No description available.

Since the discovery of the p120ctn binding partner, Kaiso, a BTB/POZ transcription factor, several studies have implicated the protein in both development and tumourigenesis. Most information about Kaiso’s function in vertebrate development has been gleaned from studies in Xenopus laevis embryos where Kaiso negatively regulates the Wnt signalling pathway. Since the Wnt signalling pathway is crucial in intestinal development, intestinal-specific Kaiso overexpressing mice were generated and characterized to elucidate Kaiso’s role in a mammalian context. Kaiso transgenic (KaisoTg/+) mice were viable and fertile but developed gross histopathological changes in the small intestine. The KaisoTg/+ mice exhibited enlarged crypts accompanied by increased secretory cell differentiation reminiscent of inhibition of the Notch pathway. Indeed, the Notch effector protein, HES1, is decreased in KaisoTg/+ mice. Additionally, KaisoTg/+ mice display a neutrophil-specific intestinal inflammation reminiscent of the knockdown of p120ctn. Interestingly, the KaisoTg/+ mice display decreased p120ctn localization at the membranes and an increase in the neutrophil adhesion molecule, ICAM-1, both of which induce neutrophilia. Notably, the KaisoTg/+ mice developed multiple crypt abscesses over time due to massive neutrophil infiltration of the epithelial cell layers. This is the first study to examine the in vivo roles of Kaiso in a mammalian context and our findings suggest a regulatory role for Kaiso in the inflammatory and Notch signalling pathways. / Thesis / Candidate in Philosophy

The adrenal gland is an important endocrine organ, protecting the body against acute and chronic stress. The adrenal cortex consists of three morphologically and functionally distinct zones: the outer zona glomerulosa (zG), the zona fasciculata (zF), and the innermost zona reticularis (zR). In rodents, zG cells produce mineralocorticoids (mainly aldosterone), while zF cells secrete glucocorticoids (mainly corticosterone). The functions of zG and zF are defined by the mutually exclusive expression of Cyp11b2 and Cyp11b1 that encode the enzymes aldosterone synthase and 11β-hydroxylase, which catalyze the terminal reactions in the production of aldosterone and corticosterone, respectively. This thesis aims to investigate the maintenance of the definitive mouse adrenal cortex. This involves studies to identify the location of adrenal stem/progenitor cells, and the mechanisms by which differentiated adrenocortical cells are replenished in the adult mice. BrdU pulse-chase studies provided valuable information about cell division and cell fate under physiological or pathophysiological stimulations. The distribution of adrenocortical cells with nuclei stained positively for BrdU and/or Ki67 was identified. Ki67 labelling marked actively dividing cells and showed that adrenocortical cells originate at or around the zG/zF interface. BrdU labelling indicated that, following cell division, cells are displaced inwards and outwards. Acute angiotensin II treatment was shown to have no significant effects on the cell proliferation or turnover in any of the adrenocortical zones. The pathophysiological effects of long-term ACTH treatment were analyzed in a mouse model of congenital adrenal hyperplasia caused by a null mutation of Cyp11b1. Cell hypertrophy was evident in all regions of the adrenal cortex due to the impaired negative-feedback of the HPA axis. Adrenocortical cell proliferation was also increased particularly in the outer zona fasciculata at the border between zG and zF where adrenocortical stem/progenitor cells might be located. The intervening steps between cell proliferation and the final differentiation into steroidogenic zG and zF cells have yet to be discovered. A visual method of monitoring levels of Cyp11b2 and Cyp11b1would offer a convenient approach to track the stages of adult stem cell differentiation that lead to normal adrenal maintenance in vivo and in vitro. In the present study an AS-mCherry-11B-EGFP BAC construct was successfully engineered, in which Cyp11b2 and Cyp11b1 were substituted by mCherry and EGFP, respectively. This BAC construct was characterized in mouse adrenocortical Y1 cells. It was determined that EGFP faithfully recapitulated the expression of Cyp11b1. Forskolin or cAMP treatment induced a rapid cell rounding effect and caused the increased expression of EGFP transgene and endogenous Cyp11b1. An attempt was made to establish a transgenic mouse model, in which zG and zF cells would be marked with mCherry and EGFP respectively, allowing the differentiation of an adrenocortical stem cell to be traced. Following microinjection of the BAC into mouse zygotes, twoAS-mCherry-11B-EGFP transgenic founder mice were identified. Unfortunately, neither of them was able to transmit the transgene through germline, suggesting the mosaicism of transgene integration. Indeed, mosaicism of the transgenic adrenals was demonstrated by RT-PCR and immunostaining, which also revealed that the exogenous EGFP expression faithfully recapitulated the endogenous Cyp11b1 in adrenals. Although it is assumed that expression of Cyp11b2 and Cyp11b1 are mutually exclusive, zG and zF cells may have the plasticity to allow the transition from one cell type into another. The AS-mCherry-11B-EGFP BAC construct is a useful tool for studying in vitro ES cell differentiation towards the adrenocortical lineage. Transgenic AS-mCherry-11B-EGFP ES cells were successfully differentiated into mesenchymal stem cells, as identified by the expression of molecular markers for the mesenchymal lineage. It has been reported that steroidogenic factor (Sf1) can promote the differentiation of MSCs into steroidogenic cells, and Shh plays an important role in Sf1 expression and the consequent adrenal development. However, Shh treatment failed to achieve transformation of mesenchymal cells into adrenocortical cells. It is thought there might be a requirement for additional factors to combine with Shh in promoting the transdifferentiation of ESC-derived mesenchymal cells. Future studies will focus on the genetic control of Cyp11b1 and Cyp11b2 in transgenic AS-mCherry-11B-EGFP ES cells. In conclusion, the location and fate of the adrenocortical progenitor cells were demonstrated by the BrdU pulse-chase studies in different mouse models. An AS-mCherry-11B-EGFP BAC construct was generated, and used to study the mutual and differential controls of Cyp11b1 and Cyp11b2 expression in adrenocortical cells in vitro and in transgenic mice in vivo.

No description available.

Aedes albopictus is an important vector of dengue and chikungunya viruses, and, over recent decades, has resisted traditional control strategies allowing it to spread from its native Southeast Asia throughout the world. In this thesis, two alternative control methods are assessed and developed: transinfection with the inherited bacteria Wolbachia, for population replacement with a refractory strain; and a genetic equivalent to the sterile insect technique, RIDL (Release of Insects carrying a Dominant Lethal), for population suppression. Wolbachia is a genus comprising maternally inherited intracellular α-proteobacteria which primarily infect arthropods. Certain strains of Wolbachia both have the ability to manipulate host reproduction through cytoplasmic incompatibility (CI) which allows Wolbachia to invade host populations by conferring a reproductive advantage on infected females, and have been shown to confer broad-spectrum pathogen resistance on their hosts. Here, a transinfection of wMel in Aedes albopictus (Uju.wMel) was generated which shows complete bidirectional CI with the natural Wolbachia infection of Ae. albopictus, in the absence of any major fitness costs and (as shown by collaborators) completely abolishes dengue and chikungunya virus transmission. It was also shown that the pathogen inhibition in Uju.wMel occurs in the absence of immune stimulation. Evidence supporting cholesterol sequestration by wMel as a mechanism for the pathogen inhibition observed in Uju.wMel was found. Previous attempts to produce a conditionally inviable genetic sexing Ae. albopictus RIDL line have resulted in a sub-optimal strain in which the construct was not sufficiently specific or repressible, resulting in a high proportion of off-target inviable mosquitoes. Here, the mating competitiveness of RIDL males was shown to be not significantly different from wild-type, confirming the potential utility of the system. Multiple truncations of the promoter were made in an attempt to reduce the off-target expression.

O mosquito Culex quinquefasciatus é considerado praga urbana e tem a capacidade de se desenvolver em águas altamente poluídas atingindo elevada densidade. Mosquitos desta espécie possuem importância vetorial na transmissão de parasitas e arboviroses. Medidas de controle químico têm se mostrado ineficazes, além de serem altamente prejudiciais ao meio ambiente. Desse modo, novas tecnologias de controle foram desenvolvidas, entre elas o SIT (Sterile Insect Technique) que utiliza radiação para esterilizar machos e liberá-los no ambiente para copular com fêmeas selvagens. Posteriormente métodos genéticos baseados nessa técnica têm sido propostos. O sistema RIDL (Liberação de Insetos Carregando Gene Letal Dominante), consiste na integração de um gene letal dominante associado a um promotor específico de fêmea, dispensando a etapa de esterilização por radiação. Nesse processo os insetos recebem dieta suplementada com um repressor químico. A expressão do gene letal dominante é mantida desligada enquanto este repressor é adicionado ao meio das larvas. Para as amostras que estariam sendo preparadas para liberação, o repressor é retirado, e o gene letal dominante é ativado, causando a morte de todas as fêmeas, restando apenas machos para liberação. Os machos homozigotos para o gene letal seriam liberados para copular com fêmeas selvagens. A progênie seria heterozigota para o gene letal, porém somente os machos sobreviveriam. Parte crucial para o sucesso deste projeto foi a adaptação do método de microinjeção de embriões para a espécie Culex quinquefasciatus tornando possível a injeção dos transgenes LA513, LA882 e LA3653 com o objetivo de obtermos linhagens transgênicas. A obtenção de linhagens transgênicas com estas construções se mostraram mais laboriosa do que o previsto, dificultando a transgenia. Porém, as aplicações práticas em controle de vetores utilizando a técnica do RIDL são imensas e pode se tornar uma importante ferramenta do Manejo Integrado de vetores. / The Culex quinquefasciatus mosquitoes are considered urban plague and are able to grown in polluted water achieving high density. This specie are important vectors in the transmission of parasites and arbovirus. Chemical control methods shown to be ineffective and highly hazards to the environment. In this way, new technologies of control have been developed, among them there is SIT (Sterile Insect Technique) that utilizes radiation to sterilize males and then release in the environment to mate with wild females. New genetic methods are been proposed based on the SIT technique, they relies on the integration of a dominant lethal gene associated to a specific promoter of female avoiding the need for radiation (RIDL). In this process the insects receive diet supplemented with a chemical repressor (tetracycline). The expression of the dominant lethal gene is kept off while this repressor is added to the larvae. For the samples that would be being prepared for release, the repressor is removed, and the dominant lethal gene is activated, causing the death of all the females, and consequently only leaving male for release. The homozygous males for the lethal gene would be set free to mate with the wild females. The lineages would be heterozygous for the lethal gene and only males would survive. Since that, the capacity of mating of the transgenic males produced by method RDIL is an important prerequisite for the success of the program. It was crucial to achieve the objectives of this project adapt the microinjection method to Culex quinquefasciatus, so it became possible inject the LA513, LA882 and LA3653 plasmids seeking for the transgenic transformation. The obtaining of the transgenic mosquito Culex quinquefasciatus seems to be more difficult then expected and its development was harmed. The practical applications using RIDL in vector control is huge and can become a useful tool in the Integrated Management of vectors.

O mosquito Culex quinquefasciatus é considerado praga urbana e tem a capacidade de se desenvolver em águas altamente poluídas atingindo elevada densidade. Mosquitos desta espécie possuem importância vetorial na transmissão de parasitas e arboviroses. Medidas de controle químico têm se mostrado ineficazes, além de serem altamente prejudiciais ao meio ambiente. Desse modo, novas tecnologias de controle foram desenvolvidas, entre elas o SIT (Sterile Insect Technique) que utiliza radiação para esterilizar machos e liberá-los no ambiente para copular com fêmeas selvagens. Posteriormente métodos genéticos baseados nessa técnica têm sido propostos. O sistema RIDL (Liberação de Insetos Carregando Gene Letal Dominante), consiste na integração de um gene letal dominante associado a um promotor específico de fêmea, dispensando a etapa de esterilização por radiação. Nesse processo os insetos recebem dieta suplementada com um repressor químico. A expressão do gene letal dominante é mantida desligada enquanto este repressor é adicionado ao meio das larvas. Para as amostras que estariam sendo preparadas para liberação, o repressor é retirado, e o gene letal dominante é ativado, causando a morte de todas as fêmeas, restando apenas machos para liberação. Os machos homozigotos para o gene letal seriam liberados para copular com fêmeas selvagens. A progênie seria heterozigota para o gene letal, porém somente os machos sobreviveriam. Parte crucial para o sucesso deste projeto foi a adaptação do método de microinjeção de embriões para a espécie Culex quinquefasciatus tornando possível a injeção dos transgenes LA513, LA882 e LA3653 com o objetivo de obtermos linhagens transgênicas. A obtenção de linhagens transgênicas com estas construções se mostraram mais laboriosa do que o previsto, dificultando a transgenia. Porém, as aplicações práticas em controle de vetores utilizando a técnica do RIDL são imensas e pode se tornar uma importante ferramenta do Manejo Integrado de vetores. / The Culex quinquefasciatus mosquitoes are considered urban plague and are able to grown in polluted water achieving high density. This specie are important vectors in the transmission of parasites and arbovirus. Chemical control methods shown to be ineffective and highly hazards to the environment. In this way, new technologies of control have been developed, among them there is SIT (Sterile Insect Technique) that utilizes radiation to sterilize males and then release in the environment to mate with wild females. New genetic methods are been proposed based on the SIT technique, they relies on the integration of a dominant lethal gene associated to a specific promoter of female avoiding the need for radiation (RIDL). In this process the insects receive diet supplemented with a chemical repressor (tetracycline). The expression of the dominant lethal gene is kept off while this repressor is added to the larvae. For the samples that would be being prepared for release, the repressor is removed, and the dominant lethal gene is activated, causing the death of all the females, and consequently only leaving male for release. The homozygous males for the lethal gene would be set free to mate with the wild females. The lineages would be heterozygous for the lethal gene and only males would survive. Since that, the capacity of mating of the transgenic males produced by method RDIL is an important prerequisite for the success of the program. It was crucial to achieve the objectives of this project adapt the microinjection method to Culex quinquefasciatus, so it became possible inject the LA513, LA882 and LA3653 plasmids seeking for the transgenic transformation. The obtaining of the transgenic mosquito Culex quinquefasciatus seems to be more difficult then expected and its development was harmed. The practical applications using RIDL in vector control is huge and can become a useful tool in the Integrated Management of vectors.

Diss. (sammanfattning) Umeå : Umeå universitet, 2007. / Härtill 4 uppsatser.

The use of genome wide ENU mutagenesis screening has uncovered vast numbers of novel genes involved in the control of the immune system. This thesis describes the characterization of a novel mutant, Captain Morgan (CPM), originally identified in an immunization screen designed to evaluate both the initial antibody response to antigen and the ability to sustain antibody production. Mapping of this mutant lead to the identification of a single base pair mutation in a novel guanine nucleotide exchange factor, dedicator of cytokinesis 8 (DOCK8). The mutation was found to result in altered gene splicing of the DOCK8 protein leading to the truncation of the protein and loss of catalytic function. The importance of understanding the role of DOCK8 in host immunity has been recently underlined by the discovery that cohorts of patients suffering from autosomal recessive forms of hyper-IgE syndrome have loss-of-function or deletions in this novel guanine nucleotide exchange factor. Disease in these patients is characterised by recurrent viral and bacterial infections mainly of the skin and lungs, with reduced levels of peripheral CD4⁺ and CD8⁺ T cells in the blood of patients. Patients also have high levels of IgE and eosinophilia in the blood and are highly atopic with increased prevalence of allergic diseases including asthma. Loss of DOCK8 function results in a number of phenotypes in CPM mice, which may help understand the immunodeficiency syndrome experienced by DOCK8 deficient patients. CPM mice, like DOCK8 deficient patients, are lymphopenic with losses of both CD4⁺ and CD8⁺ T cells in the blood and secondary lymphoid organs. Challenge of CPM mice with modified vaccina virus (MVA) and influenza strain X31 demonstrated normal primary anti-viral responses. However, similar to the loss of germinal centre B cells previously described in these mice, memory T cell responses were diminished, which may explain the susceptibility of DOCK8 deficient patients to recurrent infections. In addition to the loss of peripheral T cells, rare populations of lymphocytes such as invariant natural killer T cells (iNKT) were also reduced in the liver and thymus. Due to their roles in bacterial and viral responses and cancer immunosurveillance it is expected that loss of these cells will contribute to disease severity. Together these findings illustrate the importance of the ENU mutagenesis model for generating new mutants, which can enhance our understanding of mammalian genes and create disease models of human disease. Further characterization of DOCK8 deficiency and the molecular mechanisms of DOCK8 function will have important implications for disease diagnosis and ongoing treatment for patients.

E-cadherin (Cdh1), is a major component of epithelial adherens junctions, binds the Wnt pathway effector β-catenin and is lost at the invasive edge of colon cancers. Crypt stem cells give rise to 4 cell lineages that, with the exception of Paneth cells, travel along the crypt-villus axis over 3 days and are shed by anoikis. Gain of function of the Wnt pathway by a mutation in Adenomatous Polyposis Coli (Apc) disrupts enterocyte turnover to result in adenoma formation. Homozygote null Cdh1 is embryonic lethal, and heterozygote Cdh1 can promote Apc^1638N induced adenoma formation, but we lack models that assess additional functions of Cdh1 in the adenoma to carcinoma transition. The aim of this thesis was to evaluate the effect of Cdh1 loss of function in the murine intestine, including in the setting of Wnt pathway activation. To do this, germline and intestinal specific conditional Cdh1 loss of function models were generated. Conditional homozygous deletion of Cdh1 resulted in embryonic lethality using Villin-Cre. In adults, homozygous Cdh1 loss using the tamoxifen inducible Villin-CreER^T2 led to intestinal inflammation, bacteraemia and disrupted crypt-villus architecture. Combined conditional homozygous deletion of Cdh1 and Apc resulted in Wnt pathway upregulation assessed by β-catenin immunolabelling. Strain dependent effects of Cdh1 heterozygosity were apparent on the Apc heterozygote background: Apc^Min/+ Cdh1^+/- (C57BL/6J) had no effect on survival or adenoma phenotype compared to littermate Apc^Min/+; Cdh1^+/fl increased adenoma burden in Apc^+/fl Vil-Cre animals (B6D2/C57BL/6J). Low frequency recombination of Apc^fl/fl using Lgr5-EGFP-IRES-CreER^T2 bypasses the loss of heterozygosity event relied on in heterozygous Apc tumour models achieving a large adenoma burden within 4 weeks. Cdh1 loss did not alter survival or adenoma phenotype in this model, including the development of large caecal tumours. Immunolabelling of tumours from Apc^fl/fl Cdh1^fl/fl animals showed persistent E-cadherin protein expression, suggesting incomplete recombination or that double homozygote enterocytes failed to survive. In vitro adenoma culture was used to test whether E-cadherin loss was incompatible with enterocyte survival in the setting of Wnt activation. Apc^fl/fl Cdh1^{+/+} and Apc^fl/fl Cdh1^fl/fl adenoma were cultured in matrigel and treated with an adenovirus expressing Cre recombinase under a CMV promoter (Ad-Cre). Ad-Cre had no effect on Apc^fl/fl Cdh1^+/+ adenoma growth. Ad-Cre treated Apc^fl/fl Cdh1^fl/fl adenoma organoids showed cells where E‑cadherin loss resulted in Wnt pathway upregulation as assessed by nuclear β-catenin and Axin2 expression, and epithelial mesenchymal transition shown by upregulation of fibronectin, twist and vimentin. This work supports a role for E-cadherin in modulation of the Wnt pathway. Further investigation is required to define the cell adhesion versus Wnt regulatory functions of E-cadherin.