A poliploidia teve importante papel no melhoramento de muitas espécies de plantas, principalmente nos cereais, incluindo híbridos intergenéricos. O processo de poliploidização, geralmente, produz novos e desejáveis fenótipos ou fornece suporte para cruzamentos de plantas em programas de melhoramento. Esse trabalho apresenta o resultado da indução de poliploidia em duas cultivares de mandioca (\'Porquinho\' e \'Vassourinha\') que foram tratadas com diferentes concentrações de colchicina (0,05%, 0,10% e 0,15%), em meio de cultura, durante 48 e 96 horas. As plantas regeneradas foram analisadas citologicamente por citometria de fluxo e pela contagem do número de cromossomos. Adicionalmente, análises do tamanho e da frequência dos estômatos foram realizadas e o resultado comprovou que podem ser utilizadas como screening das plantas tratadas. O tratamento com 0,1% de colchicina, em meio de cultura, durante 96 horas apresentou maior número de plantas tetraplóides produzidas sobre o número de plantas regeneradas nas duas cultivares avaliadas. Não há diferenças entre as doses de colchicina e tempos de exposição a droga no número de tetraplóides encontrados em cada tratamento. Entretanto, foi possível verificar que existe diferença significativa na produção de tetraplóides entre as cultivares analisadas, indicando que pode haver uma resposta genética à indução da duplicação dos cromossomos com colchicina. / The polyploidy played an important role in the improvement of many species of plants, especially in cereals including intergeneric hybrids. The process of polyploidy usually produces new and desirable phenotypes or provides support for crosses of plants in breeding programs. This research work reports the result of polyploidy induction in two cassava cultivars (\'Porquinho\' and \'Vassourinha\') by different colchicine concentrations (0.05 %, 0.10 % and 0.15 %) in culture medium exposed for 48 or 96 hours. Regenerated plants were examined cytologically by flow cytometry and chromosome counting. Additionally, analysis of the size and frequency of stomata were performed and the results showed that can be used for screening of the treated plants. Treatment with 0.1% colchicine in the culture medium for 96 hours showed a higher number of tetraploid plants produced about the number of regenerated plants in both cultivars evaluated. No significant differences among colchicine doses and duration times treatment were found in the number of tetraploids found in each treatment. However, we observed a significant difference in tetraploid production between the cultivars analyzed, indicating that there may be a genetic response to induction of chromosome doubling with colchicine.

A demodicidose é uma importante dermatopatia parasitária em cães. É decorrente da proliferação excessiva de ácaros comensais do gênero Demodex sp no tegumento canino. A manifestação clínica da demodicidose juvenil generalizada têm sido associada à disfunção imune hereditária de linfócitos T específica ao parasita, enquanto que, a demodicidose do aduto pode ser consequente a doenças imunossupressoras. A resposta imune celular é considerada crucial na defesa contra o parasita e, encontra-se comprometida em cães com demodicidose. Com o escôpo de se determinar se linfócitos sanguíneos periféricos, TCD4+, TCD8+ e a razão TCD4+:TCD8+ são bons indicadores da evolução clínica da doença e do estado imune de cães demodicidose generalizada, tais parâmetros foram quantificados em 16 animais com demodicidose generalizada, na pré, trans e pós terapia, e em outros 30 animais hígidos, utilizando-se da técnica de citometria de fluxo. Para as análises estatísticas comparativas, foram padronizados quatro momentos de observação dos animais com demodicidose, a saber: primeira consulta: quando do estabelecimento do diagnóstico; segunda consulta; consulta de obtenção do primeiro exame parasitológico negativo e, finalmente, naquela de estabelecimento da alta clínica, preconizada quando da ausência de evidenciação do ácaro no exame parasitológico cutâneo, por três consultas consecutivas. Os valores absolutos médios de linfócitos totais, linfócitos TCD4+ e TCD8+ nos animais com demodicidose, mostraram-se inferiores àqueles do Grupo Controle em todos os momentos de observação. Somente os valores absolutos de linfócitos TCD4+, apresentaram diminuição significativa, em relação ao Grupo Controle, no momento da primeira consulta. Entre o Grupo Experimental, foi observado elevação signficativa entre os valores absolutos dos linfócitos totais, TCD4+ e TCD8+, entre a primeira consulta e aquela de obtenção do primeiro exame parasitológico negativo, quando estes valores se aproximaram daqueles observados no Grupo Controle. Paralelamente, evidenciou-se alta correlação da elevação dos número absoluto médio dos linfócitos TCD4+ e TCD8+, com a diminuição da contagem de ácaros. A razão TCD4+:TCD8+, não diferiu significativamente entre o Grupo Controle e Experimental. O tratamento da demodicidose não alterou a razão TCD4+: TCD8+. Não foi observado correlação entre o período necessário para o estabelecimento da alta clínica e a razão TCD4+:TCD8+. O comportamento dos linfócitos sanguíneos periféricos TCD4+, TCD8+, e da razão TCD4+:TCD8+, demonstra que a participação de outros mecanismos, que não a franca alteração destas subpopulações, sejam importantes na patogenia da doença. Em linhas gerais, não foram observadas diferenças significativas, nos valores dos linfócitos TCD4+, TCD8+ e a razão TCD4+:TCD8+, entre os animais do Grupo Controle, e os animais com demodicidose generalizada , de forma que, o uso da determinação destes parâmetros, é desaconselhado no monitoramento da evolução clínica e no estabelecimento do prognóstico, nos animais com demodicidose generalizada. / Demodicosis is a serious canine parasitic skin disease. It is caused by the presence of increasead amounts of Demodex mite in the skin. Clinical signs of juvenile-onset generalized demodicosis are associated with specific hereditary dysfunction of T lymphocytes while adult-onset can be induced by immunosuppressive diseases. The cellular immunity is crucial in keeping low numbers of skin mites and it is depressed in dogs with generalized demodicosis. The aim of this study was to verify whether CD4+, CD8+ T-lymphocytes counts and CD4+:CD8+ ratio could be good indicators of disease progression and immune status in canine demodicosis. For this, using the flow cytometry technique, the CD4+, CD8+ T-lymphocytes counts and CD4+:CD8+ ratio of 16 dogs with generalized demodicosis were evaluated at four moments: first and second consultation, first time animal was presented without mites in skin scrapings and finally, on clinical improvement. These values were then compared with those of 30 controls healthy dogs. The absolute numbers of CD4+, CD8+ and total lymphocytes were lower than control healthy dogs at all moments of analysis. Only at the first consultation CD4+ lymphocyte counts was significant lower than control group. Dogs with generalized demodicosis had signicant increased counts of CD4+, CD8+ and total lymphocytes from the first consultation until the first negative skin scraping. At this point lymphocyte counts reached levels closesth to control group ones. CD4 : CD8+ ratio didn´t differ throughout treatment of canine demodicosis neither when average level for ill dogs were compared to with those healthy ones. Furthermore CD4+, CD8+ and CD4:CD8+ ratio didn´t correlated with time taken for successfull treatment completion and so they couldn´t be used as prognosis predictor. A high correlation between increased of CD4+, CD8+ T-lymphocytes counts and decreased mites counts was observed in dogs with generalized demodicosis. Circulating lymphocyte subpopulations are therefore similar in dogs with canine demodicosis and healthy dogs and there is no correlation between clinical status or response to therapy and the lymphocytes subpopulations counts. We can than conclude that CD4+, CD8+ T-lymphocytes counts and CD4+:CD8+ ratio cannot be used as a parameters to predict progression of an individual patient in a clinical context.

La plupart des virus sont connus pour leur capacité à causer des maladies symptomatiques chez l’Homme et chez les autres animaux. Certains d’entre eux sont des grands virus à ADN nommés Virus à Grand ADN Nucléo-Cytoplasmique (NCLDV), rapportés comme infectant les cellules eucaryotiques. Au début du XXI ème siècle, quatre familles ont été définies par Iyer et al. comme ayant une origine commune (groupe monophylétique) : Asfarviridae, Phycodnaviridae, Irido-Ascoviridae et Poxviridae.En 2003, la description d’Acanthamoeba polyphaga mimivirus a cassé un paradigme dans le monde des virus. Par leur taille de particule (450nm), par leur longueur de génomes(supérieure à 1Mb) et leur contenu génique, leur découverte a changé la définition traditionnelle des virus (Lwoff). Depuis 2013 et notamment par les isolements successifs de Pandoravirus,Pithovirus et Mollivirus, ces virus ont été décrits comme possédant de nouvelles propriétés.Leur découverte a été rendue possible grâce à la méthode de co-culture utilisant des protistes, notamment des cellules du genre Acanthamoeba. Cette méthode a été de nombreuses fois modulée par différentes équipes. Dans notre cas, nous avons combiné différentes stratégies appliquées à notre co-culture : la co-culture a été couplée à la cytométrie en flux pour détecter la lyse des protistes. De plus, la cytométrie a été utilisée avec un marqueur à ADN dans le but d’identifier de façon putative le virus et de discriminer les différentes populations virales. Enfin,nous sommes capables de séparer ces populations en utilisant un appareil FACS trieur.L’ensemble de ces techniques a permis l’isolement de nouveaux virus. / Most viruses are known for their ability to cause symptomatic diseases in humans andother animals. Some of them are large DNA viruses named Nucleo-cytoplasmic Large DNAviruses (NCLDV), known for infecting eukaryotic cells. At the beginning of the 21st centuryfour families were defined by Iyer et al. as having a common origin (monophyletic group):Asfarviridae, Phycodnaviridae, Irido-Ascoviridae and Poxviridae.In 2003, the description of Acanthamoeba polyphaga Mimivirus broke this paradigmin the virus world. Because of their particles size (450 nm), their genome size (up to 1Mb),and their gene contents, their discovery changed the traditional definition of viruses (Lwoff).Since 2013 and the successive isolations of Pandoravirus, Pithovirus and Mollivirus; theseviruses have been characterized as possessing various novel properties.Their discoveries have been possible thanks to the co-culture method using protistnotably Acanthamoeba genus cells. This method went through multiple improvements and isemployed by different teams in different ways. In our case and in order to enhance thismethod we combined strategies applied in our co-culture. Indeed, this method consists inusing flow cytometry to detect lysis of protist cells (after all steps of co-culture enrichment).In addition, the flow cytometry was used with a DNA marker in order to identity viruses anddiscriminate viral populations. Then, we were able, using a FACS sorter device, to separatedifferent viral populations from our supernatants.Altogether these techniques have permitted the isolation of new viruses.

acase@tulane.edu

Photonic crystals serve as powerful building blocks for the development of lab-on-chip devices. Currently they are used for a wide range of miniaturized optical components such as extremely compact waveguides to refractive-index based optical sensors. Here we propose a new technique for analyzing and characterizing cells through the design of a micro-flow cytometer using photonic crystals. While lab scale flow cytometers have been critical to many developments in cellular biology they are not portable, difficult to use and relatively expensive. By making a miniature sensor capable of replicating the same functionality as the large scale units with photonic crystals, we hope to produce a device that can be easily integrated into a lab-on-chip and inexpensively mass produced for use outside of the lab. Using specialized FDTD software, the proposed technique has been studied, and multiple important flow cytometry functions have been established. As individual cells flow near the crystal surface, transmission of light through the photonic crystal is influenced accordingly. By analyzing the resulting changes in transmission, information such as cell counting and shape characterization have been demonstrated. Furthermore, correlations for simultaneously determining the size and refractive indices of cells has been shown by applying the statistical concepts of central moments.

This research studied recombinant DNA-derived protein expression utilising expression vectors containing IRES sequences to link the gene of interest with the gene encoding selectable marker in mammalian cell cultures. Polycistronic expression constructs utilising internal ribosome entry site (IRES) can link unrelated genes under control of a single promoter. Transient study on the IRESlinked gene expression was performed. It was possible to standardise the level of protein expression to plasmid number by determining the number of free plasmids in the cytoplasm. The expression of a selectable marker when downstream of IRES was reduced in comparison to the monocistronic construct. Importantly when IRES was used, there were no negative effects on recombinant gene expression upstream of IRES. Down-regulating the selectable marker gene expression has been shown to enhance the probability of obtaining highly expressing clones. To investigate the effects of down-regulating fusion metallothionein green fluorescent protein (MTGFP), new constructs were created to combine metal inducible M2.6 promoter to drive the expression of human growth hormone linked to MTGFP by an attenuated IRES. This resulted in less MTGFP expression, reduced survivability and mean fluorescence in the presence of heavy metal. The increased metal sensitivity lengthened the initial selection period using reduced metal concentration in comparison to cells transfected with wildtype MTGFP. FACS can be used to select for resistance conferred by MTGFP despite reduced expression. FACS enrichment and sorting increased the hGH expression, which was correlated with mean fluorescence of the population; therefore fluorescence can be used as an indication of the final recombinant protein expression. Different approaches to isolate suitable clones were also investigated. It is preferable to select the transfected pool in low metal concentration for two weeks, sort for cells of high-fluorescence, and allow for recovery and proliferation. It is then possible to amplify gene expression by culturing the clones in increasing metal, resulting in further improvement of recombinant protein expression.

Despite high remission rate in acute myeloid leukaemia (AML) after chemotherapy, relapse of the underlying disease remains a major challenge and one of the most frequent causes of treatment failure. In this study, the presence of leukaemiaassociated phenotypes (LAPs) was first studied retrospectively using our standard diagnostic protocol with 3-colour flow cytometry. LAPs were present in 54 (64%) of 84 AML patients analysed between 2002 to 2004. The presence of LAPs was correlated with failure to respond to induction chemotherapy (p <0.05) in univariate analysis. Presence of LAPs was shown to be an independent predictor for failure to respond to induction chemotherapy with a relative risk ratio of 1.6 (p < 0.05, 95% CI, 1.0-2.6) in multivariate analysis. Subsequently, in a prospective study, we used 5-colour multiparametric flow cytometry (MFC) for detection of LAPs to determine if LAPs could be detected in a greater proportion of leukaemic patients and minimal residual disease (MRD) detection could therefore be applied in more patients. In 54 consecutive, newly diagnosed AML patients from 2005 to 2007, LAPs were identified in 51 (94%). Thus, MRD studies were potentially applicable to virtually all patients. The sensitivity and specificity of MFC technique was improved by analysing 10 normal and 5 regenerating bone marrows (BM) for the presence of these LAPs and by determining maximum log difference (LD). CD7, CD19, CD2, CD11b and CD56 were the most sensitive and reliable markers for MRD studies. LAPs were rarely detected in either normal or regenerating BMs. Through dilutional experiments from 50% LAPs to 0.001%, it was determined that 1 leukaemic in 104 and 105 normal cells could be detected using the improved techniques. Of the 54 patients, 31 received chemotherapy, with 27 achieving complete remission (CR). Two were LAP negative and thus 25 were evaluable for MRD post induction and 22-post consolidation chemotherapy. Detection of MRD >0.15% was able to distinguish between two groups of patients according to relapse status. Although, the number of patients was small, detection of MRD post induction > 0.15% was shown to be an independent predictor of adverse prognosis for both relapse free survival (RFS) and overall survival (OS) in a multivariate analysis [p = 0.037 and 0.026, 95% CI (1.1-20.5 and 1.2-22.2), hazard ratio 4.7 and 5.2 respectively]. Post consolidation, there was a trend for patients with higher MRD values to show shorter RFS (p = 0.06). MFC using 5-colour allows us to detect LAPs in virtually all AML patients and our preliminary results suggest the technique is a suitable approach for MRD analysis. However, 5-colour MFC is technically challenging, resource intensive, and may not be feasible in a routine diagnostic laboratory. This led us to assess whether we could identify other potential markers for LAPs. Interleukin-3 alpha receptor- chain IL-3_ (CD123) has been suggested to be a marker of leukaemic stem cells (LSC). These cells are thought to be responsible for initiating and maintaining leukaemic cell growth post chemotherapy and hence to give rise to relapse of the disease. Therefore, we analysed 34 AML patients for expression of CD123 in the blast population and defined a population containing leukaemic stem cells using the immunophenotypic markers CD123+/CD34+/CD38-. Thirty-two (94%) of AML patients expressed CD123. We then used a molecular marker to determine whether CD123 expression was confined to the LSC. Thirtynine patients were screened for the presence of FMS-like tyrosine kinase 3 - internal tandem duplication (FLT3/ITD) as the most common molecular abnormality in AML patients. Of those, 12 (31%) were FLT3/ITD positive. In seven of them, CD34+/CD38-/CD123+ and CD34+/CD38-/CD123- populations were sorted to homogeneity by Fluorescence Activated Cell Sorting (BD FACSAriaTM Cell Sorter) and tested for FLT3/ITD. In six of seven patients with FLT3/ITD positive AML, we could not detect the mutation in the CD34+/CD38-/CD123- fraction, but the mutation was detected in the CD34+/CD38-/CD123+ fraction in all seven patients. This novel finding demonstrates that, the oncogenic event occurs in CD123 positive cells, thus supporting the concept that CD123 is a marker of the LSC in CD123 positive AML. This observation suggests novel treatment approaches employing surface marker CD123-targeting antibodies may be of use in the treatment of AML. In conclusion, we demonstrate that using five-colour MFC improves LAP detection in AML and enables MRD studies using immunophenotyping to be applied to virtually all AML patients. Additionally, it increases the sensitivity of the technique for detecting LAP populations. Moreover, evaluation of MRD post induction chemotherapy is the most sensitive time point for detection of MRD, with MRD levels >0.15% predicting relapse and worse prognosis. As an alternative to using individualised LAPs specific to each patient, CD34+/CD38-/CD123+ cells may in the future serve as a better marker for MRD studies. This marker identifies the putative LSC, which is responsible for regrowth of leukaemia and relapse of the disease. Thus, instead of looking at whole “blast” population which results in huge data analysis and interpretation for the different LAPs which may have different underlying biology, it may be more informative to look at the frequency of LSC after achieving CR using CD34+/CD38-/CD123+ as the single LAP for MRD studies. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1317088 / Thesis (Ph.D.) -- University of Adelaide, School of Medicine, 2008

<p>Transition metals such as nickel (Ni), cobalt (Co), palladium (Pd), chromium (Cr) and gold (Au) are widely used as alloys in jewelry and biomaterials such as orthodontic and orthopaedic appliances. These metals also cause cell-mediated allergic contact dermatitis (ACD) reactions in a significant proportion of the population upon prolonged direct exposure. The immune mechanisms underlying the response to these metals are not yet well defined. In the studies described in this thesis we therefore investigated the profile of cytokine responses to various metal ions <i>in vitro</i> and the relationship with the ACD reaction <i>in vivo</i>. In the first study, we investigated the relationship between the profile and magnitude of Ni²⁺-induced cytokine responses <i>in vitro</i> and the degree of <i>in vivo</i> reactivity to Ni²⁺. PBMC from Ni²⁺-reactive (ACD) and non-reactive control subjects were cultured with or without NiCl₂. The numbers of IL-4-, IL-5- and IL-13-producing cells and the concentrations of IFN-γ, IL-10 and IL-13 produced were analysed by ELISpot and ELISA respectively. Ni²⁺ elicited a mixed Th1- and Th2-type cytokine profile in PBMC from ACD subjects with a positive correlation observed between the levels of the elicited cytokines and the degree of patch test reactivity. Hence, suggesting an involvement of both Th1- and Th2-type cytokines in ACD to Ni²⁺ and a direct association between the magnitude of the Ni²⁺-induced cytokine response overall and the <i>in vivo</i> reactivity to Ni²⁺. The impact of the regulatory cytokine IL-10 on Ni²⁺-induced Th1- and Th2-type cytokine responses in human PBMC was investigated in the next study. PBMC from blood donors with a history of Ni²⁺ reactivity and non-reactive control donors were stimulated with Ni²⁺ <i>ex vivo</i> with or without addition of human recombinant IL-10 (rIL-10) or neutralising mAb to IL-10. Depletion/enrichment experiments were performed to phenotype the Ni²⁺-specific cytokine producing cells. Exogenous rIL-10 significantly down-regulated the production of all cytokines but with a more pronounced effect on IFN-γ. IL-10 neutralisation, on the other hand, enhanced the levels of Ni²⁺-induced IFN-γ only. Ni²⁺-specific cytokine-producing cells in PBMC were found to be predominantly CD4⁺ T cells. Thus, IL-10 may play a regulatory role <i>in vivo</i> by counteracting the ACD reactions mediated by CD4⁺ T cells producing Th1-type cytokines. In the third study, we investigated the relationship between <i>in vivo</i> patch test reactivity to a number of metals (Ni, Co, Pd, Cr and Au) included in the standard and/or dental patch test series and <i>in vitro</i> responses to the metals in question. PBMC from metal patch test positive and negative control subjects were stimulated with a panel of eight metal salts and cytokine responses analysed by ELISpot and/or ELISA. A mixed Th1- (IL-2 and/or IFN-γ) and Th2-type (IL-4 and/or IL-13) cytokine profile was observed in PBMC from most metal allergic subjects upon <i>in vitro</i> stimulation with the metal(s) to which the subject was patch test positive. Our data suggest that other metals included in the standard and dental patch test series, just like Ni2⁺, induce a mixed Th1- and Th2-type cytokine profile in PBMC from ACD subjects <i>in vitro</i>. We further developed a simplified ELISpot protocol utilising plates precoated with capture monoclonal antibodies (mAb) and subsequent detection in one step using enzyme-labelled mAb, for enumerating the frequency of allergen (Ni²⁺)-specific cytokine producing cells. This was compared with a regular ELISpot protocol, with an overnight incubation for capture mAb adsorbtion and detection with biotinylated mAb followed by enzyme-labelled streptavidin. PBMC from Ni²⁺-reactive and non-reactive subjects were incubated with or without NiCl₂ and the enumeration of cells producing IFN-γ, IL-4 or IL-13 by the two protocols were compared. PBMC from Ni²⁺-reactive subjects showed significantly higher Ni²⁺-induced IL-4 and IL-13 responses and the number of antigen-specific cytokine-producing cells determined by the two ELISpot protocols correlated well. In a nutshell, our data point to the potential use of <i>in vitro</i> cytokine assays as diagnostic tools in distinguishing ACD subjects sensitised to different metals and non-sensitised subjects.</p>

<p>Today the immunosuppressive treatment is kept to a level were the incidence of acute rejection is below 20% within the first year after transplantation. As a consequence, a group of transplanted patients is over-immunosuppressed and at risk for infections. There is therefore an urgent need for tools which are able to determine the cellular immune response after organ transplantation. This knowledge would facilitate the task of prospectively opimize the immunosuppressive treatment and identify patients at risk of developing rejection episodes or infections.</p><p>To address this issue, a rat-kidney transplantation model for acute rejection was developed to study immune responses to allografts. Infiltrating lymphocytes were analysed using an in vitro culture system which allowed cells to propagate from the biopsies to culture medium. The numbers of outgrowing cells were correlated with morphological and immunohistochemical signs of rejection. When immunosuppressive treatment was administered for 2 and four days after acute rejection, histology did not reveal any improvement, however cellular propagation was reduced by 50 and 75%, respectively. Using the tissue culture technique in human transplanted kidney grafts, which was originally developed for the animal model, the number of propagated cells measured was profoundly higher in grafts with acute cellular rejection than from grafts in other groups. In some cases the number of propagated cells was better correlated with the clinical outcome than the diagnosis made by morphological evaluation. To determine immune responses to cytomegalovirus (CMV), we utilised Human Leukocyte Antigen (HLA) tetramer staining and stimulation of T cells with viral antigens. Both of these techniques independently detected CMV specific T cells in immunosuppressed and healthy individuals with latent or active infection. Although the frequency of CMV specific T cells did not differ between groups, there was a functional impairment in immunosuppressed patients as evidenced by reduced interferon-gamma production. In conclusion, these techniques can be used to determine the cellular immune response to allografts and cytomegalovirus and prove valuable for the optimization of immunosuppressive protocols and antiviral treatment. </p>

Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2009.