Three strains of cyanobacteria (Anabaena PCC7120, A. variabilis and Nostoc commune), all belonging to the family Nostocaceae, were found to be capable of modulating the production and chemical composition of extracellular polysaccharides (EPS) in response to carbon and nitrogen availability as well as pH. While the carbohydrate compositions of the glycans produced by the different organisms were indicative of their recent evolutionary divergence, there were measurable differences that were dependent upon growth conditions. The EPS resulting from biofilm growth conditions was reduced in glucuronic acid levels in both Anabaena variabilis ATCC 29413 and Anabaena PCC 7120. Under planktonic conditions, the glycan from A. variabilis contained glucuronic acid when grown in nitrate-free BG-11₀ medium whereas A. PCC 7120 produced similar levels in standard BG-11 medium. This suggests that phylogeneticallyrelated cyanobacteria respond very differently to changes in their local environment. The pH of BG-11 cultures increased to 9-10 for all three strains of cyanobacteria. The increase resulted in an increase in the amount of dissolved inorganic carbon available in the medium, creating an imbalance in the carbon-nitrogen ratio, with the complete consumption of 17.65 mmol L⁻¹ nitrates raising the pH to near 10 in BG-11 medium. While increased carbon availability has been shown to induce capsulated morphologies in strains of cyanobacteria, only Nostoc commune DRH-1 exhibited this behavior, and only when grown in BG-11 medium. Carbon and nitrogen availability as well as pH modulate the monosaccharide composition of the glycan generated by cyanobacteria investigated. The different characteristics of the glycans produced can affect the survivability of the organisms and the community structure of cyanobacterial biofilms and microbial mats found in nature. As cyanobacteria are ubiquitous organism both now and in the past, they play a pivotal role in the biological and geological processes of the Earth, controlling the availability and cycling of carbon and nitrogen both actively and passively. / Master of Science in Life Sciences

Diabetic retinopathy (DR) is an incurable, chronic disease that is the leading cause of blindness in working-age adults. A prominent characteristic of DR is the extensive dysfunction within the retina microvasculature. Specialized vascular cells known as pericytes (PCs) are lost or become dysfunctional during disease progression; a thickening of the extracellular matrix (ECM) composing the vascular basement membrane (vBM) and endothelial cell (EC) tight junction disruption are also key features of this disease and contribute to its pathogenesis. PC loss is believed to be a central cue for disease initiation. However, studies inducing PC loss and observing acute changes in the vasculature did not report severe vessel damage or vBM thickening, suggesting that the effects of PC loss occur over a longer period of time. Because the chronic effects of PC loss are more difficult to ascertain, especially in a complex condition such as DR, the mechanisms underlying microvascular defects in DR remain poorly understood. The work presented in this dissertation focuses on pericyte-endothelial cell interactions and their interplay with the ECM/vBM during a variety of physiological and pathological conditions. First, we isolated and functionally validated a primary mouse embryonic PC cell line that we then applied to a co-culture model with ECs to better understand the dynamic interactions between these two critical components of the capillary wall. In the co-culture model, we found that primary PCs promoted EC organization into vessel-like structures and enhanced EC-EC junctions. To complement these in vitro studies, we analyzed animal models and human tissue for the PC-EC interactions and ECM/vBM remodeling under different conditions (physiological and pathological). Moreover, we analyzed microglia and astrocytes to enhance our understanding of the tissue-vessel interface, bolstering our experimental results and facilitating the generation of more hypotheses for future research. Overall, our work suggests that PC-EC interactions in diabetic scenarios play a crucial role in ECM/vBM remodeling; engagement with the ECM/vBM in turn impacted PC behaviors including migration away from the endothelium and induced EC loss of tight junctions, key changes in the onset and progression of DR. / Doctor of Philosophy / Diabetic retinopathy is a group of eye diseases occurring in patients suffering from diabetes and is the leading cause of adult blindness among the working-aged. About one in three people with diabetes over the age of 40 have overt signs of DR. The primary cause for this disease is long-term, high blood sugar levels that damages blood vessels systemically as well as in the eye. Current treatments for DR can prevent the condition from getting worse, but no treatment exists that results in a complete cure. This work described in this dissertation focuses on the interactions between vascular pericytes and endothelial cells, two of the main cell types that compose capillaries (i.e. the smallest blood vessels important for oxygen delivery). The studies presented herein also focus on the response of these cells to the extracellular matrix, a scaffold of proteins that surround pericytes and endothelial cells to stabilize blood vessels. We found that extracellular matrix components dramatically increase as a result of the interactions between pericytes and endothelial cells exposed to diabetic conditions. These changes in the extracellular matrix also had important effects on pericytes and endothelial cells and their engagement with their environment and other cells. Taken together, our work suggests that pericyte-endothelial cell interactions and their crosstalk with the ECM play an important role in blood vessel formation and in the accumulation of microvascular defects that fuel diabetic retinopathy progression.

Rapid rearing of dairy heifers during late prepuberty has been linked to impairments in mammary development and reductions in milk yield. Our objective was to determine how varying energy intakes between 2 and 14 wk of age affect mammary parenchymal development. At 2 wk of age, Holstein calves were assigned to 1 of 4 treatments (HH, HL, LH, and LL) with 2 levels of energy intake (High or Low) and 2 periods of growth (2 to 8 and 8 to 14 wk of age). At 14 wk, parenchyma at the stromal interface, mid-gland, and above the cistern were collected, fixed, and embedded in paraffin. Digital images of stained sections were used to determine tissue composition (% epithelium, lumen, and stroma). Immunochemistry revealed estrogen receptor (ER) and Ki67 (nuclear proliferation antigen) positive cells, type IV collagen, fibronectin (FN), and laminin. Images representing 4 increasing grades were used to quantify ECM protein deposition. Lumenal and stromal areas were 3.5 ± 1.4% higher (p<0.01) and 4.0 ± 1.7% lower (p<0.01), respectively, in HH and HL heifers. Ki67 labeling in terminal ductular units and subtending ducts was 2.1 ± 0.8% (p<0.01) and 1.4 ± 0.7% (p<0.05) lower for the same feeding level combination. FN deposition was also increased (p<0.05) in HH and HL heifers. High rates of gain between 2 and 14 wk of age resulted in greater lumenal area and reduced cell proliferation in mammary parenchyma at 14 wk of age. Changes in FN deposition could have mediated growth differences. / Master of Science

The purpose of this research was to examine the impact of cation addition on an industrial activated sludge system and to determine the applicability of the findings of a previous VPI&SU doctoral student (Higgins, 1995) to this industrial sludge. From this research, the implications on the full-scale industrial wastewater treatment system could be postulated. The addition of sodium and potassium to the feed stream of a laboratory-scale activated sludge system improved sludge volume index (SVI), but produced high supernatant solids. Sodium addition resulted in weak flocs and a deterioration in sludge dewatering. Potassium addition also resulted in poor sludge dewatering but did not significantly affect floc stability. The addition of magnesium improved SVI, supernatant solids, and floc stability, but did not significantly affect sludge dewatering. Increasing the monovalent/divalent cation ratio in the reactor feed stream, reduced SVI but produced high supernatant solids. The soluble and bound extracellular protein content was observed to increase with as this ratio increased. No trend was observed with soluble or bound extracellular polysaccharide content. Batch addition of both calcium and magnesium improved sludge settling and dewatering, although this effect was not observed when sludge with low extracellular polymer content was examined. This research confirmed that the effect of cations on activated sludge properties is highly dependent on the feed wastewater composition and starting and operating conditions. This implies the need for laboratory or pilot-scale testing before the effect of cation addition on a given sludge can be determined. For the industrial sludge studied here, the addition of 7 to 12 mM magnesium produced the most desirable sludge characteristics including improved SVI, supernatant solids and floc stability. / Master of Science

No / Streptococcus mutans was shown to produce two extracellular proteases capable of degrading both gelatin and collagen-like substrates. These enzymes have molecular masses of 52 and 50 kDa when analysed by SDS-PAGE. Both enzymes were inhibited by EDTA, but not by a range of other inhibitors with different specificities, indicating that they are metalloproteases. The activity of EDTA-inactivated enzymes could be restored by the addition of manganese and zinc. The identical inhibition and restoration profiles of the two enzymes suggest that one of the proteases may be a degradation product of the other.

Epithelial to mesenchymal transition (EMT), the transdifferentation of an epithelial cell into a mesenchymal fibroblast, is a cellular process necessary for embryonic development and wound healing. However, uncontrolled EMT can result in accumulation of myofibroblasts and excessive deposition of ECM, contributing to the pathological progression of fibrotic diseases such as pulmonary fibrosis. The ability to control EMT is important for development of novel therapeutics for fibrotic pathologies and for designing novel biomaterials for tissue engineering applications seeking to promote EMT for development of complex tissues. EMT is a highly orchestrated process involving the integration of biochemical signals from specific integrin-mediated interactions with extracellular matrix (ECM) proteins and soluble growth factors such as TGFβ. TGFβ, a potent inducer of EMT, is activated via cell contraction-mediated mechanical release of the growth factor from a macromolecular latency complex. Thus TGFβ activity and subsequent EMT may be influenced by the biochemical and biophysical state of the surrounding ECM. Based on these knowns, it was hypothesized that both changes in integrin engagement and increases in substrate rigidity would modulate EMT due to changes in epithelial cell contraction and TGFβ activation. Here we show that integrin-specific interactions with fibronectin (Fn) fragments displaying both the RGD and PHSRN binding sites facilitate cell binding through α5β1 and α3β1 integrins, and lead to maintenance of epithelial phenotype, while Fn fragments displaying only the RGD site facilitate cell binding through αv integrins and lead to EMT. An in depth investigation into α3β1 binding to Fn fragments indicates that binding is dependent on both the presence and orientation of the PHSRN site. Studies investigating the contribution of ECM stiffening on EMT responses show that increasingly rigid Fn substrates are sufficient to induce spontaneous EMT. Analysis of TGFβ-responsive genes implicate TGFβ-expression, activation or signaling as a mechanism for the observed EMT responses. Together these results suggest that the ECM micromechanical environment is a significant contributor to the onset of EMT responses and provide insights into the design of biomaterial-based microenvironments for the control of epithelial cell phenotype.

Soilborne plant pathogens are responsible for many of the major crop diseases worldwide. However, plant root tips are generally resistant to pathogen infections. The goal of this dissertation research is to understand the mechanism of this natural resistance by testing the hypothesis that root caps and root border cells control the rhizosphere community through the biological products which they deliver to the soil. Specific objectives of this dissertation project are 1) identifying, isolating, and characterizing the genes important for border cell development and for root exudates delivery, and 2) analyzing the function of extracellular macromolecules in root exudates in root tip-fungal pathogen interaction. The expression of a primary cell wall synthesis gene, PsFut1, encoding Pisum sativum fucosyltransferase, was characterized during border cell production, and the impact of silencing this gene on border cell development was examined. Another gene, BRDgal1, encoding β-galactosidase, was identified and characterized in Pisum sativum during this study. It was shown that this β-galactosidase is specifically produced in and secreted from root border cells. The microarray transcriptional profiling in M. truncatula and mRNA differential display analysis in pea plants were carried out following the induction of border cell production to gain a broader understanding of the genes which potentially influence border cell development. In order to study the commonality of border cell production across different plant species, the expression of rcpme1, the marker gene for border cell production, was compared between the garden pea and a gymnosperm species, the Norway spruce (Picea abies). To accomplish the second objective, the focus of this study was shifted from border cell development to mucilaginous root exudates excreted by border cells and root cap cells. This resulted in a breakthrough in the understanding of the mechanisms of root tip resistance. The presence of extracellular DNA in the root mucilage was discovered and its requirement for root tip resistance to fungal infection was demonstrated. Extracellular proteins in the root mucilage were identified and they were shown to be also required for the root tip resistance to fungal infection. This work provided new insights into understanding plant defense mechanisms.

Neutrophils are one of the main effector cells of innate immunity and were shown to kill bacteria by phagocytosis more than 100 years ago. Neutrophils are also capable of antimicrobial activity by producing extracellular structures named neutrophil extracellular traps (NETs). This thesis is an investigation of porcine neutrophils and their ability to produce NETs, as well as the antimicrobial ability of secretions from activated porcine neutrophils in combating a variety of common porcine pathogens. Porcine neutrophils were found to produce NET-like structures, and secretions from activated neutrophils were found to possess variable bactericidal activity against common pathogens of swine. Antimicrobial proteins dependent on elastase activity were shown to be partially responsible for the bactericidal activities of activated neutrophils. Several antimicrobial proteins and peptides were identified via proteomic techniques. This work allows for better understanding of innate immunity in swine, and identification of potential targets for addressing porcine health. / Ontario Ministry of Agriculture Food & Rural Affairs, Ontario Pork, Natural Sciences and Engineering Research Council of Canada

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2005. / Härtill 4 uppsatser.