Regulation of Spermatogenesis: Differentiation of GFP-labeled Stem Cells and the Function of Cytoplasmic Bridges

During spermatogenesis, different genes are expressed in a strictly coordinated fashion providing an excellent model to study cell differentiation. Recent identification of testis specific genes and the development of green fluorescence protein (GFP) transgene technology and an in vivo system for studying the differentiation of transplanted male germ cells in infertile testis has opened new possibilities for studying the male germ cell differentiation at molecular level. We have employed these techniques in combination with transillumination based stage recognition (Parvinen and Vanha-Perttula, 1972) and squash preparation techniques (Parvinen and Hecht, 1981) to study the regulation of male germ cell differentiation. By using transgenic mice expressing enhanced-(E)GFP as a marker we have studied the expression and hormonal regulation of beta-actin and acrosin proteins in the developmentally different living male germ cells. Beta-actin was demonstrated in all male germ cells, whereas acrosin was expressed only in late meiotic and in postmeiotic cells. Follicle stimulating hormone stimulated b-actin-EGFP expression at stages I-VI and enhanced the formation of microtubules in spermatids and this way reduced the size of the acrosomic system. When EGFP expressing spermatogonial stem cells were transplanted into infertile mouse testis differentiation and the synchronized development of male germ cells could be observed during six months observation time. Each colony developed independently and maintained typical stage-dependent cell associations. Furthermore, if more than two colonies were fused, each of them was adjusted to one stage and synchronized. By studying living spermatids we were able to demonstrate novel functions for Golgi complex and chromatoid body in material sharing between neighbor spermatids. Immunosytochemical analyses revealed a transport of haploid cell specific proteins in spermatids (TRA54 and Shippo1) and through the intercellular bridges (TRA54). Cytoskeleton inhibitor (nocodazole) demonstrated the importance of microtubules in material sharing between spermatids and in preserving the integrity of the chromatoid body. Golgi complex inhibitor, brefeldin A, revealed the great importance of Golgi complex i) in acrosomic system formation ii) TRA54 translation and in iii) granule trafficking between spermatids.

Photodamage to Oxygen Evolving Complex - An Initial Event in Photoinhibition of Photosystem II.

Photosystem II (PSII) of oxygenic photosynthesis is susceptible to photoinhibition. Photoinhibition is defined as light induced damage resulting in turnover of the D1 protein subunit of the reaction center of PSII. Both visible and ultraviolet (UV) light cause photoinhibition. Photoinhibition induced by UV light damages the oxygen evolving complex (OEC) via absorption of UV photons by the Mn ion(s) of OEC. Under visible light, most of the earlier hypotheses assume that photoinhibition occurs when the rate of photon absorption by PSII antenna exceeds the use of the absorbed energy in photosynthesis. However, photoinhibition occurs at all light intensities with the same efficiency per photon. The aim of my thesis work was to build a model of photoinhibition that fits the experimental features of photoinhibition. I studied the role of electron transfer reactions of PSII in photoinhibition and found that changing the electron transfer rate had only minor influence on photoinhibition if light intensity was kept constant. Furthermore, quenching of antenna excitations protected less efficiently than it would protect if antenna chlorophylls were the only photoreceptors of photoinhibition. To identify photoreceptors of photoinhibition, I measured the action spectrum of photoinhibition. The action spectrum showed resemblance to the absorption spectra of Mn model compounds suggesting that the Mn cluster of OEC acts as a photoreceptor of photoinhibition under visible light, too. The role of Mn in photoinhibition was further supported by experiments showing that during photoinhibition OEC is damaged before electron transfer activity at the acceptor side of PSII is lost. Mn enzymes were found to be photosensitive under visible and UV light indicating that Mn-containing compounds, including OEC, are capable of functioning as photosensitizers both in visible and UV light. The experimental results above led to the Mn hypothesis of the mechanism of continuous-light-induced photoinhibition. According to the Mn hypothesis, excitation of Mn of OEC results in inhibition of electron donation from OEC to the oxidized primary donor P680+ both under UV and visible light. P680 is oxidized by photons absorbed by chlorophyll, and if not reduced by OEC, P680+ may cause harmful oxidation of other PSII components. Photoinhibition was also induced with intense laser pulses and it was found that the photoinhibitory efficiency increased in proportion to the square of pulse intensity suggesting that laser-pulse-induced photoinhibition is a two-photon reaction. I further developed the Mn hypothesis suggesting that the initial event in photoinhibition under both continuous and pulsed light is the same: Mn excitation that leads to the inhibition of electron donation from OEC to P680+. Under laser-pulse-illumination, another Mn-mediated inhibitory photoreaction occurs within the duration of the same pulse, whereas under continuous light, secondary damage is chlorophyll mediated. A mathematical model based on the Mn hypothesis was found to explain photoinhibition under continuous light, under flash illumination and under the combination of these two.

Role and Function of c-Jun Protein Complex in Cancer Cell Behavior

Transcription factors play a crucial role in the regulation of cell behavior by modulating gene expression profiles. Previous studies have described a dual role for the AP-1 family transcription factor c-Jun in the regulation of cellular fate. In various cell types weak and transient activations of c-Jun N-terminal kinase (JNK) and c-Jun appear to contribute to proliferation and survival, whereas strong and prolonged activation of JNK and c-Jun result in apoptosis. These opposite roles played by c-Jun are cell type specific and the molecular mechanisms defining these antonymous c-Jun-mediated responses remain incompletely understood. c-Jun activity in transformed cells is regulated by signalling cascades downstream of oncoproteins such as Ras and Raf. In addition, the pro-proliferative role and the survival promoting function for c-Jun has been described in various cancer models. Furthermore, c-Jun was described to be overexpressed in different cancer types. However, the molecular mechanisms by which c-Jun exerts these oncogenic functions are not all clearly established. Therefore it is of primary interest to further identify molecular mechanisms and functions for c-Jun in cancer. Regulation of gene expression is tightly dependent on accurate protein-protein interactions. Therefore, co-factors for c-Jun may define the functions for c-Jun in cancer. Identification of protein-protein interactions promoting cancer may provide novel possibilities for cancer treatment. In this study, we show that DNA topoisomerase I (TopoI) is a transcriptional co-factor for c-Jun. Moreover, c-Jun and TopoI together promote expression of epidermal growth factor receptor (EGFR) in cancer cells. We also show that the clinically used TopoI inhibitor topotecan reduces EGFR expression. Importantly, the effect of TopoI on EGFR transcription was shown to depend on c-Jun as Jun-/- cells or cells treated with JNK inhibitor SP600125 are resistant to topotecan treatment both in regulation of EGFR expression and cell proliferation. Moreover, c-Jun regulates the nucleolar localization and the function of the ribonucleic acid (RNA) helicase DDX21, a previously identified member of c-Jun protein complex. In addition, c-Jun stimulates rRNA processing by supporting DDX21 rRNA binding. Finally, this study characterizes a DDX21 dependent expression of cyclin dependent kinase (Cdk) 6, a correlation of DDX21 expression with prostate cancer progression and a substrate binding dependency of DDX21 nucleolar localization in prostate cancer cells. Taken together, the results of this study validate the c-Jun-TopoI interaction and precise the c-Jun-DDX21 interaction. Moreover, these results show the importance for protein-protein interaction in the regulation of their cellular functions in cancer cell behavior. Finally, the results presented here disclose new exciting therapeutic opportunities for cancer treatment.

Parallella, dynamiska representationer i gymnasiefysiken : implementering av ett interaktivt datorverktyg i didaktiska situationer och uppfattning av effektivt lärande

The computer is a useful tool in the teaching of upper secondary school physics, and should not have a subordinate role in students' learning process. However, computers and computer-based tools are often not available when they could serve their purpose best in the ongoing teaching. Another problem is the fact that commercially available tools are not usable in the way the teacher wants. The aim of this thesis was to try out a novel teaching scenario in a complicated subject in physics, electrodynamics. The didactic engineering of the thesis consisted of developing a computer-based simulation and training material, implementing the tool in physics teaching and investigating its effectiveness in the learning process. The design-based research method, didactic engineering (Artigue, 1994), which is based on the theoryof didactical situations (Brousseau, 1997), was used as a frame of reference for the design of this type of teaching product. In designing the simulation tool a general spreadsheet program was used. The design was based on parallel, dynamic representations of the physics behind the function of an AC series circuit in both graphical and numerical form. The tool, which was furnished with possibilities to control the representations in an interactive way, was hypothesized to activate the students and promote the effectiveness of their learning. An effect variable was constructed in order to measure the students' and teachers' conceptions of learning effectiveness. The empirical study was twofold. Twelve physics students, who attended a course in electrodynamics in an upper secondary school, participated in a class experiment with the computer-based tool implemented in three modes of didactical situations: practice, concept introduction and assessment. The main goal of the didactical situations was to have students solve problems and study the function of AC series circuits, taking responsibility for theirown learning process. In the teacher study eighteen Swedish speaking physics teachers evaluated the didactic potential of the computer-based tool and the accompanying paper-based material without using them in their physics teaching. Quantitative and qualitative data were collected using questionnaires, observations and interviews. The result of the studies showed that both the group of students and the teachers had generally positive conceptions of learning effectiveness. The students' conceptions were more positive in the practice situation than in the concept introduction situation, a setting that was more explorative. However, it turned out that the students' conceptions were also positive in the more complex assessment situation. This had not been hypothesized. A deeper analysis of data from observations and interviews showed that one of the students in each pair was more active than the other, taking more initiative and more responsibilityfor the student-student and student-computer interaction. These active studentshad strong, positive conceptions of learning effectiveness in each of the threedidactical situations. The group of less active students had a weak but positive conception in the first iv two situations, but a negative conception in the assessment situation, thus corroborating the hypothesis ad hoc. The teacher study revealed that computers were seldom used in physics teaching and that computer programs were in short supply. The use of a computer was considered time-consuming. As long as physics teaching with computer-based tools has to take place in special computer rooms, the use of such tools will remain limited. The affordance is enhanced when the physical dimensions as well as the performance of the computer are optimised. As a consequence, the computer then becomes a real learning tool for each pair of students, smoothly integrated into the ongoing teaching in the same space where teaching normally takes place. With more interactive support from the teacher, the computer-based parallel, dynamic representations will be efficient in promoting the learning process of the students with focus on qualitative reasoning - an often neglected part of the learning process of the students in upper secondary school physics.

Perspektivmöten i skola och handledning : lärares tankar om specialpedagogisk handledning

To create a more inclusive school, an increase in multidisciplinary cooperation is needed. One possible form of collaboration could encompass the special education teacher taking on the role of a consultant for other teachers in need of support in working with heterogeneous groups of pupils. Previous research shows that special education teachers see the role as consultant as diffuse and complex. The overarching aim of the present study involves deepening the knowledge on how consultation in a special educational context can be understood and developed based on teachers’ descriptions on this particular form of activity interpreted against various perspectives on consultation. The study is qualitative in nature and rests on a hermeneutic interpretive research tradition in combination with an abductive approach. The theoretical framework consists of two different approaches to consultation: the directive and the non-directive approach. The approaches differ regarding particular emphasis on advice and reflection during the consultation and with respect to who or what should be the focus of the consultation. The two approaches are here studied through various theories such as social learning theory, Bruner's theory of scaffolding, Roger’s humanist psychology, and constructivism. Semi-structured interviews were held with eighteen special education teachers (n=9) and class teachers (n=9) working in the compulsory school. The overall interpretation of the results shows that special education consultation can be understood as three different types of consultation. Consultation as counseling which harmonizes with the directive perspective on consultation is the most prominent type. In the consultation as counseling conversation, the special educational knowledge transfer is central and the focus is placed on the pupil. Although special education knowledge transfer emerges as a unique aspect of special education consultation, there are several inherent challenges in this type of consultation that can be addressed in that teachers also describe two other types of consultation. In the reflective consultation, there is a move away from the pupil focus and toward a focus on the class teacher and the use of reflection. The reflective consultation harmonizes with the non-directive approach to consultation. This type of consultation does not as of yet have a prominent place in the Finland-Swedish school context and at this stage it is not seen as a legitimate type of consultation according to the teachers’ descriptions. Despite this, certain aspects of the reflective conversation could be given more space in the development of consultation within special educational contexts. The co-operative consultation is characterized by the teachers acting as teammates and using professional exchange as a strategy for consultation. Both teachers' knowledge is seen as central, and rather than the special education teacher acting as the expert and moderator, the teachers control the consultation together and jointly move the work along. The co-operative consultation enables the focus to move from the pupil toward the context, which can lead to the development of inclusive practices. The results indicate that this type of consultation holds potential in the development of special educational consultation that takes place between equal colleagues. The co-operative consultation opens up for a third collaborative approach to consultation, where aspects of the directive and non-directive perspective can merge and develop. The thesis concludes with the proposal that special pedagogical consultation can be understood from an integrated perspective. The characteristics of the consultation can vary depending on the type of problem or situation, while co-operative consultation can be seen as the ideal as equal colleagues meet in consultation conversations. In order to develop the co-operative consultation, both teachers are required to have knowledge of consultation as a practice, to be part of a collaborative school climate, and that teachers are provided with enough time to take part in consultations.

Species och artikelbruk i finskspråkiga grundskoleelevers inlärarsvenska. En longitudinell undersökning i årskurserna 7–9

Denna doktorsavhandling utreder hur finska grundskolelever använder de svenska substantivens bestämdhetsformer och artiklar och hur deras kunskaper utvecklas under årskurserna 7-9. Species och artikelbruk är problematiska för alla andraspråksinlärare i svenska, men de är synnerligen svåra för inlärare vars förstaspråk saknar morfologisk species. Det svenska systemet avviker också kraftigt från det motsvarande systemet i engelskan, varför tidigare kunskaper i engelska inte är till någon stor hjälp i inlärningen, låt vara att bestämdheten som begrepp redan är bekant för inläraren. Den teoretiska referensramen bygger på både grammatiska beskrivningar av den svenska grammatiken och på teorierna om grammatikinlärningen i andraspråk. Bland de sistnämnda är teorierna om tvärspråkligt inflytande, explicit respektive implicit inlärning samt helsekvensinlärning (på engelska formulaic language) av relevans. Undersökningsmaterialet består av korta texter samt inspelat muntligt material som med jämna mellanrum insamlats av finskspråkiga grundskolelever (n=67) som läser B-svenska. Undersökningen är i första hand kvantitativ, om än inmatningen av nominalfraserna i materialet samt deras formella och semantiska aspekter i analysprogrammet Microsoft Access också innebar en omfattande kvalitativ analys. Undersökningen bygger på performansanalysen och analysen av obligatoriska kontexter och beräkningen av frekvenser och korrekthetsprocent för de olika nominalfrastyperna. Informanterna använder komplext språk redan i årskurs 7. Korrekthetsprocenten stiger under undersökningstiden i de flesta frastyperna, men skillnaderna är sällan statistiskt signifikanta. Den normativa analysen visar också, att formfelen är i både det skriftliga och det muntliga materialet signifikant vanligare än speciesfelen. Det är med andra ord lättare för informanterna att välja rätt species än att bilda en korrekt nominalfras. I tidigare undersökningar i Sverige har likadana resultat nåtts. De mest centrala frastyperna i undersökningen bildar i båda typerna av materialet en inlärningsgång som upprepas i alla årskurser och kan förklaras med komplexitetsskillnaderna mellan de olika frastyperna. Informanterna behärskar bäst de frastyper, som varken innehåller artiklar eller ändelser. Näst bäst behärskar de substantivets bestämda form singularis och svagast obestämd form singularis, vars artikel är en klassisk svårighetskälla för finska svenskinlärare. Analysen av informanternas läromedel visar dock att den typiska undervisningsordningen i läromedlen inte motsvarar inlärningsgången som upptäckts i denna undersökning.

Sphingosine-1-phosphate-evoked invasion of follicular thyroid cancer cells : evidence for the involvement of HIF-Iα, MMP2 and MMP9

Sphingolipids are widely expressed molecules, which traditionally were considered to have majorly structural properties. Nowadays, however, they are implicated in a wide range of different biological processes. The bioactive lipid sphingosine 1-phosphate (S1P) has emerged during the past decade as one of the most studied molecules due to its proliferative and pro-migratory abilities both during normal physiology and in the pathology of a subset of different diseases. Migration and invasion of cancer cells require changes in cell behavior and modulation of the tissue microenvironment. Tumor aggressiveness is markedly enhanced by hypoxia, in which hypoxia inducible transcription factors 1-2α (HIF-1-2α) are activated to promote metabolism, proliferation and migration. Invasion requires degradation of the extracellular matrix (ECM) achieved by several degrading and remodeling enzymes. Matrix metalloproteinases (MMPs) are broadly expressed and well accepted as proteolytic enzymes with essential roles both in normal physiology and in pathology. Previously, S1P was shown to strongly evoke migration of follicular ML-1 thyroid cancer cells. The objective of this study was to further investigate and understand the mechanisms behind this regulation. In the first project it was demonstrated that S1P enhances the expression and activity of HIF-1α. S1P enhanced the expression of HIF-1α by increasing its synthesis and stability. The S1P-increased HIF-1α was mediated via S1P3, Gi/0, PI3K, PKCβI, ERK1/2, mTOR and translation factors p70S6K and eIF4E. Finally, it was shown that HIF-1α mediated S1P-induced migration. The ECM is constituted of a complex and coordinated assembly of many types of proteins. In order to be able to invade, cells need to break down the ECM, therefore several key players in this event were investigated in the second project. S1P increased the secretion and activity of MMP2 and MMP9 via S1P-receptor 1 and 3 and that these MMPs participated in the S1P-facilitated invasion of ML-1 cells. In this interplay, calpains and Rac1 were involved, both of which are crucial players in migration and invasion. The prognosis for some types of thyroid cancer is relatively good. However, there are forms of thyroid cancers, for which there are no treatments or the current available treatments are inefficient. Thus, new medical interventions are urgently needed. In the third project the significance of the S1P-receptor modulating drug FTY720, which is currently used for the treatment of multiple sclerosis (MS), was studied. The effect of FTY720 was tested on several thyroid cancer cell lines, and it inhibited the proliferation and invasion of all cancer cell lines tested. In ML-1 cells, FTY720 attenuated invasion by blocking signaling intermediates important for migration and invasion of the cells. Moreover, FTY720 inhibited the proliferation of ML-1 cells by increasing the expression of p21 and p27, hence, inducing cell arrest in G1 phase of the cell cycle. Thus, it can be suggested that FTY720 could be used in the treatment of thyroid cancer.