Electroporation affects the dielectric properties of cells. Dielectric measurement techniques can provide a label-free and non-invasive modality to study this phenomenon. In this thesis we introduce a dielectrophoresis (DEP) based technique to study changes in the cytoplasm conductivity of single Chinese hamster ovary (CHO) cells immediately after electroporation. Using a microfluidic chip, we study changes in the DEP response of single CHO cells a few seconds after electroporation. First, in order to quantify our DEP measurement results and relate them to the cells internal conductivity, we introduce a dielectric model for CHO cells. This is achieved by measuring the DEP response of many individual cells in the β-dispersion frequency region and curve fitting to the measured data. Second, we present quantitative results for changes in the cytoplasm conductivity of single cells subjected to pulsed electric fields with various intensities. We observe that when electroporation is performed in media with lower ionic concentration than cells cytoplasm, their internal conductivity decreases after electroporation depending on the intensity of applied pulses. We also observe that with reversible electroporation there is a limit on the decrease in the cells’ internal conductivity. We hypothesize the reason is the presence of large and relatively immobile negative ions inside the cell which attract mobile positive ions (mainly sodium and potassium) to maintain cell electrical neutrality. We monitor the temporal response of cells after electroporation to measure the time constant of changes due to ion transport and observe this ranges from seconds to tens of seconds depending on the applied pulse intensity. This result can be used to infer information about the density and resealing time of very small pores (not measurable with conventional marker molecules). Lastly, we measure the electroporation of cells in media with different conductivities. Our results show that electroporation in very low conductivity media requires stronger pulses to achieve a similar poration extent as in high conductivity media. The outcome of this thesis can be used to improve our understanding of the dynamics of electroporation as well as its modelling in order to make more accurate predictions or optimize the process for specific applications. / February 2017

Chan Oi Chi. / "May 2004." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (p. 259-310) / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

In Syrian hamsters, social behavior is mediated exclusively by chemosensory cues and circulating gonadal steroid hormones. Where these two signals are processed in the brain is unknown, but the posterior bed nucleus of the stria terminalis (pBNST) has been suggested as a candidate site. Therefore, we tested male hamsters’ preference for opposite-sex odors following excitotoxic lesions of the pBNST. Lesions of the pBNST (pBNST-X) eliminated male hamsters’ preference for opposite-sex odors. Furthermore, pBNST-X males spent significantly less time investigating female odors than clean odors and significantly less time investigating female odors than control males did. Lesions of the pBNST did not change male hamsters’ investigation of male odors. The deficits observed in pBNST-X males were not due to a failure to discriminate between odors, as pBNST-X males were able to distinguish between odors. Together, these data suggest the pBNST is critical for opposite-sex odor preference in male hamsters.

Les patients atteints de dystrophies musculaires présentent de plus en plus fréquemment des complications cardiaques, qui sont majoritairement des cardiomyopathies dilatées (CMD), et qui déterminent leur pronostic vital. Dans ce contexte, les traitements de l'atteinte cardiaque sont ceux de l'insuffisance cardiaque, qui agissent sur l'environnement fonctionnel du cœur, mais pas directement sur l'atteinte myocardique.<br /> L'objectif de cette thèse a été d'évaluer les effets d'un traitement novateur de la CMD dans les dystrophies musculaires, en utilisant le hamster de la lignée CHF147. La démarche proposée est d'induire un phénomène compensateur de la dilatation cardiaque et ainsi d'améliorer le pronostic vital, en utilisant les propriétés de l'Insulin-like Growth Factor 1 (IGF-1).<br /> Des hamsters CHF147 jeunes ont été traités pendant un temps court, de façon systémique avec la protéine recombinante IGF-1 (rhIGF-1) à faible dose. Les effets macroscopiques, histologiques et fonctionnels du traitement ont été évalués 35 jours après le début de celui-ci et les voies de signalisation impliquées dans l'induction des effets observés ont été étudiées. Une étude de survie a été réalisée afin de mesurer les effets du traitement à long terme. L'évaluation des effets à court terme de l'administration de rhIGF-1 sur la CMD du hamster CHF147 indique un ralentissement de la dilatation des cavités cardiaques ainsi que de l'extension de la fibrose myocardique, et une préservation de multiples paramètres fonctionnels, tels que le débit cardiaque, le volume d'éjection, la pression de fin de diastole et, en particulier, la contractilité myocardique. Les effets observés sont dus en partie à des modifications au niveau des protéines impliquées dans le cycle du calcium. A long terme, la survie des hamsters CHF147 traités augmente significativement d'environ 20% et est associée à une préservation partielle de la fonction cardiaque.<br />Toutefois, une augmentation du niveau sérique d'IGF-1 pourrait augmenter le risque d'effets secondaires délétères. Afin de limiter les effets systémiques d'IGF-1 et de cibler son administration au cœur, nous avons injecté localement le plasmide pCMV-IGF1 codant pour l'IGF-1 dans le myocarde des hamsters CHF147. Les effets du traitement ont été évalués après 35 jours et montrent des résultats comparables à ceux obtenus avec la protéine rhIGF-1.<br />En conclusion, ce travail de thèse a montré qu'un traitement basé sur IGF-1 permet de ralentir l'évolution de la CMD des hamsters CHF147, en préservant la structure et la fonction cardiaques, et d'améliorer significativement leur survie. IGF-1 semble donc être un candidat prometteur pour la mise au point d'une approche par thérapie génique dans l'insuffisance cardiaque, due à une CMD, dans le contexte des dystrophies musculaires.

The objective of this study was to examine the effects of a medium chain triglyceride oil mixture (MCTo), designed to increase energy expenditure and improve lipid profiles containing medium chain triglycerides, phytosterols and n-3 fatty acids in the form of flaxseed oil, versus the antioxidant alpha-lipoic acid (ALA). Forty-eight hamsters were fed (i) hypercholesterol emic (HC) control, (ii) HC MCTo, (iii) HC ALA, (iv) HC MCTo/ALA diets for 4 weeks. No effects on food intake, body weight, total body water, lean body mass, fat mass, and tissue thiobarbituric acid-reactive substances (TBARS) were observed. ALA alone had no effect on total cholesterol (TC); however, MCTo feeding increased TC with (p < 0.03) and without (p < 0.003) ALA when compared to control. ALA increased HDL levels compared to control (p 0.04) and MCTo/ALA (p < 0.007) groups. MCTo, with (p < 0.0001) or without (p < 0.006) ALA, increased non-HDL cholesterol levels versus control. The non-HDL:HDL ratio was decreased by ALA compared to MCTo (45%) and MCTo/ALA (68%) (p < 0.0001), a similar trend was seen when compared to the HC control (22%) group (p < 0.14). Triglyceride levels were not altered by any of the dietary treatments. Liver and heart tissue reduced glutathione (GSH) was increased (p < 0.05) by all three treatments when compared to control. Both tissues showed an increase (p < 0.05) in oxidized glutathione (GSSG) when fed ALA compared to all other treatments. Hamsters fed ALA had a lower (p < 0.05) GSH/GSSG ratio compared to all treatment groups. In conclusion, MCTo feeding does not elicit beneficial effects on circulating plasma lipids and measures of body composition. In addition, our results do not clearly support an improvement in oxidative status through supplementation of ALA. However, our results do support the existence of beneficial effects of ALA on circulating lipoprotein content in the hamster.

Etwa 15% aller Paare bleiben ungewollt kinderlos. Männliche Faktoren sind in circa einem Drittel der Fälle als ursächlich anzusehen. Jedoch sind die Erfolgsraten der Therapie männlicher Infertilität durch assistierte Reproduktion auch nach über 30 Jahren seit deren Einführung unbefriedigend. Bestehende Spermienaufbereitungsmethoden wie einfaches Waschen, swim up oder die Dichtegradientenzentrifugation basieren auf makroskopisch-funktionellen Parametern wie Motilität und Morphologie. Spezifische Eigenschaften wie etwa eine aktivierte Apoptosesignalkaskade der Spermien werden dabei nicht berücksichtigt. Die wesentlichen Elemente verschiedener Signalwege der aus somatischen Zellen bekannten Apoptose konnten auch am humanen ejakulierten Spermatozoon nachgewiesen werden. Über die (negativen) Auswirkungen der Apoptose auf die männliche Fruchtbarkeit gibt es einen Konsens. Ziel der vorliegenden Arbeit war es, Selektionsmethoden zu entwickeln, welche auf subzellulärer Ebene intakte Spermien mit dem größtmöglichen Fertilisationspotential aus dem Ejakulat extrahieren. In einem ersten Versuchskomplex konnte gezeigt werden, dass durch die Kombination von Dichtegradientenzentrifugation und swim-up (Standardmethoden in Reproduktionskliniken und andrologischen Laboren) zur Aufbereitung der Spermien von subfertilen Patienten eine akzeptable Reduktion der Spermien mit aktivierter Apoptosesignalkaskade erreicht werden kann. Jedoch gaben die großen interindividuellen Unterschiede im Separationseffekt Anlass zur Entwicklung innovativer Untersuchungs- und Separationsmethoden. So wurden unter anderem fluoreszenzbasierte Tests zur Evaluation von Spermiendefekten, wie beispielsweise einer gestörten Integrität des mitochondrialen Membranpotentials, eingeführt. In den Untersuchungen wurde die Praktikabilität dieser neuen Analyseverfahren im Routineeinsatz unter Standardbedingungen getestet und bestätigt. Die innovative Selektionsmethode der Annexin V-MACS Separation basiert auf der Bindung von Annexin V-MicroBeads an apoptotische Spermien, womit eine Subpopulation reifer, motiler und vitaler Spermien mit inaktivierter Apoptosesignalkaskade gezielt angereichert wird. Das Konzept wurde zudem erfolgreich auf ein (Glaswoll-) Festphasen-Filtersystem ohne frei schwimmende Microbeads übertragen. Dadurch gelang die Minimierung eines potentiellen Transmissionsrisikos der Microbeads bei der Anwendung im Rahmen der künstlichen Befruchtung. Den hohen Stellenwert dieser Verfahren belegen die Ergebnisse zweier in-vitro Modelle, an denen erstmalig gezeigt werden konnte, dass durch die Selektion von Spermien mit inaktivierter Apoptose-Signalkaskade höhere Fertilisationsraten erreichbar sind.

A leishmaniose visceral é uma infecção grave que leva a pancitopenia. Quando se trata de disfunções medulares decorrentes de infecção por Leishmania (Leishmania) infantum há poucas abordagens descrevendo as alterações na mielopoiese e os mecanismos que levam a pancitopenia na LV. Alguns estudos demonstram uma relação importante entre a pancitopenia e o fator de crescimento insulin-like growth fator-I (IGF-I), no entanto, o seu papel endógeno na hematopoiese ainda não está claro. Propomos estudar a influência desse fator na hematopoiese e sua relação com o desenvolvimento da pancitopenia em hamsteres infectados por via intra-peritoneal com 2x107 de amastigotas por L. (L.) infantum. Avaliamos em 90 e 120 dias pós-infecção a LDU (Leishman-Donovan units) no baço e fígado, quando observamos tendência à progressão da infecção. Aos 60 dias pós-infecção, os animais com LV desenvolveram a plaquetopenia como primeira alteração hematológica, e a partir dos 90 dias pós-infecção, a anemia, e a leucopenia com reduções significantes nos leucócitos totais, linfócitos e neutrófilos. Já aos 120 dias de infecção, os leucócitos totais diminuíram significantemente acompanhados por uma redução de linfócitos, monócitos e eosinófilos. A partir desses dados, focamos a análise da medula óssea nos hamsteres com 90 e 120 pós-infecção. No mielograma, vimos alterações somente nos hamsteres com LV aos 90 dias pós-infecção, com um aumento significante na proporção células mielóides imaturas: células mielóides maduras. Na biópsia da tíbia, houve um aumento significante da celularidade quando comparados com seu respectivo controle, apenas no período de 90 dias pós-infecção. Em adição, observamos uma proliferação e/ou infiltrado macrofágico significante nos hamsteres com LV, mas sem diferença estatística nos períodos de 90 e 120 dias pós-infecção. Na avaliação semi-quantitativa de fibras de reticulina, somente aos 90 dias pós-infecção, observamos aumento significante nos infectados comparados aos controles, caracterizando um quadro fibrótico. Foi medida a expressão de mRNA de IGF-I por PCR em tempo real, aos 90 e 120 dias pós-infecção, onde ocorreu um aumento significante da expressão de IGF-I nos animais infectados em relação aos controles aos 90 dias. Como expostos, os hamsteres com LV apresentam alterações hematológicas como anemia, leucopenia e plaquetopenia, e ainda alterações na medula óssea como aumento da celularidade, proliferação macrofágica e fibrose acompanhados por um aumento da expressão de IGF-I. Assim podemos concluir que nossos dados indicam que o hamster se constitui num bom modelo para o estudo da patogênese da pancitopenia e das alterações medulares decorrentes da infecção por L.(L.) infantum. Neste modelo, ocorre alteração da expressão de IGF-I durante a evolução da infecção com possível papel na patogenia da pancitopenia. / Visceral leishmaniasis is a serious infection that leads to pancytopenia. When it comes to spinal cord dysfunction resulting from infection Leishmania (Leishmania) infantum there are few approaches describing the changes in myelopoiesis and mechanisms that lead to pancytopenia in LV. Some studies have shown a significant relationship between pancytopenia and insulin-like growth factor-growth factor I (IGF-I), however, their endogenous role in hematopoiesis is still not clear. We propose to study the influence of this factor in hematopoiesis and its relationship to the development of pancytopenia in hamsters infected intraperitoneally with 2x107 amastigotes by L. (L.) infantum. Evaluated at 90 and 120 days post-infection the LDU (Leishman-Donovan units) in the spleen and liver, when we observed a tendency to progression of the infection. At 60 days post-infection, animals with LV developed thrombocytopenia as the first hematologic changes, and from 90 days post-infection, anemia, and leukopenia with significant reductions in total leukocytes, lymphocytes and neutrophils. Already at 120 days of infection, total leukocytes decreased significantly accompanied by a decrease lymphocytes, monocytes and eosinophils. From these data, we focus on the analysis of the bone marrow in hamsters with 90 and 120 post-infection. The myelogram changes seen only in hamsters with LV at 90 days post-infection, with a significant increase in the proportion immature myeloid cells: mature myeloid cells. Biopsy of the tibia, there was a significant increase in cellularity compared with their respective control, only 90 days post-infection. In addition, we observed a proliferation and / or infiltration significant macrophage in hamsters with LV, but no statistical difference in the periods of 90 and 120 days post-infection. In semi-quantitative assessment of reticulin fibers, only at 90 days post-infection, we observed a significant increase in infected compared to controls, featuring a fibrous framework. MRNA expression of IGF-I was measured by real-time PCR, at 90 and 120 days post-infection, where a significant increase in IGF-I expression in infected animals compared to controls at 90 days occurred. As set out, the present hamsters VL hematological disorders such as anemia, leukopenia and thrombocytopenia, as well as changes in bone marrow cellularity increased, macrophage proliferation and fibrosis accompanied by a reduction in IGF-I expression. Thus we can conclude that our data indicate that the hamster is a good model to study the pathogenesis of pancytopenia and marrow changes resulting from infection by L. (L.) Infantum. In this model, alteration of IGF-I expression during the course of infection with a possible role in the pathogenesis of pancytopenia.

Master of Science / Department of Psychology / Jerome Frieman / Male and female Phodopus campbelli were investigated for kin recognition abilities, assessed by kin discrimination of bedding, urine, fecal, or midventral glandular secretion odors. Using a habituation-discrimination paradigm, subjects were presented either the odors of a pair of their own brothers (C[subscript 1]) or a pair of brothers that were unrelated to them (C[subscript 2]). All subjects were then presented with a final test discrimination odor from a male donor unrelated to themselves or the first donors. All subjects differentiated between the bedding and glandular odors of two unrelated males, regardless of condition assignment. Subjects did not statistically differentiate between the odors of two brothers. These results suggest Phodopus campbelli are capable of recognizing kin based on bedding and midventral gland odors.

A leishmaniose visceral é uma infecção grave que leva a pancitopenia. Quando se trata de disfunções medulares decorrentes de infecção por Leishmania (Leishmania) infantum há poucas abordagens descrevendo as alterações na mielopoiese e os mecanismos que levam a pancitopenia na LV. Alguns estudos demonstram uma relação importante entre a pancitopenia e o fator de crescimento insulin-like growth fator-I (IGF-I), no entanto, o seu papel endógeno na hematopoiese ainda não está claro. Propomos estudar a influência desse fator na hematopoiese e sua relação com o desenvolvimento da pancitopenia em hamsteres infectados por via intra-peritoneal com 2x107 de amastigotas por L. (L.) infantum. Avaliamos em 90 e 120 dias pós-infecção a LDU (Leishman-Donovan units) no baço e fígado, quando observamos tendência à progressão da infecção. Aos 60 dias pós-infecção, os animais com LV desenvolveram a plaquetopenia como primeira alteração hematológica, e a partir dos 90 dias pós-infecção, a anemia, e a leucopenia com reduções significantes nos leucócitos totais, linfócitos e neutrófilos. Já aos 120 dias de infecção, os leucócitos totais diminuíram significantemente acompanhados por uma redução de linfócitos, monócitos e eosinófilos. A partir desses dados, focamos a análise da medula óssea nos hamsteres com 90 e 120 pós-infecção. No mielograma, vimos alterações somente nos hamsteres com LV aos 90 dias pós-infecção, com um aumento significante na proporção células mielóides imaturas: células mielóides maduras. Na biópsia da tíbia, houve um aumento significante da celularidade quando comparados com seu respectivo controle, apenas no período de 90 dias pós-infecção. Em adição, observamos uma proliferação e/ou infiltrado macrofágico significante nos hamsteres com LV, mas sem diferença estatística nos períodos de 90 e 120 dias pós-infecção. Na avaliação semi-quantitativa de fibras de reticulina, somente aos 90 dias pós-infecção, observamos aumento significante nos infectados comparados aos controles, caracterizando um quadro fibrótico. Foi medida a expressão de mRNA de IGF-I por PCR em tempo real, aos 90 e 120 dias pós-infecção, onde ocorreu um aumento significante da expressão de IGF-I nos animais infectados em relação aos controles aos 90 dias. Como expostos, os hamsteres com LV apresentam alterações hematológicas como anemia, leucopenia e plaquetopenia, e ainda alterações na medula óssea como aumento da celularidade, proliferação macrofágica e fibrose acompanhados por um aumento da expressão de IGF-I. Assim podemos concluir que nossos dados indicam que o hamster se constitui num bom modelo para o estudo da patogênese da pancitopenia e das alterações medulares decorrentes da infecção por L.(L.) infantum. Neste modelo, ocorre alteração da expressão de IGF-I durante a evolução da infecção com possível papel na patogenia da pancitopenia. / Visceral leishmaniasis is a serious infection that leads to pancytopenia. When it comes to spinal cord dysfunction resulting from infection Leishmania (Leishmania) infantum there are few approaches describing the changes in myelopoiesis and mechanisms that lead to pancytopenia in LV. Some studies have shown a significant relationship between pancytopenia and insulin-like growth factor-growth factor I (IGF-I), however, their endogenous role in hematopoiesis is still not clear. We propose to study the influence of this factor in hematopoiesis and its relationship to the development of pancytopenia in hamsters infected intraperitoneally with 2x107 amastigotes by L. (L.) infantum. Evaluated at 90 and 120 days post-infection the LDU (Leishman-Donovan units) in the spleen and liver, when we observed a tendency to progression of the infection. At 60 days post-infection, animals with LV developed thrombocytopenia as the first hematologic changes, and from 90 days post-infection, anemia, and leukopenia with significant reductions in total leukocytes, lymphocytes and neutrophils. Already at 120 days of infection, total leukocytes decreased significantly accompanied by a decrease lymphocytes, monocytes and eosinophils. From these data, we focus on the analysis of the bone marrow in hamsters with 90 and 120 post-infection. The myelogram changes seen only in hamsters with LV at 90 days post-infection, with a significant increase in the proportion immature myeloid cells: mature myeloid cells. Biopsy of the tibia, there was a significant increase in cellularity compared with their respective control, only 90 days post-infection. In addition, we observed a proliferation and / or infiltration significant macrophage in hamsters with LV, but no statistical difference in the periods of 90 and 120 days post-infection. In semi-quantitative assessment of reticulin fibers, only at 90 days post-infection, we observed a significant increase in infected compared to controls, featuring a fibrous framework. MRNA expression of IGF-I was measured by real-time PCR, at 90 and 120 days post-infection, where a significant increase in IGF-I expression in infected animals compared to controls at 90 days occurred. As set out, the present hamsters VL hematological disorders such as anemia, leukopenia and thrombocytopenia, as well as changes in bone marrow cellularity increased, macrophage proliferation and fibrosis accompanied by a reduction in IGF-I expression. Thus we can conclude that our data indicate that the hamster is a good model to study the pathogenesis of pancytopenia and marrow changes resulting from infection by L. (L.) Infantum. In this model, alteration of IGF-I expression during the course of infection with a possible role in the pathogenesis of pancytopenia.

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