The Caspian region and specifically the Apsheron peninsula of Azerbaijan is known to be polluted with a variety of environmental contaminants, making risk assessment difficult. The wetlands of Sumgayit contain particularly complex mixtures of contaminants. Flow cytometry and the micronucleus assay were used to assess chromosomal damage in aquatic turtles and frogs inhabiting contaminated wetlands in Azerbaijan. By evaluating biomarkers that are indicative of somatic effects, elevated chromosomal damage was documented at several sites in Azerbaijan relative to reference sites. Sediment samples were analyzed for polycyclic aromatic hydrocarbons (PAHs), organochlorines (OCs), and mercury to evaluate contaminant associations with genetic damage. Sediment samples revealed heterogeneous patterns of PAH and mercury concentrations throughout Sumgayit. Significant positive correlations were documented between both PAH and mercury sediment concentrations and chromosomal damage. Population genetic methods were employed to study the effects of long-term chronic contaminant exposure in marsh frogs from Sumgayit. The Sumgayit region has reduced levels of genetic diversity, likely due to environmental degradation. One of the most contaminated sites in Sumgayit, WTP, appears to be a source of new mutations as a result of an increased mutation rate. Finally, the Sumgayit region seems to act as an ecological sink, with levels of gene flow into the region exceeding gene flow out of the region. This study provides not only exposure and biomarker data, but also an integrated method for assessing the cumulative population impacts of contaminant exposure by studying both population genetic and evolutionary effects. The results presented here will be used in conjunction with those of ongoing research involving both wildlife and humans to develop comprehensive ecological and human risk assessments.

Regulation of reproduction is one central feature of social life. In particular, only a few individuals are in charge of producing offspring in eusocial species. This division of the reproductive labour is mainly mediated by pheromones emitted by the queens in social insects. These queen pheromones may signal the presence of a fertile queen so that workers react accordingly by taking care of her and not reproducing. Here I investigated two aspects of the reproduction of two polygynous ant species. The first one, Linepithema humile, is a unicolonial, highly polygynous and invasive species. It has been the focus of numerous studies about queen pheromones; in particular, it has been reported that queen cuticular hydrocarbons (CHC) profile is related to queen fertility. The other one, Tapinoma erraticum, is a multi-colonial, weakly polygynous and native species. Workers can lay haploid eggs in the absence of the queens which is impossible for Linepithema workers. The major part of my thesis dealt with the queen fertility signalling issue. In the first two chapters I demonstrate the link between queen fertility and queen pheromone output. I first study a queen releaser pheromone, the queen retrieval behaviour. This behaviour is performed by the workers who lay a chemical trail toward a queen located outside the nest. I successfully show this behaviour to be related to queen fertility, and not mating status, in L. humile and T. erraticum since only fertile queens (mated or not) induce such recruitment. I then highlight the role of queen fertility in the prevention of worker reproduction in T. erraticum. Again, mated fertile queens and unmated fertile queens are both able to induce such primer effect. In a third chapter I report that CHC profiles may discriminate female castes (workers, queens, virgin queens, and virgin egg-laying queens) in T. erraticum. Finally, chapter 4 summarizes my attempts to prove that CHC may be involved in queen retrieval or queen attraction. None of the various bioassays tested allows me to demonstrate the putative role of CHC as queen pheromone. In a second part, I was interested in the consequences of sex determination in T. erraticum. Chapter 5 presents the flow cytometry methodology (FC) which allowed me to score the number of sperm cells from spermathecae of several ant species, and to demonstrate that polygynous species (such as L. humile and T. erraticum) store less sperm than monogynous ones. FCM also allows determining the ploidy of sperm and adult somatic cells and chapter 6 which presents a large survey on the ploidy level within the species T. erraticum. This species displays diploid males that may produce diploid sperm which in turn can father a viable triploid female progeny. I report differences in the frequency of triploidy among female castes, the proportion of triploid workers being more important than triploid virgin queens whereas I never observed triploid mated fertile queens. Such results greatly suggest a putative regulatory mechanism involved in the rearing of triploid females. In the last chapter I investigated two populations that differ in the occurrence of triploid workers. I report these populations to vary in the number of queens and workers per nest. /La régulation de la reproduction est un aspect essentiel de la vie sociale. En particulier, chez les espèces eusociales, seuls quelques individus sont impliqués dans la production de la descendance. Dans les sociétés d’insectes, une telle division du travail reproducteur est principalement assurée par l’émission de phéromones par les reines. Ces phéromones royales renseignent les membres de la colonie sur la présence d’une reine fertile, de telle sorte que les ouvrières réagissent en s’occupant d’elle et en s’abstenant de se reproduire. Au cours de ce travail, je me suis intéressé à deux aspects de la reproduction au sein de deux espèces de fourmis polygynes. La première espèce, Linepithema humile, est invasive, unicoloniale et hautement polygyne dans les régions à climat méditerranéen. Elle a fait l’objet de nombreuses études portant notamment sur les phéromones royales. En particulier, il a été montré que le profil d’hydrocarbures cuticulaires (HCC) des reines est corrélé à leur fertilité. La seconde espèce, Tapinoma erraticum, est une espèce indigène, multi-coloniale et faiblement polygyne. Ses ouvrières sont capables de pondre des œufs mâles en absence de reines, ce dont sont incapables les ouvrières de Linepithema. Je me suis principalement intéressé à la question de la signalisation de la fertilité des reines. Dans les deux premiers chapitres, je démontre le lien existant entre la fertilité des reines et la production de phéromone royale. J’ai d’abord étudié une phéromone incitatrice (releaser) qui provoque un recrutement royal. Ce comportement collectif très caractéristique correspond à la mise en place d’une piste chimique en direction d’une reine découverte par les ouvrières en dehors du nid. Je montre que ce comportement est lié à la fertilité de la reine chez les espèces L. humile et T. erraticum car seules les reines fertiles (fécondées ou non) sont capables de d’induire le recrutement royal. Je mets ensuite en évidence le rôle de la fertilité des reines dans la régulation de la reproduction des ouvrières de T. erraticum. A nouveau, les reines fécondées fertiles et les reines vierges fertiles sont toutes deux capables d’induire un même effet déclencheur (primer), en l’occurrence, l’inhibition de la reproduction des ouvrières. Dans un troisième chapitre, je montre que les profils d’hydrocarbures (HCC) permettent de distinguer sans ambiguïté les différentes castes femelles (ouvrières, reines fertiles, reines vierges et reines vierges pondeuses) chez T. erraticum. Enfin, le chapitre 4 résume mes tentatives pour démontrer le rôle des HCC dans les phénomènes de recrutement royal ou d’attractivité des reines. Aucun des bio-essais réalisés ne me permet de démontrer l’implication des HCC dans la phéromone royale. Dans une seconde partie, je me suis intéressé aux conséquences du déterminisme du sexe chez T. erraticum. Le chapitre 5 présente cytométrie de flux (CF), une méthode qui me permet de compter les spermatozoïdes stockés dans les spermathèques de quelques espèces de fourmis et de montrer que les reines des espèces polygynes (telles que L. humile et T. erraticum) stockent moins de sperme que les espèces monogynes. La CF permet aussi de déterminer le niveau de ploïdie des cellules spermatiques ou somatiques chez l’adulte. Je me sers de cette application dans le chapitre 6 afin d’étudier le niveau de ploïdie au sein de l’espèce T. erraticum. Je montre que, dans les populations étudiées, il existe des mâles diploïdes et que ces mâles peuvent produire du sperme diploïde fertile, capable d’engendrer une descendance femelle triploïde. Je note des différences dans la fréquence des femelles triploïdes : la proportion d’ouvrières triploïdes est significativement plus importante que celle des reines vierges triploïdes. De plus, je n’ai jamais observé la présence de reines fécondées fertiles triploïdes. De tels résultats suggèrent fortement la présence d’un phénomène de régulation au cours de l’élevage du couvain triploïde. Dans le dernier chapitre, j’ai étudié deux populations de T. erraticum qui diffèrent au niveau de la proportion d’ouvrières triploïdes. Ces populations présentent des différences significatives dans le nombre de reines et d’ouvrières par nid.

La microcirculation est rapidement altérée dans le sepsis et la persistance de ces altérations est associée à un mauvais pronostic. La microcirculation est composée de vaisseaux invisibles à l’œil (< 100 µm), de l’endothélium, du glycocalyx, des cellules musculaires lisses et des éléments sanguins dont les GR. De nombreuses études animales et humaines ont rapporté des altérations rhéologiques des GR dans le sepsis. Ces modifications comprennent une diminution de la déformabilité, une augmentation de l’agrégation et de l’adhérence globulaire. De plus, l’altération de la déformabilité peut induire des altérations du flux microcirculatoire dans des modèles expérimentaux animaux. Ces mêmes altérations rhéologiques sont rapportées dans le diabète. Dans cette pathologie, les GR présentent une diminution du contenu membranaire en AS, comme dans les processus de sénescence. La déformabilité des GR dépend des caractéristiques cellulaires incluant surtout les propriétés de la membrane, la géométrie cellulaire et dans une moindre mesure la viscosité cellulaire. Malgré la connaissance des altérations de la rhéologie dans le sepsis, peu de travaux, au contraire du diabète, s’interessent aux modifications de la membrane. Nous avons étudié, par analogie aux altérations globulaires rapportées dans le diabète, la membrane des GR de patients admis en soins intensifs pour un sepsis, et comparé à des GR de patients non septiques et de volontaires sains. Le contenu membranaire en AS était significativement diminué chez les patients septiques par rapport aux patients non-septiques et aux volontaires sains. De plus, les GR des patients septiques, analysés par une technique de cytométrie en flux indépendante de la température de l’échantillon, étaient rapidement plus sphériques (dans les 24 heures du sepsis) et incapables de modifier leurs formes en hypoosmolalité. Cette technique de cytométrie a par ailleurs aussi été utilisée pour l’analyse de GR de patients diabétiques et en insuffisance rénale terminale. La diminution du contenu en AS est aussi rapidement observée sur la transferrine, suggérant une augmentation de la concentration et/ou de l’activité de la neuraminidase, enzyme clivant l’AS. Dans un modèle de choc septique induit chez l’ovin, nous avons confirmé la rapidité de ce phénomène. En effet, la concentration en AS libre augmente dès la 15ième heure après induction du sepsis. In-vitro, nous avons pu reproduire les modifications de forme des GR observés chez les patients septiques par incubation de GR de volontaire avec de la neuraminidase, et ce en 10 heures, quelles que soient les concentrations utilisées. Ces modifications de forme et de membrane s’accompagnent d’une augmentation significative du contenu en lactate, suggérant une stimulation de la glycolyse érythrocytaire et en 2,3-DPG, facilitant la libération de l’O2 de l’Hb vers les tissus. Toutes ces modifications touchant la membrane des GR des patients de soins intensifs, surtout septiques, peuvent être responsables des altérations de rhéologie que nous avons observé grâce au LORCA sur une large population admis aux soins intensifs. Une meilleure compréhension des mécanismes conduisant aux altérations rhéologiques des GR dans le sepsis, et ses effets potentiellement déletères sur la microcirculation, sont nécessaires avant d’envisager les GR comme cible thérapeutique.

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. 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.

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 in vitro and the relationship with the ACD reaction in vivo. In the first study, we investigated the relationship between the profile and magnitude of Ni2+-induced cytokine responses in vitro and the degree of in vivo 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 in vivo 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+ ex vivo 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 in vivo by counteracting the ACD reactions mediated by CD4+ T cells producing Th1-type cytokines. In the third study, we investigated the relationship between in vivo 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 in vitro 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 in vitro 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 in vitro. 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 in vitro cytokine assays as diagnostic tools in distinguishing ACD subjects sensitised to different metals and non-sensitised subjects.

Dendritic cells (DCs) are prime mediators of innate and adaptive immunity. In humans the DC population comprise only 0.1% of all leukocytes, making their isolation and ex vivo manipulation difficult. Since study of DC activity in vitro requires large numbers of DCs to be readily available, a cell line model, KG-1, was selected. KG-1 cells are a cytokine-responsive human CD34+ myelomonocytic cell line and can be induced to differentiate to a DC phenotype. A range of differentiation agents and protocols were compared, and differentiation efficiency was determined using both morphological features and cell surface marker expression. Expression of CD83, CD11c, CD123, CD86, HLA-DR and DC-SIGN was assessed by immunofluorescence and flow cytometry. KG-1 cells stimulated with 10 ng/ml PMA and 100 ng/ml Ionomycin were found to be the ideal model for obtaining Dendritic Like Cells (DLCs) in vitro. The effect of lactic acid bacteria on KG-1 differentiation was also tested using two immunomodulatory strains, Lactobacillus rhamnosus R0011 and Lactobacillus helveticus R0052. After 5 days of incubation with R0011 the KG-1 cells expressed DC-specific surface markers CD83, CD86, CD11c, CD123, DC-SIGN and HLA-DR. Lactobacillus rhamnosus R0011 induced a marked rise in CD83 expression with a mean fluorescence intensity of 115.3 after 5 days, suggesting this strain promoted KG-1 differentiation to DLC. Analysis of cytokine by KG-1 DLC indicated that constitutive production of pro-inflammatory cytokines TNF-α and IL-12 was minimal. However IL-10 and TGF-β were detected after TLR-agonist stimulation of R0011-differentiated KG-1s. This study aimed to develop and assess the KG-1 cell model for screening effects of mediators and microbes on DC. / UOIT

Agriculture is a valuable economic resource in western Canada, but for decades farmers have focused on intensive production practices while ignoring the long-term health and maintenance of the land. In recent years, the use of conservation agricultural techniques has been encouraged in an effort to conserve prairie landscape while sustaining cropland productivity. Sustainable agricultural practices that promote soil and water conservation and benefit wildlife and prairie biodiversity include conservation tillage and planting of winter cereal crops. Many species of wild birds nest in the ground cover provided by minimum tillage and fall seeded cropland in the spring. Although habitat quality in conservation areas is superior for birds, there is potential for eggs of ground nesting birds to be exposed to herbicides during spring weed control operations. Herbicides commonly used on the prairies to control weed growth in conservational systems include 2,4-D and Buctril-M®. Since the subtlethal effects of exposure to these herbicides may include DNA damage and immunomodulation, the overall goal of this study was to assess whether <i>in ovo</i> exposure to the herbicides 2,4-D and Buctril-M® adversely affects genetic material and/or immune system function in newly hatched domestic chickens (<i>Gallus gallus</i>) and ducks (<i>Anas platyrhynchos</i>), as surrogates for wild bird species.<p>Study design attempted to reproduce actual field exposures by use of an agricultural field spray simulator to apply formulated herbicides (as opposed to pure active ingredients) at recommended crop application rates. In three separate experiments, fertile chicken eggs were sprayed with 2,4-D ester formulation or with Buctril-M® formulation, and fertile duck eggs were sprayed with 2,4-D ester formulation, during either an early (embryonic day 6) or late (embryonic day 15 for chickens or embryonic day 21 for ducks) stage of incubation. Genotoxicity and immune system function were evaluated in the hatchlings as the main toxicological endpoints to assess potential subtle effects from herbicide exposure, but additional measures of general health and development were also evaluated. Two endpoints were used to assess subtle changes to genetic integrity. The comet assay was used to detect structural damage (strand breaks) in avian lymphocyte DNA, as an index of acute genotoxic effects. Flow cytometry was used to examine potential clastogenic effects of the herbicides, by determining if chromosomal changes resulted in variability in the DNA content of avian erythrocytes. Several endpoints were examined to evaluate potential exposure-induced effects on the immune system. Immunopathological assessment of chicks and ducklings included differential lymphocyte counts, as well as immune organ weights and histopathology. The cell-mediated and humoral immune responses in hatchlings were assessed using the delayed-type hypersensitivity test and measurement of systemic antibody production in response to immunization, respectively. Exposure of fertile chicken and duck eggs to Buctril-M® or 2,4-D had no effects on the biomarkers of genetic integrity in this study. Differences in herbicide treatment (high and low concentrations) and times of exposure (early and late incubation stages) did not translate into noticeable factor effects in final model analyses for any of the genotoxicity assay variables evaluated in newly hatched chickens exposed in ovo to 2,4-D. Similarly, comet assay outcomes in chicks exposed to Buctril-M® were not significantly associated with either herbicide treatment or time of exposure as fixed effect factors. Results of the comet assay using peripheral lymphocytes from ducklings provided evidence of potential primary genetic damage associated with the time of spray exposure in ovo. Comet tail DNA content was significantly associated (P = 0.0269) with exposure times, suggesting that ducks may be increasingly sensitive to spray exposure conditions at an early stage of embryological development. Effects of exposure timing were not attributable to herbicide treatment. Although 2,4-D exposure time was associated with DNA strand breakage in ducklings, there was no evidence of chromosomal damage. However, an association between the HPCV values (a measure of DNA content variability) and time of spray exposure was observed in the experiment where 21-day-old chickens were treated in ovo with Buctril-M®. The mean HPCV value for the early exposure group (E6) was significantly greater (P = 0.0210) than that of the group treated later in incubation (E15). However, Buctril-M® the concentration of herbicide did not have any influence on this outcome, and the reason for the difference between exposure times is uncertain, but may be attributed to stress associated with manipulations during spraying. An increase in HPCV, reflecting greater intercellular DNA variability, is indicative of increased incidence of chromosomal damage, which may be an effect of disturbance during early periods of incubation as a result of exposure conditions.<p>Among the panel of immunotoxicity tests conducted to evaluate the effects of <i>in ovo</i> exposure to 2,4-D and Buctril-M® on the developing avian immune system, only heterophil/ lymphocyte (H/L) ratios and relative immune organ weights were significantly associated with either herbicide treatment or time of spray exposure in all three experiments. In 21-day-old chicks exposed in ovo to 2,4-D, relative bursa weight was associated with the different herbicide treatments (P = 0.0006). Relative bursa weights were significantly lower in chicks in the low dose group, while the opposite effect was observed in the high dose chicks, compared with the controls. It is unlikely that the observed decrease in bursa weight in the low dose group is causally related to herbicide exposure because a consistent dose-response effect was not observed, but this outcome may be explained by a compensatory immune response. The relative spleen weights of newly hatched chickens exposed in ovo to Buctril-M® exhibited a significant association with herbicide treatment (P = 0.0137). Relative spleen weights for birds in the low dose treatment groups were significantly different than both the control (P = 0.0179) and high dose groups (P = 0.0125). However, there was no significant difference between high dose and control groups, and this outcome reduces the likelihood of a causal relationship between spleen weight and herbicide exposure. In the parallel experiment involving in ovo exposure to 2,4-D to ducklings, relative bursa weight was associated with time of spray exposure (P = 0.0434). Ducklings that hatched from eggs exposed to spray on day 6 of incubation exhibited greater mean relative bursa weights than the birds exposed to spray at a later incubation stage (E21). This result implies that spray exposure during earlier stages of development may result in conditions which affect the humoral immune response, if increased bursal weight is associated with increased B lymphocyte and antibody production. In the same experiment, mean H/L ratios in peripheral blood samples from 21-day-old ducklings were significantly different between the groups treated with the high concentration of 2,4-D and water (control) (P = 0.0395). Although ratios from the birds in the low dose groups were not significantly different from the control groups, changes in H/L ratio values demonstrate a dose dependent relationship with increasing herbicide exposure.<p>Residue analysis of chicken and duck eggs in this study measured transfer of herbicide through the shell and into the embryo 24 hours and up to 5 days (chickens only) after spraying. Mean 2,4-D residue concentrations were higher in both chicken and duck eggs from the high dose (10X) groups than in eggs exposed to the recommended field rate of herbicide application (1X). Embryo residue concentrations in the chicken eggs increased from the day following exposure to 5 days after spraying, in both low and high dose groups. This observation indicates that the risk of contaminant-induced adverse effects may continue to increase for at least several days after exposure, thereby influencing the concentration of herbicide to which the developing embryo is exposed.<p>On the Canadian prairies, wild bird eggs are potentially to be exposed to 2,4-D and Buctril-M® during various stages of embryonic development. The present study examined effects of herbicide exposure at two distinct times during incubation, and demonstrated the potential for subtle impacts on genetic integrity and the immune system. Results indicate that spray exposure during earlier stages of organogenesis may cause more significant adverse effects. Given the possible harmful consequences of the observed changes on the long-term health of wild birds, further research is needed in order to better characterize the risks of in ovo agrochemical exposure in prairie ecosystems.

The FMS-like tyrosine kinase 3- internal tandem duplication (FLT3/ITD) aberration is common in acute myeloid leukemia (AML) and associated with poor patient outcome. Inhibitors targeting FLT3/ITD are in development, but clinical responses are transient. This project focussed on elucidating molecular signalling consequences of FLT3/ITD inhibition, to identify rational drug combinations for future development. A Multicolour Phospho Flow Cytometry (MPFC) assay was developed to assess signalling events downstream of FLT3/ITD in primary patient samples, focusing on alterations in ERK, STAT5, Akt, and S6. STAT5 signalling appeared to be important exclusively in FLT3/ITD samples. MPFC accurately predicted the presence of FLT3/ITD, inhibitor sensitivity and the initial positive clinical response of a trial patient receiving a FLT3/ITD inhibitor. PI3K pathway upregulation was observed in a Sorafenib-resistant FLT3/ITD cell line established to study resistance mechanisms of FLT3 inhibition. Further, combination FLT3 and PI3K inhibition demonstrated synergy, suggesting potential clinical relevance to this therapeutic strategy.

The FMS-like tyrosine kinase 3- internal tandem duplication (FLT3/ITD) aberration is common in acute myeloid leukemia (AML) and associated with poor patient outcome. Inhibitors targeting FLT3/ITD are in development, but clinical responses are transient. This project focussed on elucidating molecular signalling consequences of FLT3/ITD inhibition, to identify rational drug combinations for future development. A Multicolour Phospho Flow Cytometry (MPFC) assay was developed to assess signalling events downstream of FLT3/ITD in primary patient samples, focusing on alterations in ERK, STAT5, Akt, and S6. STAT5 signalling appeared to be important exclusively in FLT3/ITD samples. MPFC accurately predicted the presence of FLT3/ITD, inhibitor sensitivity and the initial positive clinical response of a trial patient receiving a FLT3/ITD inhibitor. PI3K pathway upregulation was observed in a Sorafenib-resistant FLT3/ITD cell line established to study resistance mechanisms of FLT3 inhibition. Further, combination FLT3 and PI3K inhibition demonstrated synergy, suggesting potential clinical relevance to this therapeutic strategy.

Identification of pathogens living in biofilms of chronic infections has been difficult with PCR, serological, biochemical and culture techniques. The study aims at the detection of bacterial pathogens in biofilms of biological secretions using SPR analysis Biacore. The antibodies were developed by isolating mononuclear lymphocytes from the blood of the patients who sustained systemic infection. The isolated lymphocytes had antibody secreting B cells (plasma cells) which were identified using flow cytometry analysis. The antibodies produced (n=4) were used to immobilize CM5 chip of Biacore to detect the bacteria in ulcer secretions with wound secretions of healthy volunteers as controls. The results from Surface Plasmon Resonance (SPR) analysis and culture technique were compared and statistically there was no significant difference obtained. The results from present study suggest that SPR analysis could be used as an alternative system for detection of bacteria in poly-microbial samples and detect the organisms that might not be discovered by culture or PCR method.