The renal capsule is a flattened layer of cells which surround the kidney. Expression of the transcription factor Foxd1 is required for normal development of the capsule. Furthermore, current evidence suggests that during development the capsule progenitors are in a state of active hedgehog signaling. We hypothesize that hedgehog plays a role in modulating capsule morphogenesis in the embryonic kidney. To test the hypothesis hedgehog signaling was inhibited in the capsule via Foxd1Cre mediated deletion of Smoothened (Smo), the activator of the pathway. Mutant kidneys were approximately 48% smaller in volume and had a 42% decrease in nephron number. Furthermore, mutants displayed abnormal patterning of the capsule where regions on the surface of the kidney had no capsule cells. The discontinuous capsule phenotype was observed only after E13.5. Additionally, capsule cells progressively lost expression of known markers Foxd1 and Raldh2 and their proliferative capacity was decreased by 54% at E13.5.

The renal capsule is a flattened layer of cells which surround the kidney. Expression of the transcription factor Foxd1 is required for normal development of the capsule. Furthermore, current evidence suggests that during development the capsule progenitors are in a state of active hedgehog signaling. We hypothesize that hedgehog plays a role in modulating capsule morphogenesis in the embryonic kidney. To test the hypothesis hedgehog signaling was inhibited in the capsule via Foxd1Cre mediated deletion of Smoothened (Smo), the activator of the pathway. Mutant kidneys were approximately 48% smaller in volume and had a 42% decrease in nephron number. Furthermore, mutants displayed abnormal patterning of the capsule where regions on the surface of the kidney had no capsule cells. The discontinuous capsule phenotype was observed only after E13.5. Additionally, capsule cells progressively lost expression of known markers Foxd1 and Raldh2 and their proliferative capacity was decreased by 54% at E13.5.

The formation of the Salmonella extracellular matrix is a multicellular behavior important for environmental persistence. It is comprised of uniquely but ill-defined assembled thin aggregative fimbriae (Tafi), cellulose and uncharacterized polysaccharides. Consequently, investigations were launched into further clarifying Tafi assembly and the polysaccharide constituents of the extracellular matrix. In the Salmonella agfBAC Tafi operon, the transcription and role of agfC has been elusive. In this study using the clinical isolate, Salmonella Enteritidis 27655-3b, agfBAC transcripts were detected using a reverse transcriptase and transcription was not enhanced by replacement of a stem-loop structure immediately preceding agfC. AgfChis was purified, localized to the periplasm, and found to specifically bind noncrystalline cellulose suggesting an association with the extracellular matrix. An inframe ΔagfC mutant displayed an abundance of 20 nm fibers, which could be complemented with agfC in trans, in addition to Tafi and an increase in cell hydrophobicity. Depolymerization of purified 20 nm fibers required exceptionally stringent conditions to release what proved to be AgfA subunits revealing the 20 nm fibers as AgfA assemblages of unique morphology. The role of AgfC in Tafi assembly was investigated further via a novel, quantitative antibody-capture assay of in-frame agf mutants. A soluble antibody-accessible form of AgfA was captured in wt, ΔagfB and ΔagfF strains in support of the extracellular nucleation-precipitation pathway of Tafi assembly, but not in ΔagfC or ΔagfE mutants. These results suggest that AgfC and AgfE are required for AgfA’s extracellular assembly and thus may act as atypical AgfAspecific chaperones which facilitate Tafi assembly. The implications of these results are presented in an assembly model for Tafi. Additional investigations revealed that Salmonella produces an O-Antigen capsule co-regulated with the extracellular matrix. Structural analysis of purified extracellular polysaccharides (EPS) yielded a repeating oligosaccharide unit similar to iv that of lipopolysaccharide O-Antigen with modifications. Putative carbohydrate transport and regulatory operons important for capsule expression, designated emcA-H and emcIJ, were identified by screening a random transposon library with immune serum generated to the capsule. The absence of capsule was confirmed by generating various in-frame Δemc mutants where emcG and emcE were shown to be important in capsule assembly and translocation. Luciferase-based expression studies showed that, AgfD differentially regulated the emc operons in coordination with extracellular matrix genes. Survival assays demonstrated the capsule is important for desiccation tolerance. The emc genes were found to be conserved in Salmonellae and thus, the O-Antigen extracellular matrix capsule may be a conserved survival strategy important for environmental persistence. Finally, a compositionally unique acidic EPS was found associated with the extracellular matrix. In-frame ΔbcsA, ΔemcG and ΔagfA mutants but neither ΔagfAΔbcsA nor ΔagfD mutants bound calcofluor, a β-glucan binding fluorescent agent, suggesting that multicellular behavior itself and not necessarily AgfD alone was influencing EPS expression. A transposon library was screened by ELISA using serum generated against purified EPS. This identified mutations inactivating genes involved in quorum sensing AI-2 degradation, flagella repression and Tafi and TolA expression. All mutations resulted in the loss of multicellular behavior and immunologically decreased levels of Tafi. This is the first report that implicates quorum sensing AI-2 degradation and flagella repression as part of the regulatory circuit for Tafi expression. Together, the results reveal Tafi uses assembly factors to facilitate extracellular polymerization which likely assists the formation of a network of branched, amorphous fimbriae. Tafi together with EPS form the extracellular matrix: Tafi stabilizes the EPS on the microbial communities; EPS imparts it with physical properties such as hydration, charge and diffusion barriers that protect it from adverse environmental conditions such as desiccation and antimicrobials. This probably contributes to Salmonella survival in the environment and facilitates its cyclic lifestyle.

The main question that this thesis addresses is what is the optimal way of producing tissue grown in the peritoneal cavity around a foreign body for its use as a vascular graft? It is known that a foreign body implanted into the peritoneal cavity induces an inflammatory response with cells recruited from within the peritoneal cavity to encapsulate the foreign body. Over the course of two to three weeks these cells produce an organised matrix and differentiate to become myofibroblasts. Tubes of these ‗tissue capsules‘ have been transplanted into the arterial vasculature in several animal models where the tissue capsule differentiates into an arterial structure. This structure consists of a layer of smooth muscle-like cells, adventitia of dense connective tissue including vasa-vasorum and an endothelial layer of flattened mesothelial cells. In order to determine whether the tissue would further differentiate ex vivo in response to mechanical stimulus an in-vitro bioreactor system was built to house tissue capsules produced in a variety of animal models. This bioreactor system could house 4 tissue capsules under physiological conditions including standard pulse rates, pressures and temperatures experienced by an artery. Boiled blood clot (BBC) scaffolds were implanted into the peritoneal cavity of rats to produce tissue capsules. After two weeks of development in the peritoneal cavity, tissue capsules were harvested and implanted into the bioreactor. Tissue capsules grafted into the bioreactor were subjected to mechanical force for a range of time-points, pressure, pulse and flow rates. When analysing tissue immunohistochemically, elastin, myosin, αSMA and desmin were detected. This staining was not consistent across all samples and only present in small parts of some tissue tested. Western analysis did not show any expression of αSMA or myosin. Finally the morphology of the tissue also resembled that of tissue previously implanted into the arterial circulation, but development of mechanical properties were not to the extent that would make the tissue useful as a vascular graft. The bioreactor system was thus modified to be able to house tissue for a period of 3 weeks. This system successfully housed tissue capsules under mechanical force in physiological ranges. Next, a range of materials were tested for their ability to be included into the peritoneal implant device used for the large animal model. Elasteon 80A did not produce any cellular growth or peritoneal pathology in all implanted samples (n = 4). Cloisite, a pro-inflammatory material produced large tissue capsule development over a 2 week implant period in 25% of samples however this tissue was heavily adhered to the greater omentum and dependent on its vascular supply. This data suggested that Elasteon could be used to coat the outer surface of a peritoneal implant device to decrease the rate of peritoneal adhesions. Three devices were designed and fabricated for their use in generating tissue for the modified Mitrofanoff procedure which requires a length of tissue to be implanted between the umbilicus and the bladder as a fistula. In all three cases no implantable material was produced that could be used for this procedure. To modify the device that could be used to produce tissue for any surgical application, a range of devices was produced and the animal model was changed to pigs. Materials incorporated into these devices include Dexon mesh and polyethylene. These devices also did not produce any tissue that could possibly be used as a vascular graft. A novel material, polymer BD347 was then produced for use in developing tissue within the interior of the device to provide greater growth and mechanical properties for developing a vascular graft. In toxicological studies, the replacement rate of cells was unaffected after seven days of incubation of fibroblasts at confluence with the polymer. A range of mechanical properties from pig vasculature was gained so that a sheet of polymer with similar properties to that of a vascular graft could be made. This polymer was fabricated as a tube and implanted into the peritoneal cavity of rats. The implanted polymer remained free-floating with a capsule of tissue in 78% of cases. A device was designed that has the ability to impart a physiological pulsation force on the developing tissue capsule in the peritoneal cavity using a sheep model. When two devices were implanted for a period of 10 days in each animal these devices produced no complications for the animal. Upon harvest all devices were free of adhesion and did not cause any peritoneal or dermal infection. In 100% of cases this device produced tissue that was thick and consistent along the length of the implant. The quality of tissue differed greatly macroscopically between tissue produced around pulsing and non-pulsing scaffolds, but microscopically the structure of both tissues was not significantly different. Approximately 90% of cells in this tissue stained positively for CD45. Tissue in pulsing devices produced a higher amount of vimentin expression in CD45 positive staining cells than tissue in non-pulsing devices. Mechanical properties of tissue in pulsed devices were also much greater than tissue in non-pulsed devices. Two of the pulsed tissues were grafted into the carotid artery of sheep as arterial patches. In one animal tissue lasted a period of 1 week before it ruptured. In the second animal tissue lasted a period of 2 weeks at which time the animal was sacrificed. In this sheep a layer of endothelial cells had migrated to populate areas of the tissue patch. Pulsation of the implant device enhanced the development of tissue capsule in the peritoneal cavity towards arterial properties. These studies provide information on the materials and designs required to produce peritoneal-derived tissue capsules that can be used in a range of surgical applications. These studies also provide information on how this tissue responds to mechanical force and provides an in vitro system for testing this tissue. This work in this thesis has produced a device that is in the stage of pre-clinical development to be used as a potential therapy for cardiovascular disease. This device is a novel development from previous devices used for generating tissue capsules for engraftment and is a significant contribution to work in developing a replacement artery.

Thesis (M.S.)--University of Michigan, 1957.

Introduction: Knee flexion contractures have a negative impact on function for patients with osteoarthritis (OA). Those with contracture treated with total knee arthroplasty (TKA) have more post-operative pain and worse outcome. Little knowledge is available about patient demographic factors or gene expression in the knee joint capsule in the setting of contracture and severe OA. // Methods: Subjects with primary severe knee OA awaiting a TKA were recruited. We collected subject demographic factors that may be associated with preoperative knee contracture. Subjects’ posterior knee capsule was harvested intraoperatively. Capsule histological analysis was performed using light microscopy. Gene expression analysis was performed using whole genome microarray and immunohistochemistry was used for protein production analysis comparing those with contracture to those without. // Results: Twenty subjects were recruited for the demographics portion of the study (13 contractures and 7 controls), and capsules from 12 subjects (6 contractures, 6 controls) were used for histology, microarray, and IHC analyses. Contracture subjects had longer duration of OA, reduced extension in the contralateral knee, and showed a trend toward elevated body mass index. Tissue cross-sectional areas of adipose, non-adipose and synovial tissues were not statistically different histologically between the two groups. There was increased expression in the contracture group for the genes CHAD, Cyr61, and Sox9. There was a corresponding increase in protein production for CHAD and Sox9. // Conclusions: Screening for OA duration and bilateral knee range of motion (ROM) could be functionally beneficial. When a knee joint contracture is present, correcting for the resulting leg length discrepancy pre- and post-operatively could improve patient outcome. Gene protein products linking capsular cells to the ECM can influence capsular fibrosis and potentially impact ROM.

<p>For autonomous exploration of complex and unknown environments, existing Deep Reinforcement Learning (Deep RL) approaches struggle to generalize from computer simulations to real world instances. Deep RL methods typically exhibit low sample efficiency, requiring a large amount of data to develop an optimal policy function for governing an agent's behavior. RL agents expect well-shaped and frequent rewards to receive feedback for updating policies. Yet in real world instances, rewards and feedback tend to be infrequent and sparse.</p><p> </p><p>For sparse reward environments, an intrinsic reward generator can be utilized to facilitate progression towards an optimal policy function. The proposed Augmented Curiosity Modules (ACMs) extend the Intrinsic Curiosity Module (ICM) by Pathak et al. These modules utilize depth image and optical flow predictions with intrinsic rewards to improve sample efficiency. Additionally, the proposed Capsules Exploration Module (Caps-EM) pairs a Capsule Network, rather than a Convolutional Neural Network, architecture with an A2C algorithm. This provides a more compact architecture without need for intrinsic rewards, which the ICM and ACMs require. Tested using ViZDoom for experimentation in visually rich and sparse feature scenarios, both the Depth-Augmented Curiosity Module (D-ACM) and Caps-EM improve autonomous exploration performance and sample efficiency over the ICM. The Caps-EM is superior, using 44% and 83% fewer trainable network parameters than the ICM and D-ACM, respectively. On average across all “My Way Home” scenarios, the Caps-EM converges to a policy function with 1141% and 437% time improvements over the ICM and D-ACM, respectively.</p>

A unique, previously undescribed Gram-negative bacterium was isolated from several soils in Texas and extensively characterized in this study. The cells measured 1-2 by 4-6 μm. The distinguishing characteristic of the bacterium is the extraordinary capsular material which surrounds the cells. The new isolates are aerobic, mesophilic, non motile and have the ability to utilize a variety of organic compounds as the sole source of carbon and energy. The organism grows optimally at 30° C and the optimal pH lies between 7.0-8.0. The isolates produce catalase but oxidase is not produced. They do not produce indole or hydrogen sulfide. The organism can hydrolyze gelatin and Tween 80 but not starch, esculin and casein. The major cellular fatty acid is anteiso 15:0. The guanine and cytosine content is 58-62 mole%. The organism's taxonomic position was further established by specific gene probes, 16S rRNA homology, DNA homology and "ribotyping." These data showed that it was most closely related to members of the genus Paenibacillus, although somewhat divergent from other species classified in this genus. After careful evaluation of the results obtained during this study, it is proposed that this unique bacterium be named Paenibacillus velasolus sp. nov.

<div> <div> <div> <p>The motion of synthetic capsules and living cells in microchannels has been the subject of numerous studies in the last decade due to its significance in engineer- ing and biomedical applications. Cell sorting and separation are common processes that are used for various purposes such as separation of leukocytes from blood used in DNA sequencing. Isolation of rare cells in blood is needed for early diagnosis of lethal diseases such as cancer. Cell isolation and enrichment will also provide a better platform to biologists to study and analyze various properties of living cells. Thus, there is a high demand for developing techniques to precisely control trajectories of the cells and manipulate them in a desired manner. Microfluidic devices provide a platform to achieve aforementioned needs while overcoming challenges such as sample contamination, cost and complexity of the procedures. In many of these applications, the background fluid is non-Newtonian due to the presence of DNA and proteins, or polymers are added to control the trajectory of the cells. In this work, we first provide a fundamental study on the dynamics of a single deformable capsule in a viscoelastic matrix under a simple shear flow. Furthermore, we investigate the motion of a single cell and suspension of cells in microchannels. The effects of cell size, inertia, cell volume fraction, cell deformability and fluid elasticity are explored. Our findings on capsule motion in the viscoelastic medium suggest that the use of constant-viscosity viscoelastic fluid pushes the cells toward the channel centerline which can be used in microfluidic devices used for cell focusing such as cytometers. However, viscoelastic fluid with shear-thinning characteristics and drives the flowing cells toward the channel wall. Particle motion in viscoelastic matrix equilibrium positions of the particle in the microchannel for a wide range of inertial and elastic effects. These fundamental studies can provide insight on the role of rheological properties of the fluid that can be tuned to control the motion of the cells and particles for efficient design of microfluidic devices. </p> </div> </div> </div>

Recently, the deep neural network structure caps-net was proposed by Sabouret al. [11]. Capsule networks are designed to learn relative geometry betweenthe features of a layer and the features of the next layer. The Capsule network’smain building blocks are capsules, which are represented by vectors. The ideais that each capsule will represent a feature as well as traits or subfeatures ofthat feature. This allows for smart information routing. Capsules traits are usedto predict the traits of the capsules in the next layer, and information is sent toto next layer capsules on which the predictions agree. This is called routing byagreement.This thesis investigates theoretical support of new and existing routing al-gorithms as well as evaluates their performance on the MNIST [16] and CIFAR-10 [8] datasets. A variation of the dynamic routing algorithm presented in theoriginal paper [11] achieved the highest accuracy and fastest execution time.