Binding of double-strand breaks in DNA by human Rad52 protein.

Double-strand breaks (DSBs) in DNA are caused by ionizing radiation. These chromosomal breaks can kill the cell unless repaired efficiently, and inefficient or inappropriate repair can lead to mutation, gene translocation and cancer. Two proteins that participate in the repair of DSBs are Rad52 and Ku: in lower eukaryotes such as yeast, DSBs are repaired by Rad52-dependent homologous recombination, whereas vertebrates repair DSBs primarily by Ku-dependent non-homologous end-joining. The contribution of homologous recombination to vertebrate DSB repair, however, is important. Biochemical studies indicate that Ku binds to DNA ends and facilitates end-joining. Here we show that human Rad52, like Ku, binds directly to DSBs, protects them from exonuclease attack and facilitates end-to-end interactions. A model for repair is proposed in which either Ku or Rad52 binds the DSB. Ku directs DSBs into the non-homologous end-joining repair pathway, whereas Rad52 initiates repair by homologous recombination. Ku and Rad52, therefore, direct entry into alternative pathways for the repair of DNA breaks.

The protective role of transferrin in Müller glial cells after iron-induced toxicity.

PURPOSE: Transferrin (Tf) expression is enhanced by aging and inflammation in humans. We investigated the role of transferrin in glial protection. METHODS: We generated transgenic mice (Tg) carrying the complete human transferrin gene on a C57Bl/6J genetic background. We studied human (hTf) and mouse (mTf) transferrin localization in Tg and wild-type (WT) C57Bl/6J mice using immunochemistry with specific antibodies. Müller glial (MG) cells were cultured from explants and characterized using cellular retinaldehyde binding protein (CRALBP) and vimentin antibodies. They were further subcultured for study. We incubated cells with FeCl(3)-nitrilotriacetate to test for the iron-induced stress response; viability was determined by direct counting and measurement of lactate dehydrogenase (LDH) activity. Tf expression was determined by reverse transcriptase-quantitative PCR with human- or mouse-specific probes. hTf and mTf in the medium were assayed by ELISA or radioimmunoassay (RIA), respectively. RESULTS: mTf was mainly localized in retinal pigment epithelium and ganglion cell layers in retina sections of both mouse lines. hTf was abundant in MG cells. The distribution of mTf and hTf mRNA was consistent with these findings. mTf and hTf were secreted into the medium of MG cell primary cultures. Cells from Tg mice secreted hTf at a particularly high level. However, both WT and Tg cell cultures lose their ability to secrete Tf after a few passages. Tg MG cells secreting hTf were more resistant to iron-induced stress toxicity than those no longer secreted hTf. Similarly, exogenous human apo-Tf, but not human holo-Tf, conferred resistance to iron-induced stress on MG cells from WT mice. CONCLUSIONS: hTf localization in MG cells from Tg mice was reminiscent of that reported for aged human retina and age-related macular degeneration, both conditions associated with iron deposition. The role of hTf in protection against toxicity in Tg MG cells probably involves an adaptive mechanism developed in neural retina to control iron-induced stress.

Example of Monte Carlo uncertainty assessment in the field of radionuclide metrology

This chapter presents possible uses and examples of Monte Carlo methods for the evaluation of uncertainties in the field of radionuclide metrology. The method is already well documented in GUM supplement 1, but here we present a more restrictive approach, where the quantities of interest calculated by the Monte Carlo method are estimators of the expectation and standard deviation of the measurand, and the Monte Carlo method is used to propagate the uncertainties of the input parameters through the measurement model. This approach is illustrated by an example of the activity calibration of a 103Pd source by liquid scintillation counting and the calculation of a linear regression on experimental data points. An electronic supplement presents some algorithms which may be used to generate random numbers with various statistical distributions, for the implementation of this Monte Carlo calculation method.

Epäpuhtauksien optinen laskenta sulpusta

Työn tarkoituksena oli hakea mittausjärjestelmän raja-arvoja optiselle kamerapohjaiselle roskalaskentajärjestelmälle sekä testata roskalaskentajärjestelmän toimivuus käytännössä. Tavoitteena oli tuotteistaa kamerapohjainen roskalaskenta-analyysi palvelutuotteeksi, jota voitaisiin hyödyntää sihtien kuntokartoituksessa ja ongelmanratkaisuvälineenä. Teoriaosa koostui kahdesta kokonaisuudesta: sulpun epäpuhtauksista, roskalaskennan teoriasta ja epäpuhtauksien mittausmenetelmistä sekä markkinoinnista, tuotteistamis- ja lanseerausprosessista palvelutuotteen näkökulmasta. Kokeellisessa osassa selvitettiin kamerapohjaiseen roskalaskentaanalyysiin vaikuttavia tekijöitä: kameran tarkennus, kuvan terävyys, analysoitavan arkin väri, neliömassa ja roskapitoisuus, impregnointi, valonlähde, kuvan muokkaus, tiedostomuoto ja pikselimäärä. Kamerapohjaisen roskalaskenta-analyysin soveltuvuus käytäntöön testattiin tehdasesimerkin avulla. Havaittiin, että kamerapohjaista roskalaskenta-analyysiä voitaisiin käyttää lähes kaikille massatyypeille. Työssä määriteltiin kalibrointimenetelmä kameran tarkentamiseksi arkin tasoon sekä suljinnopeusanalyysi massatyypistä riippuvan suljinnopeuden selvitykseen. Kamerapohjaisessa roskalaskenta-analyysissä määritettiin käytettäväksi arkin neliömassana 60 g/m2, suljinaukkoa F5 ja terävyysasetusta 5. Tulokseksi saatiin, että analysoitavia arkkeja ei tarvitse impregnoida tai jälkikäsitellä. Korrelaatiota Somerville-erotustehokkuuteen ei löytynyt. Esimerkkitehtaasta selvitettiin primääriportaan roskapitoisuudet ja erotustehokkuudet. Tehdasesimerkin tulosten perusteella havaittiin happivaiheen ja D0-vaiheen olleen tehokkaimpia epäpuhtauksien poistajia.

Tratamento de águas de refrigeração com peróxido de hidrogênio

Hydrogen peroxide and chlorine are compared as possible disinfectants for water-cooling circuits. To this purpose, samples taken from the cooling system of a steel making plant were treated (at 25ºC and pH values of 5.5 and 8.5) with varying amounts of the two oxidizing agents (0.0 mg/L, 2.0 mg/L and 6.0 mg/L). The results were evaluated through bacterial counting and measurement of corrosion rates upon AISI1020 carbon steel coupons. Bacterial removal and corrosion effects proved to be similar and satisfactory for both reagents.

Hankintaprosessin arviointi ja kehittäminen Case: NCC Rakennus Oy

Tämän tutkimuksen tarkoituksena on löytää keinoja NCC Rakennus Oy:n hankinta- ja tilausprosessien tehostamiseen. Tutkimuksen kohteena ovat projektikohtaisten hankintojen hankintaprosessi, kausisopimuksiin perustuvat tilausprosessit sekä hankintakäytännöt. Tutkimus on toteutettu laadullisena tapaustutkimuksena ja pääasiallisena aineistonkeruumenetelmänä on käytetty teemahaastatteluita. Teemahaastattelujen avulla on pyritty selvittämään kuinka ihmiset ja järjestelmät toimivat prosesseissa ja tunnistamaan eri prosessivaiheiden mahdollisia ongelmakohtia. Mahdolliset ongelmakohdat ja niiden kehitysehdotukset on tässä tutkimuksessa jäsennetty vika- ja vaikutusanalyysin avulla. Saatujen tulosten mukaan hankinta- ja tilausprosessien tehostaminen vaatii muun muassa yhteisten toimintatapojen rakentamista, koulutusta ja yhteistyön kehittämistä eri sidosryhmien kanssa.

Sähköisten hankintamenettelyjen taloudelliset yritysvaikutukset

Pro gradu -tutkielmassa tutkitaan julkisissa hankinnoissa käyttöön otettavien uusien sähköisten menettelyjen, sähköisen huutokaupan ja dynaamisen hankintajärjestelmän, yritysvaikutuksia. Ydintutkimustehtävänä on tutkia, millaisia taloudellisia vaikutuksia sähköisillä menettelyillä tulee olemaan yrityksiin niiden tarjotessa palveluita tai tavaroita julkiselle sektorille ja mitkä ovat sähköisten menettelyjen käytön motiivit. Tutkielman teoriaosa on pohja empiiriselle tutkimukselle, jossa aineistona on käytetty suppeaa kyselytutkimusta, haastatteluja ja lisäksi materiaalia on tuotettu analysoimalla hallinnollisten kustannusten määrää sähköisissä hankintamenettelyissä. Yritysvaikutusten arvioinnissa tarkasteltiin sähköisten hankintamenettelyjen käytön motiiveja. Motiiveiksi tunnistettiin tehokkuuden tavoittelu, kustannussäästöt, ajankäytön ja henkilöresurssien käytön tehostaminen sekä avoimuus. Tutkimuksessa onnistuttiin todentamaan edellä mainittuja tekijöitä taloudellisilla malleilla. Määrälliset ja laadulliset tutkimustulokset osoittavat kokonaisuudessaan tuloksellisella tavalla sähköisten hankintamenettelyjen käytön motiiveita ja vaikutuksia yrityksiin. Sähköisillä hankintamenettelyillä voidaan perustellusti tehostaa hankintaprosessia ja saavuttaa kustannussäästöjä. Monia yleisiä yritysten julkisiin hankintoihin pääsyn esteinä pitämiä seikkoja voitaisiin vähentää tai poistaa käyttämällä sähköisiä hankintoja. Sähköistä huutokauppaa ja dynaamista hankintajärjestelmää koskevan lainsäädännön luodessa puitteet menettelyiden käytölle, tulee menettelyjen käyttöönoton motiivien ja kannustimien riittävyyttä seurata. Hallinnollisten kustannusten väheneminen ja muiden esteiden pieneneminen ei välttämättä synnytä pysyvää kannustinvaikutusta, vaan hankintaprosessia tulee pyrkiä sähköistämään kokonaisvaltaisesti myös muissa hankintalain mukaisissa hankintamenettelyissä ja kilpailuttamisen ulkopuolisissa hankinnan osa-alueissa kuten laskuttaminen, maksatus ja tilaus.

Pharmacological immunomodulation enhances peripheral nerve regeneration

To assess the effect of N-Acetylmuramyl-L-Alanyl-D-Isoglutamine MDP topically administrated on the regenerating peripheral neurons, twelve male C57BL/6J adult mice were equally distributed into three groups. Four mice underwent unilateral sciatic nerve transection and polyethylene tubulization, with a 4mm gap between the proximal and distal nerve stumps and were implanted with collagen + PBS (COL). Other four animals underwent the same surgical procedure but received collagen + MDP (COL/MDP) inside the prosthesis. Four animals were not operated and served as control group (NOR). After 4 weeks, the regenerated nerve cables were processed for total myelinated axon counting and myelinated fiber diameter measurement. The L5 dorsal root ganglion (DRG) was also removed and sectioned for sensory neurons counting and measurement. The results revealed significant difference (p<0.05) in axonal counting among the groups NOR (4,355±32), COL (1,869±289) and COL/MDP (2,430±223). There was a significant reduction in the axonal diameter in the operated groups (COL=3.38µm±1.16 and COL/MDP=3.54µm±1.16) compared to NOR (6.19µm±2.45). No difference was found in the number of DRG neurons between the experimental groups (COL=564±51; COL/MDP=514±56), which presented fewer sensory neurons compared to NOR (1,097±142). Data obtained indicate that locally applied MDP stimulates peripheral nerve regeneration in mice.

Sähköisen taloushallinnon kehitys ja ilmiön näkyvyys alan ammattilehtikirjoittelussa

Pro gradu –tutkielman ensisijaisena tavoitteena on tutkia sähköisen ta-loushallinnon kehitystä ja miten se on näkynyt alan suomalaisessa ja kansainvälisessä ammattilehtikirjoittelussa vuosina 1997 – 2013. Tarkoi-tuksena on löytää kirjoittelun perusteella sähköisen taloushallinnon kehi-tyksen edistäviä ja hidastavia tekijöitä sekä minkälaisia tulevaisuuden näkymiä artikkelit luovat sähköiselle taloushallinnolle. Lisäksi tavoitteena on löytää yhtäläisyyksiä ja eroja kansallisen ja kansainvälisen kirjoittelun välillä. Tutkielma on laadullinen tutkimus ja tutkimusmenetelminä käytetään si-sällönanalyysia, sisällönerittelyä, teemoittelua ja vertailevaa tutkimusta. Tutkielman empiirinen aineisto koostuu Tilisanomien, Balanssi–lehden ja Accountancy–lehden sähköistä taloushallintoa koskevista artikkeleista aikavälillä 1997 – 2013. Tutkimustulosten perusteella sähköisen taloushallinnon kehityskulku näkyy myös ammattilehtikirjoittelussa. Tietojärjestelmät ja niiden käytön mahdollistava lainsäädäntö ovat sähköisen taloushallinnon perusedelly-tykset. Suurimpia kehityksen edistäjiä ovat julkinen valta, verkkolaskut ja standardointi. Kehityksen hidastajina nähdään yhtenäisten standardien puute ja asenteet. Sähköisen taloushallinnon tulevaisuuden näkymiä ovat standardoidut prosessit ja käsitteet koko taloushallinnon alueella sekä viranomaisraportointi XBRL-kielen avulla. Suurimmat erot kansalli-sessa ja kansainvälisessä kehityksessä on havaittavissa verkkolaskuissa ja XBRL-raportoinnissa. Johtopäätöksenä todetaan että kirjoittelun perusteella voidaan löytää sähköisen taloushallinnon kehitystä hidastavia ja edistäviä tekijöitä ja se luo viitteitä tulevaisuuden kehitykselle. Sähköinen taloushallinto kehittyy kunkin maan valtiovallan tahtotilan ja lainsäädännön mukaan. Jatkotut-kimuskohteena tutkielmaa voisi laajentaa kansainvälisemmäksi ottamalla mukaan tarkasteluun useampia kansainvälisiä lehtiä ja kansainvälistä lainsäädäntöä.

Spontaneous Focusing on Numerosity in the Development of Early Mathematical Skills

The aim of the present set of longitudinal studies was to explore 3-7-year-old children.s Spontaneous FOcusing on Numerosity (SFON) and its relation to early mathematical development. The specific goals were to capture in method and theory the distinct process by which children focus on numerosity as a part of their activities involving exact number recognition, and individual differences in this process that may be informative in the development of more complex number skills. Over the course of conducting the five studies, fifteen novel tasks were progressively developed for the SFON assessments. In the tasks, confounding effects of insufficient number recognition, verbal comprehension, other procedural skills as well as working memory capacity were aimed to be controlled. Furthermore, how children.s individual differences in SFON are related to their development of number sequence, subitizing-based enumeration, object counting and basic arithmetic skills was explored. The effect of social interaction on SFON was tested. Study I captured the first phase of the 3-year longitudinal study with 39 children. It was investigated whether there were differences in 3-year-old children.s tendency to focus on numerosity, and whether these differences were related to the children.s development of cardinality recognition skills from the age of 3 to 4 years. It was found that the two groups of children formed on the basis of their amount of SFON tendency at the age of 3 years differed in their development of recognising and producing small numbers. The children whose SFON tendency was very predominant developed faster in cardinality related skills from the age of 3 to 4 years than the children whose SFON tendency was not as predominant. Thus, children.s development in cardinality recognition skills is related to their SFON tendency. Studies II and III were conducted to investigate, firstly, children.s individual differences in SFON, and, secondly, whether children.s SFON is related to their counting development. Altogether nine tasks were designed for the assessments of spontaneous and guided focusing on numerosity. The longitudinal data of 39 children in Study II from the age of 3.5 to 6 years showed individual differences in SFON at the ages of 4, 5 and 6 years, as well as stability in children.s SFON across tasks used at different ages. The counting skills were assessed at the ages of 3.5, 5 and 6 years. Path analyses indicated a reciprocal tendency in the relationship between SFON and counting development. In Study III, these results on the individual differences in SFON tendency, the stability of SFON across different tasks and the relationship of SFON and mathematical skills were confirmed by a larger-scale cross-sectional study of 183 on average 6.5-year-old children (range 6;0-7;0 years). The significant amount of unique variance that SFON accounted for number sequence elaboration, object counting and basic arithmetic skills stayed statistically significant (partial correlations varying from .27 to .37) when the effects of non-verbal IQ and verbal comprehension were controlled. In addition, to confirm that the SFON tasks assess SFON tendency independently from enumeration skills, guided focusing tasks were used for children who had failed in SFON tasks. It was explored whether these children were able to proceed in similar tasks to SFON tasks once they were guided to focus on number. The results showed that these children.s poor performance in the SFON tasks was not caused by their deficiency in executing the tasks but on lacking focusing on numerosity. The longitudinal Study IV of 39 children aimed at increasing the knowledge of associations between children.s long-term SFON tendency, subitizing-based enumeration and verbal counting skills. Children were tested twice at the age of 4-5 years on their SFON, and once at the age of 5 on their subitizing-based enumeration, number sequence production, as well as on their skills for counting of objects. Results showed considerable stability in SFON tendency measured at different ages, and that there is a positive direct association between SFON and number sequence production. The association between SFON and object counting skills was significantly mediated by subitizing-based enumeration. These results indicate that the associations between the child.s SFON and sub-skills of verbal counting may differ on the basis of how significant a role understanding the cardinal meanings of number words plays in learning these skills. The specific goal of Study V was to investigate whether it is possible to enhance 3-year old children.s SFON tendency, and thus start children.s deliberate practice in early mathematical skills. Participants were 3-year-old children in Finnish day care. The SFON scores and cardinality-related skills of the experimental group of 17 children were compared to the corresponding results of the 17 children in the control group. The results show an experimental effect on SFON tendency and subsequent development in cardinality-related skills during the 6-month period from pretest to delayed posttest in the children with some initial SFON tendency in the experimental group. Social interaction has an effect on children.s SFON tendency. The results of the five studies assert that within a child.s existing mathematical competence, it is possible to distinguish a separate process, which refers to the child.s tendency to spontaneously focus on numerosity. Moreover, there are significant individual differences in children.s SFON at the age of 3-7 years. Moderate stability was found in this tendency across different tasks assessed both at the same and at different ages. Furthermore, SFON tendency is related to the development of early mathematical skills. Educational implications of the findings emphasise, first, the importance of regarding focusing on numerosity as a separate, essential process in the assessments of young children.s mathematical skills. Second, the substantial individual differences in SFON tendency during the childhood years suggest that uncovering and modeling this kind of mathematically meaningful perceiving of the surroundings and tasks could be an efficient tool for promoting young children.s mathematical development, and thus prevent later failures in learning mathematical skills. It is proposed to consider focusing on numerosity as one potential sub-process of activities involving exact number recognition in future studies.

Matematiskt gestaltande i förskolan

The purpose of the thesis is to study how mathematics is experienced and used in preschool children’s activities and how preschool teachers frame their teaching of mathematical content. The studies include analyses of children’s actions in different activities from a mathematical perspective and preschool teachers’ intentions with and their teaching of mathematics. Preschool teachers’ understanding of the knowledge required in this area is also scrutinised. The theoretical points of departure are variation theory and sociocultural theory. With variation theory the focus is directed towards how mathematical content is dealt with in teaching situations where preschool teachers have chosen the learning objects. The sociocultural perspective has been chosen because children’s mathematical learning in play often takes place in interactions with others and in the encounter with culturally mediated concepts. The theoretical framework also includes didactical points of departure. The study is qualitative, with videography and phenomenography as metholological research approaches. In the study, video observations and interviews with preschool teachers have been used as data collection methods. The results show that in children’s play mathematics consists of volume, geometrical shapes, gravity, quantity and positioning. The situations also include size, patterns, proportions, counting and the creation of pairs. The preschool teachers’ intentions, planning and staging of their goal-oriented work are that all children should be given the opportunity to discern a mathematical content. This also includes making learning objects visible in here-and-now-situations. Variation and a clear focus on the mathematical content are important in this context. One of the study’s knowledge contributions concerns the didactics of mathematics in the preschool. This relates to the teaching of mathematics and includes the knowledge that preschool teachers regard as essential for their teaching. This includes theoretical and practical knowledge about children and children’s learning and didactical issues and strategies. The conclusion is that preschool teachers need to have a basic knowledge of mathematics and the didactics of mathematics.

A role for histone-like protein H1 (H-NS) in the regulation of hemolysin expression by Serratia marcescens

The histone-like protein H1 (H-NS) is an abundant structural component of the bacterial nucleoid and influences many cellular processes including recombination, transcription and transposition. Mutations in the hns gene encoding H-NS are highly pleiotropic, affecting the expression of many unrelated genes. We have studied the role of H-NS on the regulation of hemolysin gene expression in Serratia marcescens. The Escherichia coli hns mutant carrying S. marcescens hemolysin genes on a plasmid constructed by ligation of the 3.2-kb HindIII-SacI fragment of pR02 into pBluescriptIIKS, showed a high level of expression of this hemolytic factor. To determine the osmoregulation of wild-type and hns defective mutants the cells were grown to mid-logarithmic phase in LB medium with 0.06 or 0.3 M NaCl containing ampicillin and kanamycin, whereas to analyze the effect of pH on hemolysin expression, the cells were grown to late-logarithmic phase in LB medium buffered with 0.1 M Tris-HCl, pH 4.5 to 8.0. To assay growth phase-related hemolysin production, bacterial cells were grown in LB medium supplemented with ampicillin and kanamycin. The expression of S. marcescens hemolysin genes in wild-type E. coli and in an hns-defective derivative at different pH and during different growth phases indicated that, in the absence of H-NS, the expression of hemolysin did not vary with pH changes or growth phases. Furthermore, the data suggest that H-NS may play an important role in the regulation of hemolysin expression in S. marcescens and its effect may be due to changes in DNA topology influencing transcription and thus the amount of hemolysin expression. Implications for the mechanism by which H-NS influences gene expression are discussed.

How auditory temporal processing deficits relate to dyslexia

Studies have shown that dyslexic children present a deficiency in the temporal processing of auditory stimuli applied in rapid succession. However, discussion continues concerning the way this deficiency can be influenced by temporal variables of auditory processing tests. Therefore, the purpose of the present study was to analyze by auditory temporal processing tests the effect of temporal variables such as interstimulus intervals, stimulus duration and type of task on dyslexic children compared to a control group. Of the 60 children evaluated, 33 were dyslexic (mean age = 10.5 years) and 27 were normal controls (mean age = 10.8 years). Auditory processing tests assess the abilities of discrimination and ordering of stimuli in relation to their duration and frequency. Results showed a significant difference in the average accuracy of control and dyslexic groups considering each variable (interstimulus intervals: 47.9 ± 5.5 vs 37.18 ± 6.0; stimulus duration: 61.4 ± 7.6 vs 50.9 ± 9.0; type of task: 59.9 ± 7.9 vs 46.5 ± 9.0) and the dyslexic group demonstrated significantly lower performance in all situations. Moreover, there was an interactive effect between the group and the duration of stimulus variables for the frequency-pattern tests, with the dyslexic group demonstrating significantly lower results for short durations (53.4 ± 8.2 vs 48.4 ± 11.1), as opposed to no difference in performance for the control group (62.2 ± 7.1 vs 60.6 ± 7.9). These results support the hypothesis that associates dyslexia with auditory temporal processing, identifying the stimulus-duration variable as the only one that unequally influenced the performance of the two groups.

Can the fractal dimension be applied for the early diagnosis of non-proliferative diabetic retinopathy?

The fractal dimension has been employed as a useful parameter in the diagnosis of retinal disease. Avakian et al. (Curr Eye Res 2002; 24: 274-280), comparing the vascular pattern of normal patients with mild to moderate non-proliferative diabetic retinopathy (NPDR), found a significant difference between them only in the macular region. This significant difference in the box-counting fractal dimension of the macular region between normal and mild NPDR patients has been proposed as a method of precocious diagnosis of NPDR. The aim of the present study was to determine if fractal dimensions can really be used as a parameter for the early diagnosis of NPDR. Box-counting and information fractal dimensions were used to parameterize the vascular pattern of the human retina. The two methods were applied to the whole retina and to nine anatomical regions of the retina in 5 individuals with mild NPDR and in 28 diabetic but opthalmically normal individuals (controls), with age between 31 and 86 years. All images of retina were obtained from the Digital Retinal Images for Vessel Extraction (DRIVE) database. The results showed that the fractal dimension parameter was not sensitive enough to be of use for an early diagnosis of NPDR.

DNA polymerase activity in trophoblast giant cells in the mouse /

In the developing mouse embryo, the diploid trophectoderm is known to undergo a diploid to giant cell transformation. These cells arise by a process of endoreduplication, characterized by replication of the entire genome without subsequent mitosis or cell division, leading to polyploidy and the formation of giant nuclei. Studies of 13.5 day rat trophoblast derived from the parietal yolk sac have indicated a relatively low rate of DNA polymerase a activity, the noinnal eukaryotic replicase, in comparison to that of DNA polymerase g. These results have suggested that endoreduplication in trophoblast giant cells may not employ the normal replicase enzyme, DNA polymerase a. In order to determine whether a 'switch' from DNA polymerase to DNA polymerase is a necessary concomitant of the diploid to giant cell transformation, two distinct populations of trophoblast giant cells, the primary giant cell derived from the mural trophectoderm and the secondary giant cell derived from the polar trophoectoderm were used. These two populations of trophoblast giant cells can be obtained from the tissue outgrowths of 3.5da blastocysts and the extraembryonic ectoderm (EX) and ectoplacental cone (EPC) of 7.5 day embryos respectively. Tissue outgrowths were treated with aphidicolin, a specific reversible inhibitor of eukaryotic DNA polymerase a, on various days after explantation. The effect of aphidicolin treatment was assessed both qualitatively, using autoradiography and quantitatively by scintillation counting and Feulgen staining. 3 DNA synthesis was measured in control and treated cultures after a Hthymidine pulse. Scintillation counts of the embryo proper revealed that DNA synthesis was consistently inhibited by greater than 907. in the presence of aphidicolin. Inhibition of DNA synthesis in the EX and EPC varied between 81-957. and 82-987. respectively, indicating that most DNA synthesis was mediated by DNA polymerase a, but that a small but significant amount of residual synthesis was indicated. A qualitative approach was then applied to determine whether the apparent residual DNA synthesis was restricted to a subpopulation of giant cells or whether all giant cells displayed a low level of DNA synthesis. Autoradiographs of the ICM of blastocysts and the embryo proper of 7.5da embryos, which acted as diploid control population, was completely inhibited regardless of duration in explant culture. In contrast, primary trophoblast giant cells derived from blastocysts and secondary giant cells derived from the EX and EPC were observed to possess some heavily labelled cells after aphidicolin treatment. These results suggest that although DNA polymerase a is the primary replicating enzyme responsible for endoreduplication in mouse trophoblast giant cells, some nonactivity is also observed. A DNA polymerase assay employing tissue lysates of outgrown 7.5da embryo, EX and EPC tissues was used to attempt to confirm the presence of higher nonactivity in tissues possessing trophoblast giant cells. Employing a series of inhibitors of DNA polymerases, it would appear that DNA polymerase a is the major polymerase active in all tissues of the 7.5da mouse embryo. The nature of the putative residual DNA synthetic activity could not be unequivically determined in this study. Therefore, these results suggest that both primary and secondary trophoblast giant cells possess and use DNA polymerase a in endoreduplicative DNA synthesis. It would appear that the high levels of DNA polymerase g activity reported in trophoblast tissue derived from the 13.5 da rat yolk sac was not a general feature of all endoreduplication.