The biliary tree is a series of ductular tissues responsible for the drainage of bile produced by the liver and pancreatic secretions from the pancreas. The biliary tree is affected by a diversity of life- threatening diseases collectively called cholangiopathies. Cholangiopathies show regionalization, with some diseases such as biliary atresia predominantly targeting extrahepatic bile ducts (EHBDs) outside of the liver. Despite this, little is known on whether anatomical location within the biliary tree contributes to differences in functionality of biliary epithelium, especially in the EHBD compartment. Additionally, reports have demonstrated the possibility for in vitro culture of bile duct stem/progenitor cell organoids from both intrahepatic (IHBD) and EHBD sources. The relation of these organoid systems to each other, and to their tissue of origin, is largely unknown. In this dissertation, I address these major questions by combining transcriptional analyses and in vitro culture of human bile duct organoids derived from primary IHBD and EHBD epithelium. First, I show that in vitro organoids can be derived from four regions of the human biliary tree: gallbladder, common bile duct, pancreatic duct, and intrahepatic bile ducts. Characterization of these organoids demonstrated expression of adult stem cell (LGR5/PROM1) and ductal (KRT19/KRT7) markers suggesting these cultures contained cells with a biliary stem/progenitor phenotype. Further, I show that IHBD organoids are distinct from EHBD organoids requiring different conditions for sustained growth. Using RNA-Sequencing, I demonstrate that primary tissues from different regions of the extrahepatic biliary tree display unique expression profiles and identify novel tissue-specific markers. I also show that only a limited number of these tissue specific differences are maintained in the in vitro organoids and that the organoids are very different from their tissue of origin. Finally, I demonstrate that IHBD, but not EHBD organoids, express a low-level of hepatocyte-specific markers under differentiation conditions. Taken together, the work in this dissertation has uncovered regional specific markers for different anatomical regions of the human biliary tree. Further, I demonstrate that major differences exist between IHBD organoids and EHBD organoids in vitro and discover that only IHBD organoids have the capacity to express hepatocyte markers under differentiation conditions. Ultimately, these results may help to identify new targets for therapeutic development for cholangiopathies and regenerative medicine. They have also provided important insight to the understanding of both basic biliary physiology and also the field of biliary stem/progenitor cell organoids.

<p>with biliary cirrhosis are highly susceptible to acute pulmonary dysfunction and suffer from hepato-pulmonary syndrome. The mechanisms of this enhanced susceptibility remain unknown. It is well established that pulmonary intravascular macrophages (PIMs) are present in cattle, horses, goat and sheep and increase susceptibility for lung inflammation. Species such as rat and mouse also recruit PIMs especially in a bile duct ligation model of biliary cirrhosis. The contributions of recruited PIMs to lung inflammation associated with liver dysfunction remain unknown. Therefore, I characterized a bile duct ligation (BDL) model in rats to study role of recruited PIMs in lung inflammation. First, Sprague Dawley rats were subjected to BDL (N=6) or sham surgeries (N=3) and were euthanized at 4 weeks post-surgery. Five rats were used as the controls. Lung tissues were collected and processed for histology, immunohistology, immuno-electron microscopy, enzyme-linked immunosorbant assay (ELISA) and reverse transcriptase-polymerase chain reaction (RT-PCR). Light microscopy demonstrated normal lung morphology in sham-operated and control rats but showed septal recruitment of mononuclear cells, which were positive for anti-rat monocytes/macrophage antibody ED-1, in BDL rats (p=0.002). Immuno-electron microscopy confirmed localization of ED-1 in PIMs. BDL rats showed increased lung expression of monocyte chemoattractant protein-1 (MCP-1) protein and mRNA compared to the controls (p=0.017) but not of IL-1â, TNF-á, TGF-â and IL-10. Then, I treated BDL rats (N=5) with gadolinium chloride (GC; 10 mg/Kg body weight intravenous) and found reduced numbers of PIMs (p=0.061) at 48 hours post-treatment along with increased expression of TGF-â and IL-10.</p><p>I challenged control rats (N=5) and BDL rats (N=6) with Escherichia coli lipopolysaccharide (E. coli LPS; 0.1 mg/Kg body weight intravenous). All the BDL rats died within 3 hours of LPS challenge (100% mortality) while the normal LPS-treated rats were euthanized at 6 hours post-treatment. Histology and ED-1 staining showed dramatic increase in the number of septal monocytes/macrophages in BDL+LPS rats compared to normal LPS-treated rats (p=0.000). Staining of lung sections with an LPS antibody localized the LPS in lungs. RT-PCR analyses showed no differences in IL-1â transcript levels between LPS challenged BDL rats and LPS challenged control rats (p=0.746) but ELISA showed increase in IL-1â concentration in LPS challenged BDL rats compared to LPS challenged control rats (p=0.000). TNF-á mRNA (p=0.062) and protein (p=0.000) was increased in BDL+LPS rats compared to the control+LPS rats. Immuno-electron microscopy showed IL-1â and TNF-á in PIMs. BDL rats challenged with LPS showed increased expression of IL-10 mRNA and protein (p=0.000 & 0.002 respectively) in lungs compared to LPS challenged control rats. TGF-â mRNA did not change (p=0.128) but lower protein concentrations (p=0.000) were observed in LPS-treated control rats compared to BDL+LPS. </p><p> To further address the role of PIMs, I treated rats with GC at 6 hours or 48 hours (N=5 each) before LPS challenge. The mortality in the 6 hour group was 20% while all the rats in 48 hour group survived till 6 hours. Histology and ED-1 staining showed decrease in the number of intravascular cells in these groups compared to LPS treated BDL rats (p=0.039 for 6 hour group; p= 0.002 for 48 hour group). There were no differences in IL-1â mRNA in both 6 hour and 48 hour groups compared to the LPS challenged BDL rats (p=0.712 & 0.509 respectively). ELISA showed no decrease in IL-1â concentration in 6 hour GC-treated group but a decrease was noticed at 48 hours compared to LPS challenged BDL rats (p=0.455 & 0.008 respectively). TNF-á mRNA levels were not different between LPS-challenged GC-treated BDL rats and LPS-challenged BDL rats (p=0.499 & 0.297 for 6 hour & 48 hour GC groups respectively). But TNF-á concentration in 48 hour GC group (p=0.001) but not in 6 hour GC group (p=0.572) was lower in comparison to BDL+LPS group. IL-10 mRNA was decreased in both 6 hour and 48 hour GC groups (p=0.038 & 0.000 respectively) compared to LPS challenged BDL rats. ELISA showed decrease in IL-10 concentration in 48 hour GC group (p=0.030) but not in 6 hour GC group (p=0.420). TGF-â mRNA expression was decreased in 48 hour GC group (p=0.000) but not in 6 hour GC group (p=0.182). But GC treatment did not affect TGF-â concentrations. </p><p>The data from these experiments characterize a BDL model to study PIM biology, show PIMs pro-inflammatory potential and their possible role as a therapeutic target in lung inflammation.</p>

<p>with biliary cirrhosis are highly susceptible to acute pulmonary dysfunction and suffer from hepato-pulmonary syndrome. The mechanisms of this enhanced susceptibility remain unknown. It is well established that pulmonary intravascular macrophages (PIMs) are present in cattle, horses, goat and sheep and increase susceptibility for lung inflammation. Species such as rat and mouse also recruit PIMs especially in a bile duct ligation model of biliary cirrhosis. The contributions of recruited PIMs to lung inflammation associated with liver dysfunction remain unknown. Therefore, I characterized a bile duct ligation (BDL) model in rats to study role of recruited PIMs in lung inflammation. First, Sprague Dawley rats were subjected to BDL (N=6) or sham surgeries (N=3) and were euthanized at 4 weeks post-surgery. Five rats were used as the controls. Lung tissues were collected and processed for histology, immunohistology, immuno-electron microscopy, enzyme-linked immunosorbant assay (ELISA) and reverse transcriptase-polymerase chain reaction (RT-PCR). Light microscopy demonstrated normal lung morphology in sham-operated and control rats but showed septal recruitment of mononuclear cells, which were positive for anti-rat monocytes/macrophage antibody ED-1, in BDL rats (p=0.002). Immuno-electron microscopy confirmed localization of ED-1 in PIMs. BDL rats showed increased lung expression of monocyte chemoattractant protein-1 (MCP-1) protein and mRNA compared to the controls (p=0.017) but not of IL-1â, TNF-á, TGF-â and IL-10. Then, I treated BDL rats (N=5) with gadolinium chloride (GC; 10 mg/Kg body weight intravenous) and found reduced numbers of PIMs (p=0.061) at 48 hours post-treatment along with increased expression of TGF-â and IL-10.</p><p>I challenged control rats (N=5) and BDL rats (N=6) with Escherichia coli lipopolysaccharide (E. coli LPS; 0.1 mg/Kg body weight intravenous). All the BDL rats died within 3 hours of LPS challenge (100% mortality) while the normal LPS-treated rats were euthanized at 6 hours post-treatment. Histology and ED-1 staining showed dramatic increase in the number of septal monocytes/macrophages in BDL+LPS rats compared to normal LPS-treated rats (p=0.000). Staining of lung sections with an LPS antibody localized the LPS in lungs. RT-PCR analyses showed no differences in IL-1â transcript levels between LPS challenged BDL rats and LPS challenged control rats (p=0.746) but ELISA showed increase in IL-1â concentration in LPS challenged BDL rats compared to LPS challenged control rats (p=0.000). TNF-á mRNA (p=0.062) and protein (p=0.000) was increased in BDL+LPS rats compared to the control+LPS rats. Immuno-electron microscopy showed IL-1â and TNF-á in PIMs. BDL rats challenged with LPS showed increased expression of IL-10 mRNA and protein (p=0.000 & 0.002 respectively) in lungs compared to LPS challenged control rats. TGF-â mRNA did not change (p=0.128) but lower protein concentrations (p=0.000) were observed in LPS-treated control rats compared to BDL+LPS. </p><p> To further address the role of PIMs, I treated rats with GC at 6 hours or 48 hours (N=5 each) before LPS challenge. The mortality in the 6 hour group was 20% while all the rats in 48 hour group survived till 6 hours. Histology and ED-1 staining showed decrease in the number of intravascular cells in these groups compared to LPS treated BDL rats (p=0.039 for 6 hour group; p= 0.002 for 48 hour group). There were no differences in IL-1â mRNA in both 6 hour and 48 hour groups compared to the LPS challenged BDL rats (p=0.712 & 0.509 respectively). ELISA showed no decrease in IL-1â concentration in 6 hour GC-treated group but a decrease was noticed at 48 hours compared to LPS challenged BDL rats (p=0.455 & 0.008 respectively). TNF-á mRNA levels were not different between LPS-challenged GC-treated BDL rats and LPS-challenged BDL rats (p=0.499 & 0.297 for 6 hour & 48 hour GC groups respectively). But TNF-á concentration in 48 hour GC group (p=0.001) but not in 6 hour GC group (p=0.572) was lower in comparison to BDL+LPS group. IL-10 mRNA was decreased in both 6 hour and 48 hour GC groups (p=0.038 & 0.000 respectively) compared to LPS challenged BDL rats. ELISA showed decrease in IL-10 concentration in 48 hour GC group (p=0.030) but not in 6 hour GC group (p=0.420). TGF-â mRNA expression was decreased in 48 hour GC group (p=0.000) but not in 6 hour GC group (p=0.182). But GC treatment did not affect TGF-â concentrations. </p><p>The data from these experiments characterize a BDL model to study PIM biology, show PIMs pro-inflammatory potential and their possible role as a therapeutic target in lung inflammation.</p>

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The world today has large challenges in order to manage a changing climate and the consequences that the climate change has on the environment and human living standards. This climate change is largely affected by the emissions of greenhouse gases, which come from usage of fossil fuels. This is a global problem that will affect the whole world and cause an increase of mean global temperature, which would lead to drastically changes in the living environment for human beings. A large part of the use of fossil fuels is connected to electric energy production. In EU almost half of the electric energy production is based on combustion of fossil fuels like natural gas and coal. These types of energy production need to be phased out and the energy consumption needs to decrease.   With climate change as a background there is a development towards more sustainable households. Companies around the world today invest in developing products that are more environmentally friendly. Household appliance companies develop products that use less water and energy, and a company like ASKO appliances AB tries to equip their machines with a new type of drying system. This new system would mean that less energy is needed for the drying cycle and humid air would not flow out in the kitchen.   The new drying system uses an air channel mounted on the side of a machine where moist hot air passes through the channel. During the passage the hot air will exchange heat with a cold surface inside of the channel. This work is focused on finding an optimal geometry of the air channel that enhances heat transfer between hot air and a cold surface. Installing obstacles inside of the duct could alter the flow pattern, and therefore enhance heat transfer. The work is mostly computer based with simulations performed in software called COMSOL Multiphysics. The software is used to build a 3-D model, where different geometries of obstacles are placed inside of the air channel. Results from simulations are compared with results from experimental trials, thus validating the computer model. Fluid flow simulations are used to investigate the effects of heat transfer for different types of geometries and sizes of obstacles. Parameters like influence angle and obstacle distance are tested.   The study shows results that obstacles inside of an air channel enhance heat transfer between a fluid and a surface. V-shaped obstacles perform the best results in order to enhance heat exchange, this compared with other tested geometries like W-shaped, wave-shaped obstacles and geometry without obstacles.   Different influence angle and distance between obstacles affects heat transfer, the study indicates that influence angle has larger effects on heat transfer than obstacle distance.

This thesis studies the parameters that affect the sound level of signalhorns in a passenger car. The project is performed on the behalf of VolvoCars Group. Physical testing was done on Volvo S90 with and withoutmodifications. During the project, the influence of installation, frequencysignature of horns and system parameters on sound pressure wereinvestigated. Acoustic measurements were performed in semi anechoicchamber and open site using the setup specified by ECE R28 document.

Thoracic duct (TD) ligation has long been the treatment of choice for canine idiopathic chylothorax. Clear identification of all the TD branches at the surgical site is critical to facilitate complete ligation, and this can be difficult due to the highly variable nature of the TD system in number, location and patency of TD branches. Failure to ligate all of the TD branches may result in persistent flow of chyle into the pleural cavity through any missed ducts, and this is the single most common cause of failure with TD ligation. Performing direct positive contrast lymphography with a water soluble contrast medium, administered through a surgically implanted mesenteric lymphatic vessel catheter has been the conventional method used to identify TD branches. This procedure involves invasive surgery to both implant and remove the mesenteric lymphatic catheter, which increases patient risk and discomfort, as well as the diagnostic time and cost. Ultrasound (US) guided percutaneous administration of contrast medium into either a popliteal or mesenteric lymph node (LN) have been proposed as alternatives to mesenteric lymphatic vessel catheterisation, however their comparability with the conventional approach has not been assessed. Computed tomographic (CT) lymphography of the caudal canine TD was performed in seven beagles with contrast medium administered through a mesenteric lymphatic catheter, and by US guided percutaneous injection into a popliteal LN. Images of the TD system were collected using both helical and sequential CT modalities for each contrast medium administration technique. It was found that percutaneous popliteal lymphography had a total diagnostic procedure time just 46% of that found with mesenteric lymphatic vessel administration, and resulted in a time saving of 52 minutes. It also incurred only 29% of the total costs, and patients were assessed to have significantly less discomfort compared to mesenteric lymphatic vessel lymphography. There was no significant difference in the number of TD branches identified by the two contrast medium administration techniques (P = 0.256). However administration of contrast medium into a mesenteric lymphatic vessel did result in the largest TD branch having a significantly greater widest diameter (P < 0.001), cross-sectional area (P < 0.001) and mean Hounsfield unit (HU) value (P < 0.001) than popliteal administration. The significant difference in TD size and contrast medium concentration may help to explain the trend for popliteal administration of contrast medium to detect slightly fewer TD branches (CR = 0.830), however this study could not confirm this trend nor its possible causes. There was no significant difference in the number of TD branches identified by the two CT modalities (P = 0.417). However helical CT did result in the largest TD branch having a significantly greater widest diameter (P < 0.001), cross-sectional area (P < 0.001) and higher mean HU value (P < 0.001). It should be noted however that in this study sequential CT was consistently performed after the helical CT was completed, which could explain the differences seen between the two CT modalities in TD branch size and contrast medium concentration. Despite helical CT having the apparent advantage of a larger TD branch which contained a higher concentration of contrast medium, it was actually found that there was a trend for helical CT to detect slightly fewer TD branches (CR = 0.876). This is possibly due to the positive pressure breath hold that was used to minimise thoracic respiratory movement for the helical CT; while the sequential CT was performed during normal respiration and was therefore not subject to abnormally elevated intrapleural pressure. This study could not confirm the slight superiority of sequential CT in detecting TD branches nor the possible reasons for this apparent difference. / Dissertation (MMedVet)--University of Pretoria, 2010. / Companion Animal Clinical Studies / unrestricted

Objective: To evaluate the age-related changes in active pumping in thoracic duct (TD) from 24-month-old Fisher-344 rats comparing with TD pumping in 9-month rats. Methods: Lymphatic diameters, contraction amplitude and frequency, ejection fraction, and fractional pump flow were determined in isolated TD preparations. Western blot analyses were performed to evaluate relative levels of eNOS and iNOS in 9- and 24-month-old TD. Results: Stretch-dependent regulation was altered in aged TD especially at higher levels of pressure: the negative inotropy, negative chronotropy and diminished minute pumping (2- to 3-fold decrease) were observed. Physiological NO/imposed-flow-dependent inhibition was completely abolished in aged TD, yet NO-synthase blockade by l-NAME (10-4 M) increased pumping in a flow-independent manner. Western blot analyses indicated that the relative levels of eNOS were decreased ∼ 7-fold in the 24-month-old TD when compared with 9-month-old TD; whereas iNOS levels were increased ∼ 10-fold in 24-month-old TD. Conclusions: These data provide the first evidence that stretch- and imposed-flow-dependent regulatory mechanisms are greatly altered in aged TD. These alterations of active pumping mechanisms in TD appear to be related with age-related disturbances in NO-dependent regulatory pathways, and may reflect diminished lymphatic muscle contractility as well as altered lymphatic endothelium function.

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