Table of Contents

Volume 7, Issue 1

  January 1, 2008

  Pages 3 - 121

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 Issue Catalogue (MARC XML)

EXTRA VIEWS

Open Access Article

DNA damage signaling in early Xenopus embryos

Aimin Peng, Andrea L. Lewellyn and James L. Maller
Pages 3 - 6
http://dx.doi.org/10.4161/cc.7.1.5157
Abstract | PDF

In Xenopus embryos, the first 12 cell division cycles before the midblastula transition (MBT) feature rapid and synchronous alternations between DNA replication and mitosis. Moreover, embryos at this stage lack checkpoints that halt the cell cycle in response to DNA damage, whereas introduction of a threshold amount of undamaged plasmid or sperm DNA allows a DNA damage checkpoint response to be activated. In a search for the underlying mechanism, we recently discovered that the checkpoint signal initiated by damaged DNA is enhanced by the undamaged threshold DNA. Thus developmental regulation of the cell cycle checkpoint is achieved at the 12th cleavage divisions by the maternally programmed increase in the DNA-to-cytoplasmic ratio. In contrast, the apoptotic response to DNA damage is not enabled in pre-MBT embryos merely by providing a threshold DNA-to-cytoplasmic ratio. Interestingly, damaged DNA triggers changes on physically separated undamaged chromatin through release of an ATM-dependent soluble checkpoint signal. We propose that this novel mode of “in trans” regulation is employed in the DNA damage response to modulate a broad range of chromatin-associated activities in a genome-wide manner.

Open Access Article

HDAC6 a new cellular stress surveillance factor

Patrick Matthias, Minoru Yoshida and Saadi Khochbin
Pages 7 - 10
http://dx.doi.org/10.4161/cc.7.1.5186
Abstract | PDF

Less than a decade has passed since HDAC6 was first identified and regarded as an unusual histone deacetylase harbouring two catalytic domains. Early demonstration of its cytoplasmic localisation, its ubiquitin-binding and its tubulin-deacetylase activities took HDAC6 far away from everything known to involve other histone-deacetylases. Recent discoveries confirmed the very unique functions of HDAC6 among deacetylases and pointed to this protein as a master regulator of cell response to cytotoxic assaults. HDAC6 appears both as a sensor of stressful stimuli and as an effector, which, thanks to its wide range of activities, mediates and coordinates appropriate cell responses.

Open Access Article

From centriole biogenesis to cellular function: Centrioles are essential for cell division at critical developmental stages

Ana Rodrigues-Martins, Maria Riparbelli, Giuliano Callaini, David M. Glover and Monica Bettencourt-Dias
Pages 11 - 16
http://dx.doi.org/10.4161/cc.7.1.5226
Abstract | PDF

Centrioles are essential for the formation of cilia, flagella and centrosome organization. Abnormalities in centrosome structure and number in many cancers can be associated with aberrant cell division and genomic instability.1,2 Canonical centriole duplication occurs in coordination with the cell division cycle, such that a single new “daughter” centriole arises next to each “mother” centriole. If destroyed, or eliminated during development, centrioles can form de novo.3-5 Here we discuss our recent data demonstrating a molecular pathway that operates in both de novo and canonical centriole biogenesis involving SAK/PLK4, SAS-6 and SAS-4.6 We showed that centriole biogenesis is a self-assembly process locally triggered by high SAK/PLK4 activity that may or not be associated with an existing centriole. SAS-6 acts downstream of SAK/PLK4 to organize nine precentriolar units, which we call here enatosomes, fitting together laterally and longitudinally, specifying a tube-like centriole precursor.7,8 The identification of mutants impaired in centriole biogenesis has permitted the study of the physiological consequences of their absence in the whole organism. In Drosophila, centrioles are not necessary for somatic cell divisions.9,10 However, we show here that mitotic abnormalities arise in syncytial SAK/PLK4-derived mutant embryos resulting in lethality. Moreover male meiosis fails in both SAK/PLK4 and DSAS-4 mutant spermatids that have no centrioles. These results show diversity in the need for centrioles in cell division. This suggests that tissue specific constraints selected for different contributions of centrosome-independent and dependent mechanisms in spindle function. This heterogeneity should be taken into account both in reaching an understanding of spindle function and when designing drugs that target cell division.

Open Access Article

Cell cycle switch to endocycle: The nucleolus lends a hand

David M.J. Martindill and Paul R. Riley
Pages 17 - 23
http://dx.doi.org/10.4161/cc.7.1.5228
Abstract | PDF

The bHLH transcription factor Hand1 is essential for placentation and cardiac morphogenesis but how its developmental activity is regulated is largely unknown. We recently showed that Hand1 is sequestered in the nucleoli of rodent trophoblast stem (TS) cells by the I-mfa domain-containing protein HICp40 and that this is associated with their proliferation and continuing self-renewal. However when these cells commit to differentiate into trophoblast giant (TG) cells, Hand1 is phosphorylated by the polo-like kinase Plk4 (Sak) and released into the nucleus to activate downstream target genes. This event underlies the release of Hand1 from the nucleolus and represents the ‘molecular switch’ that promotes mitotic cell cycle exit and the onset of endoreduplication. In this brief discussion we examine the wider implications of these findings and address some of the unanswered questions that remain.

Open Access Article

A reciprocal relationship between Rb and Skp2: implications for restriction point control, signal transduction to the cell cycle, and cancer

Richard K. Assoian and Yuval Yung
Pages 24 - 27
http://dx.doi.org/10.4161/cc.7.1.5232
Abstract | PDF

The identification of Skp2 as a direct E2F target has allowed us to document the existence of a positive feedback loop comprised of Rb-E2F, Skp2, p27 and cyclin E-cdk2. We have termed this set of regulatory molecules the Skp2 autoinduction loop. Interference with this loop selectively regulates cell cycle progression through the restriction point. We describe here how the Skp2 autoinduction loop may interact with other regulatory controls on the restriction point, and how the reciprocal relationship between Rb and Skp2 may affect signal transduction to the cell cycle and thinking about the role of Skp2 over-expression in cancers.




PERSPECTIVES

Open Access Article

Role of the hypoxia inducible factors HIF in iron metabolism

Carole Peyssonnaux, Victor Nizet and Randall S. Johnson
Pages 28 - 32
http://dx.doi.org/10.4161/cc.7.1.5145
Abstract | PDF

Iron is an essential element in all living organisms and is required as a cofactor for oxygen-binding proteins. Iron metabolism, oxygen homeostasis and erythropoiesis are consequently strongly interconnected. Iron needs to be tightly regulated, as iron insufficiency induces a hypoferric anemia in mammals, coupled to hypoxia in tissues, whereas excess iron is toxic, and causes generation of free radicals. Given the links between oxygen transport and iron metabolism, associations between the physiology of hypoxic response, and the control of iron availability are important. Numerous lines of investigation have proven that the HIF transcription factors function as central mediators of cellular adaptation to critically low oxygen levels in both normal and compromised tissues. Several of these target genes are involved in iron homeostasis, reflecting the molecular links between oxygen homeostasis and iron metabolism.

Open Access Article

Importance of the cell cycle phase for the choice of the appropriate DSB repair pathway, for genome stability mintenance: the trans-S double-strand break repair model

Fabien Delacôte and Bernard S. Lopez
Pages 33 - 38
http://dx.doi.org/10.4161/cc.7.1.5149
Abstract | PDF

A DNA double-strand break (DSB) is a highly harmful lesion which can lead to genome rearrangements. Two main pathways compete for DSB repair: homologous recombination (HR) and nonhomologous end-joining (NHEJ). Depending on the cell cycle phase, the choice of one DSB repair pathway over the other will secure genome stability maintenance or in contrast will increase the risk of genetic instability. HR with the sister chromatid is an efficient way to maintain genome stability, for damage occurring at a post-replication stage. However, in G1 checkpoint-defective cells, DSBs produced in the G1 phase and not repaired by NHEJ, can progress through S phase and be processed by HR in late S/G2 phase. We propose the “trans-S DSB repair” model to account for these data. In this situation HR cannot use the sister chromatid (which is also broken at the same locus) and is thus forced to use ectopic homologous sequences dispersed through the genome, increasing the risk of genetic instability. This shows that the two DSB repair pathways can compete through the cell cycle and underlines the importance of the association between the cell cycle checkpoint and the appropriate DNA repair pathway for genome stability maintenance.

Open Access Article

Controlling the cell death mediators Bax and Bak: puzzles and conundrums

Jamie I. Fletcher and David C.S. Huang
Pages 39 - 44
http://dx.doi.org/10.4161/cc.7.1.5178
Abstract | PDF

In spite of the available genetic, biochemical and structural data, precisely how the initiators of apoptosis, the BH3-only proteins, trigger Bax and Bak to cause organellar damage remains highly contentious. A widely accepted model suggests that these two critical mediators of apoptosis remain inert until they are directly activated by a subclass of BH3-only proteins including Bim, Bid, and possibly Puma. However, our recent work showed that these death ligands are dispensable for apoptosis to proceed, whereas neutralization of the pro-survival Bcl 2 proteins is essential. These findings challenge current assumptions about how the BH3-only proteins provoke apoptotic cell death and have implications for the development of therapeutics to interfere with the Bcl 2 regulated apoptotic pathway for treating diseases such as cancer.

Open Access Article

Oncogene-induced plasticity and cancer stem cells

Ulf R. Rapp, Fatih Ceteci and Ralf Schreck
Pages 45 - 51
http://dx.doi.org/10.4161/cc.7.1.5203
Abstract | PDF

The cancer stem cell hypothesis is an evolving concept of oncogenesis that has recently gained wide acceptance. In its simplest form this hypothesis suggests that many if not all tumors arise by consecutive genetic changes in a small subpopulation of cells termed cancer stem cells. These cells have the capacity to sustain tumor growth and are defined by three features: self-renewal, differentiation into the cell types of the original cancer and potent tumor formation. The definition of a cancer stem cell does not necessarily imply its origin from a stem, progenitor or differentiated cell. Hence, the term tumor or cancer initiating cell is often used instead to avoid any implications. Here, we propose a model suggesting that tumor cells progressively acquire stem cell properties as a consequence of oncogene-induced plasticity. The basis of our proposal are data from several experimental in vitro and in vivo models demonstrating reprogramming events triggered by specific combinations of oncogenes. These oncogene combinations not only induce cell lineage switches but also drive the reversal of ontogeny within cell lineages during tumor progression to metastasis. In this perspective article we will summarize the experimental evidence that illustrates our concept and discuss its implications for tumor formation and tumor therapy.

Open Access Article

Ulp2 and the DNA damage response: Desumoylation enables safe passage through mitosis

Rachael Felberbaum and Mark Hochstrasser
Pages 52 - 56
http://dx.doi.org/10.4161/cc.7.1.5218
Abstract | PDF

The DNA damage checkpoint is a crucial defense mechanism used by cells to withstand DNA damage. Activation of the checkpoint halts the cell cycle at metaphase and allows time for DNA repair prior to cell division. Much effort has been placed on identifying the proteins involved in checkpoint activation and how they elicit the damage response, whereas much less is known about how the checkpoint is silenced and cell division resumes. We recently reported that Ulp2, a SUMO protease, is required for cell division following termination of the DNA damage checkpoint in budding yeast. Here we discuss potential mechanisms by which Ulp2 enables the successful completion of mitosis following DNA damage. We also suggest candidate Ulp2 substrates whose desumoylation may be necessary for cell cycle restart. Finally, given the requirement of Ulp2 for survival in the presence of various metaphase-arresting agents, we suggest that the necessity for Ulp2 following checkpoint termination may not be specific to the DNA-damage response, but rather may indicate a broader role for desumoylation following prolonged metaphase arrest.




REVIEW

Open Access Article

Lumen formation during mammary epithelial morphogenesis: insights from in vitro and in vivo models

Arnaud A. Mailleux, Michael Overholtzer and Joan S. Brugge
Pages 57 - 62
http://dx.doi.org/10.4161/cc.7.1.5150
Abstract | PDF

The mammary gland undergoes a morphogenetic program during embryogenesis and puberty that leads to the development of hollow ductal system terminating in acinar units. It later expands to generate an elaborate network to deliver milk to newborn progeny. Previous studies in our laboratory using three-dimensional basement membrane cultures of mammary epithelial cells, in which acini-like structures form from single cells, have indicated that lumen formation requires clearance of the cells in the center of the acini by apoptosis. This apoptotic program in vitro requires the pro-death mediator BIM. Recently we found that BIM is also required in the mouse mammary gland for apoptosis during lumen formation, which correlates the 3D acinar model to mammary morphogenesis in vivo. Herein we put into perspective the relevance of our in vitro and in vivo findings to discuss luminal space formation and maintenance during mammary morphogenesis.




REPORTS

Open Access Article

c-Myc protein is degraded in response to UV irradiation

Sébastien Britton, Bernard Salles and Patrick Calsou
Pages 63 - 70
http://dx.doi.org/10.4161/cc.7.1.5111
Abstract | PDF

The c-MYC proto-oncogene encodes a transcription factor that is critical for cell growth and proliferation. It is one of the genes frequently altered in cancer cells in which it exhibits constitutive activity. The half-life of c-MYC is very short in quiescent cells due to ubiquitin-mediated proteolysis. We report here the rapid and dose-dependent decline of c-MYC protein level after UV-irradiation in various human and rodent cells. This decline is due to a proteasomal degradation of c-MYC protein and does not require the binding sites for the FBW7 and SKP2 ubiquitin ligases. Together, our data exclude a prominent role for the stress-responsive kinase PAK2, for the major phosphoinositide 3-kinase related protein kinases ATR, ATM, DNA-PK and mTOR and for ERK, JNK and p38 mitogen activated protein kinases in this UV-induced degradation process. We propose that c-MYC degradation is part of the global cell response to UV-damage, complementary to the accumulation and activation of the p53 transcription factor. By contributing to the replication arrest after infliction of lesions to the genome, the induced degradation of c-MYC may be part of the safeguard mechanisms maintaining genome stability.

Open Access Article

Effects of estradiol and medroxyprogesterone acetate on expression of the cell cycle proteins cyclin D1, p21 and p27 in cultured human breast tissues

Natalija Eigeliene, Pirkko Härkönen and Risto Erkkola
Pages 71 - 80
http://dx.doi.org/10.4161/cc.7.1.5102
Abstract | PDF

Estrogen and progesterone are key regulators of normal breast epithelial cell proliferation and differentiation. They are also involved in the initiation and progression of breast tumorigenesis. Several experimental studies have demonstrated that steroid hormones affect cell cycle proteins associated with tumor initiation and progression. Hormone replacement therapy (HT) is widely used to alleviate climacteric symptoms among postmenopausal women. Little is known, however, about cell cycle protein regulation during hormonal treatment of human breast tissue (HBT). In this study we aimed to evaluate the effects of 17β-estradiol (E2) and medroxyprogesterone acetate (MPA) on cultured HBTs representing samples from reduction mammoplasty of premenopausal (pre-HBT) and postmenopausal (postm-HBT) women, and from peritumoral tissue (peritum-HBT) after breast tumor surgery among postmenopausal patients. Explants of HBT were cultured for 14 days in medium supplemented with E2, MPA or E2+MPA. Expression of cyclin D1, p21 and p27 was assessed by immunohistochemical staining of explants cultured for 2 and 14 days. Further, Ki-67 staining was performed to evaluate correlation between proliferation and cell cycle regulatory protein expression. Our results showed that HBTs studied were positive for ERα, ERβ and PR (≥10% of the cells stained). The level of p21 was lower (p<0.001) in pre-HBT than in postm-HBT, whereas p27 levels were higher (p<0.05) in pre-HBT than in postm- and peritum-HBT. The level of Ki67 positive cells was higher in pre-HBT than in post-HBT. Interestingly, level of p21 positive cells showed an opposite pattern. Treatment with E2 increased the relative number of cyclin D1-staining cells and decreased that of p27-staining cells in postm-HBT (p<0.05), but not in pre-HBT. All hormone regimens (E2, MPA, E2+MPA) increased the number of p21-positive cells in postm-HBTs at 14 days and E2 even at 2 days. In pre-HBT p21 staining was increased (p<0.05) in explants cultured with E2 for 14 days but no response was observed in cyclin D1 and p27. The number of cyclin D1-staining cells was clearly higher (p<0.05) in peritum-HBT than in non-tumorous pre- or postm-HBT, but the response cyclin D1 to all hormonal treatments in peritum-HBT was the same as in postm-HBT. Moreover, we found that E2, MPA, E2+MPA in vitro increased numbers of Ki-67 positive cells in post-HBTs at 14 days and E2 also in pre-HBT. Stimulated proliferation rate was associated with increase of cyclin D1 and p21-positive cells and reduced numbers of p27, especially in post-HBTs. Taken together, our results suggest that cell cycle regulatory proteins are more sensitive to exogenous hormone treatment in postm-HBT than in pre-HBT.

Open Access Article

NCX-4016, a nitro-derivative of aspirin, inhibits EGFR and STAT3 signaling and mdulates Bcl-2 proteins in cisplatin-resistant human ovarian cancer cells and xenografts

Karuppaiyah Selvendiran, Anna Bratasz, Liyue Tong, Louis J. Ignarro and Periannan Kuppusamy
Pages 81 - 88
http://dx.doi.org/10.4161/cc.7.1.5103
Abstract | PDF

We have previously reported the inhibitory effect of NCX-4016, a nitro derivative of aspirin, on the proliferation of cisplatin-resistant human ovarian cancer cells, in vitro (Bratasz et al., Proc Natl Acad Sci USA 2006; 103:3914-9). In this report we present the results of our study on the mechanistic aspects of drug action including the molecular and signaling pathways involved in an in vitro cell line, as well as in a murine tumor xenograft. We report, for the first time, that NCX-4016 significantly inhibited the growth of cisplatin-resistant human ovarian cancer xenografts in mice. We observed that the inhibitory effect of NCX-4016 on cell proliferation was associated with G1 phase cell-cycle arrest with increased activity of p53, p21 and p27 proteins. NCX-4016 modulated the Bcl-2 family of proteins, and induced apoptosis by activating Bax and cytochrome c release in a time-dependent manner. In addition, NCX-4016 selectively down-regulated the phosphorylated forms of EGFR (Tyr845, Tyr992), pAkt (Ser473, Thr305), and STAT3 (Tyr705, Ser727), in vitro and in vivo. Taken together, the results clearly suggested that NCX-4016 causes significant induction of cell-cycle arrest and apoptosis in cisplatin-resistant human ovarian cancer cells via down-regulation of EGFR/PI3K/STAT3 signaling and modulation of Bcl-2 family proteins. Thus, NCX-4016 appears to be a potential therapeutic agent for treating recurrent human ovarian carcinoma.

Open Access Article

Catalytic activities of recA protein are dependent on the lattice length of the single-strand DNA ligand

Raquel Sussman, Shashi K. Sharma and Alan Kuzirian
Pages 89 - 95
http://dx.doi.org/10.4161/cc.7.1.5109
Abstract | PDF

recA protein (RecA) performs diverse catalytic activities that require a complex with single-stranded DNA and an NTP. A subset of these functions shows optimal activity at a high DNA/protein ratio and requires NTP hydrolysis, whereas other catalytic activities are optimal in RecA-saturated complexes that require NTP, but do not hydrolyze it. To analyze the mechanism of catalytic discrimination, we investigated the properties of RecA bound to small oligonucleotides (oligos) of defined sizes. We show that RecA bound to (dT)₁₆ is optimal for co-protease activity and not active as ATPase whereas the complex with (dT)₂₄ is competent in ATP hydrolysis but impaired as a co-protease. Thermodynamic measurements of the equilibrium-binding properties of these complexes showed that (dT)₂₄ promoted a more salt sensitive complex than the one formed with (dT)₁₆, indicating more ionic interactions between RecA and DNA in the former. X-ray pictures show that the oligo complexes form helixes. We propose that RecA may change its conformation as a function of the number of phosphates available to the monomer in the interacting DNA lattice, thus promoting an allosteric change in catalytic activities. This model offers explanations for the observed inhibition of co-protease activity by excess ssDNA.

Open Access Article

Yeast Chfr homologs retard cell cycle at G₁ and G₂/M via Ubc4 and Ubc13/Mms2-dependent ubiquitination

Greta L. Loring, Kathryn C. Christensen, Scott A. Gerber and Charles Brenner
Pages 96 - 105
http://dx.doi.org/10.4161/cc.7.1.5113
Abstract | PDF

Checkpoint with forkhead-associated and RING (Chfr) is a ubiquitin ligase (E3) that establishes an antephase or prometaphase checkpoint in response to mitotic stress. Though ubiquitination is essential for checkpoint function, the sites, linkages and ubiquitin conjugating enzyme (E2) specificity are controversial. Here we dissect the function of the two Chfr homologs in S. cerevisiae, Chf1 and Chf2, overexpression of which retard cell cycle at both G₁ and G₂. Using a genetic assay, we establish that Ubc4 is required for Chf2-dependent G₁ cell cycle delay and Chf protein turnover. In contrast, Ubc13/Mms2 is required for G₂ delay and does not contribute to Chf protein turnover. By reconstituting cis and trans-ubiquitination activities of Chf proteins in purified systems and characterizing sites modified and linkages formed by tandem mass spectrometry, we discovered that Ubc13/Mms2-dependent modifications are a distinct subset of those catalyzed by Ubc4. Mutagenesis of Lys residues identified in vitro indicates that site-specific Ubc4-dependent Chf protein autoubiquitination is responsible for Chf protein turnover. Thus, combined genetic and biochemical analyses indicate that Chf proteins have dual E2 specificity accounting for different functions in the cell cycle.

Open Access Article

Troy binding to lymphotoxin-α activates NFκB mediated transcription

Tsuyoshi Hashimoto, David Schlessinger and Chang-Yi Cui
Pages 106 - 111
http://dx.doi.org/10.4161/cc.7.1.5135
Abstract | PDF

Troy is a TNFR superfamily member that is highly expressed in developing hair follicles. Its possible function and ligand in skin have, however, been unknown. Here we demonstrate that an immunomodulatory cytokine, lymphotoxin-α (LTα), is a functional ligand of Troy by 3 biochemical approaches: (1) Immunoprecipitation assays revealed that LTα, but not LTβ or any obligate combination of LTα and LTβ, binds to Troy. (2) Co-transfection of LTα with Troy sharply upregulated NFκB reporter transcription, whereas LTβ or a combination of LTα and LTβ did not. (3) Recombinant LTα protein upregulated NFκB activity through Troy in a dosedependent manner. We further found that LTα is expressed in dermal papillae of developing hair follicles, whereas Troy was expressed in adjacent matrix region. This suggested involvement of LTα-Troy signaling in mesenchyme-epithelium interactions during hair follicle development. However, in Troy mutant mice that we generated, hair subtype composition and morphology were altered slightly if at all. The present study thus suggested a subtle function of the newly identified LTα-Troy pathway in skin appendage development, however, it may have an additional action compensated by a parallel EDA signaling pathway.

Open Access Article

Regulation of the p14ARF promoter by DNA methylation

Vinay Badal, Sergio Ménendez, David Coomber and David P. Lane
Pages 112 - 119
http://dx.doi.org/10.4161/cc.7.1.5137
Abstract | PDF

The INK4a/ARF locus encodes p14ARF which plays an important role in the p53 pathway. Interestingly, methylation of the INK4a/ARF locus is a common event in carcinogenesis. In this study we analyzed the effect of epigenetic alteration on the p14ARF promoter and its direct link to the expression of the p14ARF mRNA and protein. The osteosarcoma cell line U2OS was used as a model to study the regulation of the ARF promoter by DNA methylation. Treatment of U2OS cells with the demethylating agent 5-aza-2’-deoxycytidine (5-aza-dC) showed a marked induction of the p14ARF mRNA and protein. A novel quantitative method described here, using restriction enzyme digestion followed by real time PCR, allowed the analysis of the level of methylation over a defined region of DNA on the p14ARF promoter. The change in the methylation level of the promoter closely corresponded to the increase in the transcription of p14ARF mRNA and protein. Upon removal of 5-aza-CdR the methylation pattern on the p14ARF promoter was re-laid with a concomitant decrease in the levels of p14ARF mRNA and protein. The increase in the levels of p14ARF was concomitant with an induction of G1-G2 cell cycle arrest and an induction of p21 protein. No increase in the levels of p53 was observed. However, induction of p14ARF upon treatment with 5-aza-CdR led to the sequestering of MDM2 to the nucleolus. Additionally, we could show a dependency between the demethylation of the p14ARF promoter, the induction of p14ARF mRNA and protein and the effect of 5-aza-CdR on cell cycle.




LETTER TO THE EDITOR

Open Access Article

Bacteriophage Mu Mom protein responsible for DNA modification is a new member of the acyltransferase superfamily

Katarzyna H. Kaminska and Janusz M. Bujnicki
Pages 120 - 121
http://dx.doi.org/10.4161/cc.7.1.5158
Abstract | PDF

Not available.