Cyclin D1 mediates epithelial proliferation and links {\it ras\/} activation to the cell cycle

The studies presented in this thesis focus on two aspects of the involvement of cyclin D1 in epithelial proliferation. Since cyclin D1 has been identified as a target for genetic alterations and deregulation in a variety of human cancers, we studied cyclin D1 expression in two experimental models of epithelial carcinogenesis. These studies provided evidence that cyclin D1 was a potential target of the activating mutation of the Ha-ras gene characteristic of the experimental protocol. In addition, evidence from two independent in vitro models suggested that cyclin D1 was indeed part of the primary cellular response to activated ras, and at least partly responsible for the increase in proliferation observed in ras-transformed cells.^ Cyclin D1 has also been described as a key regulator of the passage through the G1 phase of the cell cycle. Cyclin D1 is induced in response to mitogens in a variety of cell lines, and cells engineered to overexpress cyclin D1 show accelerated G1 transit. In order to study the involvement of cyclin D1 in epithelial cell growth and differentiation, we generated transgenic mice that constitutively overexpress cyclin D1 in stratified epithelia. These mice developed thymic hyperplasia and skin hyperproliferation, providing in vivo evidence of the potential of cyclin D1 to regulate growth of epithelial cells. ^

Neocortical hyperexcitability defect in a mutant mouse model of spike-wave epilepsy, {\it stargazer}

Single-locus mutations in mice can express epileptic phenotypes and provide critical insights into the naturally occurring defects that alter excitability and mediate synchronization in the central nervous system (CNS). One such recessive mutation (on chromosome (Chr) 15), stargazer(stg/stg) expresses frequent bilateral 6-7 cycles per second (c/sec) spike-wave seizures associated with behavioral arrest, and provides a valuable opportunity to examine the inherited lesion associated with spike-wave synchronization.^ The existence of distinct and heterogeneous defects mediating spike-wave discharge (SWD) generation has been demonstrated by the presence of multiple genetic loci expressing generalized spike-wave activity and the differential effects of pharmacological agents on SWDs in different spike-wave epilepsy models. Attempts at understanding the different basic mechanisms underlying spike-wave synchronization have focused on $\gamma$-aminobutyric acid (GABA) receptor-, low threshold T-type Ca$\sp{2+}$ channel-, and N-methyl-D-aspartate receptor (NMDA-R)-mediated transmission. It is believed that defects in these modes of transmission can mediate the conversion of normal oscillations in a trisynaptic circuit, which includes the neocortex, reticular nucleus and thalamus, into spike-wave activity. However, the underlying lesions involved in spike-wave synchronization have not been clearly identified.^ The purpose of this research project was to locate and characterize a distinct neuronal hyperexcitability defect favoring spike-wave synchronization in the stargazer brain. One experimental approach for anatomically locating areas of synchronization and hyperexcitability involved an attempt to map patterns of hypersynchronous activity with antibodies to activity-induced proteins.^ A second approach to characterizing the neuronal defect involved examining the neuronal responses in the mutant following application of pharmacological agents with well known sites of action.^ In order to test the hypothesis that an NMDA receptor mediated hyperexcitability defect exists in stargazer neocortex, extracellular field recordings were used to examine the effects of CPP and MK-801 on coronal neocortical brain slices of stargazer and wild type perfused with 0 Mg$\sp{2+}$ artificial cerebral spinal fluid (aCSF).^ To study how NMDA receptor antagonists might promote increased excitability in stargazer neocortex, two basic hypotheses were tested: (1) NMDA receptor antagonists directly activate deep layer principal pyramidal cells in the neocortex of stargazer, presumably by opening NMDA receptor channels altered by the stg mutation; and (2) NMDA receptor antagonists disinhibit the neocortical network by blocking recurrent excitatory synaptic inputs onto inhibitory interneurons in the deep layers of stargazer neocortex.^ In order to test whether CPP might disinhibit the 0 Mg$\sp{2+}$ bursting network in the mutant by acting on inhibitory interneurons, the inhibitory inputs were pharmacologically removed by application of GABA receptor antagonists to the cortical network, and the effects of CPP under 0 Mg$\sp{2+}$aCSF perfusion in layer V of stg/stg were then compared with those found in +/+ neocortex using in vitro extracellular field recordings. (Abstract shortened by UMI.) ^

Growth regulatory role of R -PTPmu in normal and neoplastic epithelial cells

Cellular oncogenes and tumor suppressor genes regulate cellular adhesion and proliferation, two important events in malignant transformation. Even though receptor-like protein tyrosine phosphatases (R-PTPs) can influence these events, their role in malignant transformation has not been studied. The major goal of this study was to determine whether downregulation of R-PTP$\mu$ expression in lung epithelial cells is associated with or causal to neoplastic transformation. Examination of R-PTP$\mu$ expression in normal and carcinoma cells demonstrated that lung epithelial cells expressed R-PTP$\mu$ whereas lung carcinoma cells did not, and that incubation with TGF-$\alpha$ and HGF induced a two fold increase in R-PTP$\mu$ mRNA expression. To associate the expression of R-PTP$\mu$ with neoplastic transformation, we transfected lung epithelial cells with the H-ras oncogene. Transformation resulted in the activation of the MAPK signal transduction pathway, the hyperphosphorylation of c-met, and the production of HGF. Upon analysis of R-PTP$\mu$ expression, we observed a significant decrease in R-PTP$\mu$ mRNA and protein levels suggesting that transformation can directly or indirectly downregulate the expression of R-PTP$\mu.$ TGF-$\beta$ reversed the H-ras transformed phenotype, an event directly correlated with upregulation of R-PTP$\mu.$ To provide a casual relationship between R-PTP$\mu$ and cessation of tumor cell growth, we transfected carcinoma cells with the wild type R-PTP$\mu$ cDNA. Transiently expressing cells were selected by FACS using the mAb 3D7 and plated into individual wells. Carcinoma cells positive for R-PTP$\mu$ expression did not grow into colonies whereas non-R-PTP$\mu$ expressing carcinoma cells did, suggesting that expression of R-PTP$\mu$ arrested cell growth. To better understand the growth arrest induced by R-PTP$\mu$, we transfected the H-ras transformed lung epithelial cell line (MvLu-1-ras) with R-PTP$\mu$ (MvLu-1-ras/R-PTP$\mu$). Examination of growth factor receptor phosphorylation revealed significant inhibition of c-met and EGF-R. Furthermore, these cells underwent apoptosis in the absence of serum. Taken together the data demonstrate that the downregulation of R-PTP$\mu$ expression is an important step in neoplastic transformation of lung epithelial cells and that its presence can induce apoptosis and inhibit the signaling of c-met and EGF-R, two major growth factor receptors in lung carcinoma. In conclusion, the expression of R-PTP$\mu$ is inversely correlated with neoplastic transformation, growth and survival of tumor cells. ^

The effects of IL -10 producing monocytes on T-cell activation in patients with epithelial ovarian cancer

A newly described subset of monocytes has been identified in peritoneal exudate cells (PEC) from the malignant ascites of patients with ovarian cancer. These cells were characterized by the production of IL-10 and TGF-β2, but not IL-12, IL-1α, or TNF-α, and expressed CD14, CD16, and CD54, but not HLA-DR, CD80, CD86, CD11a, CD11b, or CD25 cell surface antigens. Since this subset of monocytes could affect the modulation of tumor immune responses in vivo, studies were undertaken to determine their effect on the activation and proliferation of autologous T-cells from the peritoneal cavity of patients with ovarian carcinoma. Cytokine transcripts, including IL-2, GM-CSF, and IFN-γ were detected in T-cells isolated from patient specimens that also contained the IL-10 producing monocytes, although the IFN-γ and IL-2 proteins could not be detected in T-cells co-incubated with the IL-10 producing monocytes in vitro. Additionally, IL-10 producing monocytes co-cultured with autologous T-cells inhibited the proliferation of the T-cells in response to PHA. T-cell proliferation and cytokine protein production could be restored by the addition of neutralizing antibodies to IL-10R and TGF-β to the co-culture system. These results suggested that this subset of monocytes may modulate antitumor immune responses by inhibiting T-cell proliferation and cytokine protein production. Further studies determined that the precursors to the inhibitory monocytes were tumor-associated and only present in the peripheral blood of patients with ovarian cancer and not present in the peripheral blood of healthy donors. These precursors could be induced to the suppressor phenotype by the addition of IL-2 and GM-CSF, two cytokines detected in the peritoneal cavity of ovarian cancer patients. Lastly, it was shown that the suppressor monocytes from the peritoneal cavity of ovarian cancer patients could be differentiated to a non-inhibitory phenotype by the addition of TNF-α and IFN-γ to the culture system. The differentiated monocytes did not produce IL-10, expressed the activation antigens HLA-DR, CD80, and CD86, and were able to stimulate autologous T-cells in vitro. Since a concomitant reduction in immune function is associated with tumor growth and progression, the effects of these monocytes are of considerable importance in the context of tumor immunotherapy. ^

Defective antitumor function of monocyte -derived macrophages from epithelial ovarian cancer patients

An abundance of monocytes and macrophages (MO/MA) in the microenvironment of epithelial ovarian cancer (EOC) suggests possible dual roles for these cells. Certain MO/MA subpopulations may inhibit tumor growth by antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, or stimulation of adaptive immunity. In contrast, other MO/MA subpopulations may support tumor growth by immunosuppressive or pro-angiogenic cytokine production. A better understanding of the phenotype and activity of MO/MA in EOC should lead to greater insight into their role in the immunopathobiology of EOC and hence suggest targets for treatment. We have found differences in the proportions of MO/MA subpopulations in the peripheral blood and ascites of EOC patients compared to normal donors, and differences in MO/MA surface phenotype in the associated tumor environment compared to the systemic circulation. We also demonstrate that, following their activation in vitro, monocyte-derived macrophages (MDM) from the peripheral blood and ascites of EOC patients exhibit antitumor effector activities that are different from the behavior of normal donor cells. The phenotypic characteristics and antitumor activity of CD14+ MO/MA and an isolated subpopulation of CD14brightCD16 −HLA-DR+ MO/MA were compared in samples of normal donor peripheral blood and the peripheral blood and ascites from EOC patients. MDM were cultured with macrophage colony-stimulating factor (M-CSF) and activated with lipopolysaccharide (LPS) or a combination of LPS plus recombinant interferon-gamma. We determined that MO/MA from EOC patients had altered morphology and significantly less ADCC and phagocytic activity than did MO/MA from normal donors. ADCC and phagocytosis are mediated by receptors for the Fe portion of IgG (FcγRs), the expression of which were also found to be deficient on EOC MDM from peripheral blood and ascites. Anti-tumor functions not mediated by the FcγRs, such as macrophage mediated cytotoxicity and cytostasis, were not impaired in EOC MDM compared to normal donor MDM. Our findings also showed that MDM from both EOC patients and normal donors produce M-CSF-stimulated cytokines, including interleukin-8, tumor necrosis factor alpha, and interleukin-6, which have the potential to support ovarian tumor growth and metastasis. These findings may be relevant to the pathogenesis of EOC and to the development of future bioimmunotherapeutic strategies. ^

Influence of termal fluctuations in radiation damage cascade production and defect dynamics in tungsten

The detailed study of the deterioration suffered by the materials of the components of a nuclear facility, in particular those forming part of the reactor core, is a topic of great interest which importance derives in large technological and economic implications. Since changes in the atomic-structural properties of relevant components pose a risk to the smooth operation with clear consequences for security and life of the plant, controlling these factors is essential in any development of engineering design and implementation. In recent times, tungsten has been proposed as a structural material based on its good resistance to radiation, but still needs to be done an extensive study on the influence of temperature on the behavior of this material under radiation damage. This work aims to contribute in this regard. Molecular Dynamics (MD) simulations were carried out to determine the influence of temperature fluctuations on radiation damage production and evolution in Tungsten. We have particularly focused our study in the dynamics of defect creation, recombination, and diffusion properties. PKA energies were sampled in a range from 5 to 50 KeV. Three different temperature scenarios were analyzed, from very low temperatures (0-200K), up to high temperature conditions (300-500 K). We studied the creation of defects, vacancies and interstitials, recombination rates, diffusion properties, cluster formation, their size and evolution. Simulations were performed using Lammps and the Zhou EAM potential for W

Sample Size vs. Bias in Defect Prediction

Most empirical disciplines promote the reuse and sharing of datasets, as it leads to greater possibility of replication. While this is increasingly the case in Empirical Software Engineering, some of the most popular bug-fix datasets are now known to be biased. This raises two significants concerns: first, that sample bias may lead to underperforming prediction models, and second, that the external validity of the studies based on biased datasets may be suspect. This issue has raised considerable consternation in the ESE literature in recent years. However, there is a confounding factor of these datasets that has not been examined carefully: size. Biased datasets are sampling only some of the data that could be sampled, and doing so in a biased fashion; but biased samples could be smaller, or larger. Smaller data sets in general provide less reliable bases for estimating models, and thus could lead to inferior model performance. In this setting, we ask the question, what affects performance more? bias, or size? We conduct a detailed, large-scale meta-analysis, using simulated datasets sampled with bias from a high-quality dataset which is relatively free of bias. Our results suggest that size always matters just as much bias direction, and in fact much more than bias direction when considering information-retrieval measures such as AUC and F-score. This indicates that at least for prediction models, even when dealing with sampling bias, simply finding larger samples can sometimes be sufficient. Our analysis also exposes the complexity of the bias issue, and raises further issues to be explored in the future.

Defect engineering during the contact co-firing step in an industrial belt furnace

This work implements an optimization of the phosphorus gettering effect during the contact co-firing step by means of both simulations and experiments in an industrial belt furnace. An optimized temperature profile, named ‘extended co-firing step’, is presented. Simulations show that the effect of the short annealing on the final interstitial iron concentration depends strongly on the initial contamination level of the material and that the ‘extended co-firing’ temperature profile can enhance the gettering effect within a small additional time. Experimental results using sister wafers from the same multicrystalline silicon ingot confirm these trends and show the potential of this new defect engineering tool to improve the solar cell efficiency.

Hexagons and spiral defect chaos in non-Boussinesq convection at low Prandtl numbers

We study the stability and dynamics of non-Boussinesq convection in pure gases ?CO2 and SF6? with Prandtl numbers near Pr? 1 and in a H2-Xe mixture with Pr= 0.17. Focusing on the strongly nonlinear regime we employ Galerkin stability analyses and direct numerical simulations of the Navier-Stokes equations. For Pr ? 1 and intermediate non-Boussinesq effects we find reentrance of stable hexagons as the Rayleigh number is increased. For stronger non-Boussinesq effects the usual, transverse side-band instability is superseded by a longitudinal side-band instability. Moreover, the hexagons do not exhibit any amplitude instability to rolls. Seemingly, this result contradicts the experimentally observed transition from hexagons to rolls. We resolve this discrepancy by including the effect of the lateral walls. Non-Boussinesq effects modify the spiral defect chaos observed for larger Rayleigh numbers. For convection in SF6 we find that non-Boussinesq effects strongly increase the number of small, compact convection cells and with it enhance the cellular character of the patterns. In H2-Xe, closer to threshold, we find instead an enhanced tendency toward roll-like structures. In both cases the number of spirals and of targetlike components is reduced. We quantify these effects using recently developed diagnostics of the geometric properties of the patterns.

Geometric diagnostics of complex patterns: Spiral defect chaos

Motivated by the observation of spiral patterns in a wide range of physical, chemical, and biological systems, we present an automated approach that aims at characterizing quantitatively spiral-like elements in complex stripelike patterns. The approach provides the location of the spiral tip and the size of the spiral arms in terms of their arc length and their winding number. In addition, it yields the number of pattern components (Betti number of order 1), as well as their size and certain aspects of their shape. We apply the method to spiral defect chaos in thermally driven Rayleigh- Bénard convection and find that the arc length of spirals decreases monotonically with decreasing Prandtl number of the fluid and increasing heating. By contrast, the winding number of the spirals is nonmonotonic in the heating. The distribution function for the number of spirals is significantly narrower than a Poisson distribution. The distribution function for the winding number shows approximately an exponential decay. It depends only weakly on the heating, but strongly on the Prandtl number. Large spirals arise only for larger Prandtl numbers. In this regime the joint distribution for the spiral length and the winding number exhibits a three-peak structure, indicating the dominance of Archimedean spirals of opposite sign and relatively straight sections. For small Prandtl numbers the distribution function reveals a large number of small compact pattern components.

Defect chaos and bursts: Hexagonal rotating convection and the complex Ginzburg-Landau equation

We employ numerical computations of the full Navier-Stokes equations to investigate non-Boussinesq convection in a rotating system using water as the working fluid. We identify two regimes. For weak non- Boussinesq effects the Hopf bifurcation from steady to oscillating (whirling) hexagons is supercritical and typical states exhibit defect chaos that is systematically described by the cubic complex Ginzburg-Landau equation. For stronger non-Boussinesq effects the Hopf bifurcation becomes subcritical and the oscil- lations exhibit localized chaotic bursting, which is modeled by a quintic complex Ginzburg-Landau equation.

MMonCa: An Object Kinetic Monte Carlo simulator for damage irradiation evolution and defect diffusion

In this work, we introduce the Object Kinetic Monte Carlo (OKMC) simulator MMonCa and simulate the defect evolution in three different materials. We start by explaining the theory of OKMC and showing some details of how such theory is implemented by creating generic structures and algorithms in the objects that we want to simulate. Then we successfully reproduce simulated results for defect evolution in iron, silicon and tungsten using our simulator and compare with available experimental data and similar simulations. The comparisons validate MMonCa showing that it is powerful and flexible enough to be customized and used to study the damage evolution of defects in a wide range of solid materials.

About the origin of low wafer performance and crystal defect generation on seed-cast growth of industrial mono-like silicon ingots

The era of the seed-cast grown monocrystalline-based silicon ingots is coming. Mono-like, pseudomono or quasimono wafers are product labels that can be nowadays found in the market, as a critical innovation for the photovoltaic industry. They integrate some of the most favorable features of the conventional silicon substrates for solar cells, so far, such as the high solar cell efficiency offered by the monocrystalline Czochralski-Si (Cz-Si) wafers and the lower cost, high productivity and full square-shape that characterize the well-known multicrystalline casting growth method. Nevertheless, this innovative crystal growth approach still faces a number of mass scale problems that need to be resolved, in order to gain a deep, 100% reliable and worldwide market: (i) extended defects formation during the growth process; (ii) optimization of the seed recycling; and (iii) parts of the ingots giving low solar cells performance, which directly affect the production costs and yield of this approach. Therefore, this paper presents a series of casting crystal growth experiments and characterization studies from ingots, wafers and cells manufactured in an industrial approach, showing the main sources of crystal defect formation, impurity enrichment and potential consequences at solar cell level. The previously mentioned technological drawbacks are directly addressed, proposing industrial actions to pave the way of this new wafer technology to high efficiency solar cells.

Choroid plexus epithelial expression of MDR1 P glycoprotein and multidrug resistance-associated protein contribute to the blood–cerebrospinal-fluid drug-permeability barrier

The blood–brain barrier and a blood–cerebrospinal-fluid (CSF) barrier function together to isolate the brain from circulating drugs, toxins, and xenobiotics. The blood–CSF drug-permeability barrier is localized to the epithelium of the choroid plexus (CP). However, the molecular mechanisms regulating drug permeability across the CP epithelium are defined poorly. Herein, we describe a drug-permeability barrier in human and rodent CP mediated by epithelial-specific expression of the MDR1 (multidrug resistance) P glycoprotein (Pgp) and the multidrug resistance-associated protein (MRP). Noninvasive single-photon-emission computed tomography with 99mTc-sestamibi, a membrane-permeant radiopharmaceutical whose transport is mediated by both Pgp and MRP, shows a large blood-to-CSF concentration gradient across intact CP epithelium in humans in vivo. In rats, pharmacokinetic analysis with 99mTc-sestamibi determined the concentration gradient to be greater than 100-fold. In membrane fractions of isolated native CP from rat, mouse, and human, the 170-kDa Pgp and 190-kDa MRP are identified readily. Furthermore, the murine proteins are absent in CP isolated from their respective mdr1a/1b(−/−) and mrp(−/−) gene knockout littermates. As determined by immunohistochemical and drug-transport analysis of native CP and polarized epithelial cell cultures derived from neonatal rat CP, Pgp localizes subapically, conferring an apical-to-basal transepithelial permeation barrier to radiolabeled drugs. Conversely, MRP localizes basolaterally, conferring an opposing basal-to-apical drug-permeation barrier. Together, these transporters may coordinate secretion and reabsorption of natural product substrates and therapeutic drugs, including chemotherapeutic agents, antipsychotics, and HIV protease inhibitors, into and out of the central nervous system.

Msh2 status modulates both apoptosis and mutation frequency in the murine small intestine

Deficiency in genes involved in DNA mismatch repair increases susceptibility to cancer, particularly of the colorectal epithelium. Using Msh2 null mice, we demonstrate that this genetic defect renders normal intestinal epithelial cells susceptible to mutation in vivo at the Dlb-1 locus. Compared with wild-type mice, Msh2-deficient animals had higher basal levels of mutation and were more sensitive to the mutagenic effects of temozolomide. Experiments using Msh2-deficient cells in vitro suggest that an element of this effect is attributable to increased clonogenicity. Indeed, we show that Msh2 plays a role in the in vivo initiation of apoptosis after treatment with temozolomide, N-methyl-N′-nitro-N-nitrosoguanidine, and cisplatin. This was not influenced by the in vivo depletion of O6-alkylguanine-DNA-alkyltransferase after administration of O6-benzylguanine . By analyzing mice mutant for both Msh2 and p53, we found that the Msh2-dependent apoptotic response was primarily mediated through a p53-dependent pathway. Msh2 also was required to signal delayed p53-independent death. Taken together, these studies characterize an in vivo Msh2-dependent apoptotic response to methylating agents and raise the possibility that Msh2 deficiency may predispose to malignancy not only through failed repair of mismatch DNA lesions but also through the failure to engage apoptosis.