The cancer stem cell hypothesis has recently re-emerged as a compelling paradigm for the development and progression of neoplastic disease. The hypothesis proposes that a specific subset of “cancer stem cells” (CSC), believed to share many features with normal stem cells, is exclusively responsible for maintaining tumour growth and driving progression. If the CSC hypothesis applies, it may require re-evaluation of the clinical approach to neoplasia. Spontaneous cancer in the domestic dog represents a significant welfare problem, with dogs developing many tumours strongly reminiscent of those affecting humans. This study sought to investigate whether cells with characteristics of CSC are identifiable in canine cancer. Assays to identify, isolate and characterise CSC were adapted to the canine system, and cancer cell lines and spontaneous tumours of diverse origin evaluated for the presence of candidate populations. Whilst analysis of surface expression patterns did not identify specific subpopulations within canine cancer cell lines, these were detectable in cells derived directly from primary tumours. Assays for stem cellassociated drug resistance mechanisms could also be used to identify subsets of putative canine CSC. Formation of “tumourspheres” by canine cancer cell lines was found to be highly density-dependent, so a potentially unreliable method of isolating CSC. Expression of the cell surface glycoprotein CD44 was associated with cellular proliferation status, although it may not represent a stable canine CSC marker. The NFκB survival pathway, associated with apoptosis resistance of some putative CSC, was constitutively active in canine cancer cell lines; suppression using specific inhibitors could reduce cell viability, indicating that this may represent a rational therapeutic target. Overall, these studies demonstrated that CSC assays may be adapted to the canine model system, although they require rigorous interrogation to distinguish apparent CSC attributes from basic biological properties. Cell lines have provided a stable background upon which to optimise assays, but appear less likely to demonstrate discrete CSC subpopulations. Putative CSC subsets may be more readily identifiable within heterogeneous primary tumour cells. The application of some of these adapted assays within a clinical setting may enable further characterisation of individual patients’ tumours, and inform therapeutic regimes for improved treatment outcomes.

Introduction: Hemostasis is a complex system in the body that maintains blood flow and prevents bleeding. Patients with platelet disorders are at the risk of mucocutaneous bleedings and at Clinical chemistry in Linköping platelet function is measured with flow cytometry. The platelet response to various agonists is measured with fluorescently labeled platelet antibodies. The aim of this study was to evaluate the stability of the frozen reagents used for platelet function testing. Further the durability of platelet antibodies after freezing and freeze-drying was tested.   Method: Platelet antibodies were prepared for freezing/freeze-drying in buffer and were analyzed at three different occasions using blood from 2 to 3 individuals with flow cytometry. Agonists and blood were added on the day of analysis. The reagent stability test was evaluated statistically using one-way ANOVA with Bonferronis post-hoc test. Results: The slight drop in percent positive platelets and median fluorescence intensity (MFI) seen over time in the reagent stability test was not statistically significant (p>0,05). All platelet antibodies could be used after freezing/freeze-drying. The results are showing a declining trend, especially for MFI values. Conclusion: The frozen reagents used for platelet function testing is stabile up to 36 months. The results from the freeze-drying/freezing indicate that all platelet antibodies keep some activity but that some are more sensitive than others. Some antibodies could not be evaluated due to concentration of agonist in the sample being too low to induce activation. Due to individual variations further studies with more participants and more agonists in their optimal concentrations are needed. / Bakgrund: Hemostas är ett komplext system i kroppen som upprätthåller blodflödet och förhindrar blödning. Mukokutana blödningar kan uppstå hos patienter som har problem i den primära hemostasen vilket kan bero på trombocydefekter. På Klinisk kemi i Linköping mäts trombocytfunktion med flödescytometri. Trombocyterna aktiveras med olika agonister och svaret på stimuli mäts genom detektion av fluoroforkonjugerade antikroppar. Syftet med denna studie var att utvärdera långtidsstabiliteten av de frysta reagens som används för trombocytfunktionsutredningen, att testa om nya trombocytantikroppar klarar att frysas och att utvärdera reagensens hållbarhet för frystorkning. Metod: Antikroppar frystorkades/frystes i buffert och analyserades vid tre tillfällen med blod från 2 till 3 personer med flödescytometri. Agonist och blod tillsattes på analysdagen. Långtidsstabilitetstestet utvärderades statistiskt med  one-way ANOVA med Bonferronis post-hoc test. Resultat: En svag nedgång över tid i procent positiva trombocyter och MFI i långtidsstabilitetstestet var inte statistiskt signifikant (p>0,05). Alla antikroppar gav signal efter frysning och frystorkning. Främst för MFI syns en nedåtgående trend över tid. Slutsats: Reagenset för trombocytfunktionsutredningen är stabilt upp till 36 månader. Resultat från frystorkningen tyder på att alla antikroppar klarar att frystorkas/frysas men vissa är känsligare än andra. Vissa antikroppar kunde inte utvärderas p.g.a. för låg agonistkoncentration för att inducera aktivering. Ytterligare försök måste göras med fler individer och fler agonister i optimal koncentration p.g.a. individuella skillnader i svar för agonister. Frystorkningsprocessen kan optimeras.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

Although the genus Nymphaea (waterlily) includes only two native species (N. alba and N. candida) in Central Europe, it poses a great challenge to taxonomy and biosystematics. The determination of both species is hampered by their phenotypic similarities, and species boundaries can be further blurred by interspecific hybridization. In addition, ornamental cultivars of different parentage often escape from cultivation and make the situation even more complex. To get insight into the caryological and phenotypic variability of czech waterlilies, the DNA flow cytometry and both distance-based and geometric morphometrics were used. Collections showed two different groups of fluorescence intensities, corresponding to N. alba and N. candida, respectively. In addition, intermediate values of nuclear DNA amount were found in some plants from South Bohemia, indicating their hybrid origin. Surprisingly, ornamental cultivars possessed the smallest genome sizes. The amount of nuclear DNA therefore seems to be a promising species-specific marker that enables not only native species but also cultivars to be distinguished. Cytometrically-proven individuals have been subjected to multivariate morphometrics, and high differenciation was discovered especially between native species. More complicated seems the distinction...

Micro-irradiation systems are used to analyse the effect of ionizing radiation at the cellular and tissue level, targeting individual cells within a population with a controlled low dose. Cell survival experiments using micro-irradiation systems are limited by factors including: 1) the radiation attenuation and optical properties of the chosen cell dish substrate, 2) the registration of the cell dish before and after irradiation or between multiple imaging modalities and 3) the analysis of the cell or colony growth after irradiation. In this thesis, a set of tools have been developed to improve micro-irradiation experiments and to increase the accuracy of information provided by the cell survival data. The first contribution, the substrate cell dish evaluation, provides a set of characteristics defining the substrates used for micro-irradiation experiments based on minimal energy loss and optical clarity using unstained cell imaging. The second contribution was the development of a novel and low cost fiducial marking device for micro-irradiation experiments using an 808 nm laser and providing marks suitable for imaging with multiple modalities. The minimum focused spot diameter was calculated as 22.9 urn and the device was used to create fiducial marks with diameters ranging from 20 urn to 130 urn. The third contribution, the development of a cell counting methodology for use with a lens-free imaging device, has been shown to accurately count thousands of cells suitable for immediate analysis. Approximately 1000 cell colonies, containing 17 729 cells on 11 cell dishes were used for testing and training for automatic cell counting. Validation of the cell counting method showed that 76% and 89% of the cell colonies were counted within a ± 20% and ± 30% error of the ground truth, respectively. Further development of the fiducial marking device, by modifying the choice of laser and making it suitable for multiple types of cell dish substrates, would increase the applications of the device. Development of the cell counting methodology for different cells line, and for cells grown on multiple types of substrates, would make the system suitable for analysis of a wide variety of cell survival studies. The cell counting methodology, applied to the CyMap lens-free imaging device, has the potential to be an extremely useful and cost effective tool for cell survival studies.

Master of Science / Department of Clinical Sciences / Maria S. Ferrer / Presence of anti-sperm antibodies (ASA) is associated with infertility in many species, including bulls but there is no standardized direct technique that allows detection of ASA bound to the sperm surface. The overall objective was to standardize a flow cytometric technique for detection of IgG and IgA directly attached to bovine sperm. The effects of fixation using phosphate buffer solution (PBS) or diluted formalin buffer solution (dFBS), exclusion of dead cells from the analysis, and aliquot variability were assessed using healthy bulls classified as Satisfactory Potential Breeders (SPB, n=9) and bulls with experimentally induced ASA (n=4) (Experiment1). The effect of freezing on the percentage of IgG- and IgA- bound sperm was assessed in samples from immunized bulls (n=4) (Experiment 2). Anti-sperm antibodies on the sperm surface were induced in yearling bulls by intramuscular injection of autologous semen and an adjuvant. Fixation of sperm cells did not affect the percentage of IgG- or IgA-bound sperm in any group of bulls. Exclusion of dead cell from the analysis did not affect the percentage of IgG-bound sperm (p= 0.0922 and p= 0.1525 for immunized and reproductively normal bulls, respectively). The exclusion of dead cells significantly increased the percentage of IgA-bound sperm in semen samples from immunized bulls (p= 0.0152) and significantly decreased the percentage of IgA- bound sperm in semen samples from reproductively normal bulls (p= 0.0012). Variability was < 10% in samples from immunized and reproductively normal bulls for percentage of IgG- and IgA-bound sperm. Freezing did not affect the percentage of IgG- (p=0.1287) or IgA-bound sperm (p=0.4175). Based on these results, fixation is neither necessary nor detrimental for analysis, and the percentage of antibody-bound cell should be calculated gated on the population of live cells only, especially when evaluating IgA binding. The percentage of ASA-bound sperm can be assessed on frozen-thawed samples. The development of this technique allows for further studies on ASA-bound sperm in populations of normal and abnormal bulls.

A research report submitted to the faculty of Medicine, University of the Witwatersrand, Johannesburg, in part fulfillment of the requirements for the degree of Master of Medicine (in the branch of Haematology) Johannesburg 1992. / The observation by Haldane in 1949 that the distribution of malaria and certain thalassaemias were similar and that the former disease must be a selective force tor the continued existence of the latter by preservation of the heterozygotes. This theory which later became known as lithe malaria hypothesis" has been applied to other inherited conditions such as G6PD deficiency, membranopathies, certain blood group polymorphisms, other heamoglobinopathies such as sickle cell disease, blood group polymorphisms and more recently HLA phenotypes. It has been shown that the Duffy blood group antigens are the receptors for. Plasmodium vivax and since these antigens are lacking in most black Africans this species of malaria is virtually absent in Africa. It has also been shown that the glycophorins are at least in part the receptors for Pfalciparum. Several variants of the glycophorins exist and the biochemistry and, where known, the molecular mechanisms by which these arise is reviewed. Experimental work is carried out to establish the growth characteristics of Pfalciparum in an in vitro culture system using cells with glycophorin variants on their membranes. Three such variants were compared to normal cells and two (S~s-U-and Dantu) were found to be partially resistant to invasion by Pfalciparum merozoites whereas the third (Henshaw) was found to be no different to controls. / MT2018

[Abstract] This thesis is aimed at better understanding of cytotype co-existence in mixed- ploidy populations with an emphasis on a microevolutionary processes behind it. Our past knowledge was based on a few thoroughly investigated model taxa like Chamerion angustifolium and Heuchera grossulariifolia, but some generalizations seem to be premature in the light of new findings. A detailed research of other taxa included in the thesis showed that polyploid complexes can vary dramatically in their ability to cope with the co-existence of cytotypes in mixed-ploidy popu- lations. Whereas mixed-ploidy populations are virtually lacking in some species (an example being Vicia cracca, Paper III.), ploidy-heterogeneous populations are very common in others, maintained by free mating interactions and the absence of reproductive isolation among cytotypes (e.g. Pilosella echioides, Paper II.). The strenght and cumulative effect of various breeding barriers (both pre- or post- zygotic) govern the position of a particular multi-ploidy complex between these two extremes and co-determine the type of cytotype co-existence in its mixed- ploidy populations. Despite the fact that the number of studies revealing cytotype co-existence has been increasing rapidly, evolutionary background and consequences of such co-...

Under vissa omständigheter kan bakterier kan ta sig in i blodbanan där de kan orsaka allvarliga infektioner (bakteriemi). Metoden som används för identifiering och resistensbestämning av bakterier i blododlingar kräver minst 16 timmars inkubation. Fram tills en resistensbestämning utförts kan empirisk antibiotikabehandling användas, men med ökande resistensutbredning blir det alltmer osäkert om denna behandling är verksam. Nyligen har en lappdiffusionsmetod för resistensbestämning direkt från blododling validerats, som kan läsas av efter 4, 6 och/eller 8 timmars inkubation. Det finns även publicerade arbeten där flödescytometriassisterad resistensbestämning används, men då krävs att bakterierna finns i tillräckligt hög koncentration, befinner sig i tillväxtfas och finns som renkultur. Syftet med examensarbetet var att optimera hantering av blododlingsflaskor så att bakterier från blododlingsflaskorna kunde isoleras med tillräcklig kvalitet och koncentration för att kunna utföra resistensbestämning med flödescytometri. Upprening av bakterierna utfördes med olika tvättbuffertar och sedan utfördes resistensbestämning med flödescytometri och  referensmetoden buljongspädning. Resultaten från uppreningen visade att sterilt vatten och Tween20 gynnade bakteriernas återhämtningsförmåga mest. Resistensbestämning utfördes med Klebsiella pneumoniae ATCC700603,  K. pneumoniae CCUG56233 och K. pneumoniae ATCC13882, som tvättats med sterilt vatten och Tween20. För CCUG56233 skiljde 1 spädningssteg i koncentrationsskalan mellan metoderna. ATCC-isolaten erhöll likartade MIC-värden (minimum inhibitory concentration) vid alla analyser men där fanns en skillnad på 2 spädningssteg mellan buljongspädning och analys med flödescytometri. Detta kan förklaras av skillnaden i inkubationstid mellan metoderna. Slutsatsen som kan dras är därför att resultaten från de två metoderna vid resistensbestämning är likartade och att sterilt vatten är mest lämpligt att använda vid upprening av bakterier. Fler undersökningar bör dock utföras.

A mudança anual de penas é um componente crítico do ciclo biológico de todas as espécies de aves e pode modificar as respostas imunológicas individuais, sendo que suas consequências são pouco estudadas. Neste contexto, uma vez que são escassos os estudos acerca do perfil clínico e parâmetros imunológicos de pinguins-de-Magalhães em diferentes fases da muda, este estudo propôs avaliar determinados aspectos relacionados a esses perfis, em animais mantidos em ambiente controlado. Foram estudados 21 indivíduos adultos (dez machos e 11 fêmeas) e os parâmetros avaliados foram: teste de hipersensibilidade cutânea tardia à fitohemaglutinina (FHA) com a avaliação histopatológica de biópsias que incluíram a análise qualitativa e quantitativa dos tipos celulares presentes na reação e no controle às 6, 24 e 48 horas; parâmetros clínicos (massa corpórea e hematologia); fagocitose e burst oxidativo; fenotipagem e resposta linfoproliferativa. O teste de FHA demonstrou diferença significativa na espessura da membrana interdigital de ambas as patas inoculadas com FHA e com o controle (PBS) quando comparadas antes da inoculação e antes da biópsia às 24 e 48 horas. A análise histomorfométrica revelou não haver diferenças significativas na frequência relativa de granulócitos (heterófilos/ eosinófilos), linfócitos, macrófagos ou trombócitos entre as patas inoculadas com FHA e PBS em nenhum dos momentos analisados. Este resultado sugere que a diferença na espessura da membrana interdigital das patas inoculadas seja em decorrência do edema e não do infiltrado inflamatório. Para analisar os efeitos do ciclo da muda nos parâmetros clínicos e hematológicos, os animais foram divididos em três grupos: pré-muda (-5 a 0 dias da muda), muda (10&ordm; ao 21&ordm; dia da muda) e padrão basal (&gt;120 dias da muda). Os resultados mostraram que em relação ao momento basal: houve aumento significativo na massa corpórea dos animais no momento da pré-muda; diminuição significativa de proteínas totais, hematócrito, eritrócitos totais e leucócitos totais na pré-muda e muda. Não houve diferença no número total de monócitos e heterófilos, contudo, houve eosinofilia e linfopenia na pré-muda e muda, respectivamente. Com relação aos parâmetros imunológicos, houve aumento no burst oxidativo gerado pelo estímulo biológico (Staphylococcus aureus) na pré-muda em relação à muda e ao padrão basal; ocorreu aumento do burst oxidativo gerado pelo estímulo químico (éster de forbol) na pré-muda em relação à muda. Em relação à fagocitose, houve diminuição desta capacidade dos leucócitos dos animais na pré-muda e muda em relação ao padrão basal quando o agente foi bacteriano; entretanto, o mesmo não foi observado para a fagocitose de leveduras (Zymosan A); houve diminuição de linfócitos T CD4+ circulantes na pré-muda e muda em relação ao padrão basal e o índice de proliferação de linfócitos aumentou durante o período de muda em relação ao basal. Esses resultados tomados em conjunto sugerem que as mudanças fisiológicas observadas durante o ciclo da muda interferem na modulação de parâmetros imunológicos e são sugestivas de serem consequência do prejuízo energético sofrido, mesmo em animais mantidos em ambientes controlados / The annual replacement of feathers is a critical component of the biological cycle of all bird species. Molt can modify their individual immune response, and the consequences of this are not well known. Within this context, and the few studies on the clinical profile and immunological parameters of Magellanic penguins at different stages of their molt, this study aimed to evaluate certain aspects related to the clinical profiles of animals kept in a controlled environment. A total of 21 adult individuals (ten males and 11 females) was analyzed and the parameters evaluated in this study were: delayed-type hypersensitivity test to phytohemagglutinin (PHA) based on the histopathological evaluation of foot web biopsies that included qualitative and quantitative analysis of cell types present in the essay and control at 6, 24 and 48 hours; clinical parameters (body mass and hematology); phagocytosis and oxidative burst; phenotyping and lymphoproliferative response. The PHA test showed significant differences in the thickness of the webbing of both feet inoculated with PHA and control (PBS), when compared before inoculation and before biopsy at 24 and 48 hours. Histomorphometric analysis revealed no significant differences in the relative frequencies of granulocytes (heterophils/ eosinophils), lymphocytes, macrophages, thrombocytes, or between the feet inoculated with PHA and with PBS in any of the samples. This result suggests that the difference in thickness of the foot webbing is due to edema and not an inflammatory infiltrate. To analyze the effects of the molting cycle on clinical and hematological parameters, the birds were divided into three groups: pre-molt (from -5 days to the start of the molt, day 0), molt (10th to 21st days of molt) and baseline (&gt;120 days after the completion of the molt). The results showed that in comparison with baseline values: a significant increase in body mass occurred during pre-molt; and a significant decrease in total protein, hematocrit, total erythrocytes and total leukocytes occurred during pre-molt and molt. There was no difference in the total numbers of monocytes and heterophils, however, there were eosinophilia and lymphopenia during pre-molt and molt, respectively. There was an increase in the oxidative burst generated by the biological stimulus (Staphylococcus aureus) in the pre-molt compared to molt and baseline levels; and there was an increased oxidative burst generated by the chemical stimuli (phorbol esters) in the pre-molt in relation to the molt. There was a decreased capacity of phagocytosis for the leukocytes in animals during pre-molt and molt compared to baseline when the agent was bacterial; however, this was not observed for the phagocytosis of yeast (Zymosan A). There was a decrease of circulating CD4+ T lymphocytes in the pre-molt and molt compared to baseline, and the lymphocyte proliferation index increased during the molt compared to baseline. The results suggest that the physiological changes observed during the molt cycle does interfere with the modulation of immune parameters, and are suggestive of resulting as a consequence of from the high energy demands of molt, even for birds kept in controlled environments