereafter their application is straightforward and they can be used to reduce, partly replace and refine further experimental studies. Similarly, cell culture models may enable absorption and toxicity testing of drug candidates with continuously growing cells of human origin, and thereby reduce the need for animal experiments. The cornea is the main route of ocular drug absorption after topical administration, and the corneal epithelium is the most important barrier to drug permeation. Membrane transporter proteins play an important role in the general pharmacokinetics and toxicology. However, their role in ocular pharmacokinetics is still poorly understood. Based on literature analysis many ocular drugs seem to be substrates of transporters, but the expression of these proteins in the eye is largely unknown. The goal of this work was to develop and evaluate cellular and computational tools for ocular pharmacokinetics and toxicology, and to characterise the active drug transporters in the corneal epithelium. The expression of monocarboxylate transporters and ATP-binding cassette (ABC) class efflux proteins was studied in the corneal epithelium and human corneal epithelial (HCE) cell model. Human corneal epithelium expressed monocarboxylate transporters 1 and 4 (MCT1 and MCT4), efflux transporters multidrug resistance-associated protein 1 and 5 (MRP1 and MRP5), and breast cancer resistance protein (BCRP). Cultured human corneal epithelial cells over-expressed several ABC class efflux proteins and MCT1 and MCT4. The functionality of efflux and monocarboxylate transport was demonstrated in HCE cells and in the rabbit cornea ex vivo. The MTT test is a widely used cytotoxicity test in cell research. It was demonstrated that substrates and inhibitors of ABC class efflux proteins may interfere with the MTT test, presumably by inhibiting dye efflux from the cells. This may lead to an underestimation of toxicity in the MTT test. Quantitative structure property relationship (QSPR) models are commonly used in early drug discovery to predict ADME properties of novel compounds. Multivariate analysis was used to develop QSPR models for in silico prediction of the corneal permeability. Two factors, the distribution coefficient (logD7.4 /logD8.0) and hydrogen binding potential, were shown to be the parameters that determine the transcorneal permeability of a compound. These models were able to predict intracameral steady state drug concentrations in rabbit eyes. In conclusion, the new in silico QSPR model can make reliable predictions for passive drug permeability in the cornea, while the HCE model seems to over-express some membrane transporters as compared to the normal human corneal epithelium. Even if these investigated methods have some restrictions they are still very useful tools for drug discovery purposes.
  • Sipilä, Sampsa (Helsingin yliopisto, 2006)
    Distinct endogenous network events, generated independently of sensory input, are a general feature of various structures of the immature central nervous system. In the immature hippocampus, these type of events are seen as "giant depolarizing potentials" (GDPs) in intracellular recordings in vitro. GABA, the major inhibitory neurotransmitter of the adult brain, has a depolarizing action in immature neurons, and GDPs have been proposed to be driven by GABAergic transmission. Moreover, GDPs have been thought to reflect an early pattern that disappears during development in parallel with the maturation of hyperpolarizing GABAergic inhibition. However, the adult hippocampus in vivo also generates endogenous network events known as sharp (positive) waves (SPWs), which reflect synchronous discharges of CA3 pyramidal neurons and are thought to be involved in cognitive functions. In this thesis, mechanisms of GDP generation were studied with intra- and extracellular recordings in the neonatal rat hippocampus in vitro and in vivo. Immature CA3 pyramidal neurons were found to generate intrinsic bursts of spikes and to act as cellular pacemakers for GDP activity whereas depolarizing GABAergic signalling was found to have a temporally non-patterned facilitatory role in the generation of the network events. Furthermore, the data indicate that the intrinsic bursts of neonatal CA3 pyramidal neurons and, consequently, GDPs are driven by a persistent Na+ current and terminated by a slow Ca2+-dependent K+ current. Gramicidin-perforated patch recordings showed that the depolarizing driving force for GABAA receptor-mediated actions is provided by Cl- uptake via the Na-K-C1 cotransporter, NKCC1, in the immature CA3 pyramids. A specific blocker of NKCC1, bumetanide, inhibited SPWs and GDPs in the neonatal rat hippocampus in vivo and in vitro, respectively. Finally, pharmacological blockade of the GABA transporter-1 prolonged the decay of the large GDP-associated GABA transients but not of single postsynaptic GABAA receptor-mediated currents. As a whole the data in this thesis indicate that the mechanism of GDP generation, based on the interconnected network of bursting CA3 pyramidal neurons, is similar to that involved in adult SPW activity. Hence, GDPs do not reflect a network pattern that disappears during development but they are the in vitro counterpart of neonatal SPWs.
  • Linder, Matts (Helsingin yliopisto, 2009)
    One of the most important factors determining the development of atherosclerosis is the amount of LDL particles in the circulation. In general, LDL particles are clinically regarded as “bad cholesterol” since these particles get entrapped within the vascular wall, leading to atherosclerosis. Circulating HDL particles are conversely regarded as “good cholesterol” because of their ability to transport cholesterol from peripheral tissues to the liver for secretion as bile salts. Once inside the artery wall LDL particles are engulfed by macrophages, resulting in macrophage foam cells. If the macrophage foam cells are not able to efflux the cholesterol back into the bloodstream, the excessive cholesterol ultimately leads to cell death, and the deposition of cellular debris within the atherosclerotic lesion. The cells ability to secrete cholesterol is mainly dependent on the ABCA1 transporter (ATP-binding cassette transporter A1) which transfers cellular cholesterol to extracellular apoA-I (apolipoprotein A-I) particles, leading to the generation of nascent HDL particles. The process of atherosclerotic plaque development is therefore to a large extent a cellular one, in which the capacity of the macrophages in handling the excessive cholesterol load determines the progression of lesion development. In this work we have studied the cellular mechanisms that regulate the trafficking of LDL-derived cholesterol from endosomal compartments to other parts of the cell. As a basis for the study we have utilized cells from patients with Niemann-Pick type C disease, a genetic disorder resulting from mutations in the NPC1 and NPC2 genes. In these cells, cholesterol is entrapped within the endosomal compartment, and is not available for efflux. By identifying proteins that bypass the cholesterol trafficking defect, we were able to identify the small GTPase Rab8 as an important protein involved in ABCA1 dependent cholesterol efflux. In the study, we show that Rab8 regulates cholesterol efflux in human macrophages by facilitating intracellular cholesterol transport, as well as by regulating the plasma membrane availability of ABCA1. Collectively, these results give new insight in to atherosclerotic lesion development and intracellular cholesterol processing.
  • Kirjavainen, Anna (Helsingin yliopisto, 2014)
    Development of the sensory epithelia of the inner ear and their primary cell types, hair cells and supporting cells, is a complex process under tight molecular regulation. These cells arise from common progenitors that are guided to follow cell-type-specific differentiation program, and undergo prominent structural changes to reach mature morphologies. The mechanisms regulating this cellular differentiation in the developing inner ear are not fully understood. The focus of this thesis has been in understanding the molecular control of the stepwise development of hair cells and supporting cells. Sequential expression of transcription factors has a central role in the control of development of the cells and tissues. Here we show that transcription factor Prox1 participates in the molecular cascade directing cellular differentiation in the inner ear. During early development, Prox1 is expressed in the progenitors of hair cells and supporting cells, and later maintained only in the supporting cells. We found novel interactions between Prox1 and hair cell-specific transcription factors Atoh1, the master regulator of hair cell development, and Gfi1, an essential survival factor of the cochlear hair cells. When overexpressed in hair cells, Prox1 suppressed the expression of Atoh1 and Gfi1, illustrating the possibility of transcriptional reprogramming of hair cells. This downregulation had functional consequences, resulting in auditory hair cell death during a restricted period at late-embryogenesis. Furthermore, when we studied Gfi1-knock-in mice, the model in which auditory hair cells die shortly after differentiation, we found positive interaction between Gfi1 and p57Kip2. Thus, p57Kip2 is introduced as a new candidate to mediate the survival-promoting function of Gfi1 in the auditory hair cells. Rho GTPases integrate signals from different molecular pathways to regulate cell cytoskeleton, intercellular junctions and polarity, all properties that are heavily modulated in the epithelial cells of the developing inner ear. A member of Rho GTPase family, Cdc42, was found to be expressed in the developing auditory sensory epithelium. Analysis of Cdc42 mutant mice revealed a versatile role of this protein, demonstrating its importance in 1) the formation of proper cellular patterning in the auditory sensory epithelium, 2) the regulation of apical-basal and planar polarities of the sensory epithelial cells, and 3) the regulation of apical cytoskeleton in these cells. In the absence of Cdc42, mechanosensory hair bundles at the apices of hair cells failed to develop normally, indicating Cdc42 s significance in hearing function. In addition, Cdc42 regulates the maturation of adherens junctions and apical actin cytoskeleton in postnatal supporting cells. Cdc42-deficient supporting cells lacked the ability for normal wound healing, showing that properly developed apical module is needed for epithelium repair following injury to the hearing organ. This thesis presents new pieces to the molecular network controlling cellular differentiation of the inner ear sensory epithelia. Understanding the regulation of this stepwise development may have therapeutic value. It may help to explain the fundamental reasons why mammalian hair cells do not regenerate and, to identify the mechanisms and factors that could be applied to promote hair cell regeneration in the future.
  • Li, Xiao-Dong (Helsingin yliopisto, 2005)