Desmin: molecular interactions and putative functions of the muscle intermediate filament protein

Desmin is the intermediate filament (IF) protein occurring exclusively in muscle and endothelial cells. There are other IF proteins in muscle such as nestin, peripherin, and vimentin, besides the ubiquitous lamins, but they are not unique to muscle. Desmin was purified in 1977, the desmin gene was characterized in 1989, and knock-out animals were generated in 1996. Several isoforms have been described. Desmin IFs are present throughout smooth, cardiac and skeletal muscle cells, but can be more concentrated in some particular structures, such as dense bodies, around the nuclei, around the Z-line or in costameres. Desmin is up-regulated in muscle-derived cellular adaptations, including conductive fibers in the heart, electric organs, some myopathies, and experimental treatments with drugs that induce muscle degeneration, like phorbol esters. Many molecules have been reported to associate with desmin, such as other IF proteins (including members of the membrane dystroglycan complex), nebulin, the actin and tubulin binding protein plectin, the molecular motor dynein, the gene regulatory protein MyoD, DNA, the chaperone alphaB-crystallin, and proteases such as calpain and caspase. Desmin has an important medical role, since it is used as a marker of tumors' origin. More recently, several myopathies have been described, with accumulation of desmin deposits. Yet, after almost 30 years since its identification, the function of desmin is still unclear. Suggested functions include myofibrillogenesis, mechanical support for the muscle, mitochondrial localization, gene expression regulation, and intracellular signaling. This review focuses on the biochemical interactions of desmin, with a discussion of its putative functions.

Disintegrins: integrin selective ligands which activate integrin-coupled signaling and modulate leukocyte functions

Extracellular matrix proteins and cell adhesion receptors (integrins) play essential roles in the regulation of cell adhesion and migration. Interactions of integrins with the extracellular matrix proteins lead to phosphorylation of several intracellular proteins such as focal adhesion kinase, activating different signaling pathways responsible for the regulation of a variety of cell functions, including cytoskeleton mobilization. Once leukocytes are guided to sites of infection, inflammation, or antigen presentation, integrins can participate in the initiation, maintenance, or termination of the immune and inflammatory responses. The modulation of neutrophil activation through integrin-mediated pathways is important in the homeostatic control of the resolution of inflammatory states. In addition, during recirculation, T lymphocyte movement through distinct microenvironments is mediated by integrins, which are critical for cell cycle, differentiation and gene expression. Disintegrins are a family of low-molecular weight, cysteine-rich peptides first identified in snake venom, usually containing an RGD (Arg-Gly-Asp) motif, which confers the ability to selectively bind to integrins, inhibiting integrin-related functions in different cell systems. In this review we show that, depending on the cell type and the microenvironment, disintegrins are able to antagonize the effects of integrins or to act agonistically by activating integrin-mediated signaling. Disintegrins have proven useful as tools to improve the understanding of the molecular events regulated by integrin signaling in leukocytes and prototypes in order to design therapies able to interfere with integrin-mediated effects.

Unraveling the functional divergence of membrane-bound pyrophosphatases

Inorganic pyrophosphatases (PPases) are enzymes that hydrolyze pyrophosphate (PPi)which is produced as a byproduct in many important growth related processes e.g. in the biosynthesis of DNA, proteins and lipids. PPases can be either soluble or membranebound. Membrane-bound PPases (mPPases) are ion transporters that couple the energy released during PPi hydrolysis to Na+ or H+ transport. When I started the project, only three Na+-transporting mPPases were known to exist. In this study, I aimed to confirm if Na+-transport is a common function of mPPases. Furthermore, the amino acid residues responsible for determining the transporter specificity were unknown. I constructed a phylogenetic tree for mPPases and selected the representative bacterial and archaeal mPPases to be investigated. I expressed different prokaryotic mPPases in Escherichia coli, isolated these as inverted membrane vesicles and characterized their functions. In the first project I identified four new Na+-PPases, two K+-dependent H+-PPases and one K+-independent mPPase. The residues determining the transporter specificity were identified by site-directed mutagenesis. I showed that the conserved glutamate residues are important for specificity, though are not the only residues that influence it. This research clarified the ion transport specificities throughout the mPPase phylogenetic tree, and revealed that Na+ transport is a widespread function of mPPases. In addition, it became clear that the transporter specificity can be predicted from the amino acid sequence in combination with a phylogenetic analysis. In the second project, I identified a novel class of mPPases, which is capable of transporting both Na+ and H+ ions and is mainly found in bacteria of the human gastrointestinal tract. The physiological role of these novel enzymes may be to help the bacteria survive in the demanding conditions of the host. In the third project, I characterized the Chlorobium limicola Na+-PPase and found that this and related mPPases are able to transport H+ ions at subphysiological Na+ concentrations. In addition, the H+-transport activity was shown to be a common function of all studied Na+-PPases at low Na+ concentrations. I observed that mutating gate-lysine to asparagine eliminated the H+ but not the Na+ ion transport function, indicating the important role of the residue in the transport of H+. In the fourth project, I characterized the unknown and evolutionary divergent mPPase clade of the phylogenetic tree. The enzymes belonging to this clade are able to transport H+ ions and, based on their sequence, were expected to be K+- and Na+-independent. The sequences of membrane-bound PPase are usually highly conserved, but the enzymes belonging to this clade are more divergent and usually contain 100−150 extra amino acid residues compared to other known mPPases. Despite the vast sequence differences, these mPPases have the full set of important residues and, surprisingly, are regulated by Na+ and K+ ions. These enzymes are mainly of bacterial origin.

Arduino-pohjainen OBD2-lukija SAE J1850 VPW protokollatuella

Kandidaatintyössä toteutetaan OBD2 (On-Board Diagnostics 2) -lukija ajoneuvon päästöjenhallintajärjestelmän diagnostiikkatiedoille yleiskäyttöisellä mikro-ohjaimella. Lukija tukee tiedonsiirtoprotokollana SAE J1850 VPW protokollaa. Mikro-ohjaimena on Atmel Corporationin AVR ATMega328. Työn tavoitteena on havainnoida vastaantulevia käytännön ongelmia ja haasteita mikro-ohjaimen käytöllä tiedonsiirtoprotokollan toteutukseen, ja verrata toteutettua järjestelmää kaupallisiin OBD2-lukijoihin. Työn johtopäätöksenä havaitaan mikro-ohjaimen suorituskyvyn rajoitteet ja sen tuomat toiminnan epävarmuustekijät. Työssä myös todetaan, että mikro-ohjain soveltuu tiedonsiirtoprotokollan toteutukseen kun rajoitteet otetaan huomioon. Kaupallisiin lukijoihin verrattuna yleiskäyttöiseen mikro-ohjaimeen perustuva toteutettu järjestelmä on kalliimpi ja toiminnoiltaan suppeampi. Mikro-ohjaimeen perustuva järjestelmä on kuitenkin muokattavissa ja laajennettavissa tarvittaessa, jolloin toteutukseen voidaan saada kaupallisista järjestelmistä mahdollisesti puuttuvia ominaisuuksia, kuten valmistajakohtaisia protokollia ja toimintoja, joita ei ole määritelty OBD2:ssa. Yhtenä esimerkkinä tällaisesta toiminnosta voi mainita ajoneuvoissa yleistyvän sähköisen käsijarrun säätöä ohjaavat komennot jarruhuoltoa varten.

A new model for the population pharmacokinetics of didanosine in healthy subjects

Didanosine (ddI) is a component of highly active antiretroviral therapy drug combinations, used especially in resource-limited settings and in zidovudine-resistant patients. The population pharmacokinetics of ddI was evaluated in 48 healthy volunteers enrolled in two bioequivalence studies. These data, along with a set of co-variates, were the subject of a nonlinear mixed-effect modeling analysis using the NONMEM program. A two-compartment model with first order absorption (ADVAN3 TRANS3) was fitted to the serum ddI concentration data. Final pharmacokinetic parameters, expressed as functions of the co-variates gender and creatinine clearance (CL CR), were: oral clearance (CL = 55.1 + 240 x CL CR + 16.6 L/h for males and CL = 55.1 + 240 x CL CR for females), central volume (V2 = 9.8 L), intercompartmental clearance (Q = 40.9 L/h), peripheral volume (V3 = 62.7 + 22.9 L for males and V3 = 62.7 L for females), absorption rate constant (Ka = 1.51/h), and dissolution time of the tablet (D = 0.43 h). The intraindividual (residual) variability expressed as coefficient of variation was 13.0%, whereas the interindividual variability of CL, Q, V3, Ka, and D was 20.1, 75.8, 20.6, 18.9, and 38.2%, respectively. The relatively high (>30%) interindividual variability for some of these parameters, observed under the controlled experimental settings of bioequivalence trials in healthy volunteers, may result from genetic variability of the processes involved in ddI absorption and disposition.

Pipeline for macro- and microarray analyses

The pipeline for macro- and microarray analyses (PMmA) is a set of scripts with a web interface developed to analyze DNA array data generated by array image quantification software. PMmA is designed for use with single- or double-color array data and to work as a pipeline in five classes (data format, normalization, data analysis, clustering, and array maps). It can also be used as a plugin in the BioArray Software Environment, an open-source database for array analysis, or used in a local version of the web service. All scripts in PMmA were developed in the PERL programming language and statistical analysis functions were implemented in the R statistical language. Consequently, our package is a platform-independent software. Our algorithms can correctly select almost 90% of the differentially expressed genes, showing a superior performance compared to other methods of analysis. The pipeline software has been applied to 1536 expressed sequence tags macroarray public data of sugarcane exposed to cold for 3 to 48 h. PMmA identified thirty cold-responsive genes previously unidentified in this public dataset. Fourteen genes were up-regulated, two had a variable expression and the other fourteen were down-regulated in the treatments. These new findings certainly were a consequence of using a superior statistical analysis approach, since the original study did not take into account the dependence of data variability on the average signal intensity of each gene. The web interface, supplementary information, and the package source code are available, free, to non-commercial users at http://ipe.cbmeg.unicamp.br/pub/PMmA.

Synergistic control of forearm based on accelerometer data and artificial neural networks

In the present study, we modeled a reaching task as a two-link mechanism. The upper arm and forearm motion trajectories during vertical arm movements were estimated from the measured angular accelerations with dual-axis accelerometers. A data set of reaching synergies from able-bodied individuals was used to train a radial basis function artificial neural network with upper arm/forearm tangential angular accelerations. The trained radial basis function artificial neural network for the specific movements predicted forearm motion from new upper arm trajectories with high correlation (mean, 0.9149-0.941). For all other movements, prediction was low (range, 0.0316-0.8302). Results suggest that the proposed algorithm is successful in generalization over similar motions and subjects. Such networks may be used as a high-level controller that could predict forearm kinematics from voluntary movements of the upper arm. This methodology is suitable for restoring the upper limb functions of individuals with motor disabilities of the forearm, but not of the upper arm. The developed control paradigm is applicable to upper-limb orthotic systems employing functional electrical stimulation. The proposed approach is of great significance particularly for humans with spinal cord injuries in a free-living environment. The implication of a measurement system with dual-axis accelerometers, developed for this study, is further seen in the evaluation of movement during the course of rehabilitation. For this purpose, training-related changes in synergies apparent from movement kinematics during rehabilitation would characterize the extent and the course of recovery. As such, a simple system using this methodology is of particular importance for stroke patients. The results underlie the important issue of upper-limb coordination.

Prokaryotic expression of a novel mouse pro-apoptosis protein PNAS-4 and application of its polyclonal antibodies

Mouse PNAS-4 (mPNAS-4) has 96% identity with human PNAS-4 (hPNAS-4) in primary sequence and has been reported to be involved in the apoptotic response to DNA damage. However, there have been no studies reported of the biological functions of mPNAS-4. In studies conducted by our group (unpublished data), it was interesting to note that overexpression of mPNAS-4 promoted apoptotic death in Lewis lung carcinoma cells (LL2) and colon carcinoma cells (CT26) of mice both in vitro and in vivo. In our studies, mPNAS-4 was cloned into the pGEX-6P-1 vector with GST tag at N-terminal in Escherichia coli strain BL21(DE3). The soluble and insoluble expression of recombinant protein mPNAS-4 (rmPNAS-4) was temperature-dependent. The majority of rmPNAS-4 was insoluble at 37°C, while it was almost exclusively expressed in soluble form at 20°C. The soluble rmPNAS-4 was purified by one-step affinity purification, using a glutathione Sepharose 4B column. The rmPNAS-4 protein was further identified by electrospray ionization-mass spectrometry analysis. The search parameters of the parent and fragment mass error tolerance were set at 0.1 and 0.05 kDa, respectively, and the sequence coverage of search result was 28%. The purified rmPNAS-4 was further used as immunogen to raise polyclonal antibodies in New Zealand white rabbit, which were suitable to detect both the recombinant and the endogenous mPNAS-4 in mouse brain tissue and LL2 cells after immunoblotting and/or immunostaining. The purified rmPNAS-4 and our prepared anti-mPNAS-4 polyclonal antibodies may provide useful tools for future biological function studies for mPNAS.

Nitric oxide synthesis and biological functions of nitric oxide released from ruthenium compounds

During three decades, an enormous number of studies have demonstrated the critical role of nitric oxide (NO) as a second messenger engaged in the activation of many systems including vascular smooth muscle relaxation. The underlying cellular mechanisms involved in vasodilatation are essentially due to soluble guanylyl-cyclase (sGC) modulation in the cytoplasm of vascular smooth cells. sGC activation culminates in cyclic GMP (cGMP) production, which in turn leads to protein kinase G (PKG) activation. NO binds to the sGC heme moiety, thereby activating this enzyme. Activation of the NO-sGC-cGMP-PKG pathway entails Ca2+ signaling reduction and vasodilatation. Endothelium dysfunction leads to decreased production or bioavailability of endogenous NO that could contribute to vascular diseases. Nitrosyl ruthenium complexes have been studied as a new class of NO donors with potential therapeutic use in order to supply the NO deficiency. In this context, this article shall provide a brief review of the effects exerted by the NO that is enzymatically produced via endothelial NO-synthase (eNOS) activation and by the NO released from NO donor compounds in the vascular smooth muscle cells on both conduit and resistance arteries, as well as veins. In addition, the involvement of the nitrite molecule as an endogenous NO reservoir engaged in vasodilatation will be described.

Silencing HIF-1α reduces the adhesion and secretion functions of acute leukemia hBMSCs

Hypoxia inducible factor-1α (HIF-1α) is an important transcription factor, which plays a critical role in the formation of solid tumor and its microenviroment. The objective of the present study was to evaluate the expression and function of HIF-1α in human leukemia bone marrow stromal cells (BMSCs) and to identify the downstream targets of HIF-1α. HIF-1α expression was detected at both the RNA and protein levels using real-time PCR and immunohistochemistry, respectively. Vascular endothelial growth factor (VEGF) and stromal cell-derived factor-1α (SDF-1α) were detected in stromal cells by enzyme-linked immunosorbent assay. HIF-1α was blocked by constructing the lentiviral RNAi vector system and infecting the BMSCs. The Jurkat cell/BMSC co-cultured system was constructed by putting the two cells into the same suitable cultured media and conditions. Cell adhesion and secretion functions of stromal cells were evaluated after transfection with the lentiviral RNAi vector of HIF-1α. Increased HIF-1α mRNA and protein was detected in the nucleus of the acute myeloblastic and acute lymphoblastic leukemia compared with normal BMSCs. The lentiviral RANi vector for HIF-1α was successfully constructed and was applied to block the expression of HIF-1α. When HIF-1α of BMSCs was blocked, the expression of VEGF and SDF-1 secreted by stromal cells were decreased. When HIF-1α was blocked, the co-cultured Jurkat cell’s adhesion and migration functions were also decreased. Taken together, these results suggest that HIF-1α acts as an important transcription factor and can significantly affect the secretion and adhesion functions of leukemia BMSCs.

Players of Th-cell differentiation and immune dysregulation – focus on GIMAP family genes

The balance of T helper (Th) cell differentiation is the fundamental process that ensures that the immune system functions correctly and effectively. The differentiation is a fine tuned event, the outcome of which is driven by activation of the T-cell in response to recognition of the specific antigen presented. The co-stimulatory signals from the surrounding cytokine milieu help to determine the outcome. An impairment in the differentiation processes may lead to an imbalance in immune responses and lead to immune-mediated pathologies. An over-representation of Th1 type cytokine producing cells leads to tissue-specific inflammation and autoimmunity, and excessive Th2 response is causative for atopy, asthma and allergy. The major factors of Th-cell differentiation and in the related disease mechanisms have been extensively studied, but the fine tuning of these processes by the other factors cannot be discarded. In the work presented in this thesis, the association of T-cell receptor costimulatory molecules CTLA4 and ICOS with autoimmune diabetes were studied. The underlying aspect of the study was to explore the polymorphism in these genes with the different disease rates observed in two geographically close populations. The main focus of this thesis was set on a GTPase of the immunity associated protein (GIMAP) family of small GTPases. GIMAP genes and proteins are differentially regulated during human Th-cell differentiation and have been linked to immune-mediated disorders. GIMAP4 is believed to contribute to the immunological balance via its role in T-cell survival. To elucidate the function of GIMAP4 and GIMAP5 and their role in human immunity, a study combining genetic association in different immunological diseases and complementing functional analyses was conducted. The study revealed interesting connections with the high susceptibility risk genes. In addition, the role of GIMAP4 during Th1-cell differentiation was investigated. A novel function of GIMAP4 in relation to cytokine secretion was discovered. Further assessment of GIMAP4 and GIMAP5 effect for the transcriptomic profile of differentiating Th1-cells revealed new insights for GIMAP4 and GIMAP5 function.

Socially shared metacognitive regulation during collaborative learning processes in student dyads and small groups

Traditionally metacognition has been theorised, methodologically studied and empirically tested from the standpoint mainly of individuals and their learning contexts. In this dissertation the emergence of metacognition is analysed more broadly. The aim of the dissertation was to explore socially shared metacognitive regulation (SSMR) as part of collaborative learning processes taking place in student dyads and small learning groups. The specific aims were to extend the concept of individual metacognition to SSMR, to develop methods to capture and analyse SSMR and to validate the usefulness of the concept of SSMR in two different learning contexts; in face-to-face student dyads solving mathematical word problems and also in small groups taking part in inquiry-based science learning in an asynchronous computer-supported collaborative learning (CSCL) environment. This dissertation is comprised of four studies. In Study I, the main aim was to explore if and how metacognition emerges during problem solving in student dyads and then to develop a method for analysing the social level of awareness, monitoring, and regulatory processes emerging during the problem solving. Two dyads comprised of 10-year-old students who were high-achieving especially in mathematical word problem solving and reading comprehension were involved in the study. An in-depth case analysis was conducted. Data consisted of over 16 (30–45 minutes) videotaped and transcribed face-to-face sessions. The dyads solved altogether 151 mathematical word problems of different difficulty levels in a game-format learning environment. The interaction flowchart was used in the analysis to uncover socially shared metacognition. Interviews (also stimulated recall interviews) were conducted in order to obtain further information about socially shared metacognition. The findings showed the emergence of metacognition in a collaborative learning context in a way that cannot solely be explained by individual conception. The concept of socially-shared metacognition (SSMR) was proposed. The results highlighted the emergence of socially shared metacognition specifically in problems where dyads encountered challenges. Small verbal and nonverbal signals between students also triggered the emergence of socially shared metacognition. Additionally, one dyad implemented a system whereby they shared metacognitive regulation based on their strengths in learning. Overall, the findings suggested that in order to discover patterns of socially shared metacognition, it is important to investigate metacognition over time. However, it was concluded that more research on socially shared metacognition, from larger data sets, is needed. These findings formed the basis of the second study. In Study II, the specific aim was to investigate whether socially shared metacognition can be reliably identified from a large dataset of collaborative face-to-face mathematical word problem solving sessions by student dyads. We specifically examined different difficulty levels of tasks as well as the function and focus of socially shared metacognition. Furthermore, the presence of observable metacognitive experiences at the beginning of socially shared metacognition was explored. Four dyads participated in the study. Each dyad was comprised of high-achieving 10-year-old students, ranked in the top 11% of their fourth grade peers (n=393). Dyads were from the same data set as in Study I. The dyads worked face-to-face in a computer-supported, game-format learning environment. Problem-solving processes for 251 tasks at three difficulty levels taking place during 56 (30–45 minutes) lessons were video-taped and analysed. Baseline data for this study were 14 675 turns of transcribed verbal and nonverbal behaviours observed in four study dyads. The micro-level analysis illustrated how participants moved between different channels of communication (individual and interpersonal). The unit of analysis was a set of turns, referred to as an ‘episode’. The results indicated that socially shared metacognition and its function and focus, as well as the appearance of metacognitive experiences can be defined in a reliable way from a larger data set by independent coders. A comparison of the different difficulty levels of the problems suggested that in order to trigger socially shared metacognition in small groups, the problems should be more difficult, as opposed to moderately difficult or easy. Although socially shared metacognition was found in collaborative face-to-face problem solving among high-achieving student dyads, more research is needed in different contexts. This consideration created the basis of the research on socially shared metacognition in Studies III and IV. In Study III, the aim was to expand the research on SSMR from face-to-face mathematical problem solving in student dyads to inquiry-based science learning among small groups in an asynchronous computer-supported collaborative learning (CSCL) environment. The specific aims were to investigate SSMR’s evolvement and functions in a CSCL environment and to explore how SSMR emerges at different phases of the inquiry process. Finally, individual student participation in SSMR during the process was studied. An in-depth explanatory case study of one small group of four girls aged 12 years was carried out. The girls attended a class that has an entrance examination and conducts a language-enriched curriculum. The small group solved complex science problems in an asynchronous CSCL environment, participating in research-like processes of inquiry during 22 lessons (á 45–minute). Students’ network discussion were recorded in written notes (N=640) which were used as study data. A set of notes, referred to here as a ‘thread’, was used as the unit of analysis. The inter-coder agreement was regarded as substantial. The results indicated that SSMR emerges in a small group’s asynchronous CSCL inquiry process in the science domain. Hence, the results of Study III were in line with the previous Study I and Study II and revealed that metacognition cannot be reduced to the individual level alone. The findings also confirm that SSMR should be examined as a process, since SSMR can evolve during different phases and that different SSMR threads overlapped and intertwined. Although the classification of SSMR’s functions was applicable in the context of CSCL in a small group, the dominant function was different in the asynchronous CSCL inquiry in the small group in a science activity than in mathematical word problem solving among student dyads (Study II). Further, the use of different analytical methods provided complementary findings about students’ participation in SSMR. The findings suggest that it is not enough to code just a single written note or simply to examine who has the largest number of notes in the SSMR thread but also to examine the connections between the notes. As the findings of the present study are based on an in-depth analysis of a single small group, further cases were examined in Study IV, as well as looking at the SSMR’s focus, which was also studied in a face-to-face context. In Study IV, the general aim was to investigate the emergence of SSMR with a larger data set from an asynchronous CSCL inquiry process in small student groups carrying out science activities. The specific aims were to study the emergence of SSMR in the different phases of the process, students’ participation in SSMR, and the relation of SSMR’s focus to the quality of outcomes, which was not explored in previous studies. The participants were 12-year-old students from the same class as in Study III. Five small groups consisting of four students and one of five students (N=25) were involved in the study. The small groups solved ill-defined science problems in an asynchronous CSCL environment, participating in research-like processes of inquiry over a total period of 22 hours. Written notes (N=4088) detailed the network discussions of the small groups and these constituted the study data. With these notes, SSMR threads were explored. As in Study III, the thread was used as the unit of analysis. In total, 332 notes were classified as forming 41 SSMR threads. Inter-coder agreement was assessed by three coders in the different phases of the analysis and found to be reliable. Multiple methods of analysis were used. Results showed that SSMR emerged in all the asynchronous CSCL inquiry processes in the small groups. However, the findings did not reveal any significantly changing trend in the emergence of SSMR during the process. As a main trend, the number of notes included in SSMR threads differed significantly in different phases of the process and small groups differed from each other. Although student participation was seen as highly dispersed between the students, there were differences between students and small groups. Furthermore, the findings indicated that the amount of SSMR during the process or participation structure did not explain the differences in the quality of outcomes for the groups. Rather, when SSMRs were focused on understanding and procedural matters, it was associated with achieving high quality learning outcomes. In turn, when SSMRs were focused on incidental and procedural matters, it was associated with low level learning outcomes. Hence, the findings imply that the focus of any emerging SSMR is crucial to the quality of the learning outcomes. Moreover, the findings encourage the use of multiple research methods for studying SSMR. In total, the four studies convincingly indicate that a phenomenon of socially shared metacognitive regulation also exists. This means that it was possible to define the concept of SSMR theoretically, to investigate it methodologically and to validate it empirically in two different learning contexts across dyads and small groups. In-depth micro-level case analysis in Studies I and III showed the possibility to capture and analyse in detail SSMR during the collaborative process, while in Studies II and IV, the analysis validated the emergence of SSMR in larger data sets. Hence, validation was tested both between two environments and within the same environments with further cases. As a part of this dissertation, SSMR’s detailed functions and foci were revealed. Moreover, the findings showed the important role of observable metacognitive experiences as the starting point of SSMRs. It was apparent that problems dealt with by the groups should be rather difficult if SSMR is to be made clearly visible. Further, individual students’ participation was found to differ between students and groups. The multiple research methods employed revealed supplementary findings regarding SSMR. Finally, when SSMR was focused on understanding and procedural matters, this was seen to lead to higher quality learning outcomes. Socially shared metacognition regulation should therefore be taken into consideration in students’ collaborative learning at school similarly to how an individual’s metacognition is taken into account in individual learning.