Diagnostics of classical and dusty plasmas w ith collecting and emissive langmuir probes = Diagnosis de plasmas clásicos y granulares mediante sondas colectoras y emisoras de Langmuir

Las sondas eléctricas se emplean habitualmente en la diagnosis de plasmas. La presente tesis aborda la operación de las sondas colectoras y emisoras de Langmuir en plasmas fríos de baja densidad. El estudio se ha centrado en la determinación del potencial de plasma, Vsp, mediante el potencial flotante de una sonda emisora. Esta técnica consiste en la medida del potencial de la sonda correspondiente a la condición de corriente neta igual a cero, el cual se denomina potencial flotante, VF. Este potencial se desplaza hacia el potencial del plasma según aumenta la emisión termoiónica de la sonda, hasta que se satura cerca de Vsp. Los experimentos llevados a cabo en la pluma de plasma de un motor iónico y en un plasma de descarga de glow muestran que la corriente de electrones termoiónicos es mayor que la corriente de electrones recogidos para una sonda polarizada por debajo del potencial del plasma, resultado inconsistente con la teoría tradicionalmente aceptada. Para investigar estos resultados se ha introducido el parámetro R, definido como el cociente entre la corriente de electrones emitidos y recogidos por la sonda. Este parámetro, que está relacionado con la diferencia de potencial VF - Vsp, también es útil para la descripción de los modos de operación de la sonda emisora (débil, fuerte y más allá del fuerte). Los resultados experimentales evidencian que, al contrario de lo que indica la teoría, R es mayor que la unidad. Esta discrepancia se puede solucionar introduciendo una población efectiva de electrones. Con dicha población, el nuevo modelo para la corriente total de la sonda reproduce los datos experimentales. El origen de este grupo electrónico es todavía una cuestión abierta, pero podría estar originada por una nueva estructura de potencial cerca de la sonda cuando ésta trabaja en el régimen de emisión fuerte. Para explicar dicha estructura de potencial, se propone un modelo unidimensional compuesto por un mínimo de potencial cerca de la superficie de la sonda. El análisis numérico indica que este pozo de potencial aparece para muy altas temperaturas de la sonda, reduciendo la cantidad de electrones emitidos que alcanzan el plasma y evitando así cualquier posible perturbación de éste. Los aspectos experimentales involucrados en el método del potencial flotante también se han estudiado, incluyendo cuestiones como las diferentes técnicas de obtención del VF, el cociente señal-ruido, el acoplamiento de la señal de los equipos utilizados para la obtención de las curvas I-V o la evidencia experimental de los diferentes modos de operación de la sonda. Estas evidencias empíricas se encuentran en todos los aspectos de operación de la sonda: la recolección de electrones, el potencial flotante, la precisión en las curvas I-V y la emisión electrónica. Ésta última también se estudia en la tesis, debido a que un fenómeno de super emisión tiene lugar en el régimen de emisión fuerte. En este modo de operación, las medidas experimentales indican que las corrientes termoiónicas de electrones son mayores que aquéllas predichas por la ecuación de Richardson-Dushman clásica. Por último, la diagnosis de plasmas usando sondas eléctrica bajo presencia de granos de polvo (plasmas granulares) en plasmas fríos de baja densidad también se ha estudiado, mediante la aplicación numérica de la técnica del potencial flotante de la sonda emisora en un plasma no convencional. Los resultados apuntan a que el potencial flotante de una sonda emisora se vería afectado por altas densidades de polvo o grandes partículas. ABSTRACT Electric probes are widely employed for plasma diagnostics. This dissertation concerns the operation of collecting and emissive Langmuir probes in low density cold plasmas. The study is focused on the determination of the plasma potential, Vsp, by means of the floating potential of emissive probes. This technique consists of the measurement of the probe potential, corresponding to the zero net probe current, which is the so-called floating potential, VF . This potential displaces towards the plasma potential as the thermionic electron emission increases, until it saturates near Vsp. Experiments carried out in the plasma plume of an ion thruster and in a glow discharge plasma show the thermionic electron current of the emissive Langmuir probe is higher than the collected electron current, for a probe with a bias potential below Vsp, which is inconsistent with the traditional accepted theory. To investigate these results, a parameter R is introduced as the ratio between the emitted and the collected electron current. This parameter, which is related to the difference VF - Vsp, is also useful for the description of the operation modes of the emissive Langmuir probe (weak, strong and beyond strong). The experimental results give an inconsistency of R > 1, which is solved by a modification of the theory for emissive probes, with the introduction of an effective electron population. With this new electron group, the new model for the total probe current agrees with the experimental data. The origin of this electron group remains an open question, but it might be originated by a new potential structure near the emissive probe when it operates in the strong emission regime. A simple one-dimension model composed by a minimum of potential near the probe surface is discussed for strongly emitting emissive probes. The results indicate that this complex potential structure appears for very high probe temperatures and the potential well might reduce the emitted electrons population reaching the plasma bulk. The experimental issues involved in the floating potential method are also studied, as the different obtaining techniques of VF, the signal-to-noise ratio, the signal coupling of the I-V curve measurement system or the experimental evidence of the probe operation modes. These empirical proofs concern all the probe operation aspects: the electron collection, the floating potential, the I-V curve accuracy as well as the electron emission. This last issue is also investigated in this dissertation, because a super emission takes place in the strong emission regime. In this operation mode, the experimental results indicate that the thermionic electron currents might be higher than those predicted by the classical Richardson-Dushman equation. Finally, plasma diagnosis using electric probes in the presence of dust grains (dusty plasmas) in low density cold plasmas is also addressed. The application of the floating potential technique of the emissive probe in a non-conventional complex plasma is numerically investigated, whose results point out the floating potential of the emissive probe might be shifted for high dust density or large dust particles.

Classical solutions for a logarithmic fractional diffusion equation

We prove global existence and uniqueness of strong solutions to the logarithmic porous medium type equation with fractional diffusion ?tu + (?)1/2 log(1 + u) = 0, posed for x ? R, with nonnegative initial data in some function space of LlogL type. The solutions are shown to become bounded and C? smooth in (x, t) for all positive times. We also reformulate this equation as a transport equation with nonlocal velocity and critical viscosity, a topic of current relevance. Interesting functional inequalities are involved.

Loss of transformed phenotype in cancer cells by overexpression of the uteroglobin gene

Uteroglobin (UG) is a multifunctional, secreted protein that has receptor-mediated functions. The human UG (hUG) gene is mapped to chromosome 11q12.2–13.1, a region frequently rearranged or deleted in many cancers. Although high levels of hUG expression are characteristic of the mucosal epithelia of many organs, hUG expression is either drastically reduced or totally absent in adenocarcinomas and in viral-transformed epithelial cells derived from the same organs. In agreement with these findings, in an ongoing study to evaluate the effects of aging on UG-knockout mice, 16/16 animals developed malignant tumors, whereas the wild-type littermates (n = 25) remained apparently healthy even after 1½ years. In the present investigation, we sought to determine the effects of induced-expression of hUG in human cancer cells by transfecting several cell lines derived from adenocarcinomas of various organs with an hUG-cDNA construct. We demonstrate that induced hUG expression reverses at least two of the most important characteristics of the transformed phenotype (i.e., anchorage-independent growth on soft agar and extracellular matrix invasion) of only those cancer cells that also express the hUG receptor. Similarly, treatment of the nontransfected, receptor-positive adenocarcinoma cells with purified recombinant hUG yielded identical results. Taken together, these data define receptor-mediated, autocrine and paracrine pathways through which hUG reverses the transformed phenotype of cancer cells and consequently, may have tumor suppressor-like effects.

A phenotype-based screen for embryonic lethal mutations in the mouse

The genetic pathways that control development of the early mammalian embryo have remained poorly understood, in part because the systematic mutant screens that have been so successful in the identification of genes and pathways that direct embryonic development in Drosophila, Caenorhabditis elegans, and zebrafish have not been applied to mammalian embryogenesis. Here we demonstrate that chemical mutagenesis with ethylnitrosourea can be combined with the resources of mouse genomics to identify new genes that are essential for mammalian embryogenesis. A pilot screen for abnormal morphological phenotypes of midgestation embryos identified five mutant lines; the phenotypes of four of the lines are caused by recessive traits that map to single regions of the genome. Three mutant lines display defects in neural tube closure: one is caused by an allele of the open brain (opb) locus, one defines a previously unknown locus, and one has a complex genetic basis. Two mutations produce novel early phenotypes and map to regions of the genome not previously implicated in embryonic patterning.

Overexpression of DNA polymerase β in cell results in a mutator phenotype and a decreased sensitivity to anticancer drugs

DNA polymerase β (pol β) is the most error prone of all known eukaryotic DNA polymerases tested in vitro. Here, we show that cells overexpressing pol β cDNA have acquired a spontaneous mutator phenotype. By measuring the appearance of mutational events using three independent assays, we found that genetic instability increased in the cell lines that overexpressed pol β. In addition, these cells displayed a decreased sensitivity to cancer chemotherapeutic, bifunctional, DNA-damaging agents such as cisplatin, melphalan, and mechlorethamine, resulting in enhanced mutagenesis compared with control cells. By using cell-free extracts and modified DNA substrates, we present data in support of error-prone translesion replication as one of the key determinants of tolerance phenotype. These results have implications for the potential role of pol β overexpression in cancer predisposition and tumor progression during chemotherapy.

Abrogation of the alternative complement pathway by targeted deletion of murine factor B

To investigate the role of complement protein factor B (Bf) and alternative pathway activity in vivo, and to test the hypothesized potential genetic lethal effect of Bf deficiency, the murine Bf gene was interrupted by exchange of exon 3 through exon 7 (including the factor D cleaving site) with the neor gene. Mice heterozygous for the targeted Bf allele were interbred, yielding Bf-deficient offspring after the F1 generation at a frequency suggesting that Bf deficiency alone has no major effect on fertility or fetal development. However, in the context of one or more genes derived from the 129 mouse strain, offspring homozygous for Bf deficiency were generated at less than expected numbers (P = 0.012). Bf-deficient mice showed no gross phenotypic difference from wild-type littermates. Sera from Bf-deficient mice lacked detectable alternative complement pathway activity; purified mouse Bf overcame the deficit. Classical pathway-dependent total hemolytic activity was lower in Bf-deficient than wild-type mice, possibly reflecting loss of the alternative pathway amplification loop. Lymphoid organ structure and IgG1 antibody response to a T-dependent antigen appeared normal in Bf-deficient mice. Sensitivity to lethal endotoxic shock was not significantly altered in Bf-deficient mice. Thus, deficiency of Bf and alternative complement activation pathway led to a less dramatic phenotype than expected. Nevertheless, these mice provide an excellent model for the assessment of the role of Bf and the alternative pathway in host defense and other functions in vivo.

Genetic and root phenotype diversity in Sri Lankan rice landraces may be related to drought resistance

Acknowledgements Mayuri Munasinghe was supported by a Commonwealth Scholarship (ref no. LKCS-2009-384). The development and use of the SNP chip was funded by a BBSRC grant BB/J003336/1. The authors thank Owen Price (University of Wollongong, Australia) for producing the coloured province map of Sri Lanka, Gareth Norton (Aberdeen) for merging the RDP1 SNP data with the Sri Lankan data and Tony Travis (Aberdeen) for help with PCA.

Gene-based approach to human gene-phenotype correlations

Elucidating the genetic basis of human phenotypes is a major goal of contemporary geneticists. Logically, two fundamental and contrasting approaches are available, one that begins with a phenotype and concludes with the identification of a responsible gene or genes; the other that begins with a gene and works toward identifying one or more phenotypes resulting from allelic variation of it. This paper provides a conceptual overview of phenotype-based vs. gene-based procedures with emphasis on gene-based methods. A key feature of a gene-based approach is that laboratory effort first is devoted to developing an assay for mutations in the gene under regard; the assay then is applied to the evaluation of large numbers of unrelated individuals with a variety of phenotypes that are deemed potentially resulting from alleles at the gene. No effort is directed toward chromosomally mapping the loci responsible for the phenotypes scanned. Example is made of my laboratory’s successful use of a gene-based approach to identify genes causing hereditary diseases of the retina such as retinitis pigmentosa. Reductions in the cost and improvements in the speed of scanning individuals for DNA sequence anomalies may make a gene-based approach an efficient alternative to phenotype-based approaches to correlating genes with phenotypes.

Phenotype-genotype studies in kuru: Implications for new variant Creutzfeldt–Jakob disease

The PRNP polymorphic (methionine/valine) codon 129 genotype influences the phenotypic features of transmissible spongiform encephalopathy. All tested cases of new variant Creutzfeldt–Jakob disease (nvCJD) have been homozygous for methionine, and it is conjectural whether different genotypes, if they appear, might have distinctive phenotypes and implications for the future “epidemic curve” of nvCJD. Genotype-phenotype studies of kuru, the only other orally transmitted transmissible spongiform encephalopathy, might be instructive in predicting the answers to these questions. We therefore extracted DNA from blood clots or sera from 92 kuru patients, and analyzed their codon 129 PRNP genotypes with respect to the age at onset and duration of illness and, in nine cases, to detailed clinical and neuropathology data. Homozygosity at codon 129 (particularly for methionine) was associated with an earlier age at onset and a shorter duration of illness than was heterozygosity, but other clinical characteristics were similar for all genotypes. In the nine neuropathologically examined cases, the presence of histologically recognizable plaques was limited to cases carrying at least one methionine allele (three homozygotes and one heterozygote). If nvCJD behaves like kuru, future cases (with longer incubation periods) may begin to occur in older individuals with heterozygous codon 129 genotypes and signal a maturing evolution of the nvCJD “epidemic.” The clinical phenotype of such cases should be similar to that of homozygous cases, but may have less (or at least less readily identified) amyloid plaque formation.

The Tabby phenotype is caused by mutation in a mouse homologue of the EDA gene that reveals novel mouse and human exons and encodes a protein (ectodysplasin-A) with collagenous domains

Mouse Tabby (Ta) and X chromosome-linked human EDA share the features of hypoplastic hair, teeth, and eccrine sweat glands. We have cloned the Ta gene and find it to be homologous to the EDA gene. The gene is altered in two Ta alleles with a point mutation or a deletion. The gene is expressed in developing teeth and epidermis; no expression is seen in corresponding tissues from Ta mice. Ta and EDA genes both encode alternatively spliced forms; novel exons now extend the 3′ end of the EDA gene. All transcripts recovered have the same 5′ exon. The longest Ta cDNA encodes a 391-residue transmembrane protein, ectodysplasin-A, containing 19 Gly-Xaa-Yaa repeats. The isoforms of ectodysplasin-A may correlate with differential roles during embryonic development.

A deletion in an indole synthase gene is responsible for the DIMBOA-deficient phenotype of bxbx maize

The biosynthesis of DIMBOA, a pesticidal secondary metabolite of maize, branches off the tryptophan pathway. We have previously demonstrated that indole is the last intermediate common to both the tryptophan and hydroxamic acid pathways. The earliest discovered mutant in the DIMBOA pathway, bxbx (benzoxazineless), is deficient in the production of DIMBOA and related compounds. This paper presents evidence that a gene identified by Kramer and Koziel [Kramer, V. C. & Koziel, M. G. (1995) Plant Mol. Biol. 27, 1183–1188] as maize tryptophan synthase α (TSA) is the site of the genetic lesion in the DIMBOA-deficient mutant maize line bxbx. We demonstrate that the TSA gene has sustained a 924-bp deletion in bxbx compared with its counterpart in wild-type maize. We report that the TSA gene maps to the same location as the bxbx mutation, on the short arm of chromosome 4. We present evidence that the very early and very high level of expression of TSA corresponds to the timing and level of DIMBOA biosynthesis but is strikingly different from the expression of the maize tryptophan synthase β (TSB) genes. We show that feeding indole to bxbx seedlings restores their ability to synthesize DIMBOA. We conclude that the maize enzyme initially named tryptophan synthase α in fact is a DIMBOA biosynthetic enzyme, and we propose that it be renamed indole synthase. This work confirms and enlarges upon the findings of Frey et al. [Frey, M. Chomet, P., Glawischniq, E., Stettner, C., Grün, S., Winklmair, A., Eisenreich, W., Bacher, A., Meeley, R. B., Briggs, S. P., Simcox, K. & Gierl, A. (1997) Science 277, 696–699], which appeared while the present paper was in review.

Tissue phenotype depends on reciprocal interactions between the extracellular matrix and the structural organization of the nucleus

What determines the nuclear organization within a cell and whether this organization itself can impose cellular function within a tissue remains unknown. To explore the relationship between nuclear organization and tissue architecture and function, we used a model of human mammary epithelial cell acinar morphogenesis. When cultured within a reconstituted basement membrane (rBM), HMT-3522 cells form polarized and growth-arrested tissue-like acini with a central lumen and deposit an endogenous BM. We show that rBM-induced morphogenesis is accompanied by relocalization of the nuclear matrix proteins NuMA, splicing factor SRm160, and cell cycle regulator Rb. These proteins had distinct distribution patterns specific for proliferation, growth arrest, and acini formation, whereas the distribution of the nuclear lamina protein, lamin B, remained unchanged. NuMA relocalized to foci, which coalesced into larger assemblies as morphogenesis progressed. Perturbation of histone acetylation in the acini by trichostatin A treatment altered chromatin structure, disrupted NuMA foci, and induced cell proliferation. Moreover, treatment of transiently permeabilized acini with a NuMA antibody led to the disruption of NuMA foci, alteration of histone acetylation, activation of metalloproteases, and breakdown of the endogenous BM. These results experimentally demonstrate a dynamic interaction between the extracellular matrix, nuclear organization, and tissue phenotype. They further show that rather than passively reflecting changes in gene expression, nuclear organization itself can modulate the cellular and tissue phenotype.

A bcr-3 isoform of RARα-PML potentiates the development of PML-RARα-driven acute promyelocytic leukemia

Acute promyelocytic leukemia (APML) most often is associated with the balanced reciprocal translocation t(15;17) (q22;q11.2) and the expression of both the PML-RARα and RARα-PML fusion cDNAs that are formed by this translocation. In this report, we investigated the biological role of a bcr-3 isoform of RARα-PML for the development of APML in a transgenic mouse model. Expression of RARα-PML alone in the early myeloid cells of transgenic mice did not alter myeloid development or cause APML, but its expression significantly increased the penetrance of APML in mice expressing a bcr-1 isoform of PML-RARα (15% of animals developed APML with PML-RARα alone vs. 57% with both transgenes, P < 0.001). The latency of APML development was not altered substantially by the expression of RARα-PML, suggesting that it does not behave as a classical “second hit” for development of the disease. Leukemias that arose from doubly transgenic mice were less mature than those from PML-RARα transgenic mice, but they both responded to all-trans retinoic acid in vitro. These findings suggest that PML-RARα drives the development of APML and defines its basic phenotype, whereas RARα-PML potentiates this phenotype via mechanisms that are not yet understood.

Molecular characterization of a mutable pigmentation phenotype and isolation of the first active transposable element from Sorghum bicolor

Accumulation of red phlobaphene pigments in sorghum grain pericarp is under the control of the Y gene. A mutable allele of Y, designated as y-cs (y-candystripe), produces a variegated pericarp phenotype. Using probes from the maize p1 gene that cross-hybridize with the sorghum Y gene, we isolated the y-cs allele containing a large insertion element. Our results show that the Y gene is a member of the MYB-transcription factor family. The insertion element, named Candystripe1 (Cs1), is present in the second intron of the Y gene and shares features of the CACTA superfamily of transposons. Cs1 is 23,018 bp in size and is bordered by 20-bp terminal inverted repeat sequences. It generated a 3-bp target site duplication upon insertion within the Y gene and excised from y-cs, leaving a 2-bp footprint in two cases analyzed. Reinsertion of the excised copy of Cs1 was identified by Southern hybridization in the genome of each of seven red pericarp revertant lines tested. Cs1 is the first active transposable element isolated from sorghum. Our analysis suggests that Cs1-homologous sequences are present in low copy number in sorghum and other grasses, including sudangrass, maize, rice, teosinte, and sugarcane. The low copy number and high transposition frequency of Cs1 imply that this transposon could prove to be an efficient gene isolation tool in sorghum.