Chromatoid body mediated RNA regulation in mouse male germline

Male germ cell differentiation, spermatogenesis is an exceptional developmental process that produces a massive amount of genetically unique spermatozoa. The complexity of this process along with the technical limitations in the germline research has left many aspects of spermatogenesis poorly understood. Post-meiotic haploid round spermatids possess the most complex transcriptomes of the whole body. Correspondingly, efficient and accurate control mechanisms are necessary to deal with the huge diversity of transcribed RNAs in these cells. The high transcriptional activity in round spermatids is accompanied by the presence of an uncommonly large cytoplasmic ribonucleoprotein granule, called the chromatoid body (CB) that is conjectured to participate in the RNA post-transcriptional regulation. However, very little is known about the possible mechanisms of the CB function. The development of a procedure to isolate CBs from mouse testes was this study’s objective. Anti-MVH immunoprecipitation of cross-linked CBs from a fractionated testicular cell lysate was optimized to yield considerable quantities of pure and intact CBs from mice testes. This protocol produced reliable and reproducible data from the subsequent analysis of CB’s protein and RNA components. We found that the majority of the CB’s proteome consists of RNA-binding proteins that associate functionally with different pathways. We also demonstrated notable localization patterns of one of the CB transient components, SAM68 and showed that its ablation does not change the general composition or structure of the CB. CB-associated RNA analysis revealed a strong accumulation of PIWI-interacting RNAs (piRNAs), mRNAs and long non-coding RNAs (lncRNAs) in the CB. When the CB transcriptome and proteome analysis results were combined, the most pronounced molecular functions in the CB were related to piRNA pathway, RNA post-transcriptional processing and CB structural scaffolding. In addition, we demonstrated that the CB is a target for the main RNA flux from the nucleus throughout all steps of round spermatid development. Moreover, we provided preliminary evidence that those isolated CBs slice target RNAs in vitro in an ATPdependent manner. Altogether, these results make a strong suggestion that the CB functions involve RNA-related and RNA-mediated mechanisms. All the existing data supports the hypothesis that the CB coordinates the highly complex haploid transcriptome during the preparation of the male gametes for fertilization. Thereby, this study provides a fundamental basis for the future functional analyses of ribonucleoprotein granules and offers also important insights into the mechanisms governing male fertility.

Factors Regulating Chondrogenic Differentiation

Chondrogenesis is a co-ordinated differentiation process in which mesenchymal cells condensate, differentiate into chondrocytes and begin to secrete molecules that form the extracellular matrix. It is regulated in a spatio-temporal manner by cellular interactions and growth and differentiation factors that modulate cellular signalling pathways and transcription of specific genes. Moreover, post-transcriptional regulation by microRNAs (miRNAs) has appeared to play a central role in diverse biological processes, but their role in skeletal development is not fully understood. Mesenchymal stromal cells (MSCs) are multipotent cells present in a variety of adult tissues, including bone marrow and adipose tissue. They can be isolated, expanded and, under defined conditions, induced to differentiate into multiple cell lineages including chondrocytes, osteoblasts and adipocytes in vitro and in vivo. Owing to their intrinsic capability to self-renew and differentiate into functional cell types, MSCs provide a promising source for cell-based therapeutic strategies for various degenerative diseases, such as osteoarthritis (OA). Due to the potential therapeutic applications, it is of importance to better understand the MSC biology and the regulatory mechanisms of their differentiation. In this study, an in vitro assay for chondrogenic differentiation of mouse MSCs (mMSCs) was developed for the screening of various factors for their chondrogenic potential. Conditions were optimized for pellet cultures by inducing mMSC with different bone morphogenetic proteins (BMPs) that were selected based on their known chondrogenic relevance. Characterization of the surface epitope profile, differentiation capacity and molecular signature of mMSCs illustrated the importance of cell population composition and the interaction between different populations in the cell fate determination and differentiation of MSCs. Regulation of Wnt signalling activity by Wnt antagonist sFRP-1 was elucidated as a potential modulator of lineage commitment. Delta-like 1 (dlk1), a factor regulating adipogenesis and osteogenesis, was shown to exhibit stage-specific expression during embryonic chondrogenesis and identified as a novel regulator of chondrogenesis, possibly through mediating the effect of TGF-beta1. Moreover, miRNA profiling demonstrated that MSCs differentiating into a certain lineage exhibit a specific miRNA expression profile. The complex regulatory network between miRNAs and transcription factors is suggested to play a crucial role in fine-tuning the differentiation of MSCs. These results demonstrate that commitment of mesenchymal stromal cells and further differentiation into specific lineages is regulated by interactions between MSCs, various growth and transcription factors, and miRNA-mediated translational repression of lineage-specific genes.

Regulation of HSF2 and its function in mitosis

The cell is continuously subjected to various forms of external and intrinsic proteindamaging stresses, including hyperthermia, pathophysiological states, as well as cell differentiation and proliferation. Proteindamaging stresses result in denaturation and improper folding of proteins, leading to the formation of toxic aggregates that are detrimental for various pathological conditions, including Alzheimer’s and Huntington’s diseases. In order to maintain protein homeostasis, cells have developed different cytoprotective mechanisms, one of which is the evolutionary well-conserved heat shock response. The heat shock response results in the expression of heat shock proteins (Hsps), which act as molecular chaperones that bind to misfolded proteins, facilitate their refolding and prevent the formation of protein aggregates. Stress-induced expression of Hsps is mediated by a family of transcription factors, the heat shock factors, HSFs. Of the four HSFs found in vertebrates, HSF1-4, HSF1 is the major stress-responsive factor that is required for the induction of the heat shock response. HSF2 cannot alone induce Hsps, but modulates the heat shock response by forming heterotrimers with HSF1. HSFs are not only involved in the heat shock response, but they have also been found to have a function in development, neurodegenerative disorders, cancer, and longevity. Therefore, insight into how HSFs are regulated is important for the understanding of both normal physiological and disease processes. The activity of HSF1 is mainly regulated by intricate post-translational modifications, whereas the activity of HSF2 is concentrationdependent. However, there is only limited understanding of how the abundance of HSF2 is regulated. This study describes two different means of how HSF2 levels are regulated. In the first study it was shown that microRNA miR-18, a member of the miR-17~92 cluster, directly regulates Hsf2 mRNA stability and thus protein levels. HSF2 has earlier been shown to play a profound role in the regulation of male germ cell maturation during the spermatogenesis. The effect on miR-18 on HSF2 was examined in vivo by transfecting intact seminiferous tubules, and it was found that inhibition of miR-18 resulted in increased HSF2 levels and modified expression of the HSF2 targets Ssty2 and Speer4a. HSF2 has earlier been reported to modulate the heat shock response by forming heterotrimers with HSF1. In the second study, it was shown that HSF2 is cleared off the Hsp70 promoter and degraded by the ubiquitinproteasome pathway upon acute stress. By silencing components of the anaphase promoting complex/cyclosome (APC/C), including the co-activators Cdc20 and Cdh1, it was shown that APC/C mediates the heatinduced ubiquitylation of HSF2. Furthermore, down-regulation of Cdc20 was shown to alter the expression of heat shock-responsive genes. Next, we studied if APC/C-Cdc20, which controls cell cycle progression, also regulates HSF2 during the cell cycle. We found that both HSF2 mRNA and protein levels decreased during mitosis in several but not all human cell lines, indicating that HSF2 has a function in mitotic cells. Interestingly, although transcription is globally repressed during mitosis, mainly due to the displacement of RNA polymerase II and transcription factors, including HSF1, from the mitotic chromatin, HSF2 is capable of binding DNA during mitosis. Thus, during mitosis the heat shock response is impaired, leaving mitotic cells vulnerable to proteotoxic stress. However, in HSF2-deficient mitotic cells the Hsp70 promoter is accessible to both HSF1 and RNA polymerase II, allowing for stress-inducible Hsp expression to occur. As a consequence HSF2-deficient mitotic cells have a survival advantage upon acute heat stress. The results, presented in this thesis contribute to the understanding of the regulatory mechanisms of HSF2 and its function in the heat shock response in both interphase and mitotic cells.

Regulation of B Cell Gene Expression and Function by Ikaros, Helios and Bcl6

B lymphocytes constitute a key branch of adaptive immunity by providing specificity to recognize a vast variety of antigens by B cell antigen receptors (BCR) and secreted antibodies. Antigen recognition activates the cells and can produce antibody secreting plasma cells via germinal center reaction that leads to the maturation of antigen recognition affinity and switching of antibody effector class. The specificity of antigen recognition is achieved through a multistep developmental pathway that is organized by interplay of transcription factors and signals through BCR. Lymphoid malignancies arise from different stages of development in abnormal function of transcriptional regulation. To understand the B cell development and the function of B cells, a thorough understanding of the regulation of gene expression is important. The transcription factors of the Ikaros family and Bcl6 are frequently associated with lymphoma generation. The aim of this study was to reveal the targets of Ikaros, Helios and Bcl6 mediated gene regulation and to find out the function of Ikaros and Helios in B cells. This study uses gene targeted DT40 B cell lines and establishes a role for Ikaros family factors Ikaros and Helios in the regulation of BCR signaling that is important at developmental checkpoints, for cell survival and in activation. Ikaros and Helios had opposing roles in the regulation of BCR signals. Ikaros was found to directly repress the SHIP gene that encodes a signaling lipid-metabolizing enzyme, whereas Helios had activating effect on SHIP expression. The findings demonstrate a balancing function for these two Ikaros family transcription factors in the regulation of BCR signaling as well as in the regulation of gene expression. Bcl6 was found to repress plasma cell gene expression program while maintaining gene expression profile of B cells. Analysis of direct Bcl6 target genes suggested novel mechanisms for Bcl6-mediated suppression of plasma cell differentiation and promoting germinal center phenotype.

Differential Regulation of c-FLIP Isoforms Through Post-translational Modifications

Cells are constantly responding to signals from the surrounding tissues and the environment. To dispose of infected and potentially dangerous cells, to ensure the optimal execution of developmental processes and to maintain tissue homeostasis, a multicellular organism needs to tightly control both the number and the quality of its cells. Apoptosis is a form of active cellular self-destruction that enables an organism to regulate its cell number by deleting damaged or potentially dangerous cells. Apoptosis can be induced by death ligands, which bind to death receptors on the cell surface. Ligation of the receptors leads to the formation of an intracellular death inducing signaling complex (DISC). One of the DISC components is caspase-8, a protease that triggers the caspase cascade and is thereby a key initiator of programmed cell death. The activation of caspase-8 is controlled by the cellular FLICE-inhibitory proteins (c-FLIPs). Consequently, sensitivity towards receptor-mediated apoptosis is determined by the amount of c-FLIP, and the c-FLIP levels are actively regulated for example during erythroid differentiation of K562 erythroleukemia cells and by hyperthermia in Jurkat leukemia cells. The aim of my thesis was to investigate how c-FLIP is regulated during these processes. We found that c-FLIP isoforms are short-lived proteins, although c-FLIPS had an even shorter half-life than c-FLIPL. In both experimental models, increased death receptor sensitivity correlated with induced ubiquitylation and consequent proteasomal degradation of c-FLIP. Furthermore, we elucidated how phosphorylation regulates the biological functions and the turnover of c-FLIP, thereby contributing to death receptor sensitivity. We mapped the first phosphorylation sites on c-FLIP and dissected how their phosphorylation affects c-FLIP. Moreover, we demonstrated that phosphorylation of serine 193, a phosphorylated residue common to all c-FLIPs, is primarily mediated by the classical PKC. Furthermore, we discovered a novel connection between the phosphorylation and ubiquitylation of c-FLIP: phosphorylation of S193 protects c-FLIP from ubiquitylation. Surprisingly, although all c-FLIP isoforms are phosphorylated on this conserved residue, the biological outcome is different for the long and short isoforms, since S193 specifically prolongs the half-lives of the short c-FLIP isoforms, but not c-FLIPL. To summarize, we show that c-FLIP proteins are modified by ubiquitylation and phosphorylation, and that the biological outcomes of these modifications are isoform-specifically determined.

Transcriptional and Epigenetic Regulation of Human CD4+ T Helper Lineage Specification

Activated T helper (Th) cells have ability to differentiate into functionally distinct Th1, Th2 and Th17 subsets through a series of overlapping networks that include signaling and transcriptional control and the epigenetic mechanisms to direct immune responses. However, inappropriate execution in the differentiation process and abnormal function of these Th cells can lead to the development of several immune mediated diseases. Therefore, the thesis aimed at identifying genes and gene regulatory mechanisms responsible for Th17 differentiation and to study epigenetic changes associated with early stage of Th1/Th2 cell differentiation. Genome wide transcriptional profiling during early stages of human Th17 cell differentiation demonstrated differential regulation of several novel and currently known genes associated with Th17 differentiation. Selected candidate genes were further validated at protein level and their specificity for Th17 as compared to other T helper subsets was analyzed. Moreover, combination of RNA interference-mediated downregulation of gene expression, genome-wide transcriptome profiling and chromatin immunoprecipitation followed by massive parallel sequencing (ChIP-seq), combined with computational data integration lead to the identification of direct and indirect target genes of STAT3, which is a pivotal upstream transcription factor for Th17 cell polarization. Results indicated that STAT3 directly regulates the expression of several genes that are known to play a role in activation, differentiation, proliferation, and survival of Th17 cells. These results provide a basis for constructing a network regulating gene expression during early human Th17 differentiation. Th1 and Th2 lineage specific enhancers were identified from genome-wide maps of histone modifications generated from the cells differentiating towards Th1 and Th2 lineages at 72h. Further analysis of lineage-specific enhancers revealed known and novel transcription factors that potentially control lineage-specific gene expression. Finally, we found an overlap of a subset of enhancers with SNPs associated with autoimmune diseases through GWASs suggesting a potential role for enhancer elements in the disease development. In conclusion, the results obtained have extended our knowledge of Th differentiation and provided new mechanistic insights into dysregulation of Th cell differentiation in human immune mediated diseases.

Genetic regulatory networks in avian B cells

Biology is turning into an information science. The science of systems biology seeks to understand the genetic networks that govern organism development and functions. In this study the chicken was used as a model organism in the study of B cell regulatory factors. These studies open new avenues for plasma cell research by connecting the down regulation of the B cell gene expression program directly to the initiation of plasma cell differentiation. The unique advantages of the DT40 avian B cell model system, specifically its high homologous recombination rate, were utilized to study gene regulation in Pax5 knock out cell lines and to gain new insights into the B cell to plasma cell transitions that underlie the secretion of antibodies as part of the adaptive immune response. The Pax5 transcription factor is central to the commitment, development and maintenance of the B cell phenotype. Mice lacking the Pax5 gene have an arrest in development at the pro-B lymphocyte stage while DT40 cells have been derived from cells at a more mature stage of development. The DT40 Pax5-/- cells exhibited gene expression similarities with primary chicken plasma cells. The expression of the plasma cell transcription factors Blimp-1 and XBP-1 were significantly upregulated while the expression of the germinal centre factor BCL6 was diminished in Pax5-/- cells, and this alteration was normalized by Pax5 re-introduction. The Pax5-deficient cells further manifested substantially elevated secretion of IgM into the supernatant, another characteristic of plasma cells. These results for the first time indicated that the downregulation of the Pax5 gene in B cells promotes plasma cell differentiation. Cross-species meta-analysis of chicken and mouse Pax5 gene knockout studies uncovers genes and pathways whose regulatory relationship to Pax5 has remained unchanged for over 300 million years. Restriction of the hematopoietic stem cell fate to produce T, B and NK cell lineages is dependent on the Ikaros and its molecular partners, the closely related Helios and Aiolos. Ikaros family members are zinc finger proteins which act as transcriptional repressors while helping to activate lymphoid genes. Helios in mice is expressed from the hematopoietic stem cell level onwards, although later in development its expression seems to predominate in the T cell lineage. This study establishes the emergence and sequence of the chicken Ikaros family members. Helios expression in the bursa of Fabricius, germinal centres and B cell lines suggested a role for Helios in the avian B-cell lineage, too. Phylogenetic studies of the Ikaros family connect the expansion of the Ikaros family, and thus possibly the emergence of the adaptive immune system, with the second round of genome duplications originally proposed by Ohno. Paralogs that have arisen as a result of genome-wide duplications are sometimes termed ohnologs – Ikaros family proteins appear to fit that definition. This study highlighted the opportunities afforded by the genome sequencing efforts and somatic cell reverse genetics approaches using the DT40 cell line. The DT40 cell line and the avian model system promise to remain a fruitful model for mechanistic insight in the post-genomic era as well.

Transcriptional Profiling of Organ‐Specific Autoimmunity in Human

Our understanding of the pathogenesis of organ‐specific autoinflammation has been restricted by limited access to the target organs. Peripheral blood, however, as a preferred transportation route for immune cells, provides a window to assess the entire immune system throughout the body. Transcriptional profiling with RNA stabilizing blood collection tubes reflects in vivo expression profiles at the time the blood is drawn, allowing detection of the disease activity in different samples or within the same sample over time. The main objective of this Ph.D. study was to apply gene‐expression microarrays in the characterization of peripheral blood transcriptional profiles in patients with autoimmune diseases. To achieve this goal a custom cDNA microarray targeted for gene‐expression profiling of human immune system was designed and produced. Sample collection and preparation was then optimized to allow gene‐expression profiling from whole‐blood samples. To overcome challenges resulting from minute amounts of sample material, RNA amplification was successfully applied to study pregnancy related immunosuppression in patients with multiple sclerosis (MS). Furthermore, similar sample preparation was applied to characterize longitudinal genome‐wide expression profiles in children with type 1 diabetes (T1D) associated autoantibodies and eventually clinical T1D. Blood transcriptome analyses, using both the ImmunoChip cDNA microarray with targeted probe selection and genome‐wide Affymetrix U133 Plus 2.0 oligonucleotide array, enabled monitoring of autoimmune activity. Novel disease related genes and general autoimmune signatures were identified. Notably, down‐regulation of the HLA class Ib molecules in peripheral blood was associated with disease activity in both MS and T1D. Taken together, these studies demonstrate the potential of peripheral blood transcriptional profiling in biomedical research and diagnostics. Imbalances in peripheral blood transcriptional activity may reveal dynamic changes that are relevant for the disease but might be completely missed in conventional cross‐sectional studies.

Regulation of cell fate by c-FLIP phosphorylation

Programmed cell death is an important physiological cellular process that maintains homeostasis and protects multicellular organisms from diseases. Apoptosis is the principal mode of cell death, which eliminates unwanted cells and an enormous effort has been made to understand the molecular mechanisms of the signaling pathway and its regulatory systems. Irregular apoptosis often has life-threatening consequences to humans, including cancer, autoimmune diseases and degenerative diseases. In cancer for example, cell death is an attractive target to eradicate uncontrollably proliferating cells that have disregard pro-apoptotic signaling. Targeted therapeutic approaches are not as effective as once expected, since now we know that the cell death pathways are not sole entities in cells, but are highly associated with various cellular processes. Proteins that regulate apoptosis can also control non-apoptotic signaling pathways. For example, c-FLIP is a protein that can either inhibit or promote caspase-8 activation, which is required to induce apoptosis. Not only has c-FLIP opposing effects on initiating apoptosis, but it also regulates various pro-survival signaling pathways in the cell. It is well known that protein expression level is a determinant of how c-FLIP can regulate different signaling pathways, but other regulatory mechanisms potentially affecting the role of c-FLIP are less well understood. This work addresses novel insights into the mechanisms of c-FLIP post-translational modifications and their functional consequences. We have identified that phosphorylation is an important inception for subcellular localization of c-FLIP, thereby dictating which apoptotic and non-apoptotic signaling pathways c-FLIP could regulate to promote cell survival. Furthermore, we have constructed mathematical models to unite independent studies to establish more systematic c-FLIP signaling pathways to understand the dynamics of extrinsically-induced apoptosis.

Characterization of Leaf-Type Ferredoxin-NADP+ Oxidoreductase (FNR) Isoforms in Arabidopsis thaliana

Life on earth is based on sunlight, which is captured in chemical form by photosynthetic reactions. In the chloroplasts of plants, light reactions of photosynthesis take place at thylakoid membranes, whereas carbon assimilation reactions occur in the soluble stroma. The products of linear electron transfer (LET), highly-energetic ATP molecules, and reducing power in the form of NADPH molecules, are further used in the fixation of inorganic CO2 molecules into organic sugars. Ferredoxin-NADP+ oxidoreductase (FNR) catalyzes the last of the light reactions by transferring electrons from ferredoxin (FD) to NADP+. In addition to LET, FNR has been suggested to play a role in cyclic electron transfer (CET), which produces ATP without the accumulation of reducing equivalents. CET is proposed to occur via two putative routes, the PGR5- route and the NDH-route. In this thesis, the leaf-type FNR (LFNR) isoforms LFNR1 and LFNR2 of a model organism, Arabidopsis thaliana, were characterized. The physiological roles of LFNRs were investigated using single and double mutant plants. The viability of the single mutants indicates functionality of both isoforms, with neither appearing to play a specific role in CET. The more severe phenotype of low-temperature adapted fnr2 plants compared to both wild-type (WT) and fnr1 plants suggests a specific role for LFNR2 under unfavorable growth conditions. The more severe phenotype of the fnr1 x fnr2 (F1 generation) plants compared to single mutants reflects down-regulated photosynthetic capacity, whereas slightly higher excitation pressure indicates mild over-excitation of electron transfer chain (ETC). However, induction of CET and various photoprotective mechanisms enable adaptation of fnr1 x fnr2 plants to scarcity of LFNR. The fnr1 fnr2 plants (F2 generation), without detectable levels of LFNR, were viable only under heterotrophic conditions. Moreover, drought stress induced acceleration of the rate of P700 + re-reduction in darkness was accompanied by a concomitant up-regulation of the PGR5-route specific components, PGR5 and PGRL1, demonstrating the induction of CET via the PGR5-route. The up-regulation of relative transcriptional expression of the FD1 gene indicates that the FD1 isoform may have a specific function in CET, while no such role could be defined for either of the LFNR isoforms. Both the membrane-bound and soluble LFNR1 and LFNR2 each appear as two distinct spots after 2D-PAGE with different isoelectric points (pIs), indicating the existence of post-translational modifications (PTMs) which do not determine the membrane attachment of LFNR. The possibility of phosphorylation and glycosylation PTMs were excluded, but all four LFNR forms were shown to contain acetylated lysine residues as well as alternative N-termini. N-terminal acetylation was shown to shift the pI of both LFNRs to be more acidic. In addition, all four LFNR forms were demonstrated to interact both with FD1 and FD2 in vitro

University students’ regulation of learning and text processing – examples from medical and teacher education

The general aim of the thesis was to study university students’ learning from the perspective of regulation of learning and text processing. The data were collected from the two academic disciplines of medical and teacher education, which share the features of highly scheduled study, a multidisciplinary character, a complex relationship between theory and practice and a professional nature. Contemporary information society poses new challenges for learning, as it is not possible to learn all the information needed in a profession during a study programme. Therefore, it is increasingly important to learn how to think and learn independently, how to recognise gaps in and update one’s knowledge and how to deal with the huge amount of constantly changing information. In other words, it is critical to regulate one’s learning and to process text effectively. The thesis comprises five sub-studies that employed cross-sectional, longitudinal and experimental designs and multiple methods, from surveys to eye tracking. Study I examined the connections between students’ study orientations and the ways they regulate their learning. In total, 410 second-, fourth- and sixth-year medical students from two Finnish medical schools participated in the study by completing a questionnaire measuring both general study orientations and regulation strategies. The students were generally deeply oriented towards their studies. However, they regulated their studying externally. Several interesting and theoretically reasonable connections between the variables were found. For instance, self-regulation was positively correlated with deep orientation and achievement orientation and was negatively correlated with non-commitment. However, external regulation was likewise positively correlated with deep orientation and achievement orientation but also with surface orientation and systematic orientation. It is argued that external regulation might function as an effective coping strategy in the cognitively loaded medical curriculum. Study II focused on medical students’ regulation of learning and their conceptions of the learning environment in an innovative medical course where traditional lectures were combined wth problem-based learning (PBL) group work. First-year medical and dental students (N = 153) completed a questionnaire assessing their regulation strategies of learning and views about the PBL group work. The results indicated that external regulation and self-regulation of the learning content were the most typical regulation strategies among the participants. In line with previous studies, self-regulation wasconnected with study success. Strictly organised PBL sessions were not considered as useful as lectures, although the students’ views of the teacher/tutor and the group were mainly positive. Therefore, developers of teaching methods are challenged to think of new solutions that facilitate reflection of one’s learning and that improve the development of self-regulation. In Study III, a person-centred approach to studying regulation strategies was employed, in contrast to the traditional variable-centred approach used in Study I and Study II. The aim of Study III was to identify different regulation strategy profiles among medical students (N = 162) across time and to examine to what extent these profiles predict study success in preclinical studies. Four regulation strategy profiles were identified, and connections with study success were found. Students with the lowest self-regulation and with an increasing lack of regulation performed worse than the other groups. As the person-centred approach enables us to individualise students with diverse regulation patterns, it could be used in supporting student learning and in facilitating the early diagnosis of learning difficulties. In Study IV, 91 student teachers participated in a pre-test/post-test design where they answered open-ended questions about a complex science concept both before and after reading either a traditional, expository science text or a refutational text that prompted the reader to change his/her beliefs according to scientific beliefs about the phenomenon. The student teachers completed a questionnaire concerning their regulation and processing strategies. The results showed that the students’ understanding improved after text reading intervention and that refutational text promoted understanding better than the traditional text. Additionally, regulation and processing strategies were found to be connected with understanding the science phenomenon. A weak trend showed that weaker learners would benefit more from the refutational text. It seems that learners with effective learning strategies are able to pick out the relevant content regardless of the text type, whereas weaker learners might benefit from refutational parts that contrast the most typical misconceptions with scientific views. The purpose of Study V was to use eye tracking to determine how third-year medical studets (n = 39) and internal medicine residents (n = 13) read and solve patient case texts. The results revealed differences between medical students and residents in processing patient case texts; compared to the students, the residents were more accurate in their diagnoses and processed the texts significantly faster and with a lower number of fixations. Different reading patterns were also found. The observed differences between medical students and residents in processing patient case texts could be used in medical education to model expert reasoning and to teach how a good medical text should be constructed. The main findings of the thesis indicate that even among very selected student populations, such as high-achieving medical students or student teachers, there seems to be a lot of variation in regulation strategies of learning and text processing. As these learning strategies are related to successful studying, students enter educational programmes with rather different chances of managing and achieving success. Further, the ways of engaging in learning seldom centre on a single strategy or approach; rather, students seem to combine several strategies to a certain degree. Sometimes, it can be a matter of perspective of which way of learning can be considered best; therefore, the reality of studying in higher education is often more complicated than the simplistic view of self-regulation as a good quality and external regulation as a harmful quality. The beginning of university studies may be stressful for many, as the gap between high school and university studies is huge and those strategies that were adequate during high school might not work as well in higher education. Therefore, it is important to map students’ learning strategies and to encourage them to engage in using high-quality learning strategies from the beginning. Instead of separate courses on learning skills, the integration of these skills into course contents should be considered. Furthermore, learning complex scientific phenomena could be facilitated by paying attention to high-quality learning materials and texts and other support from the learning environment also in the university. Eye tracking seems to have great potential in evaluating performance and growing diagnostic expertise in text processing, although more research using texts as stimulus is needed. Both medical and teacher education programmes and the professions themselves are challenging in terms of their multidisciplinary nature and increasing amounts of information and therefore require good lifelong learning skills during the study period and later in work life.