Characterization of alpha thalassemic genotypes by multiplex ligation-dependent probe amplification in the Brazilian population

Alpha-thalassemia is the most common inherited disorder of hemoglobin synthesis. Genomic deletions involving the alpha-globin gene cluster on chromosome 16p13.3 are the most frequent molecular causes of the disease. Although common deletions can be detected by a single multiplex gap-PCR, the rare and novel deletions depend on more laborious techniques for their identification. The multiplex ligation-dependent probe amplification (MLPA) technique has recently been used for this purpose and was successfully used in the present study to detect the molecular alterations responsible for the alpha-thalassemic phenotypes in 8 unrelated individuals (3 males and 5 females; age, 4 months to 30 years) in whom the molecular basis of the disease could not be determined by conventional methods. A total of 44 probe pairs were used for MLPA, covering approximately 800 kb from the telomere to the MSLN gene in the 16p13.3 region. Eight deletions were detected. Four of these varied in size from 240 to 720 kb and affected a large region including the entire alpha-globin gene cluster and its upstream regulatory element (alpha-MRE), while the other four varied in size from 0.4 to 100 kb and were limited to a region containing this element. This study is the first in Brazil to use the MLPA method to determine the molecular basis of alpha-thalassemia. The variety of rearrangements identified highlights the need to investigate all cases presenting microcytosis and hypochromia, but without iron deficiency or elevated hemoglobin A2 levels and suggests that these rearrangements may be more frequent in our population than previously estimated.

Chromogenic in situ hybridization compared with other approaches to evaluate HER2/neu status in breast carcinomas

Human epidermal growth factor receptor 2 (HER2) has been evaluated in breast cancer patients to identify those most likely to benefit from herceptin-targeted therapy. HER2 amplification, detected in 20-30% of invasive breast tumors, is associated with reduced survival and metastasis. The most frequently used technique for evaluating HER2 protein status as a routine procedure is immunohistochemistry (IHC). HER2 copy number alterations have also been evaluated by fluorescence in situ hybridization (FISH) in moderate immunoexpression (IHC 2+) cases. An alternative procedure to evaluate gene amplification is chromogenic in situhybridization (CISH), which has some advantages over FISH, including the correlation between HER2 status and morphological features. Other methodologies have also been used, such as silver-enhanced in situ hybridization (SISH) and quantitative real-time RT-PCR, to determine the number of HER2 gene copies and expression, respectively. Here we will present a short and comprehensive review of the current advances concerning HER2 evaluation in human breast cancer.

Role of a GenoType MTBDRplus line probe assay in early detection of multidrug-resistant tuberculosis at a Brazilian reference center

Resistance to Mycobacterium tuberculosis is a reality worldwide, and its diagnosis continues to be difficult and time consuming. To face this challenge, the World Health Organization has recommended the use of rapid molecular tests. We evaluated the routine use (once a week) of a line probe assay (Genotype MTBDRplus) for early diagnosis of resistance and for assessment of the main related risk factors over 2 years. A total of 170 samples were tested: 15 (8.8%) were resistant, and multidrug resistance was detected in 10 (5.9%). The sensitivity profile took 3 weeks (2 weeks for culture and 1 week for rapid testing). Previous treatment for tuberculosis and the persistence of positive acid-fast smears after 4 months of supervised treatment were the major risk factors observed. The use of molecular tests enabled early diagnosis of drug-resistant bacilli and led to appropriate treatment of the disease. This information has the potential to interrupt the transmission chain of resistant M. tuberculosis.

Peak Force Kelvin Probe Force Microscopy Investigation of ZrO2 Nanocomposites for Hygroelectricity Power Element

ZrO2 nanocomposites were investigated considering their perspective application in hygroelectric power elements. Scanning probe microscopy (SPM) techniques allowed to visualize the surface topography and electrical properties. In this work was compared spacial charge behaviour of sample in humid and dry air conditions. Also different SPM modes were compared. Kelvin probe force microscopy (KPFM) was applied to characterize the spacial charge distribution on surface of the sample. Measurements showed, that trapped charge is not dissipated and can be manipulated with low voltages. Humidity influence on the electric potential of the sample was shown.

Cloning, sequencing and characterization of a chitin synthase gene fragment from the fungus Phascolomyces articulosus /

Phascolomyces articulosus genomic DNA was isolated from 48 h old hyphae and was used for amplification of a chitin synthase fragment by the polymerase chain reaction method. The primers used in the amplification corresponded to two widely conserved amino acid regions found in chitin synthases of many fimgi. Amphfication resulted in four bands (820, 900, 1000 and 1500 bp, approximately) as visualized in a 1.2% agarose gel. The lowest band (820 bp) was selected as a candidate for chitin synthase because most amplified regions from other fimgi so far exhibited similar sizes (600-750 bp). The selected fragment was extracted from the gel and cloned in the Hinc n site of pUC19. The derived plasmid and insert were designated ^\5C\9'PaCHS and PaCHS respectively. The plasmid pUC19-PaC/fS was digested by several restriction enzymes and was found to contain BamHl and HincU sites. Sequencing of PaCHS revealed two intron sequences and a total open reading frame of 200 amino acids. The derived polypeptide was compared with other related sequences from the EMBL database (Heidelberg, Germany) and was matched to 36 other fiilly or partially sequenced fimgal chitin synthase genes. The closest resemblance was with two genes (74.5% and 73.1% identity) from Rhizopus oligosporus. Southern hybridization with the cloned fragment as a probe to the PCR reaction showed a strong signal at the fragment selected for cloning and weaker signals at the other two fragments. Southern hybridization with partially digested Phascolomyces articulosus genomic DNA showed a single band. The amino acid sequence was compared with sequences from other chitin synthase gene classes using the CLUSTALW program. The chitin synthase fragment from Phascolomyces articulosus was initially grouped in class n along with chitin synthase fragments from Rhizopus oligosporus and Phycomyces blakesleeanus which also belong to the same class, Zygomycetes. Bootstrap analysis using the neighbor-joining method available by CLUSTALW verified such classification. Comparison of PaCHS revealed conservation of intron positions that are characteristic of chitin synthase gene fragments of zygomycetous fungi.

Cloning of actin genes from a genomic library of the newt, Notophthalmus viridescens

The regenerating urodele limb is a useful model system in which to study, in vivo, the controls of cell proliferation and differentiation. Techniques are available which enable one to experimentally manipulate mitogenic influences upon the blastema, as well the morphogenesis of the regenerating 11mb. Although classical regeneration studies have generated a wealth of knowledge concerning tissue interactions, little 1s known about the process at the level of gene expression. The aim of this project was to clone potentially developmentally regulated genes from a newt genomic library for use in future studies of gene expression during limb regeneration. We decided to clone the cytoskeletal actin gene for the following reasons: 1. its expression reflects the proliferative and differentiatlve states of cells in other systems 2. the high copy number of cytoplasmic actin pseudogenes in other vertebrates and the high degree of evolutionary sequence conservation among actin genes increased the chance of cloning one of the newt cytoplasmic actin genes. 3. Preliminary experiments indicated that a newt actin could probably be identified using an available chick ~-actln gene for a molecular probe. Two independent recombinant phage clones, containing actin homologous inserts, were isolated from a newt genomic library by hybridization with the chick actin probe. Restriction mapping identified actin homologous sequences within the newt DNA inserts which were subcloned into the plasmid pTZ19R. The recombinant plasmids were transformed into the Escherichia coli strain, DHsa. Detailed restriction maps were produced of the 5.7Kb and 3.1Kb newt DNA inserts in the plasmids, designated pTNAl and pTNA2. The short «1.3 Kb) length of the actin homologous sequence in pTNA2 indicated that it was possibly a reverse transcript pseudogene. Problems associated with molecular cloning of DNA sequences from N. viridescens are discussed with respect to the large genome size and abundant highly repetitive DNA sequences.

Selected topics in fast atom bombardment (FAB) mass spectrometry a)specially designed probe tips and ion generation, b)structures of vitamin B6 schiff base complexes

This thesis can be broken down into two sections. Section one is a study . of the ionization mechanisms and the ion source optimization for Fast Atom Bombardment (FAB) ionization. For this study, several specially designed probe tips were created and tested under various experimental conditions. The aIm of this section is to understand the operating characteristics of a FAB IOn source better. The second section involves the study of several Vitamin B6 Schiff Base complexes using both positive and negative ion FAB MS. This section is an exploration of the usefulness of FAB MS as a structure probe for the metalcoordination complexes of Vitamin B6.

Localization of monoterpenoid indole alkaloid (MIA) biosynthesis by in situ hybridization (ISH)

Monoterpenoid indole alkaloids (MIA) are among the largest and most complex group of nitrogen containing secondary metabolites that are characteristic of the Apocynaceae plant family including the most notable Catharanthus roseus. These compounds have demonstrated activity as successful drugs for treating various cancers, neurological disorders and cardiovascular conditions. Due to the low yields of these compounds and high pharmacological value, their biosynthesis is a major topic of study. Previous work highlighting the leaf epidermis and leaf surface as a highly active area in MIA biosynthesis and MIA accumulation has made the epidermis a major focus of this thesis. This thesis provides an in-depth analysis of the valuable technique of RNA in situ hybridization (ISH) and demonstrates the application of the technique to analyze the location of the biosynthetic steps involved in the production of MIAs. The work presented in this thesis demonstrates that most of the MIAs of Eurasian Vinca minor, African Tabernaemontana e/egans and five Amsonia species, including North American Amsonia hubrichitii and Mediterranean A. orienta/is, accumulate in leaf wax exudates, while the rest of the leaf is almost devoid of alkaloids. Biochemical studies on Vinca minor displayed high tryptophan decarboxylase (TOe) enzyme activity and protein expression in the leaf epidermis compared to whole leaves. ISH studies aimed at localizing TOe and strictosidine synthase suggest the upper and lower epidermis of V. minor and T. e/egans as probable significant production sites for MIAs that will accumulate on the leaf surface, however the results don't eliminate the possibility of the involvement of other cell types. The monoterpenoid precursor to all MIAs, secologanin, is produced through the MEP pathway occurring in two cell types, the IPAP cells (Gl0H) and epidermal cells (LAMT and SLS). The work presented in this thesis, localizes a novel enzymatic step, UDPG-7-deoxyloganetic acid glucosyltransferase (UGT8) to the IPAP cells of Catharanthus longifolius. These results enable the suggestion that all steps from Gl0H up to and including UGT8 occur in the IPAP cells of the leaf, making the IPAP cells the main site for the majority of secologanin biosynthesis. It also makes the IPAP cells a likely cell type to begin searching for the gene of the uncharacterized steps between Gl0H and UGT8. It also narrows the compound to be transported from the IPAP cells to either 7-deoxyloganic acid or loganic acid, which aids in the identification of the transportation mechanism.

Probe-level linear model fitting and mixture modeling results in high accuracy detection of differential gene expression

Affiliation: Institut de recherche en immunologie et en cancérologie, Université de Montréal

Évaluation de la fréquence des micronoyaux et du potentiel clastogène et/ou aneugène du benzo-a-pyrène suite à une exposition in vitro des lymphocytes humains

Le benzo-a-pyrène (BaP) est un cancérogène reconnu pour l'homme, contaminant présent dans notre environnement. Il cause des dommages à l'ADN que nous avons mesurés dans les lymphocytes exposés à de faibles concentrations de BaP, provenant de 20 jeunes volontaires non fumeurs et en santé. Suite à l’exposition, la fréquence des micronoyaux (MN) augmente significativement et décrit une courbe dose-réponse non linéaire, suggérant le déclenchement du processus de détoxification et la réparation de l’ADN. Des différences entre les individus et entre les sexes sont présentes dans la réponse génotoxique produite par le BaP. Le test des aberrations chromosomiques montre que le pourcentage de chromosomes cassés augmente significativement dans les cellules exposées au BaP. Combinés avec l'augmentation de la fréquence des MN, nos résultats confirment l'effet clastogène du BaP déjà rapporté dans la littérature. L’hybridation in situ en fluorescence (FISH) des MN avec une sonde pancentromérique est aussi utilisée pour établir leur mécanisme de formation. La FISH révèle que la majorité des MN formés après une exposition au BaP contient un centromère et plus, ce qui est significativement différent de la condition non exposée. Plus précisément, dans nos conditions expérimentales, les MN induits par le BaP contiennent surtout trois centromères et plus, indiquant également la présence d'un effet aneugène. L'effet clastogène du BaP est relié à son rôle d'initiateur dans la cancérogenèse, alors que l'effet aneugène le relierait à l'étape de progression. Ces résultats sont importants puisque l'exposition aux composés de la classe du BaP est de longue durée (cigarette, air pollué).

Étude de prévalence et associations des gènes de virulence et résistance aux antimicrobiens d’Escherichia coli de la flore intestinale du poulet sain

Les Escherichia coli pathogènes de la volaille (APEC) font partie des E. coli extra-intestinaux pathogènes (ExPEC) et seraient un réservoir possible de gènes de virulence et de résistance aux antimicrobiens (RAM) des ExPEC chez l’humain. L’objectif de cette étude était d’évaluer l’effet d’un prébiotique et d’un mélange d’acide organique et d’huiles essentielles encapsulés sur la prévalence des gènes de virulence des ExPEC et de RAM, ainsi que les associations entre ces gènes chez E. coli de l’intestin du poulet sain. Des échantillons de contenus caecaux de poulets de 29 jours d’âge ayant reçu un de ces ingrédients alimentaires comparativement à des témoins ont été analysés pour la présence des gènes de virulence iucD, tsh, papC et des gènes de RAM blaTEM, blaSHV, tetA, tetC, blaCMY-2, aadA1, aac3 par PCR. La prévalence d’iucD était supérieure dans le groupe témoin comparativement aux groupes «prébiotique» et «acide organique» et la prévalence de papC était affectée dans le groupe «acide organique». La prévalence d’isolats d’E.coli positifs pour blaCMY-2 était supérieure dans le groupe témoin comparée aux groupes «prébiotique» et «acide organique», tel que démontré par la technique d’hybridation de l’ADN sur HGMF (Hydrophobic Grid Membrane Filter). De plus, la prévalence des isolats d’E. coli positifs pour tetA, blaTEM, aadA1 ou tsh était affectée par les ingrédients alimentaires. Dans l’ensemble, des associations entre la présence de tsh et iucD, blaTEM et aadA1, et iucD et blaCMY-2 ont été observées. .Cette étude démontre l’utilité de certains ingrédients alimentaires pour dimunier le risque d’exposition en santé publique.

Laser Induced Fluorescence Based Optical Fiber Probe for Analyzing Bacteria

Optical fiber based laser induced fluorescence (LIF) measurements were carried out using Rhodamine B to analyze two different species of bacteria , a Gram-positive bacteria namely Bacillus smithii , and fibrin alginolvticus, a Gram- negative bacteria . The fiber sensor was clearly able to distinguish between the two species of bacteria . Quenching effect of the dye Rhodamine B by Bacillus smithii was observed . The effect of dye on the samples was also studied in detail.

Characterization of radio frequency plasma using Langmuir probe and optical emission spectroscopy

The radio frequency plasma generated during the sputtering of Indium Tin Oxide target using Argon was analyzed by Langmuir probe and optical-emission spectroscopy. The basic plasma parameters such as electron temperature and ion density were evaluated. These studies were carried out by varying the RF power from 20 to 50 W. A linear increase in ion density and an exponential decrease in electron temperature with rf power were observed. The measured plasma parameters were then correlated with the properties of ITO thin films deposited under similar plasma conditions.

Laser induced fluorescence based optical fiber probe for analyzing bacteria

Optical fiber based laser induced fluorescence (LIF) measurements were carried out using Rhodamine B to analyze two different species of bacteria , a Gram-positive bacteria namely Bacillus .cmithii , and fibrin alginolvticus, a Gram-' negative bacteria . The fiber sensor was clearly able to distinguish between the two species of bacteria . Quenching effect of the dye Rhodamine B by Bacillus smitltii was observed . The effect of dye on the samples was also studied in detail.

Studies on Laser Induced and RF Sputtered Plasma using Optical Emission Spectroscopy and Langmuir Probe

The main objective of the present study is to understand different mechanisms involved in the production and evolution of plasma by the pulsed laser ablation and radio frequency magnetron sputtering. These two methods are of particular interest, as these are well accomplished methods used for surface coatings, nanostructure fabrications and other thin film devices fabrications. Material science researchers all over the world are involved in the development of devices based on transparent conducting oxide (TCO) thin films. Our laboratory has been involved in the development of TCO devices like thin film diodes using zinc oxide (ZnO) and zinc magnesium oxide (ZnMgO), thin film transistors (TFT's) using zinc indium oxide and zinc indium tin oxide, and some electroluminescent (EL) devices by pulsed laser ablation and RF magnetron sputtering.In contrast to the extensive literature relating to pure ZnO and other thin films produced by various deposition techniques, there appears to have been relatively little effort directed towards the characterization of plasmas from which such films are produced. The knowledge of plasma dynamics corresponding to the variations in the input parameters of ablation and sputtering, with the kind of laser/magnetron used for the generation of plasma, is limited. To improve the quality of the deposited films for desired application, a sound understanding of the plume dynamics, physical and chemical properties of the species in the plume is required. Generally, there is a correlation between the plume dynamics and the structural properties of the films deposited. Thus the study of the characteristics of the plume contributes to a better understanding and control of the deposition process itself. The hydrodynamic expansion of the plume, the composition, and SIze distribution of clusters depend not only on initial conditions of plasma production but also on the ambient gas composition and pressure. The growth and deposition of the films are detennined by the thermodynamic parameters of the target material and initial conditions such as electron temperature and density of the plasma.For optimizing the deposition parameters of various films (stoichiometric or otherwise), in-situ or ex-situ monitoring of plasma plume dynamics become necessary for the purpose of repeatability and reliability. With this in mind, the plume dynamics and compositions of laser ablated and RF magnetron sputtered zinc oxide plasmas have been investigated. The plasmas studied were produced at conditions employed typically for the deposition of ZnO films by both methods. Apart from this two component ZnO plasma, a multi-component material (lead zirconium titanate) was ablated and plasma was characterized.