Dissertations / Theses: 'Genetics, Experimental'

1

Dahlman, Ingrid. "Genetic dissection of experimental autoimmune neuroinflammatory diseases in rats /." Stockholm, 1999. http://diss.kib.ki.se/1999/91-628-3768-0/.

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2

Jagodić, Maja. "The complex genetics of experimental autoimmune neuroinflammation /." Stockholm, 2004. http://diss.kib.ki.se/2004/91-7140-157-1/.

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3

Lux, Melissa McNeil. "A laboratory course in experimental genetics for the biology major." Thesis, University of North Texas, 2001. https://digital.library.unt.edu/ark:/67531/metadc3007/.

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This manual has been designed for a class of twenty- four students concurrently enrolled in the lecture course. The laboratory aids in the learning process and fosters an interest in the science of genetics. This manual and the experiments contained within are both informative and fun. The manual correlates with and expands upon the genetics course. Each investigation, with the exception of the Drosophila melanogaster project, can be completed in a 3-4 hour timeframe. This manual provides a “hands on” experience of theories simply discussed in the lecture course. This manual is intended to be a one-source manual where each investigation is designed to include an adequate introduction. Special attention has been given for each investigation with both the student and instructor in mind.

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4

Reodica, Mayfebelle Biotechnology &amp Biomolecular Sciences Faculty of Science UNSW. "Transcriptional repression mechanisms of sporulation-specific genes in saccharomyces cerevisiae." Awarded by:University of New South Wales. School of Biotechnology and Biomolecular Sciences, 2006. http://handle.unsw.edu.au/1959.4/32731.

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For organisms undergoing a developmental process it is ideal that specific genes are induced and repressed at the correct time and to the correct level in a coordinated manner. The process of meiosis and spore formation (collectively known as sporulation) in Saccharomyces cerevisiae provides a convenient system to elucidate transcriptional mechanisms of gene repression and the contribution such repression mechanisms offer to cells capable of undergoing a developmental process. This thesis focuses on transcriptional repression of sporulation-specific genes during both vegetative/mitotic conditions and sporulation. The fitness contribution of transcriptional repressors that regulate sporulationspecific genes during vegetative growth were investigated considering the similarities between meiosis and mitosis such as DNA replication, chromosome segregation and cytokinesis. Well-characterised sporulation genes of different functions were expressed in vegetative cells and ectopic expression of these genes was found not to be lethal. It was ascertained through strain competition studies that ectopic expression of the genes IME1, SMK1, SPR3 and DIT1 during mitotic growth did not affect cellular fitness. The expression of NDT80 in vegetative cells, however, caused a marked reduction in fitness and cells were also further compromised in the absence of the Sum1p repressor that regulates NDT80 transcription. The role of NDT80 as a transcriptional activator of middle sporulation genes, rather than the over-expression of NDT80 as a protein, caused the reduction of cell viability. Transcriptional regulation of the middle sporulation-specific gene SPR3 by the meiosis-specific Set3p repressor complex was investigated using synchronous sporulation cultures of the W303a/?? strain commonly used for sporulation studies. In a mutant W303a/?? ??set3/??set3 strain, lacking a key component of the Set3p repression complex, the transcription of SPR3 was uncharacteristically expressed at higher levels and derepressed during late sporulation. This SPR3 expression was consistent for both SPR3 transcript and SPR3::lacZ reporter protein studies. This preliminary work will enable future studies, using SPR3 promoter deletions fused to a lacZ reporter, aimed at determining the region of the SPR3 promoter that the Set3p complex may interact with to transcriptionally repress the gene during sporulation.

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5

Johnson, Sarah J. "Sinusoidal cell responses to experimental liver injury." Thesis, University of Newcastle Upon Tyne, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.320389.

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6

Ono, Jasmine. "Genetics of adaptation in experimental populations of yeast." Thesis, University of British Columbia, 2017. http://hdl.handle.net/2429/64159.

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Evolution proceeds through genetic changes to individuals, which are either propagated or disappear over generations. Adaptation is one of the main mechanisms driving these changes in genetic composition. Speciation can also result from different, and incompatible, genetic changes occurring in different populations. This thesis furthers our knowledge of the genetics of adaptation and speciation using the budding yeast Saccharomyces cerevisiae. My work on the genetic basis of adaptation to high concentrations of copper, when contrasted with a similar experiment using the fungicide nystatin, showed that the environment has a strong influence on both the number of genes that are the targets of selection and the types of potentially beneficial mutations. These results have implications for the repeatability of genetic evolution. In a second study, I found that genetic interactions between individually isolated single-step beneficial mutations from the same selective environment often exhibited the type of epistasis that underlies speciation even though these mutations occurred within a single biosynthetic pathway. These results support the mutation-order model of speciation by adaptation, where the chance order of mutations in separated populations leads to divergence and the build-up of reproductive isolation due to genetic incompatibility. Negative genetic interactions became positive when the level of stress was increased, indicating that genetically-based reproductive isolation can also be environment-dependent. Finally, I found that diploid yeast were generally not able to adapt to a level of fungicide to which haploid yeast can adapt. Diploids have been found to adapt to a lower concentration of the same drug, indicating that the exact environment (type and concentration) and ploidy can have an impact on the likelihood of genetic rescue. Together, these results have implications for our understanding of the genetic basis of adaptation in different types of environments and different levels of the same environmental stressor.
Science, Faculty of
Zoology, Department of
Graduate

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7

Herath, Shanti Irene. "Characterisation of CD+ T cells in experimental cutaneous Leishmaniasis." Thesis, Imperial College London, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.269692.

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8

Sinha, S. "The role of oncogenes in experimental rat liver cancer." Thesis, Open University, 1989. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.234898.

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9

Gifford, Danna R. "Population genetics of rifampicin-resistant Pseudomonas aeruginosa." Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:044b9258-4f10-4e77-9ff6-aa4035cec33b.

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Antibiotic resistance is generally associated with a cost in terms of reduced competitive fitness in the absence of antibiotics. Despite this 'cost of resistance', the cessation of antibiotic treatment does not result in significant reductions in the prevalence of resistance. The maintenance of resistance, in spite of the costs, has been attributed to the rarity of reversion mutations, relative to compensatory mutations at other loci in the genome. However, the large size of bacteria populations, and the potential for migration, suggest that reversion mutations should occasionally be introduced to resistant populations. In this thesis, I show that additional mechanisms can prevent fixation of reversion mutations even if they do occur. Using an experimental evolution approach, with rifampicin resistance in Pseudomonas aeruginosa as a model system, I measured the costs of resistance in several environments and followed the adaptive dynamics of resistant populations where a sensitive lineage had invaded by migration. The results suggest that several additional mechanisms contribute to the maintenance of antibiotic resistance. Most rifampicin resistance mutations are not unconditionally costly in all environments, suggesting that migration between environments could maintain a resistant reservoir population. In environments where resistance is initially costly, the fixation of a revertant is not guaranteed, even if introduced through migration. Revertant fixation was impeded or prevented by clonal interference from adaptation in the resistant strain. Revertants that did successfully replace the resistant strain were forced to adapt to do so. Contrary to assumptions in the existing literature, fitness in the resistant strains was not recovered by general compensatory mutations, but instead by adaptive mutations specific to the environment. The data challenge several assumptions about the maintenance of antibiotic resistance: that resistance mutations are always costly, that the rarity of back mutations prevents the reversion of resistance, and that resistant strains recover fitness by compensatory mutations.

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10

DIAS, VIVIANE L. "Aspectos da resistencia a infeccao experimental com Trypanosoma cruzi." reponame:Repositório Institucional do IPEN, 2010. http://repositorio.ipen.br:8080/xmlui/handle/123456789/9609.

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Dissertacao (Mestrado)
IPEN/D
Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

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11

Li, Luosheng. "Molecular genetics of type 2 diabetes /." Stockholm, 2002. http://diss.kib.ki.se/2002/91-7349-194-2/.

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12

Carroll, Scott. "Functional and genetic dissection of susceptibility to experimental «Cryptococcus neoformans» infection." Thesis, McGill University, 2010. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=95084.

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This thesis examines the functional and genetic factors that control the inbred mouse host response to Cryptococcus neoformans. The inbred mouse strains C57BL/6J and C3H/HeN were susceptible, and the inbred mouse strains CBA/J and SJL/J were resistant, to experimental cryptococcal pneumonia. This observation led to three general hypotheses: (1) early innate immune differences between inbred strains cause differential susceptibility to cryptococcal pneumonia; (2) natural variation in susceptibility among inbred strains is a complex trait under the control of specific genetic loci; and (3) allergic airway disease is a common phenotype among susceptible inbred strains. Functional characterization of the innate immune response in C57BL/6J and SJL/J mice revealed a heightened pro-inflammatory response in SJL/J as early as three hours post-infection. This polarization continued throughout the course of infection; C57BL/6J mice presented an allergic, Th2 immune response, and SJL/J mice presented a Th1 immune response. Intracellular signaling analysis in vitro revealed that the enhanced pro-inflammatory response observed in SJL/J mice was dependent on the prolonged activation of the NF-κB and phosphatidylinositol 3 kinase pathways. Next, a quantitative trait loci (QTL) analysis for cryptococcal pneumonia susceptibility in a segregating F2 population bred from the parental inbred strains C57BL/6J and CBA/J was performed. This revealed a sex-effect in the quantitative lung fungal burden that warranted a stratified QTL analysis approach. Two significant novel QTL were identified in the female F2 intercross: Cnes1 on chromosome 1, and Cnes2 on chromosome 17. Cnes3 was identified in the male F2 intercross as a unique QTL distal to Cnes2. Furthermore, a genome-wide pairwise analysis revealed significant QTL interactions in both the female and male F2 intercrosses that collectively explained 43.8 and 19.5% of the phenotypic variance in each sex, respectively. Finally, characte
Cette thèse étudie les facteurs fonctionnels et génétiques contrôlant la réponse de l'hôte aux infections fongiques causées par la levure Cryptococcus neoformans. Les lignées de souris consanguines C57BL/6J et C3H/HeN sont sensibles à la pneumonie expérimentale à cryptocoques, alors que les lignées CBA/J et SJL/J y sont résistantes. En se basant sur ces observations, trois hypothèses ont été formulées: (1) des différences du système immunitaire inné expliquent la sensibilité différentielle à la pneumonie à cryptocoques des lignées de souris consanguines; (2) la variation naturelle de la sensibilité entre lignées consanguines est un trait génétique complexe sous le contrôle de locus génétiques spécifiques; et (3) la maladie allergique respiratoire est un phénotype commun associé à une sensibilité à la pneumonie à cryptocoques progressive chez les lignées consanguines. La caractérisation fonctionnelle de la réaction immunitaire innée chez les souris C57BL/6J et SJL/J a révélé une réponse pro-inflammatoire accrue chez les souris SJL/J trois heures après l'infection. Cette polarisation a continué tout au long de l'infection, les souris C57BL/6J développant une réponse immunitaire allergique Th2, alors que les souris SJL/J ont présenté une réponse immunitaire Th1. Une analyse in vitro de la signalisation intracellulaire a révélé que la réaction pro-inflammatoire observée chez les souris SJL/J dépend de l'activation prolongée des cascades de signalisation NF-κB et PI3K (phosphatidylinositol 3 kinase). L'étude des liaisons de traits complexes (QTL) chez des souris F2 (C57BL/6J x CBA/J) a révélé un effet du sexe sur le phénotype, justifiant ainsi une analyse séparée. Deux locus de susceptibilité ont été identifiés chez les femelles: Cnes1 (chromosome 1), et Cnes2 (chromosome 17). Chez les mâles, seul le locus Cnes3 (chromosome 17, en aval de Cnes2) a été identifié. Une analyse QTL sur 2 locus a

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13

Kennedy, James Andrew. "Characterization and modulation of Kupffer cell function in experimental obstructive jaundice." Thesis, Queen's University Belfast, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.387894.

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14

Xinmin, Li. "Experimental studies of pleiotropy at the Adh locus of Drosophila melanogaster." Thesis, University of Nottingham, 1990. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.276173.

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15

Chan, A. C. Y. R. "The genetics of vaccine responses in experimental lines of chickens." Thesis, University of Cambridge, 2015. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.709442.

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16

Mortimer, Sandra 1981. "Experimental analysis of trans-splicing of an ascidian troponin I gene." Thesis, McGill University, 2007. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=101643.

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I investigated SL trans-splicing in the troponin I gene of Ciona intestinalis. Experimental mutation of the AG dinucleotide adjacent to the natural trans-splice acceptor site (-64) in CiTnI/nuclacZ constructs eliminated trans-splicing to that site in Ciona embryos but activated trans-splicing at cryptic acceptor sites at -76 and -39, adjacent to the nearest AG dinucleotides. However, not all AG dinucleotides specify cryptic acceptor sites because outron internal deletions or 3'truncation mutants were trans-spliced at a far-upstream AG-adjacent cryptic site (-346), leaving many AGs in the retained outron segments. Thus, additional sequence elements that are present only in the -346 and -76/-64/-39 regions are required for cryptic acceptor activity. All mutant constructs generated detectable beta-gal enzyme expression, although the mutant with the longest retained-outron segment appeared less active. Therefore, mRNA accumulation and translation do not require trans-splicing to the natural acceptor site, although they may be facilitated by the normal removal of the outron during trans-splicing.

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17

Ferreira, Lívia Carvalho [UNESP]. "Efeito da curcumina na angiogênese em modelo experimental de câncer de mama." Universidade Estadual Paulista (UNESP), 2014. http://hdl.handle.net/11449/111000.

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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
O câncer de mama representa a neoplasia mais comum entre as mulheres, sendo classificado como o segundo mais frequente no mundo. O crescimento do tumor requer a formação de novos vasos que são estimulados por fatores angiogênicos e seus receptores. As células neoplásicas são capazes de modificar seu fenótipo para promover alterações funcionais e estimular a produção de proteínas envolvidas na angiogênese. A curcumina, componente do extrato de Curcuma longa L, é utilizada como alimento e na medicina tradicional, no entanto algumas evidências indicam que esse extrato possui ação oncostática em diferentes tipos de câncer. Assim, o objetivo do presente estudo foi avaliar os efeitos do tratamento com curcumina na progressão tumoral e angiogênese em modelo experimental de câncer de mama. Células da linhagem de câncer de mama receptor triplo negativo (MDA-MB-231) foram cultivadas e a taxa de viabilidade celular verificada pelo ensaio MTT após tratamento com diferentes doses de curcumina. Para o estudo in vivo, as células foram implantadas em camundongos nude atímicos, os quais foram aleatoriamente divididos em animais tratados (n = 5) e controles (n = 8). Os animais receberam diariamente 300 mg/kg de curcumina ou veículo administrados intraperitonealmente durante 21 dias, cinco vezes por semana. O tamanho do tumor foi avaliado semanalmente com paquímetro digital. Ao final do tratamento, a angiogênese foi verificada in vivo pela técnica de tomografia computadorizada por emissão de fóton único (SPECT), com o radiotraçador Tc-99m acoplado a proteína recombinante VEGF-c, que possui afinidade pelos receptores VEGFR2/3. Além disso, a expressão de VEGF-A, VEGF-C, VEGFR2/3, marcador de proliferação celular (Ki-67) e o fator de Von Willebrand (vWF) foram verificados pelo procedimento imuno-histoquímico. No estudo in vitro, houve diminuição da taxa de viabilidade das células tratadas com curcumina, quando comparadas ...
Breast cancer is the most common cancer in women being rated as the second most common in the world. Tumor growth requires the formation of new vessels that are stimulated by angiogenic factors and their receptors. The neoplastic cells are able to modify their phenotype to promote functional changes and stimulate the production of proteins involved in angiogenesis. Curcumin, a component of the extract of Curcuma longa L, is used both as food and in traditional medicine, however some evidence indicates that this extract has oncostatic effects in different types of cancer. The aim of this study was to evaluate the effects of treatment with curcumin in tumor progression and angiogenesis in an experimental model of breast cancer. The cell line of human breast cancer (MDA-MB-231) was cultured and the rate of cell viability was measured by MTT assay after treatment with different doses of curcumin. For in vivo study, the cells were implanted in female athymic nude mice, which were randomly divided into treated (n = 5) and control animals (n = 8). The animals received daily 300 mg/kg of curcumin or vehicle daily, starting in the same day as tumor implantation and continued for 21 days five times per week. Tumor size was measured weekly with a digital caliper. The end of treatment, angiogenesis was assessed in vivo by the technique of computed tomography single photon emission tomography (SPECT) with the radiotracer Tc-99m-HYNIC-VEGF-c protein coupled recombining, which has affinity for VEGFR2/3. Furthermore, the expression of VEGF-A, VEGF-C e VEGFR2/3 marker of cell proliferation (Ki-67) and von Willebrand factor (vWF) receptors were verified by immunohistochemical. Curcumin treatment in vitro was able to significantly decrease cell viability (p < 0.05) Animals treated with curcumin showed less tumor volume (232.5 ± 53.2 mm 3) compared to control animals (282.0 ± 88.5 mm3), however, no significant difference (p > 0,05). The radioactivity of ...

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18

Ferreira, Lívia Carvalho. "Efeito da curcumina na angiogênese em modelo experimental de câncer de mama /." São José do Rio Preto, 2014. http://hdl.handle.net/11449/111000.

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Orientador: Debora Aparecida Pires de Campos Zuccari
Banca: Sônia Maria Oliani
Banca: Angelo Gustavo Zucca Matthes
Resumo: O câncer de mama representa a neoplasia mais comum entre as mulheres, sendo classificado como o segundo mais frequente no mundo. O crescimento do tumor requer a formação de novos vasos que são estimulados por fatores angiogênicos e seus receptores. As células neoplásicas são capazes de modificar seu fenótipo para promover alterações funcionais e estimular a produção de proteínas envolvidas na angiogênese. A curcumina, componente do extrato de Curcuma longa L, é utilizada como alimento e na medicina tradicional, no entanto algumas evidências indicam que esse extrato possui ação oncostática em diferentes tipos de câncer. Assim, o objetivo do presente estudo foi avaliar os efeitos do tratamento com curcumina na progressão tumoral e angiogênese em modelo experimental de câncer de mama. Células da linhagem de câncer de mama receptor triplo negativo (MDA-MB-231) foram cultivadas e a taxa de viabilidade celular verificada pelo ensaio MTT após tratamento com diferentes doses de curcumina. Para o estudo in vivo, as células foram implantadas em camundongos nude atímicos, os quais foram aleatoriamente divididos em animais tratados (n = 5) e controles (n = 8). Os animais receberam diariamente 300 mg/kg de curcumina ou veículo administrados intraperitonealmente durante 21 dias, cinco vezes por semana. O tamanho do tumor foi avaliado semanalmente com paquímetro digital. Ao final do tratamento, a angiogênese foi verificada in vivo pela técnica de tomografia computadorizada por emissão de fóton único (SPECT), com o radiotraçador Tc-99m acoplado a proteína recombinante VEGF-c, que possui afinidade pelos receptores VEGFR2/3. Além disso, a expressão de VEGF-A, VEGF-C, VEGFR2/3, marcador de proliferação celular (Ki-67) e o fator de Von Willebrand (vWF) foram verificados pelo procedimento imuno-histoquímico. No estudo in vitro, houve diminuição da taxa de viabilidade das células tratadas com curcumina, quando comparadas ...
Abstract: Breast cancer is the most common cancer in women being rated as the second most common in the world. Tumor growth requires the formation of new vessels that are stimulated by angiogenic factors and their receptors. The neoplastic cells are able to modify their phenotype to promote functional changes and stimulate the production of proteins involved in angiogenesis. Curcumin, a component of the extract of Curcuma longa L, is used both as food and in traditional medicine, however some evidence indicates that this extract has oncostatic effects in different types of cancer. The aim of this study was to evaluate the effects of treatment with curcumin in tumor progression and angiogenesis in an experimental model of breast cancer. The cell line of human breast cancer (MDA-MB-231) was cultured and the rate of cell viability was measured by MTT assay after treatment with different doses of curcumin. For in vivo study, the cells were implanted in female athymic nude mice, which were randomly divided into treated (n = 5) and control animals (n = 8). The animals received daily 300 mg/kg of curcumin or vehicle daily, starting in the same day as tumor implantation and continued for 21 days five times per week. Tumor size was measured weekly with a digital caliper. The end of treatment, angiogenesis was assessed in vivo by the technique of computed tomography single photon emission tomography (SPECT) with the radiotracer Tc-99m-HYNIC-VEGF-c protein coupled recombining, which has affinity for VEGFR2/3. Furthermore, the expression of VEGF-A, VEGF-C e VEGFR2/3 marker of cell proliferation (Ki-67) and von Willebrand factor (vWF) receptors were verified by immunohistochemical. Curcumin treatment in vitro was able to significantly decrease cell viability (p < 0.05) Animals treated with curcumin showed less tumor volume (232.5 ± 53.2 mm 3) compared to control animals (282.0 ± 88.5 mm3), however, no significant difference (p > 0,05). The radioactivity of ...
Mestre

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19

Mustafa, Elwaleed Ibrahim. "Experimental autoimmune sialadenitis : studies of immunopathogenesis, cellular signaling and MHC genetics /." Stockholm, 2001. http://diss.kib.ki.se/2001/91-628-4791-0/.

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20

Thorsell, Annika. "Central neuropeptide Y (NPY) expression and function : role in stress, experimental anxiety, and cognition /." Stockholm, 2000. http://diss.kib.ki.se/2000/91-628-4256-0/.

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21

Dutta, Ranendra Nath. "Experimental Test of Solitary Wave Theory in Viral Populations." University of Toledo Health Science Campus / OhioLINK, 2008. http://rave.ohiolink.edu/etdc/view?acc_num=mco1226950654.

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22

Jironkin, Aleksey. "Computational and experimental analysis of plant promoters : identifying functional elements." Thesis, University of Warwick, 2013. http://wrap.warwick.ac.uk/60669/.

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Understanding the regulatory DNA sequences are becoming increasingly important in understanding the way plants integrate signalling cues mediated through the actions of the transcription factors (TFs). This thesis presents an interdisciplinary investigations into regulatory elements found in the promoter regions of a model organism Arabidopsis thaliana. The intergenic DNA sequences are studied between sets of orthologous genes in A. thaliana and 3 other related species to uncover hundreds of evolutionary conserved noncoding sequences (CNSs). The CNSs are found to be more skewed towards the annotated transcription start sites (TSSs) and enriched in previously identified transcription factors binding motifs. Furthermore, the nucleosomes are predicted to have strong presence in the uncovered CNS than random intergenic sequences alone. Altogether the evidence presented in the thesis points to the functional nature of the CNSs. Then, the promoters of genes thought to be co-regulated together and transcriptionally active during infection with fungal pathogen Botrytis cinerea are experimentally tested for direct protein-DNA interaction using high-throughput Yeast One-Hybrid (Y1H) library screens against the TFs found in A. thaliana. The resulting predictions were further validated using pairwise Y1H screen to suggest potential common regulation by ORA59, PIF7, ESE1, At4g38900 and ERF14, and uncovering a complex gene regulatory network (GRN) associated with the tested genes. The promoter fragments together with the predictions from the Y1H screens were used in the computational analysis to establish transcription factor specific binding motifs. Some of the newly predicted motifs were mutated and tested again for altered binding of the associated TFs. Furthermore, in planta mutations of the TFs predicted to be interacting with the promoters of the genes in the Y1H screens were found to have significant impact on the susceptibility of A. thaliana to infection with B. cinerea, further informing gene regulatory network active in response to biotic stress.

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23

Chao, Vincent 1973. "Ecological and sexual divergence in experimental populations of Chlamydomonas." Thesis, McGill University, 2001. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=32982.

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Laboratory studies on speciation have revealed that selection must be disruptively applied on traits related to the mating system in order to produce deviations from random mating in experimental populations. One problem with these experiments, however, has been the complexity of the model organism used, most frequently Drosophila species. Due to the multi genic nature of the mating systems of such organisms, it has been difficult to obtain the necessary gene combinations that result in complete sexual isolation. In the present study, I have used a simple sexual organism, the unicellular green algae Chlamydomonas reinhardtii, as a model for ecological and sexual differentiation. Disruptive selection was applied on the flagella, by selecting simultaneously for photo taxis and mating, behaviours for which these organelles are of fundamental importance. An asymmetric response to selection for photo taxis and zygote production was obtained in populations selected for conditions at opposite ends of the environmental spectrum used, differentiating these two populations in both movement capacity and mating efficiency. These results are discussed in relation to previous experiments on speciation and to the implications of future experimental studies on the same subject.

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24

Hietpas, Ryan T. "Experimental Illumination of Comprehensive Fitness Landscapes: A Dissertation." eScholarship@UMMS, 2006. http://escholarship.umassmed.edu/gsbs_diss/667.

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Evolution is the single cohesive logical framework in which all biological processes may exist simultaneously. Incremental changes in phenotype over imperceptibly large timescales have given rise to the enormous diversity of life we witness on earth both presently and through the natural record. The basic unit of evolution is mutation, and by perturbing biological processes, mutations may alter the fitness of an individual. However, the fitness effect of a mutation is difficult to infer from historical record, and complex to obtain experimentally in an efficient and accurate manner. We have recently developed a high throughput method to iteratively mutagenize regions of essential genes in yeast and subsequently analyze individual mutant fitness termed Exceedingly Methodical and Parallel Investigation of Randomized Individual Codons (EMPIRIC). Utilizing this technique as exemplified in Chapters II and III, it is possible to determine the fitness effects of all possible point mutations in parallel through growth competition followed by a high throughput sequencing readout. We have employed this technique to determine the distribution of fitness effects in a nine amino acid region of the Hsp90 gene of S. cerevisiae under elevated temperature, and found the bimodal distribution of fitness effects to be remarkably consistent with near-neutral theory. Comparing the measured fitness effects of mutants to the natural record, phylogenetic alignments appear to be a poor predictor of experimental fitness. In Chapter IV, to further interrogate the properties of this region, library competition under conditions of elevated temperature and salinity were performed to study the potential of protein adaptation. Strikingly, whereas both optimal and elevated temperatures produced no statistically significant beneficial mutations, under conditions of elevated salinity, adaptive mutations appear with fitness advantages up to 8% greater than wild type. Of particular interest, mutations conferring fitness benefits under conditions of elevated salinity almost always experience a fitness defect in other experimental conditions, indicating these mutations are environmentally specialized. Applying the experimental fitness measurements to long standing theoretical predictions of adaptation, our results are remarkably consistent with Fisher’s Geometric Model of protein evolution. Epistasis between mutations can have profound effects on evolutionary trajectories. Although the importance of epistasis has been realized since the early 1900s, the interdependence of mutations is difficult to study in vivo due to the stochastic and constant nature of background mutations. In Chapter V, utilizing the EMPIRIC methodology allows us to study the distribution of fitness effects in the context of mutant genetic backgrounds with minimal influence from unintended background mutations. By analyzing intragenic epistatic interactions, we uncovered a complex interplay between solvent shielded structural residues and solvent exposed hydrophobic surface in the amino acid 582-590 region of Hsp90. Additionally, negative epistasis appears to be negatively correlated with mutational promiscuity while additive interactions are positively correlated, indicating potential avenues for proteins to navigate fitness ‘valleys’. In summary, the work presented in this dissertation is focused on applying experimental context to the theory-rich field of evolutionary biology. The development and implementation of a novel methodology for the rapid and accurate assessment of organismal fitness has allowed us to address some of the most basic processes of evolution including adaptation and protein expression level. Through the work presented here and by investigators across the world, the application of experimental data to evolutionary theory has the potential to improve drug design and human health in general, as well as allow for predictive medicine in the coming era of personalized medicine.

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25

Hietpas, Ryan T. "Experimental Illumination of Comprehensive Fitness Landscapes: A Dissertation." eScholarship@UMMS, 2013. https://escholarship.umassmed.edu/gsbs_diss/667.

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Evolution is the single cohesive logical framework in which all biological processes may exist simultaneously. Incremental changes in phenotype over imperceptibly large timescales have given rise to the enormous diversity of life we witness on earth both presently and through the natural record. The basic unit of evolution is mutation, and by perturbing biological processes, mutations may alter the fitness of an individual. However, the fitness effect of a mutation is difficult to infer from historical record, and complex to obtain experimentally in an efficient and accurate manner. We have recently developed a high throughput method to iteratively mutagenize regions of essential genes in yeast and subsequently analyze individual mutant fitness termed Exceedingly Methodical and Parallel Investigation of Randomized Individual Codons (EMPIRIC). Utilizing this technique as exemplified in Chapters II and III, it is possible to determine the fitness effects of all possible point mutations in parallel through growth competition followed by a high throughput sequencing readout. We have employed this technique to determine the distribution of fitness effects in a nine amino acid region of the Hsp90 gene of S. cerevisiae under elevated temperature, and found the bimodal distribution of fitness effects to be remarkably consistent with near-neutral theory. Comparing the measured fitness effects of mutants to the natural record, phylogenetic alignments appear to be a poor predictor of experimental fitness. In Chapter IV, to further interrogate the properties of this region, library competition under conditions of elevated temperature and salinity were performed to study the potential of protein adaptation. Strikingly, whereas both optimal and elevated temperatures produced no statistically significant beneficial mutations, under conditions of elevated salinity, adaptive mutations appear with fitness advantages up to 8% greater than wild type. Of particular interest, mutations conferring fitness benefits under conditions of elevated salinity almost always experience a fitness defect in other experimental conditions, indicating these mutations are environmentally specialized. Applying the experimental fitness measurements to long standing theoretical predictions of adaptation, our results are remarkably consistent with Fisher’s Geometric Model of protein evolution. Epistasis between mutations can have profound effects on evolutionary trajectories. Although the importance of epistasis has been realized since the early 1900s, the interdependence of mutations is difficult to study in vivo due to the stochastic and constant nature of background mutations. In Chapter V, utilizing the EMPIRIC methodology allows us to study the distribution of fitness effects in the context of mutant genetic backgrounds with minimal influence from unintended background mutations. By analyzing intragenic epistatic interactions, we uncovered a complex interplay between solvent shielded structural residues and solvent exposed hydrophobic surface in the amino acid 582-590 region of Hsp90. Additionally, negative epistasis appears to be negatively correlated with mutational promiscuity while additive interactions are positively correlated, indicating potential avenues for proteins to navigate fitness ‘valleys’. In summary, the work presented in this dissertation is focused on applying experimental context to the theory-rich field of evolutionary biology. The development and implementation of a novel methodology for the rapid and accurate assessment of organismal fitness has allowed us to address some of the most basic processes of evolution including adaptation and protein expression level. Through the work presented here and by investigators across the world, the application of experimental data to evolutionary theory has the potential to improve drug design and human health in general, as well as allow for predictive medicine in the coming era of personalized medicine.

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26

Baldwin-Brown, James. "Identifying selection in differentiated populations through simulation, experimental evolution, and whole genome sequencing." Thesis, University of California, Irvine, 2017. http://pqdtopen.proquest.com/#viewpdf?dispub=10245933.

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Population differentiation is both one of the central processes underlying the diversity that we observe in the natural world, and a mechanism that can be used to differentiate between evolutionary forces both at the level of the polymorphism, and at the level of the entire genome. Here, I use simulated evolution to analyze the statistical power to detect signals of selection in artificially selected laboratory populations, and use genomic data from wild populations of the clam shrimp Eulimnadia texana to identify genomic signals of selection in wild populations. Several loci in the wild populations appear to be under selection, and I analyze the types of genes that appear to contribute to differentiation of these populations. Additionally, I describe an analysis of genome assembly techniques that allowed for the creation of a highly contiguous genome assembly in the clam shrimp. I find that a pipeline that uses custom software to combine the results of several different genome assemblers is capable of producing genomes using long-read genomic sequencing data that are orders of magnitude more contiguous that pre-long-read methods. Simulations of experimental evolution indicated that extremely high levels of replication were necessary in order to achieve high power to detect signals of selection in experimental evolution. To this end, I describe a set of replicate experimentally evolved populations of E. texana that can be used to identify regions under selection with much higher power than could be accomplished with earlier experimental evolution schemes.

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27

Frenk, Mora Silvia Elena. "Embryology in its relation to genetics and evolution : experimental analysis and historical perspectives." Thesis, University of Cambridge, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.283901.

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28

Immonen, Elina. "Evolutionary genetics and genomics of the female side of sexual interactions in Drosophila." Thesis, University of St Andrews, 2012. http://hdl.handle.net/10023/3224.

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Sexual interactions play an important role in generating sexual selection and antagonistic co-evolution. These forces can shape differences between the sexes, but also have the potential to generate population divergence and contribute to speciation. The aim in this thesis was to provide new insights into the genes involved in different stages of female sexual interactions, using Drosophila as a model system. In chapter 2 I tested whether a candidate gene (period) that influences species-specific rhythmic characteristics in male courtship song in D. melanogaster also has a pleiotropic effect on female song preference. Using mutant and transgenic strains I found support for this. In chapter 3 I examined further how females respond to the song at the level of gene expression, using microarrays. Expression profiles revealed modest changes in transcripts abundance overall, which were dominated by antennal olfactory genes, neuropeptide encoding genes and immunity genes. Many of these have previously been found to respond to mating. In chapter 4 I therefore studied further two of these genes, TurandotM and TurandotC and their role in female post-mating fitness. Using RNA interference I found that knocking down these genes influenced immediate fecundity. In chapter 5 I focused on analysing post-mating gene expression patterns in relation to sexual selection in D. pseudoobscura using microarrays. I explored the consequences of experimental variation in female promiscuity on gene expression divergence as a whole, and in response to mating. I found large-scale expression divergence between monandrous and polyandrous females after 100 generations of experimental evolution. Experimental polyandry increased the expression of genes that show female-biased expression in wild-type individuals and decreased male- biased gene expression. Females experiencing no sexual selection showed the opposite pattern. Out of the genes affected by mating, the majority showed increased expression in polyandrous compared to monandrous females, with enrichment e.g. in oogenesis-related genes.

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Jamall, Siddiqua. "Factors influencing the regulation of cytoplasmic expression of IL-2 mRNA from a eukaryotic expression vector : an experimental and image-based study." Thesis, King's College London (University of London), 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.246853.

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30

Garmendia, Eva. "A Unified Multitude : Experimental Studies of Bacterial Chromosome Organization." Doctoral thesis, Uppsala universitet, Institutionen för medicinsk biokemi och mikrobiologi, 2017. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-332471.

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Bacteria are many, old and varied; different bacterial species have been evolving for millions of years and show many disparate life-styles and types of metabolism. Nevertheless, some of the characteristics regarding how bacteria organize their chromosomes are relatively conserved, suggesting that they might be both ancient and important, and that selective pressures inhibit their modification. This thesis aims to study some of these characteristics experimentally, assessing how changes affect bacterial growth, and how, after changing conserved features, bacteria might evolve. First, we experimentally tested what are the constraints on the horizontal transfer of a gene highly important for bacterial growth. Second, we investigated the significance of the location and orientation of a highly expressed and essential operon; and we experimentally evolved strains with suboptimal locations and orientations to assess how bacteria could adapt to these changes. Thirdly, we sought to understand the accessibility of different regions of the bacterial chromosome to engage in homologous recombination. And lastly, we constructed bacterial strains with chromosomal inversions to assess what effect the inversions had on growth rate, and how bacteria carrying costly inversions could evolve to reduce these costs. The results provide evidence for different selective forces acting to conserve these chromosome organizational traits. Accordingly, we found that evolutionary distance, functional conservation, suboptimal expression and impaired network connectivity of a gene can affect the successful transfer of genes between bacterial species. We determined that relative location of an essential and highly expressed operon is critical for supporting fast growth rate, and that its location seems to be more important than its orientation. We also found that both the location, and relative orientation of separated duplicate sequences can affect recombination rates between these sequences in different regions of the chromosome. Finally, the data suggest that the importance of having the two arms of a circular bacterial chromosome approximately equal in size is a strong selective force acting against certain type of chromosomal inversions.

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31

Truman, Julie. "Experimental evolution of parasite life history in bacteriophage Φ2." Thesis, University of Liverpool, 2014. http://livrepository.liverpool.ac.uk/2007429/.

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Parasite life history theory predicts that lifetime reproductive success evolves through differential allocation of energy to life history traits constrained by trade-offs. These life history traits govern the characteristics of parasites such as their virulence, transmission and infection phenotypes, so understanding their evolution is a key concern for infectious disease prediction and management. This thesis uses the powerful tool of experimental evolution to gain a fuller understanding of the factors and constraints involved in parasite life history evolution, using bacteriophage Φ2 as a model. I found that the evolution of life history in this phage is sensitive to spatial structure, UV-C exposure and coparasitism with plasmids, and evolution can be mediated by co-evolution with the host. The high levels of variance I observed here suggest that evolution of parasite life history is more complex than a single trajectory towards a predicted optimum, and likely involves some degree of epistasis or pleiotropy with genes elsewhere on the genome. There was some degree of independent evolution of individual life-history traits, indicating that simple direct trade-offs were not in operation. I demonstrated that co-evolution with the host provided additional mutational input, resulting in a greater degree of evolution in co-evolved populations than those evolved to a static host. Furthermore, I note that co-parasitism with phage and plasmid may provide the necessary conditions for plasmid persistence under fluctuating selection for plasmid-encoded traits, and that the efficacy and suitability of phage as therapeutic agents against plasmid-encoded antibiotic resistance is complicated. No direct link between mutation and phenotype could be elucidated in this study, suggesting that evolution in life history is either governed by genes not examined in this thesis, or involves epistasis and pleiotropy with genes elsewhere on the genome. I concluded that it is important to consider the specific ecology of the focal parasite, its host and any co-occuring symbionts in order to make informed predictions of life history evolution, and general predictions may not be achievable.

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32

Bäckman, Ulrika. "Treatment of Experimental Neuroblastoma with Angiogenic Inhibitors." Doctoral thesis, Uppsala University, Department of Medical Cell Biology, 2003. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-3536.

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Neuroblastoma is a childhood cancer that originates from neuroblasts in the peripheral nervous system. Neuroblastoma show considerable heterogeneity with respect to location, responsiveness to treatment and prognosis. Since current therapy involves drugs with risk of serious side effects in the growing child, there is a clinical need for more effective and less toxic treatment strategies.

Angiogenesis, the formation of new blood vessels, is critical for tumor progression. Specific inhibition of tumor-induced angiogenesis should restrict growth of most solid tumors and thereby provide a new treatment strategy. The aim of this study was to investigate the effects of angiogenic inhibition in experimental neuroblastoma in mice.

We found that experimental neuroblastomas expressed the perhaps most potent angiogenic growth factor, VEGF-A, and that plasma VEGF-A levels correlated with tumor size. SU5416, a novel antagonist of VEGFR-1 and 2, reduced angiogenesis and tumor growth in our model. We also investigated the properties of SU11657, a new, orally available, synthetic small molecule multi-targeted tyrosine kinase inhibitor. SU11657, at a well-tolerated dose, was more potent than SU5416 in reducing tumor growth rate and angiogenesis, even in MYCN-amplified tumors. Chemotherapeutics can also inhibit angiogenesis, when administrated daily in a non-toxic dose. CHS 828, a new chemotherapeutic, given orally, alone induced complete neuroblastoma regression in 44 % of the animals. Furthermore, the bisphosphonate zoledronic acid, developed to reduce bone resorption, showed anti-tumor activity in our model. Zoledronic acid was more potent than the angiogenic inhibitor TNP-470. Thus bisphosphonates may have other beneficial properties in patients with cancer apart from preventing bone resorption.

In conclusion, SU5416, SU11657, CHS 828, and zoledronic acid represent new drugs with potent anti-tumor effects. Angiogenic inhibition as single therapy or in combination with chemotherapeutics may be beneficial in the treatment of rapidly growing and highly vascularized solid tumors of childhood such as neuroblastoma.

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33

Manning, Lauren Brooke. "Experimental Evaluation of Discoid Domain Receptor 2 as an Ideal Target for Development of Disease-Modifying Osteoarthritis Drugs." Thesis, Harvard University, 2015. http://nrs.harvard.edu/urn-3:HUL.InstRepos:17331959.

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Abstract: Osteoarthritis (OA) affects 250 million people worldwide. Currently, no targets for disease-modifying osteoarthritis drugs exist. Matrix metalloproteinase-13 (MMP-13) would make it an ideal target; however, its broad biological effects restrict its application as a target enzyme of inhibitory drugs in the treatment of OA. The expression and activation of discoidin domain receptor 2 (DDR2) is increased in human OA tissues and mouse models of OA and was co-localized with elevated expression of MMP-13 in degenerative articular cartilages. In healthy articular cartilage, DDR2 is kept inactivated by the pericellular matrix, which separates the receptor from its ligand, type II collagen. Once enzymes capable of degrading the pericellular molecules expose chondrocytes to type II collagen, DDR2 is activated and induces expression of MMP-13 leading to degradation of type II collagen and proteoglycans resulting in joint destruction and OA. We tested the hypothesis that complete removal of Ddr2 from the knee joint of mouse adult articular cartilage can delay progression of osteoarthritis prior to or after initiation of articular cartilage degeneration. To accomplish this goal, conditional knock out techniques were used with Aggrecan-CreERT2 mice and floxed Ddr2 mice, Ddr2 was removed from articular cartilage of knee joints in mice at 8 weeks of age via intraperitoneal Tamoxifen injection (2mg/10g body weight) for 5 consecutive days (Group A). Mice were subjected to destabilization of the medial meniscus (DMM) or sham surgery at 10 weeks of age. An additional experimental group was subjected to DMM or sham surgery at 10 weeks of age and then DDR2 was removed by intraperitoneal Tamoxifen injection 8 weeks later (Group B). Knee joints from mice in Group A and their corresponding controls were harvested at 8 weeks or 16 weeks post-surgery and mice from Group B and their controls were harvested at 16 weeks post surgery. Histology was performed and the OARSI Modified Mankin Score was used to evaluate articular cartilage degeneration. Statistically significant differences were determined via T-test. We found the average modified score for Group A 8 week control was 1.64 (n=7) whereas with Ddr2 removed was 0.64 (n=7) [P<0.05]. 2) The average modified score for Group A 16 week control was 4.67 (n=7) and with Ddr2 removed was 1.27 (n=9) [P<0.05]. 3) The average modified score for Group B was 1.1 (n=5). In conclusion, conditional removal of Ddr2 in articular cartilage attenuated articular cartilage degeneration in mature knee joints of mouse models of OA.

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34

Strand, Magnus. "Estrogen signaling in stroke : genetic and experimental studies." Doctoral thesis, Umeå : Univ, 2007. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-1397.

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35

Korbmacher, François. "Towards functional assignment of Plasmodium membrane transport proteins: an experimental genetics study on four diverse proteins." Doctoral thesis, Humboldt-Universität zu Berlin, 2021. http://dx.doi.org/10.18452/23029.

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Etliche Membran Transport Proteine (MTP) sind essentiell in den Plasmodium Blutstadien, und geraten zunehmend in den Fokus der Wirkstoffentwicklung. Die physiologischen Rollen der Transporter sind jedoch oft ungeklärt. In dieser Arbeit wurden mittels experimenteller Genetik funktionelle Charakteristika der MTPs untersucht. Am Maus Parasiten Plasmodium berghei und der Plasmodium falciparum Blutstadien-Kultur wurden vier MTPs ausgewählt: ein konservierter Folat Transporter (FT2), sowie eine P. falciparum-spezifisches P-Typ ATPase und zwei essentielle MTPs (CRT und ATP4). Diese Auswahl verkörpert ein breites Spektrum an MTP Kandidaten und reflektieren zudem das Potenzial und die Grenzen funktioneller Analysen von Plasmodium MTPs mittels reverser Genetik. Für den Folat Transporter 2 (FT2) wurde eine Kombination von transgenen Strategien auf P. berghei angewandt. Durch ein endogenes tag von FT2 wurde die Lokalisierung im Apicoplast, sowie dessen Expression über fast den kompletten Zyklus hinweg gezeigt. Nach der Deletion von FT2, wiesen die Parasiten einen Defekt während der Sporulation auf. Demzufolge bilden sich nur nicht infektiöse Sporozoiten, was letztendlich zur Unterbrechung des Lebenszyklus der Parasiten führt. Eine Aminophospholipid P-Typ ATPase, wurde mittels CRISPR/Cas9 in P. falciparum genetisch deletiert und die Mutante analysiert. Im Gegensatz zu den meisten vitalen P-Typ ATPasen erweist sich das Gen in den asexuellen Blutstadien als entbehrlich. Des Weiteren bilden die MTPs ATP4 und CRT einen einflussreichen Faktor bei Malaria-Therapien. Eine umfassende Analyse von räumlichen und zeitlichen Expressionsmustern von transgenen Parasiten mit mCherry-getaggten Proteinen zeigt ein Expression der beiden MTPs über die Blutstadien hinaus, was auf zusätzliche Funktionen in den jeweiligen Stadien verweist. Diese Studie trägt, basierend auf Lokalisation, Expression und funktioneller Deletion, zur funktionellen Entschlüsselung der vier untersuchten MTPs bei.
Many membrane transport proteins (MTP) are essential for Plasmodium infection and gain importance as candidate drug targets in malaria therapy, whereas the physiological functions often remain enigmatic. In this thesis, we applied experimental genetics to determine key characteristics of four Plasmodium MTPs. We employed the murine malaria model parasite Plasmodium berghei and in vitro blood cultures of Plasmodium falciparum. We selected one conserved MTP called FT2, which was previously shown to transport folate, a P-type ATPase that is specific for P. falciparum as well as two essential MTPs, CRT and ATP4. These targets exemplify the range of druggable candidates and illustrate the potential and limitations of reverse genetics to decipher their physiological roles. A combination of transgenic and knockout strategies was applied to the P. berghei folate transporter 2 (FT2). We show that endogenously tagged FT2 localises to the apicoplast membranes, and is broadly expressed throughout the parasite’s life cycle. Analysis of FT2-deficient parasites revealed a severe sporulation defect in the vector; the vast majority of ft2– oocysts form large intracellular vesicles which displace the cytoplasm. Very few sporozoites are generated and these are non-infectious to the mammalian host, resulting in a complete arrest of Plasmodium transmission. A candidate aminophospholipid P-type ATPase, was assessed by a CRISPR/Cas9-mediated gene disruption. Compared to many vital P-type ATPases this gene is dispensable for asexual blood replication. Two MTPs, ATP4 and CRT are prime targets for antimalarial therapies. A comprehensive spatio-temporal expression analysis of transgenic parasites expressing mCherry-tagged proteins revealed expression beyond blood infection, indicative of functions in additional parasite stages. The findings of this study contribute towards a better understanding of the roles of the four MTPs based on localisation, expression and functional deletion.

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36

Andersen, Malin. "Computational and experimental approaches to regulatory genetic variation." Doctoral thesis, Stockholm : Bioteknologi, Kungliga Tekniska högskolan, 2007. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-4593.

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37

Wong, Ka-ho, and 王家豪. "Transgenic chlamydomonas reinhardtii as an experimental system to study the regulation of carotenoid biosynthesis in green microalgae." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2006. http://hub.hku.hk/bib/B37728337.

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38

Wallberg, Peter. "A clinical and experimental study of basal cell carcinoma : aspects on epidemiology, genetics and microphysiology /." Stockholm, 2000. http://diss.kib.ki.se/2000/91-628-3993-4/.

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39

Silva, Adelmo Resende da. "Análise genética de caracteres quantitativos em milho com o delineamento III e marcadores moleculares." Universidade de São Paulo, 2002. http://www.teses.usp.br/teses/disponiveis/11/11137/tde-26072002-180545/.

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Caracteres importantes em espécies vegetais estão, em sua maior parte, sob o controle de vários locos gênicos, denominados locos de caracteres quantitativos (QTLs). A expressão fenotípica desses caracteres produz uma distribuição contínua de valores devido à segregação genotípica, aos efeitos ambientais e à interação genótipos por ambientes. Com o advento dos marcadores moleculares e de modelos estatístico-genéticos adequados tornou-se possível a detecção e o mapeamento de regiões cromossômicas que controlam estes caracteres. Este trabalho foi realizado para analisar caracteres quantitativos em uma população de milho tropical com os objetivos: (1) obter estimativas das variâncias genéticas aditiva e de dominância, e do grau médio de dominância; (2) construção de um mapa genético utilizando marcador molecular do tipo microssatélite; (3) detecção de QTLs e estimação de seus efeitos genéticos, e de suas interações com ambientes, utilizando o delineamento III. Neste trabalho foram utilizadas 250 progênies F2:3 retrocruzadas para ambos os genitores, totalizando 500 progênies, que foram avaliadas em seis ambientes para a obtenção de dados fenotípicos. Foi realizada a genotipagem com 140 marcadores moleculares para as 250 plantas F2 genitoras. O mapa genético foi construído utilizando o programa MAPMAKER/EXP V.3.0. Os componentes das variâncias genéticas e o grau de dominância dos caracteres foram estimados utilizando-se o delineamento III. A detecção de QTLs e as estimativas de seus efeitos genéticos foram efetuados utilizando a metodologia de Cockerham & Zeng (1996). Os resultados das análises de variâncias evidenciaram sobredominância para produção de grãos e dominância parcial para os outros nove caracteres. Por causa do desequilíbrio de ligação nesta população, estas estimativas estão, provavelmente, superestimadas. O mapa genético com 140 marcadores tem 1.730,1 centiMorgans (cM) de extensão e intervalo médio de 12,4 cM entre marcadores adjacentes. Foram detectados QTLs em todos os cromossomos para todos os caracteres, indicando que os locos que controlam estes caracteres estão distribuídos pelo genoma. A análise genética utilizada permitiu a estimação dos efeitos aditivos, dominantes e epistáticos dos QTLs. Efeitos aditivos, dominantes e epistáticos foram detectados para todos os caracteres. As magnitudes, em módulo, dos efeitos aditivos e dominantes foram maiores do que os efeitos epistáticos para todos os caracteres, exceto para posição relativa da espiga onde os efeitos epistáticos foram um pouco maiores do que os efeitos dominantes. A porcentagem de marcadores que detectaram efeitos epistáticos variou de 10,71% para teor de umidade nos grãos a 50,71% para número de folhas. Para produção de grãos, os efeitos epistáticos foram detectados em 32,14% dos marcadores, sendo encontrados valores altos para alguns marcadores. Os efeitos dos QTLs detectados variaram muito em magnitude e sinal entre marcadores adjacentes, demonstrando que os QTLs contribuem de forma diferenciada para as expressões dos caracteres. Os efeitos dos QTLs tiveram pequena ou nenhuma interação genótipos por ambientes para os caracteres avaliados, exceto para teor de umidade nos grãos.
Most of the agronomic traits in crop species are under the control of unknown number of loci, which have been termed quantitative trait loci (QTL). Because of genotypic segregation, environmental effects, and of genotype by environment interaction, the phenotypic values of these traits present continuous variation. The advent of molecular markers, and of sophisticated statistical-genetic models allowed the analysis of quantitative traits at the DNA level, by detecting and mapping chromosomal regions with loci affecting those traits. This research was carried out to analyze quantitative traits in a tropical maize population by (1) estimating genetic parameters as additive and dominance genetic variance, and the level of dominance; (2) developing a genetic map with microsatellites as molecular markers; (3) detecting and estimating genetic effects of QTLs as well as QTLs by environment interaction by using the Design III methodology. The genetic material was a set of 250 F2:3 progenies developed from a cross of two inbred lines. The F2 plants, parents of the F2:3 progenies, were genotyped with microsatellites and a genetic map was developed by using MAPMAKER/EXP V.3.0 software. The F2: 3 progenies were backcrossed to both parental inbred lines giving rise to 500 backcrossed progenies, which were evaluated in six environments. The components of genetic variances and the level of dominance of the traits were estimated by using the Design III approach. The detection of QTLs and estimates of their genetic effects were computed by using Cockerham & Zeng (1996) methodology. Average level of dominance was overdominance for grain yield (GY), and partial dominance for the other nine traits. Because of the linkage disequilibrium in this population, probably these estimates are overestimated. The genetic map with 140 markers spanned 1,730.1 centimorgans (cM) in length with an average interval of 12.4 cM between adjacent markers. QTLs were detected at all chromosomes for all traits, indicating that the genes controlling the quantitative traits assessed are spread in the genome. The genetic analysis used allowed the estimation of additive, dominance and epistatic effects of the QTLs. Additive, dominance and epistatic effects were detected for all traits, although the magnitudes, in module, of additive and dominance effects were larger than epistatic effects for all traits, except for ear placement where epistatic effects were slightly larger than dominance effects. The percentage of markers that detected significant epistatic effects ranged from 10.71% for grain moisture to 50.71% for number of leaves. For GY, the dominance effects were more important than additive effects; epistatic effects were detected in 32.14% of the markers, whereas in some markers high values were found. The magnitudes and signs of the QTLs effects were highly variable among the markers, showing that the contribution of the QTLs for the expression of the traits was very different, and that epistatic effects are important for the expression of all traits. Also, fewer QTL effects by environment interactions were detected for all traits, except for grain moisture.

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40

Petkiewicz, Stephanie L. "The Met receptor tyrosine kinase in mammary gland tumorigenesis and development /." Thesis, McGill University, 2007. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=103278.

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The Met receptor tyrosine kinase (RTK) is expressed in the mammary gland under both normal and neoplastic conditions. Overexpression of the Met receptor is found in 15--20% of human breast cancers and is correlated with shortened disease-free interval and overall survival. In order to explore the role of dysregulated Met receptor signaling on the development of mammary tumors I have characterized a transgenic mouse model that expresses either wild type or a dysregulated Met receptor in the mammary epithelium under the control of the mouse mammary tumor virus promoter/enhancer (MMTV-Met). The Met receptor variants contained a mutation that results in decreased receptor ubiquitination and prolonged receptor signaling (Y1003F) or an activating mutation that was originally observed in patients with papillary renal carcinoma (M1250T) or both mutations (YF/MT). In vitro and in vivo transformation assays demonstrated that each mutation singly is weakly transforming, however, there was an additive effect on transformation when both mutations were present. This additive effect was observed in the transgenic mice where multiparous MMTV-Met-YF/MT mice developed tumors earlier and with much greater penetrance than did mice expressing either of the single mutants. This provides the first in vivo model that demonstrates a role for ubiquitination in suppression of transforming activity of an RTK. MMTV-Met-YF/MT tumors displayed a range of histological phenotypes but were mainly comprised of luminal lineage cells. Notably, MMTV-Met-M1250T tumors contained cells from both the basal and luminal populations, suggesting transformation of a progenitor cell. Progenitor cell transformation in RTK transgenic mouse models is uncommon and highlights distinct signaling differences and potentially lineage specificity of the two Met mutants.
Through assays of overexpression in vivo and inhibition in vitro, Met receptor signaling has been correlated with the development of the mammary gland. To examine the effects of loss of Met receptor signaling on mammary gland development I have utilized the Cre/LoxP1 recombination system to knock-out the Met receptor from the mammary epithelium. Mammary-specific Cre recombinase efficiently excised floxed DNA as visualized by activation of a beta-galactosidase reporter In Met+/+ glands, however, few beta-galactosidase positive cells are retained In the Mefl/fl glands and an intermediate number are retained in the Met fl/+ glands. This indicates that Met-null cells are selected against and supports a role for Met in the development of the mammary gland.

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Pavelescu, Irina. "Brassinosteroids role in arabidopsis root development : theoretical and experimental approaches." Doctoral thesis, Universitat de Barcelona, 2016. http://hdl.handle.net/10803/396085.

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This PhD thesis represents an advance in the present understanding of the spatiotemporal control of model plant Arabidopsis thaliana root growth and development. The size and structure of a living organism are tightly controlled by the coordination between several highly dynamic molecular and cellular processes, such as cell division, movement, growth and deformation. At tissue level, a mesoscopic description of the system and these processes can be used, in terms of mechanical forces and energy minimization (see (Hamant & Traas, 2010) for a review focused on plants). How cells decide to switch from a cellular process to another is a fundamental question to understand the growth and shape of an organ. Because of the thermal fluctuations and finite number of molecules involved in the molecular reactions, cells take presumably these decisions in a stochastic manner, which makes it challenging to understand how morphogenesis generates organs with characteristic shapes and sizes. Plant roots grow due to cell division in the meristem and subsequent cell elongation up to terminal differentiation. The pleiotropic phenotypes of the short-root mutants available make it difficult to univocally assess which mechanism sets the transition from elongation to final differentiation. To elucidate it, in this thesis we use a novel approach based on the quantitative information associated to the phenotypic variability of wild type roots together with computational modeling of different mechanisms. In Chapter 1 we introduced the already published work in the field of root and meristem growth, at experimental and computational level. In Chapter 2 we have employed theoretical and computational models to analyze individual isogenic Arabidopsis seedlings and to quantify their heterogeneity, which we have quantified, together with their mean values. The quantification of heterogeneity has been crucial since it allowed the identification of dynamical mechanisms involved in Arabidopsis root growth. By analyzing these mechanisms in WT plants and Brassinosteroids (BRs) mutants, we found that growth defects in the BRs loss of function mutant are generated by defects related to cell differentiation. To deepen into this result, in Chapter 3 we investigated the mechanism through which cells decide to differentiate and achieve their final length. In this sense, we adopted a computational approach, combined with plant variability analysis, to test three putative mechanisms: Ruler (Band et al, 2012; De Vos et al, 2014), Timer (De Vos et al, 2014; Mähönen et al, 2014) and Sizer (Grieneisen et al, 2012). We compared the simulated data, based on the values extracted in Chapter 2, with experiments, and we found that Arabidopsis thaliana primary root uses a Sizer mechanism based on measuring cell sizes for final cell differentiation. We show this mechanism translates into specific correlations among phenotypic traits and explains why root growth is proportional to the meristem activity and displays mature cells of stereotyped length. We challenged our model by evaluating such correlations in a well-known BR signaling short-root mutant. We further show that BR signaling at the meristem is sufficient to recover some of the correlation slopes and hence root growth, yet it alters the mechanism. Together, our results establish a theoretical quantitative framework for stationary root growth and underscore the value of using computational modeling together with quantitative data. In Chapter 4 we analyzed the coupling between meristematic activity and telomere length by applying a novel quantitative fluorescence in situ hybridization to measure telomere length with tissue resolution in the primary root. The implementation of a new image analysis protocol contributed to revealing a telomere distribution map, with telomere length gradients along the meristem, and the longest telomeres localized in the stem cell niche (Gonzalez-Garcia et al, 2015). We applied this method to WT plants, several generations of telomerase deficient mutants, mutants with larger telomeres and cell differentiation mutants. Furthermore, we generated transgenic plants to check the localization of telomerase and we evaluated the relationship between telomere length and resistance to DNA damage. We also evaluated computationally the telomere distributions observed in WT and telomerase deficient mutants and we simulated the telomere dynamics which can generate such distributions. The conclusions of this thesis were contextualized in Chapter 5.
El tamaño y la estructura de un organismo vivo son el resultado de una coordinación entre procesos moleculares y celulares, altamente dinámicos, como la división, el movimiento, el crecimiento y la deformación. A nivel de tejido se puede usar una descripción mesoscópica del sistema y estos procesos, habitualmente en términos de fuerzas mecánicas y minimización de la energía (dirigirse a (Hamant & Traas, 2010) para una revisión sobre plantas). Por tanto, la morfogénesis y formación de órganos en Eucariotas son investigadas tanto por la Biología de desarrollo, como por la Física de la materia blanda (Cross & Greenside, 2009; Cross & Hohenberg, 1993; Murray, 2002). El crecimiento global de una planta es fuertemente relacionado con el crecimiento y desarrollo de su raíz. Las raíces crecen debido a sucesivas divisiones celulares en el meristemo, seguidas por elongación y diferenciación celular. Para poder estudiar el desarrollo de la raíz es imprescindible conocer qué determina a las células tomar las decisiones de parar de dividir y elongarse o parar de elongarse y diferenciarse. Debido a las fluctuaciones térmicas y el número finito de moléculas que participan en las reacciones moleculares, es de esperar que estas decisiones no son tomadas por todas las células a la vez, sino de una manera estocástica, lo que hace dificil entender cómo la morfogénesis basada en un comportamiento celular estocástico puede generar formas y tamaños característicos de órganos. En este contexto, esta tesis usa modelos matemáticos para cuantificar y generar predicciones sobre la dinámica de crecimiento de la raíz de Arabidopsis thaliana, que han sido testeadas mediante un abordaje experimental. En el Capítulo 2 de esta tesis hemos diseñado un marco teórico para describir el crecimiento estacionario de la raíz y hemos analizado la variabilidad existente entre raíces isogénicas. En el Capítulo 3 hemos usado un modelo matemático para investigar el mecanismo que las células usan para decidir cuándo parar de elongarse y adquirir su tamaño final. Basándonos en las predicciones de este modelo, hemos analizado la variabilidad intrínseca de las plantas silvestres y hemos identificado relaciones específicas entre los parámetros de crecimiento, que nos ayudaron a descartar posibles modelos. En el Capítulo 4 hemos cuantificado la longitud telomérica en las células de la raíz y evaluado funcionalidades biológicas. Nuestro análisis mostró una distribución heterogénea, que impulsó la modelización matemática de la dinámica telomérica, basada en las fluctuaciones y el comportamiento dinámico de la longitud telomérica.

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Lieber, Daniel Solomon. "Computational and Experimental Approaches For Evaluating the Genetic Basis of Mitochondrial Disorders." Thesis, Harvard University, 2013. http://dissertations.umi.com/gsas.harvard:10830.

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Mitochondria are responsible for many fundamental biological pathways and metabolic processes, including aerobic ATP production by the mitochondrial respiratory chain. In humans, mitochondrial dysfunction can lead to severe disorders of energy metabolism, which are collectively referred to as mitochondrial disorders and affect approximately 1:5,000 individuals. These disorders are clinically heterogeneous and can affect multiple organ systems, often within a single individual. Symptoms can include myopathy, exercise intolerance, hearing loss, blindness, stroke, seizures, diabetes, and GI dysmotility. Mutations in over 150 genes in the mitochondrial DNA (mtDNA) and nuclear genome are known to cause mitochondrial diseases and an additional ~1,000 nuclear-encoded mitochondrial proteins have the potential to underlie mitochondrial disorders but have not yet been linked to human disease. As a result, determining a molecular diagnosis for patients with suspected mitochondrial disorders remains a challenge.

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Randall, Ryan Nicole. "Experimental phylogenetics: a benchmark for ancestral sequence reconstruction." Thesis, Georgia Institute of Technology, 2012. http://hdl.handle.net/1853/48998.

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The field of molecular evolution has benefited greatly from the use of ancestral sequence reconstruction as a methodology to better understand the molecular mechanisms associated with functional divergence. The method of ancestral sequence reconstruction has never been experimentally validated despite the method being exploited to generate high profile publications and gaining wider use in many laboratories. The failure to validate such a method is a consequence of 1) our inability to travel back in time to document evolutionary transitions and 2) the slow pace of natural evolutionary processes that prevent biologists from ‘witnessing’ evolution in action (pace viruses). In this thesis research, we have generated an experimentally known phylogeny of fluorescent proteins in order to benchmark ancestral sequence reconstruction methods. The tips/leaves of the fluorescent protein experimental phylogeny are used to determine the performances of various ASR methods. This is the first example of combining experimental phylogenetics and ancestral sequence reconstruction.

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44

Zeyl, Clifford. "Sex, parasitic DNA and adaptation in experimental populations of Saccharomyces cerevisiae and Chlamydomonas reinhardtii." Thesis, McGill University, 1996. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=40475.

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The widespread occurrence among eukaryotes of sex and of mobile DNA sequences requires an evolutionary explanation, since both appear to reduce individual fitness. Both phenomena have been hypothesized to provide fitness advantages to populations, but such explanations require rather than explain the initial establishment of mobile elements and genes for sex. Genes encoding sexuality may invade asexual populations as molecular parasites, whose success then allows mobile elements to spread as parasites of sexual genomes. The prediction that mobile elements can invade only sexual populations was tested using isogenic sexual and asexual populations of Saccharomyces cerevisiae and the retrotransposon Ty3. Active Ty3 elements more consistently invaded sexual than asexual populations. In subsequent experiments involving selection on media containing ethanol as a carbon source or $ beta$-glycerophosphate as a limiting phosphorus source, transposition by galactose-induced Ty3 elements produced none of the mutations involved in adaptation to these media, and conferred no adaptive advantage among competing populations. The mean copy numbers of two mobile elements were unchanged by long-term sexual or asexual propagation of Chlamydomonas reinhardtii populations, because transposition by these elements occurred very rarely or had no effect on fitness. Sexual and asexual S. cerevisiae populations did not differ in their adaptation to galactose media, but sexual populations maintained on glucose had higher growth rates on both media than did asexual populations maintained on glucose, implying that selection against deleterious mutations was more effective in sexual populations.

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Ponzo, Marisa Grace 1980. "Gene expression profiling of Met receptor tyrosine kinase-induced mouse mammary tumors." Thesis, McGill University, 2009. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=115881.

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Breast cancer is a heterogeneous disease comprised of distinct biological entities that correlate with diverse clinical outcomes. Gene expression profiling has divided this heterogeneity into luminal, ERBB2+ and basal molecular subtypes. Basal breast cancers are difficult to treat as they lack expression of candidates suitable for targeted therapies and are associated with poor outcome.
Elevated protein level of the hepatocyte growth factor receptor, MET, is observed in 20% of human breast cancers and correlates with poor prognosis. However, the role of MET in mammary tumorigenesis is poorly understood. To address this, we generated a murine model that expresses weakly oncogenic mutants of Met (Metmt) in the mammary epithelium under the transcriptional control of the mouse mammary tumor virus promoter. We demonstrate that Metmt induces mammary carcinomas with diverse phenotypes and used gene expression microarrays to elucidate gene expression changes induced by Met. Since mammary tumors contained variable contents of epithelium and stroma, we used laser capture microdissection to procure epithelial cells for microarray analysis. Based on immunohistochemistry and expression profiling, we show that Metmt produces tumors with luminal or basal characteristics. From hierarchical clustering, Metmt-induced basal tumors clustered with murine models that share features of epithelial to mesenchymal transition and human basal breast cancers. Moreover, Metmt basal tumors clustered with human basal breast cancer. The status of MET among the human breast cancer subtypes has not previously been addressed. We demonstrate that MET levels are variable across molecular subtypes but show elevation in the basal subtype and correlates with poor outcome. We used a candidate gene approach derived from microarray data to gain an understanding of signals required for Met-dependent tumorigenesis. We investigated Nck adaptor proteins and demonstrate a role for Nck in cell motility and actin dynamics of Met-dependent breast carcinoma cells and show elevated expression in human basal breast cancers. By generating a unique mouse model in which Met is expressed in mammary epithelia, with the examination of MET levels in human breast cancer, we have established a novel link between MET and basal breast cancer. This work identifies poor outcome basal breast cancers that may benefit from anti-MET therapies.

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46

Barlow, Vicki. "The development of enhanced experimental strategies for the DNA analysis of low-template or compromised forensic sample types." Thesis, Northumbria University, 2015. http://nrl.northumbria.ac.uk/30231/.

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Single-cell DNA analysis is not routinely carried out in a forensic setting as it is considered unreliable due to challenges associated with DNA amplification, contamination and profile interpretation. In light of the development of increasingly sensitive techniques, the question of the reliability of single-cell DNA analysis in terms of both processing and interpretation is addressed in the first part of this thesis. Optimising all stages of the DNA analysis process has provided a sensitive method which facilitates the successful outcome of a useable profile from single-cells. Although no consensus profile can be generated for this sample type, interpretation guidelines have been set to enable the robust analysis of single cells. It has been concluded that single-cells can be reliably amplified and profiled for forensic purposes. Both DNA and textile fibres have a proven track record in forensic casework yet their analysis is rarely combined. As an application of the aforementioned single-cell DNA analysis, this project explores the possibility that when fibres are transferred from one surface to another, they could also be acting as a vector for the wearer’s own DNA, through cells that have adhered to the fibre surfaces. Fluorescent staining and microscopy is used to detect the cells in situ on the fibre surface, which are then recovered and processed for DNA using the previously optimised single-cell analysis methods, along with a newly developed DNA assay designed for the amplification of low DNA template samples. The results of this study have demonstrated that cells can be visualised in situ on the fibre surface and that there is potential for cell transfer to occur. It has been concluded however, that from a casework point of view, targeting transferred fibres for cells may not be the best approach as it is time consuming and has not been shown to be effective in this study. The final part of this thesis is focused on the efficacy of massively parallel sequencing (MPS) technology for samples that are expected to be severely degraded due to age or exposure to a hostile environment. The ability of both the recently launched Illumina ForenSeq™ DNA Signature Prep Kit for nuclear DNA markers and an in-house method for the sequencing of degraded mitochondrial DNA, have been tested to determine if MPS offers a more comprehensive evaluation of degraded material than the traditional PCR-CE methods. The results of the ForenSeq kit have demonstrated the effectiveness of its low molecular weight STR and SNP markers for amplifying low template, degraded DNA samples, with alleles amplified using less than 20 pg total DNA input. This kit has also therefore shown application in the field of bioarchaeology, as it can provide the biological sex of the sample, biogeographic ancestry information and also aids detection of sample/control contamination. The in-house mitochondrial DNA assay resulted in the successful amplification and sequencing of samples for which no nuclear DNA was amplified. The high depth of read coverage in these samples, average of 18,000, allowed for the identification of even low level variants.

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47

Rossdeutscher, Lionel Philip David. "The role of tumoral 1,25 dihydroxyvitamin D3 in inhibition of tumor growth and progression in the PyVMT MMTV#634 transgenic breast cancer model /." Thesis, McGill University, 2007. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=112354.

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Vitamin D3 must be metabolically activated by the liver to 25-hydroxyvitamin D3 (25OHD3) and then by the kidney 1alphahydroxylase (1alphaOHase) to become 1,25dihydroxyvitamin D 3 (1,25(OH)2D3). 1,25(OH)2 D3 is a potent inhibitor of tumor growth in vitro and in vivo. Recent studies indicate that metabolic activation of 1,25(OH) 2D3 also occurs in cancer cells such as breast cancer. Consequently, the major objective of this project was to determine if tumoral 25OHD 3-1alphahydroxylase modulates any or all of the stages of breast tumor progression without inducing the hypercalcemic side effects of 1,25(OH) 2D3. For this purpose we used the PyVMT breast cancer mouse model in which the oncoprotein, polyomamiddle T antigen (PyMT) is under the control of mouse mammary tumor virus LTR (MMTV LTR). Mice exhibited tumors restricted to the mammary epithelium progressing to the various stages of breast cancer. Animals were treated with either vehicle, 25OHD3 (2000 pM/24h) or 1,25(OH)2D3 (12pM/24h). Mice treated with the vitamin D precursor, 25OHD3, exhibited a marked reduction in tumor onset and growth comparable to the 1,25(OH)2D3 treated group. Furthermore, biomarkers of tumor progression were markedly reduced in 25OHD3 and 1,25(OH)2D3 animals as compared to vehicle-treated animals. However, mean circulating calcium concentrations remained unchanged in 25OHD3 treated animals but increased significantly in 1,25(OH)2D3 treated animals as compared to controls. Tumoral levels of 1,25(OH)2D3 in mice treated with 25OHD3 were increased 79% in comparison to vehicle control mice. Additionally, 25OHD3 and 1,25(OH)2D 3 treated animals had a significant decrease in the mean number of lung metastases per animal as compared to vehicle treated control animals. This study therefore suggests an important autocrine role of 1alphaOHase expression in breast tumor cells. Furthermore, accumulation of intra-tumoral 1,25(OH) 2D3 in response to 25OHD3 administration strongly suggests that locally produced 1,25(OH)2D3 plays a significant role in restraining tumor growth without inducing the hypercalcemic side effects associated with 1,25(OH)2D3.

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Korbmacher, François [Verfasser]. "Towards functional assignment of Plasmodium membrane transport proteins: an experimental genetics study on four diverse proteins / François Korbmacher." Berlin : Humboldt-Universität zu Berlin, 2021. http://d-nb.info/1237268516/34.

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49

Mattsson, Göran. "Experimental Studies on the Vasculature of Endogenous and Transplanted Islets of Langerhans." Doctoral thesis, Uppsala University, Department of Medical Cell Biology, 2003. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-3596.

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The blood vessels of the pancreatic islets are of crucial importance for oxygen and metabolite supply as well as dispersal of secreted hormones. In addition to this, endothelial cells have an important role in the revascularization process after islet transplantation. Previous studies have reported signs of poor engraftment of transplanted islets, presumably due to impaired revascularization. The aims of this thesis were to investigate the revascularization process of transplanted islets and to examine the role of islet endothelial cells. In this context, the lectin Bandeiraea simplicifolia was found to stain endothelium of both endogenous and transplanted pancreatic islets. By using this lectin we investigated the vascular density of both endogenous and islets transplanted syngeneically beneath the renal capsule, into the spleen or intraportally into the liver of normoglycemic C57BL/6 mice. One month post-transplantation, a time point when the grafts are assumed to be completely revascularized, the vascular density was decreased at all three implantation sites when compared to endogenous islets. Furthermore, most of the blood vessels were located in the graft connective tissue stroma. Similar results were obtained when islet transplant vascular density was determined six months post-transplantation and in cured diabetic animals after one month. In order to evaluate the function of intraportally transplanted islets, we developed a method to retrieve such islets. We treated the implantation organ (liver) first enzymatically (collagenase) and then mechanically, thereafter we could re-isolate the transplanted islets for further in vitro studies. The retrieved islets had a decreased insulin relase, insulin content and glucose oxidation rate when compared to non-transplanted control islets. To understand the role of islet endothelium in the revascularization of transplanted islets we performed angiogenesis GEArray studies on islet endothelial cells, from non-cultured, cultured and transplanted islets. We found that the islet endothelium expressed mRNA for both inhibitors and inducers of angiogenesis, and that this expression differed with time. The functional consequences of this remain to be determined. In summary, the results presented above provide a useful platform for future studies of the morphology and function of islet endothelial cells, especially with a view for elucidating changes induced by islet transplantation.

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50

Nordmark, Gunnel. "Clinical and Experimental Studies in Primary Sjögren’s Syndrome and Systemic Lupus Erythematosus." Doctoral thesis, Uppsala University, Department of Medical Sciences, 2005. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-5943.

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Autoimmune mechanisms and genetic susceptibility contribute to the pathogenesis of primary Sjögren’s syndrome and SLE. These chronic systemic autoimmune diseases have many serological and clinical features in common and have an impact on daily life. The studies in this thesis aim to elucidate their autoimmune mechanisms, define susceptibility genes and evaluate effects of androgen supplement on health-related quality of life.

Autoantibodies against α-fodrin, a widely distributed cytoskeletal protein, were detected at similar frequencies in sera from patients with primary and secondary Sjögren’s syndrome and SLE. Consequently, testing for antibodies against α-fodrin would not add diagnostic value compared to conventional serological analysis and does not discriminate between these diseases.

The type I interferon (IFN) system was found to be activated in primary Sjögren’s syndrome. IFN-α containing cells were detected in minor salivary gland biopsies, while sera from patients with primary Sjögren’s syndrome induced IFN-α production in the presence of apoptotic and necrotic cell material. This ability of sera correlated with the presence of antibodies against RNA-binding proteins and IFN-α production was dependent on RNA in immune complexes. The natural interferon producing cells/plasmacytoid dendritic cells (NIPC/PDC) were the IFN-α producers and blocking of FcγRIIa inhibited the production. Single nucleotide polymorphisms (SNPs) in two genes in the type I IFN signalling pathway, those for tyrosine kinase 2 and interferon regulatory factor 5, were strongly associated with SLE in a Swedish, Finnish and Icelandic population. The minor allele frequencies were lower in SLE patients than in healthy controls. These SNPs may decrease the function of the type I IFN system, thereby conferring protection against SLE.

Supplementation with dehydroepiandrosterone (DHEA) in glucocorticoid treated women with SLE led to mild improvements in health-related quality of life in respect of mental well-being and sexuality, whereas physical well-being was unaffected.

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Dissertations / Theses on the topic 'Genetics, Experimental'

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