Telomere length dynamics in human lymphocyte subpopulations measured by flow cytometry.

To measure the average length of telomere repeats at chromosome ends in individual cells we developed a flow cytometry method using fluorescence in situ hybridization (flow FISH) with labeled peptide nucleic acid (PNA) probes. Results of flow FISH measurements correlated with results of conventional telomere length measurements by Southern blot analysis (R = 0.9). Consistent differences in telomere length in CD8+ T-cell subsets were identified. Naive and memory CD4+ T lymphocytes in normal adults differed by around 2.5 kb in telomere length, in agreement with known replicative shortening of telomeres in lymphocytes in vivo. T-cell clones grown in vitro showed stabilization of telomere length after an initial decline and rare clones capable of growing beyond 100 population doublings showed variable telomere length. These results show that flow FISH can be used to measure specific nucleotide repeat sequences in single cells and indicate that the very large replicative potential of lymphocytes is only indirectly related to telomere length.

The gene for the ligand binding chain of the human interferon gamma receptor.

In order to characterize the gene encoding the ligand binding (1(st); alpha) chain of the human IFN-gamma receptor, two overlapping cosmid clones were analyzed. The gene spans over 25 kilobases (kb) of the genomic DNA and has seven exons. The extracellular domain is encoded by exons 1 to 5 and by part of exon 6. The transmembrane region is also encoded by exon 6. Exon 7 encodes the intracellular domain and the 3' untranslated portion. The gene was located on chromosome 6q23.1, as determined by in situ hybridization. The 4 kb region upstream (5') of the gene was sequenced and analyzed for promoter activity. No consensus-matching TATA or CAAT boxes in the 5' region were found. Potential binding sites for Sp1, AP-1, AP-2, and CREB nuclear factors were identified. Compatible with the presence of the Sp1/AP-2 sites and the lack of TATA box, S1-nuclease mapping experiments showed multiple transcription initiation sites. Promoter activity of the 5' flanking region was analyzed with two different reporter genes: the Escherichia coli chloramphenicol acetyltransferase and human growth hormone. The smallest 5' region of the gene that still had full promoter activity was 692 base pairs in length. In addition, we found sequences belonging to the oldest family of Alu repeats, 2 - 3 kb upstream of the gene, which could be useful for genetic studies.

Supply chain management as a competitive advantage in the Spanish grocery sector

Adversarial relationships have long dominated business relationships,but Supply Chain Management (SCM) entails a new perspective. SCM requiresa movement away from arms-length relationships toward partnership stylerelations. SCM involves integration, co-ordination and collaborationacross organisations and throughout the supply chain. It means that SCMrequires internal (intraorganisational) and external (interorganisational)integration. This paper analyses the relationship between internal andexternal integration processes, their effect on firms performance andtheir contribution to the achievement of a competitive advantage.Performance improvements are analysed through costs, stock out and leadtime reductions. And, the achievement of a better competitive positionis measured by comparing the firm s performance with its competitors performance. To analyse this, an empirical study has been conducted inthe Spanish grocery sector.

Length of activity season drives geographic variation in body size of a widely distributed lizard

Understanding the factors that drive geographic variation in life history is an important challenge in evolutionary ecology. Here, we analyze what predicts geographic variation in life-history traits of the common lizard, Zootoca vivipara, which has the globally largest distribution range of all terrestrial reptile species. Variation in body size was predicted by differences in the length of activity season, while we found no effects of environmental temperature per se. Females experiencing relatively short activity season mature at a larger size and remain larger on average than females in populations with relatively long activity seasons. Interpopulation variation in fecundity was largely explained by mean body size of females and reproductive mode, with viviparous populations having larger clutch size than oviparous populations. Finally, body size-fecundity relationship differs between viviparous and oviparous populations, with relatively lower reproductive investment for a given body size in oviparous populations. While the phylogenetic signal was weak overall, the patterns of variation showed spatial effects, perhaps reflecting genetic divergence or geographic variation in additional biotic and abiotic factors. Our findings emphasize that time constraints imposed by the environment rather than ambient temperature play a major role in shaping life histories in the common lizard. This might be attributed to the fact that lizards can attain their preferred body temperature via behavioral thermoregulation across different thermal environments. Length of activity season, defining the maximum time available for lizards to maintain optimal performance, is thus the main environmental factor constraining growth rate and annual rates of mortality. Our results suggest that this factor may partly explain variation in the extent to which different taxa follow ecogeographic rules.

A natural structural variant of the mouse TCR beta-chain displays intrinsic receptor function and antigen specificity.

The Cbeta0 alternate cassette exon is located between the Jbeta1 and Cbeta1 genes in the mouse TCR beta-locus. In T cells with a VDJbeta1 rearrangement, the Cbeta0 exon may be included in TCRbeta transcripts (herein called TCRbeta-Cbeta0 transcripts), potentially inserting an additional 24 aa between the V and C domains of the TCR beta-chain. These TCRbeta splice isoforms may be differentially regulated after Ag activation, because we detected TCRbeta-Cbeta0 transcripts in a high proportion (>60%) of immature and mature T cells having VDJbeta1 rearrangements but found a substantially reduced frequency (<35%) of TCRbeta-Cbeta0 expression among CD8 T cells selected by Ag in vivo. To study the potential activity of the TCRbeta-Cbeta0 splice variant, we cloned full-length TCR cDNAs by single-cell RT-PCR into retroviral expression vectors. We found that the TCRbeta-Cbeta0 splice isoform can function during an early stage of T cell development normally dependent on TCR beta-chain expression. We also demonstrate that T hybridoma-derived cells expressing a TCRbeta-Cbeta0 isoform together with the clonally associated TCR alpha-chain recognize the same cognate peptide-MHC ligand as the corresponding normal alphabetaTCR. This maintenance of receptor function and specificity upon insertion of the Cbeta0 peptide cassette signifies a remarkable adaptability for the TCR beta-chain, and our findings open the possibility that this splice isoform may function in vivo.

Length Change of P. C. Concrete Due to Moisture Content, MLR-85-09, 1987

Portland cement concrete is an outstanding structural material but stresses and cracks often occur in large structures due to drying shrinkage. The objective of this research was to determine the change in length due to loss of moisture from placement through complete drying of portland cement concrete. The drying shrinkage was determined for four different combinations of Iowa DOT structural concrete mix proportions and materials. The two mix proportions used were an Iowa DOT D57 (bridge deck mix proportions) and a water reduced modified C4 mix. Three 4"x 4"x 18" beams were made for each mix. After moist curing for three days, all beams were maintained in laboratory dry air and the length and weight were measured at 73°F ± 3°F. The temperature was cycled on alternate days from 73°F to 90°F through four months. From four months through six months, the temperature was cycled one day at 73°F and six days at 130°F. It took approximately six months for the concrete to reach a dry condition with these temperatures. The total drying shrinkage for the four mixes varied from .0106 in. to .0133 in. with an average of .0120 in. The rate of shrinkage was approximately .014% shrinkage per 1% moisture loss for all four mixes. The rate and total shrinkage for all four mixes was very similar and did not seem to depend on the type of coarse aggregate or the use of a retarder.

Long sequence duplications, repeats, and palindromes in HIV-1 gp120: length variation in V4 as the product of misalignment mechanism.

We have shown that indels in gp120 V4 are associated to the presence of duplicated and palindromic sequences, suggesting that they may be produced by strand-slippage misalignment mechanism. Indels in V4 involved region-specific duplications 9 to 15 bp long, and repeats of various lengths, associated to trinucleotides AAT. No duplications were found in V3 and C3. The frequency of palindromic sequences in individual genes was found to be significantly higher in gp120 (p < or = 3.00E-7), and significantly lower in Tat (p < or = 9.00E-7) than the average frequency calculated over the full genome. The finding of elements of misalignment in association with indels in V4 suggests that these mutations may occur in proviral DNA after integration of HIV into the host genome. It also implies that occurrence of large indels in gp120 is not random but is directed by the presence and distribution of elements of misalignment in the HIV genome.

Length variation in HIV-1 gp120 as the product of DNA misalignment mechanism

Background: To determine whether misalignment structures such as duplications, repeats, and palindromes are associated to insertions/deletions (indels) in gp120, indicating that indels are indeed frameshift mutations generated by DNA misalignment mechanism. Methods: Cloning and sequencing of a fragment of HIV-1 gp120 spanning C2-C4 derived from plasma RNA in 12 patients with early chronic disease and naïve to antiretroviral therapy. Results: Indels in V4 involved always insertion and deletion of duplicated nucleotide segments, and AAT repeats, and were associated to the presence of palindromic sequences. No duplications were detected in V3 and C3. Palindromic sequences occurred with similar frequencies in V3, C3 and V4; the frequency of palindromes in individual genes was found to be significantly higher in structural (gp120, p ≤ 3.00E-7) and significantly lower in regulatory (Tat, p ≤ 9.00E-7) genes, as compared to the average frequency calculated over the full genome. Discussion: Indels in V4 are associated to misalignment structures (i.e. duplications repeat and palindromes) indicating DNA misalignment as the mechanism underlying length variation in V4. The finding that indels in V4 are caused by DNA misalignment has some very important implications: 1) indels in V4 are likely to occur in proviral DNA (and not in RNA), after integration of HIV into the host genome; 2) they are likely to occur as progressive modifications of the early founder virus during chronic infection, as more and more cells get infected; 3) frameshift mutations involving any number of base pairs are likely to occur evenly across gp120; however, only those mutants carrying a functional gp120 (indels as multiples of three base pairs) will be able to perpetuate the virus cycle and to keep spreading through the population.

Telomere length dynamics in normal individuals and in patients with hematopoietic stem cell-associated disorders.

The telomere length in nucleated peripheral blood (PB) cells indirectly reflects the mitotic history of their precursors: the hematopoietic stem cells (HSCs). The average length of telomeres in PB leukocytes can be measured using fluorescence in situ hybridization and flow cytometry (flow FISH). We previously used flow FISH to characterize the age-related turnover of HSCs in healthy individuals. In this review, we describe results of recent flow FISH studies in patients with selected hematopoietic stem cell-associated disorders: chronic myelogenous leukemia (CML) and several bone marrow failure syndromes. CML is characterized by a marked expansion of myeloid Philadelphia chromosome positive (Ph+) cells. Nevertheless, nonmalignant (Ph-) HSCs typically coexist in the bone marrow of CML patients. We analyzed the telomere length in > 150 peripheral blood leukocytes (PBLs) and bone marrow samples of patients with CML as well as samples of Ph- T-lymphocytes. Compared to normal controls, the overall telomere fluorescence in PBLs of patients with CML was significantly reduced. However, no telomere shortening was observed in Ph- T-lymphocytes. Patients in late chronic phase (CP) had significantly shorter telomeres than those assessed earlier in CP. Our data suggest that progressive telomere shortening is correlated with disease progression in CML. Within the group of patients with bone marrow failure syndromes, we only found significantly shortened telomeres (compared to age-adjusted controls) in granulocytes from patients with aplastic anemia (AA). Strikingly, the telomere length in granulocytes from AA patients who had recovered after immunosuppressive therapy (recAA) did not differ significantly from controls, whereas untreated patients and nonresponders with persistent severe pancytopenia (sAANR) showed marked and significant telomere shortening compared to healthy donors and patients with recAA. Furthermore, an inverse correlation between age-adjusted telomere length and peripheral blood counts was found in support of a model in which the degree of cytopenia and the amount of telomere shortening are correlated. These results support the concept of extensive proliferation of HSCs in subgroups of AA patients and suggest a potential use of telomere-length measurements as a prognostic tool in this group of disorders as well.

Expanding view of phenotype and oxidative stress in Friedreich's ataxia patients with and without idebenone

Summary: Friedreich's ataxia (FRDA), the most common autosomal recessive ataxia, is characterised by progressive ataxia with dysarthria of speech, loss of deep-tendon reflexes, impaired vibratory and proprioceptive sensations and corticospinal weakness with a Babinski's sign. Patients eventually also develop kyphoscoliosis, cardiomyopathy and diabetes mellitus. The disease is a GAA repeat disorder resulting in severely reduced levels of frataxin, with secondary increased sensitivity to oxidative stress. The anti-oxidative drug, idebenone, is effective against FRDA-associated cardiomyopathy. We provide detailed clinical, electrophysiological and biochemical data from 20 genetically confirmed FRDA patients and have analysed the relation-ship between phenotype, genotype and malondialdehyde (MDA), which is a marker of superoxide formation. We assessed the effects of idebenone biochemically by measuring blood M DA and clinically by serial measurements of the International Cooperative Ataxia Rating Scale (ICARS). The GAA repeat length influenced the age at onset (p <0.001), the severity of ataxia (p= 0.02), the presence of cardiomyopathy (p =0.04) and of low-frequency hearing loss (p = 0.009). Multilinear regression analysis showed (p = 0.006) that ICARS was dependent on the two variables of disease duration (p = 0.01) and size of the GAA expansion (p = 0.02). We found no correlation to bilateral palpebral ptosis visual impairment, diabetes mellitus or skeletal deformities, all of which appear to be signs of disease progression rather than severity. We discuss more thoroughly two underrecognised clinical findings: palpebral ptosis and GAA length-dependent low-frequency hearing loss. The average ICARS remained unchanged in 10 patients for whom follow-up on treatment was available (mean 2.9 years), whereas most patients treated with idebenone reported an improvement in dysarthria (63%), hand dexterity (.58%) and fatigue (47%) after taking the drug for several weeks or months. Oxidative stress analysis showed an unexpected increase in blood MDA levels in patients on idebenone (p = 0.04), and we discuss the putative underlying mechanism for this result, which could then explain the unique efficacy of idebenone in treating the FRDA-associated cardiomyopathy, as opposed to other antioxidative drugs. Indeed, idebenone is not only a powerful stimulator of complexes II and III of the respiratory chain, but also an inhibitor of complex I activity, then promoting superoxide formation. Our preliminary clinical observations are the first to date supporting an effect of idebenone in delaying neurological worsening. Our MDA results point to the dual effect of idebenone on oxidative stress and to the need for controlled studies to assess its potential toxicity at high doses on the one hand, and to revisit the exact mechanisms underlying the .physiopathology of Friedreich's ataxia on the other hand, while recent reports suggest non-oxidative pathophysiology of the disease.

Quantitative developmental response to the length of exposure to long photoperiod in wheat and barley

The present study was designed to analyse the effect of the length of exposure to a long photoperiod imposed c. 3 weeks after sowing in spring wheat (cv. UQ189) and barley (cv. Arapiles) to (i) establish whether the response to the number of cycles of exposure is quantitative or qualitative, (ii) determine the existence of a commitment to particular stages well before the stage has been observable, and (iii) study the interrelationships between the effects on final leaf number and phyllochron when the stimulus is provided several days after seedling emergence. Both wheat and barley seemed to respond quantitatively to the number of long-day cycles they were exposed to. However, wheat showed a requirement of approximately 4 long-day cycles to be able to produce a significant response in time to heading. The barley cultivar used in the study was responsive to the minimum length of exposure. The response to extended photoperiod cycles during the stem elongation phase was due to the ‘ memory’ photoperiod effects being related, in the case of wheat, to the fact that the pre-terminal spikelet appearance phase saturated its photoperiod response well before that stage was reached. Therefore, the commitment to the terminal spikelet appearance in wheat may be reached well before this stage could be recognized. As the response in duration to heading exceeded that of the final leaf number, and the stem elongation phase responded to memory effects of photoperiod, the phyllochron of both cereals was responsive to the treatments accelerating the average phyllochron when exposed to longer periods of long days. The response in average phyllochron was due to a switch from bi-linear to linear models of leaf number v. time when the conditions were increasingly inductive, with the phyllochron of the initial (6–8) leaves being similar for all treatments (within each species), and from then on increased.

Puupellettien tuotanto-, jakelu- ja käyttöketjun tekniikka, talous ja markkinat

Puupelletti on puumurskeesta puristamalla valmistettu polttoainejaloste, jonka raaka- aineita ovat mekaanisen metsäteollisuuden sivutuotteet kutterinlastu ja sahanpuru. Sylinterin muotoiset pelletit ovat yleensä halkaisijaltaan 6-10 mm ja pituudeltaan 10 - 30 mm. Tässä diplomityössä tarkastellaan pellettiketjua valmistuksesta loppukäyttöön. Työ sisältää valmistuksen, kuljetuksen ja käytön niin tekniset puolet, kuin taloudelliset näkökohdatkin. Myös muiden maiden pellettimarkkinoiden tilanne tuodaan esille. Työn lopussa kuvataan muutama case-esimerkki onnistuneista pellettiketjuista. Suomessa puupellettimarkkinoiden kehitys alkoi 1998 Vöyrissä auenneen pellettitehtaan myötä. Pellettien tuotanto ja kulutus ovat sen jälkeen kasvaneet nopeasti ja nyt vuonna 2001 pellettien tuotantokapasiteetti on 150 000 t/a. Pellettimarkkinoiden nopea kehitys Suomessa perustuu öljyn korkeaan hintaan, käytön helppouteen ja ympäristöystävällisyyteen. Käyttölaitteistojen yleistyessä ja jakelun tehostuessa pellettiliiketoiminta tulee vahvistamaan asemaansa entisestään Suomessa

Working capital management in the value chain of automotive industry

The objective of this Master’s thesis is to examine working capital management in the automotive industry in years 2006-2008. The study is conducted by the analysis of financial statements. The sample consists of 65 companies that represent different stages in the value chain of automotive industry beginning from raw material suppliers and ending to car dealers. Working capital management is studied by the cash conversion cycle (CCC). The results show that the average CCC of the value chain is 67 days. Car manufacturers had the longest CCC, 106 days, whereas the CCC of oil companies was the shortest, 22 days. The findings suggest that the cycle time of working capital usually follows the cycle time of inventories, since the changes in cycle times of accounts receivable and payable compensate each other. Improvements in working capital management could be achieved by sharing more accurate information in the chain for example about inventory levels and order points of customer. It could also be discussed within the automotive industry, if the long credit periods, which tie up working capital, are really needed. New technologies enable faster payments, which would reduce the cash conversion cycles, improve the profitability of companies, and increase the competitiveness of the value chain. Working capital should not be reduced at the expense of value chain partners, because nowadays the competition is rather between the value chains than between the companies. Similar research design is applied earlier to study working capital management in the value chain of pulp and paper industry. Even if the industries and the structures of the chains differ from each other, results were surprisingly similar. In future research, working capital management in other industries’ value chains could still be studied and compared to previous studies. ICT industry, for example, could be an interesting object.

Käyttöpääoman sitoutuminen ja kiertoajat lääkejakeluketjussa

Työn tavoitteenaan oli selvittää suomalaisen lääkejakeluketjun rakenne sekä saada selkeä kuva ketjun toiminnan tehokkuudesta käyttöpääoman sitoutumisen ja kiertoaikojen osalta. Työn alussa käydään läpi teoriaa ja tutkimusta käyttöpääomasta ja sen erien kiertoajoista sekä erityisesti niiden huomioimisesta arvoketjussa. Lisäksi esitellään Suomen lääkemarkkinoiden toimintaa sekä sen toimijoita. Työn empiirinen osa suoritettiin kahdessa päävaiheessa. Ensin analysoitiin Voitto+ -ohjelmasta saatuja valittujen lääkejakeluketjun yritysten julkisia tilinpäätöstietoja ajalta 2005- 2011. Analysointi perustui käyttöpääoman sekä sen erien myyntisaamisten, ostovelkojen ja vaihto-omaisuuden kiertoaikojen laskemiseen. Ketjun yrityksiä vertailtiin toisiinsa ketjun osien keskimääräisten kiertoaikojen osalta ja saatiin selville niiden suhteellinen sijoittuminen toisiinsa nähden. Työn toisessa päävaiheessa haastateltiin esimerkkitoimijaa käsitellyn lääkejakeluketjun jokaisesta osasta: lääketehdas, tukkujakelija ja apteekki. Työssä huomattiin, että käyttöpääoman kiertoaikaan lääkejakeluketjussa vaikuttaa ensisijaisesti ketjun osan toiminnan luonne ja käyttöpääoman hallinnan tehokkuus lääkejakeluketjun toimijoiden välillä vaihtelee osaamisen ja resurssien mukaan. Ketjun eri osissa painottuvat eri käyttöpääoman erät ja ketjun toiminta on optimoitunut aikojen saatossa hyvin stabiiliin tilaan, jota ketjun jäsenillä ei näytä olevan halua muuttaa. Lääkejakeluketjun käyttöpääomaan hallintaa voitaisiin tehostaa varastonhallintaa parantamalla sekä informaation jakamisella saattamalla kysyntätiedot nopeammin koko ketjun tietoon.

Supply chain performance management system

The purpose of the study is to determine general features of the supply chain performance management system, to assess the current state of performance management in the case company mills and to make proposals for improvement – how the future state of performance management system would look like. The study covers four phases which consist of theory and case company parts. Theoretical review gives understanding about performance management and measurement. Current state analysis assesses the current state of performance management in the mills. Results and proposals for improvement are derived from current state analysis and finally the conclusions with answers to research questions are presented. Supply chain performance management system consists of five areas: perfor-mance measurement and metrics, action plans, performance tracking, performance dialogue and rewards, consequences and actions. The result of the study revealed that all mills were quite average level in performance management and there is a room for improvement. Created performance improvement matrix served as a tool in assessing current performance management and could work also as a tool in the future in mapping the current state after transformation process. Limited harmonization was revealed as there were different ways to work and manage performance in the mills. Lots of good ideas existed though actions are needed to make a progress. There is also need to harmonize KPI structure.