A limnological study of the Dundas Marsh region, involving the main physical, chemical and biological factors, and their interrelationships, as investigated during the summer seasons of 1946 to 1948, inclusive. The account includes a review of pertinent historical and descriptive material on the entire region. / Thesis / Master of Arts (MA)

Bone defects are a pervasive complication arising from many clinical conditions, both mechanical and pathological. Current treatments for large bony defects focus on applying bone grafts or synthetic materials to the defect area. Cell-based—and especially stem-cell—therapies have advanced greatly thanks to increasing attention focused on their ability to generate new tissues in situ with biomechanical properties approaching that of native tissue, but they suffer from their own shortcomings as well. Exosomes have been shown to play critical roles in cell-signaling and tissue regeneration and are therefore potentially ideal therapeutic vehicles for treating bone defects. Exosomes are small microvesicles counted amongst stem cells’ paracrine factors capable of delivering nucleic acid and enzymatic protein cargoes in a targeted 2 manner. Our previous studies have shown that hMSC-Exosomes are both proliferative and chemotactic, inhibit inflammatory cytokine production, and suppress osteoclast differentiation. Our long term goal is to develop hMSC-Exosome as a clinical therapy for bone regeneration. The objectives of this study were to determine the ability of hMSC-Exosome to enhance bone healing in a rat calvarial defect model, and to further investigate the integrity of the exosome under certain storage conditions. The specific aims of this study were: 1) To determine the osteogenic potential of hMSC-Exosomes in rat calvarial defects, and 2) To determine the impact of variable storage conditions on the integrity of exosomes. To investigate in vivo regenerative potential, rats with surgically-created craniotomy defects were treated with hMSC-Exosome suspension via a collagen gel matrix. After 4 weeks, the calvaria were harvested and analyzed via micro-CT. Volumetric micro-CT analysis showed that hMSC-Exosome could significantly enhance center healing, structural integrity, and growth uniformity in a calvarial defect model. Western blot and TEM showed thorough destruction of surface protein markers and decreased membrane integrity in lyophilized exosome fraction; moderate progressive surface protein marker loss and aggregation were observed with increasing freeze-thaw cycles. In summary, hMSC-Exosome is a promising therapeutic for treatment of bone defects.

No description available.

After a wild-type isolate of Epicoccum purpurascens was exposed to shortwave ultraviolet light, several new strains were recovered which were improved in sporulation, fungicide tolerance, and performance in suppression of white mold caused by Sclerotinia sclerotiorum. The efficacy of E. purpurascens in controlling white mold of snap bean (Phaseolus vulgaris) was assessed in greenhouse and field trials. White mold was significantly reduced in both greenhouse and field trials when 2-4 sprays of E. purpurascens conidial suspensions (in 1% malt extract) were sprayed onto the plant surface during the flowering period. Germination of E. purpurascens conidia on senescent petals was greater than on younger flowers. Addition of malt extract to conidial suspensions improved germination on flowers and increased colonization of emerging flowers. Application of E. purpurascens did not accelerate senescence of bean leaves or affect pod yield of bean in greenhouse trials. Mycoparasitism of S. sclerotiorum by E. purpurascens was found only rarely in in vitro tests and was not observed on flower disks. Production of inhibitory compounds by E. purpurascens was the most important mechanism in suppression of white mold but competition for nutrients also appeared to play a role in biocontrol. The influence of nutrients on conidial germination, growth, sporulation and production of antifungal compounds by E. purpurascens were also investigated.

The aim is to describe the development of selected demographic characteristics and their comparison in the long term or in selected years characterizing the babies and their mothers in the Czech Republic. The indicators are divided according to what they described to the biological factors, social factors and health factors. The analysis of biological characteristics includes age, frequency, order of birth and mothers parity. Social characteristics examined include marital status and education. Health characteristics in the thesis deal with pregnancy and childbirth, addictive substances use and childlessness. This thesis also describe the evolution of the mentioned characteristics and their combinations for the region NUTS II. in the Czech Republic supported by cluster analysis, which confirmed the regional differentiation. The major finding is that mothers still continuing moving to higher age groups, increasing the proportion of births outside marriage, increasing the representation of women in higher education and increasing the proportion of childbearing by caesarean section. In conclusion, this work also includes a description of the current typical woman, who has a children

<p>The aim of this essay was to investigate if different variables affected the distribution of fresh water pulmonate in Babati District, Tanzania. Can the absence of intermediate host be explained by basic vegetation evaluation, pH, conductivity and temperature? Or can it be explained by other factors as animals and vegetation in the surrounding? The study was carried out in Babati District, in Lake Babati, Kiongozi/Farahani River and the irrigation schemes in Matufa and Gichameda from the 23td of February until 7th of March, 2009. The species found during the survey were <em>Biomphalaria, Bulinus, </em>and <em>Lymnea. </em>Only <em>Biomphalaria pfeifferi </em>were present in the genus <em>Biomphalaria</em>. In <em>Bulinus </em>spp.<em>, B. globosus, B. forskalii, </em>and <em>B. africanus </em>were present. <em>Lymnea spp</em>. was represented by <em>L. natalensis</em>. Statistical tests were carried out with logistic models. The results of the statistical analysis revealed different significant results for the different snail species present. <em>L. natalensis </em>showed a significantly positive effect of the water temperature and was distributed in water temperatures ranging from 20.9°C to 24.3°C, which is in the lower range in this study. <em>Biomphalaria pfeifferi </em>and <em>Bulinus spp</em>. were significantly affected to an increase in conductivity. L. natalensis did show a significant effect of the type of bottom in the water body, and found muddy bottoms more suitable. Animal activity (livestock) did show a significant effect on the distribution of <em>L. natalensis </em>which found habitats without animals more suitable. Both <em>B. pfeifferi </em>and <em>L. natalensis </em>were significantly affected by vegetation in the surrounding and found habitats with grass, shrubs and trees more favourable before cultivated areas and forests. The statistical analysis made on the data collected in Babati District showed that temperature, conductivity, bottom in water body and vegetation in the surrounding, in general, significantly affected the fresh water pulmonate. Several variables as pH, water flow, canopy cover, vegetation in the water, however, were not significantly affecting the distribution of the snails. Further investigations of interactive effects of variables, however, are necessary to prevent high infection rates of trematodes infecting the pulmonate present in Babati District, Tanzania.</p>

The aim of this essay was to investigate if different variables affected the distribution of fresh water pulmonate in Babati District, Tanzania. Can the absence of intermediate host be explained by basic vegetation evaluation, pH, conductivity and temperature? Or can it be explained by other factors as animals and vegetation in the surrounding? The study was carried out in Babati District, in Lake Babati, Kiongozi/Farahani River and the irrigation schemes in Matufa and Gichameda from the 23td of February until 7th of March, 2009. The species found during the survey were Biomphalaria, Bulinus, and Lymnea. Only Biomphalaria pfeifferi were present in the genus Biomphalaria. In Bulinus spp., B. globosus, B. forskalii, and B. africanus were present. Lymnea spp. was represented by L. natalensis. Statistical tests were carried out with logistic models. The results of the statistical analysis revealed different significant results for the different snail species present. L. natalensis showed a significantly positive effect of the water temperature and was distributed in water temperatures ranging from 20.9°C to 24.3°C, which is in the lower range in this study. Biomphalaria pfeifferi and Bulinus spp. were significantly affected to an increase in conductivity. L. natalensis did show a significant effect of the type of bottom in the water body, and found muddy bottoms more suitable. Animal activity (livestock) did show a significant effect on the distribution of L. natalensis which found habitats without animals more suitable. Both B. pfeifferi and L. natalensis were significantly affected by vegetation in the surrounding and found habitats with grass, shrubs and trees more favourable before cultivated areas and forests. The statistical analysis made on the data collected in Babati District showed that temperature, conductivity, bottom in water body and vegetation in the surrounding, in general, significantly affected the fresh water pulmonate. Several variables as pH, water flow, canopy cover, vegetation in the water, however, were not significantly affecting the distribution of the snails. Further investigations of interactive effects of variables, however, are necessary to prevent high infection rates of trematodes infecting the pulmonate present in Babati District, Tanzania.

The cell division of Saccharomyces cerevisiae is controlled by the action of pheromones at the G1 phase of the cell cycle. A general method was developed for the isolation of constitutive mutants in the pheromone response pathway. Recessive alleles of the SCG1 gene (encoding the α subunit of a G protein) were isolated as well as a dominant mutation in the STE4 gene (encoding the β subunit of a G protein). Analysis of double mutants suggested that the STE4 gene product functions after the SCG1 product but before the STE5 gene product. Double mutants carrying either scg1 or STE4Hp1 constitutive alleles together with the temperature-sensitive unresponsive mutation, ste5-3ts, showed arrest and recovery when shifted from 34° C to 22° C. Recovery from the constitutive signal was independent of the receptor. The STE4Hp1 sst2 ste5ts triple mutant was not able to recover from arrest, suggesting that an SST2-dependent mechanism is involved in recovery of the STE4Hp1 mutant from constitutive arrest. In contrast, the scg1-7 sst2 ste5ts triple mutant recovered only partially suggesting that even though SST2 gene product is probably involved in recovery of the scg1-7 mutant, this mutant can recover by an SST2-independent mechanism. This implies existence of another, SST2-independent postreceptor recovery mechanism. The scg1-null mutant do not recover from constitutive arrest (J. Hirschman, personal communication). Both recovery mechanisms probably operate at the G protein step. Isolation of a constitutive allele of STE5 allowed the definition of its site of action as being after the STE4-controlled step. In addition, constitutive activation of the pheromone pathway by STE5Hp1 mutation was found to be partially dependent on the STE4 and STE18 gene products, the β and γ subunits of a G protein. A comprehensive genetic model is presented to explain the mechanisms of signal transduction and recovery.

CD4+ T cells play a central role in immunity. During virus infections, CD4+ T cells provide the necessary help for B cells to secrete anti-viral antibody and may act as effector cells themselves through the secretion of anti-viral cytokines such as IFN-γ and TNF-α. Recent studies in the lymphocytic choriomeningitis virus (LCMV) system have shown that CD4+ T cells are required to maintain the clearance of persistent viral infections as well as maintain virus-specific memory CD8+ cytotoxic T lymphocytes (CTL). Despite these important functions, surprisingly little information exists concerning the longevity, magnitude, and stability of the CD4+ T cell response following a virus infection. This thesis takes advantage of the well-studied LCMV system to address the above issues as well as to examine the role CD4+ T cells play during heterologous virus infections and to determine the fate of CD4+T cells following a high-dose LCMV infection. The cell surface phenotype of the CD4+ T cells was first examined in C57BL/6 mice acutely infected with LCMV. FACS analysis revealed the modulation of several activation markers on CD4+ T cells during an acute infection with LCMV, consistent with an activated cell phenotype. In addition, 25% of the CD4+ T cells were blast-sized by day 7 post-infection (p.i.) even though the total number of CD4+ T cells did not increase in the spleen during the acute infection. Additional studies were performed using CZ-1, a novel monoclonal antibody (mAb) previously generated in our laboratory that defines a sialic acid-dependent CD45RB-associated epitope. Examination of the expression of the CZ-1 antigen on CD4+ T cells following LCMV infection revealed that the blast-sized CD4+ T cells at day 6 p.i. were CZ-1 +. Further cell surface phenotyping showed that those blast cells activated at day 6 p.i. were CD45RB1oCD44hiCD62L-. This contrasts with the CZ-1-CD45RBhiCD441oCD62L+ resting cell population prior to infection. To determine if memory CD4+ T cells continued to express the CZ-1 epitope long after resolution of the LCMV infection, CD4+CZ-1+ and CD4+CZ-1- populations were purified by cell sorting and placed into an in vitro proliferation assay with LCMV-infected antigen-presenting cells (APC). It was found that the CD4+CZ-1+ population contained virtually all of the virus-specific memory. Thus, these studies indicate that the CZ-1 epitope defines a novel activation and memory marker for murine CD4+T cells. Examination of virus-specific cytokine production using ELISPOT assays showed a significant increase in the number of IFN-γ-secreting cells in the spleen during an acute LCMV-infection. CD8+ T cells made up the majority of the IFN-γ-producing cells, but analysis of the cell culture supernatants by ELISA revealed that the CD4+T cells produced more IFN-γ on a per cell basis. No significant increase in IL-4 levels was detected under these experimental conditions. These data suggest that LCMV infection induces primarily a virus-specific Th1 response that is characterized by increased IFN-γ production. No quantitative information was known about the frequency and longevity of the LCMV-specific CD4+ T cell response. Using limiting dilution assays (LDA), I examined the CD4+ T cell precursor (Thp) frequency in C57BL/6 mice infected with LCMV. The virus-specific CD4+ Thp frequency increased from <1/100,000 in uninfected mice to a peak of approximately 1/600 in FACS-purified splenic CD4+ T cell populations by 10 days p.i. with LCMV. After the peak of the response, the CD4+ Thp frequency decreased only about 2-fold per CD4+ T cell to approximately 1/1200 and remained stable into long-term memory. The CD4+ Thp frequency to each of the two known LCMV major histocompatibility complex (MHC) class II-restricted peptides dropped only 2- to 7-fold from the peak of the acute LCMV response into long-term memory. Thus, the CD4+T cell frequencies remain elevated after the acute infection subsides and remain extremely stable throughout long-term immunity. The above results show that LDA can account for +T cells as being virus-specific following LCMV infection. However, using newer, more sensitive assays based on intracellular cytokine production, >20% of the CD4+ T cells secreted IFN-γ after stimulation with phorbol myristic acid and ionomycin during the peak of the acute CD4+ T cell response. In addition, >10% of the CD4+ T cells secreted IFN-γ after stimulation with the LCMV MHC class II-restricted CD4 peptides. Thus, these new sensitive assays reveal a heretofore unappreciated, yet profound antigen-specific CD4+T cell response during LCMV infection. Infection of mice with a series of unrelated viruses, termed heterologous viruses, causes the reduction of memory CD8+ T cells specific to earlier infections. In order to examine the fate of CD4+ T cells under these conditions, I examined cytokine production and followed the CD4+ Thp frequency following heterologous virus infections. Challenge of LCMV-immune mice with vaccinia virus (VV) resulted in a significant increase in both the amount of IFN-γ protein and the frequency of IFN-γ-producing cells in the peritoneal cavity 3 days after infection as compared to control non-immune mice acutely infected with VV or to LCMV-immune mice alone. Intracellular IFN-γ staining revealed that both CD4+ and CD8+ T cells contributed to this increased IFN-γ production. LDA analysis of the LCMV-specific CD4+ Thp frequency following multiple heterologous virus infections or protein antigen immunizations, revealed that the CD4+ Thp frequency remains stable even under conditions that reduce the LCMV-specific CD8+ CTLp frequency. Additional studies using high-dose LCMV Clone 13 demonstrated that, like CD8+ T cells, there is a decline in detectable LCMV-specific CD4+Thp during overwhelming virus infections. The data presented in this thesis help provide a better understanding of the CD4+ T cell response during virus infections. I make several novel observations, including the demonstration that mAb CZ-1 defines a novel activation and memory marker for CD4+ T cells, that the LCMV-specific memory CD4+ Thp frequency remains extremely stable into long-term immunity, and that heterologous virus infections do not disturb the stable memory CD4+ T cell pool following a virus infection. I also provide data using new sensitive assays based on intracellular cytokine production that there is a much more profound antigen-specific CD4+ T cell response during viral infections than has previously been realized. Finally, I provide evidence that the virus-specific CD4+ T cells become unresponsive following a high-dose LCMV Clone 13 infection. Thus, the data presented in this thesis highlight some important similarities and differences between the CD4+ and CD8+ T cell responses during acute viral infections.

Glia are non-excitable cells found in nervous tissue, and have an important role in synaptic plasticity and the maintenance of neuronal environment, as well as the activity, development, degeneration, and repair of neurons. Glial cells are interconnected via gap junctions to form a multicellular syncytium and utilize intercellular and intracellular Ca2+signals to regulate their functions. Glial Ca2+ signals regulate important cell functions that include gene expression, cell proliferation, metabolism, ion transport systems, release of cell products, and cell death. Consequently, significant alterations of glial Ca2+ signals are associated with pathological processes such as epilepsy, Alzheimer's disease and stroke. Two major forms of Ca2+ signals, intercellular Ca2+ waves and intracellular Ca2+ oscillations occur within glia. Intercellular Ca2+ waves consist of the propagation of elevations in intracellular calcium concentration ([Ca2+]i) between neighboring cells, while intracellular Ca2+ oscillations consist of repetitive elevations in [Ca2+]i that remain confined to single cells. Ca2+ signals are initiated by either a localized chemical, mechanical and electrical stimuli. However, the exact mechanism of their initiation, propagation and modulation is not fully understood. Previous studies have led to the hypothesis that mechanically-induced intercellular Ca2+ waves in glia are mediated by the diffusion of second messenger inositol (1,4,5)-trisphosphate (IP3) through the gap junctions (GJ). However, intracellular Ca2+ may also diffuse between cells during the spread of intercellular Ca2+ wave. Alternatively, Ca2+ waves may be mediated by the release of extracellular messengers, e.g. ATP, that act via phospholipase C (PLC) -linked receptors, e.g. P2y receptors. It is also unknown if the propagation of Ca2+waves requires the regeneration of the signaling message by each cell. An interesting consequence of the propagation of an intercellular Ca2+ wave in glia is that they induce intracellular Ca2+ oscillations in cells that participate in its propagation. These intracellular Ca2+ oscillations may serve to resolve information contained in the position and strength of a local stimulus that induces intercellular Ca2+ wave propagation. Although the mechanism by which Ca2+ waves initiate Ca2+ oscillations is unknown it would seem likely that the mechanism of wave propagation is linked to the mechanism of initiation of Ca2+ oscillations. Guided by previous findings, I hypothesized that intercellular Ca2+ waves propagate by the diffusion of IP3 via gap junctions between neighboring cells to establish an intercellular gradient of IP3 concentration ([IP3]i that within individual cells initiates distinct intracellular Ca2+ oscillations. Two specific aims were investigated to test this hypothesis. The First Specific Aim was to determine if intercellular Ca2+ waves in glia are initiated by the generation of IP3 within a stimulated cell, and propagated by diffusion of IP3 molecules between neighboring cells via gap junctions. The Second Specific Aim, was to determine if intercellular Ca2+ waves induce distinct intracellular Ca2+ oscillations by establishing a specific gradient of oscillation-promoting [IP3]i within the glial syncytium. The initiation and propagation of intercellular Ca2+ waves and intracellular Ca2+ oscillations were examined in primary cultures of rat neonatal cortical glia, utilizing the techniques of a) the intracellular measurement of [Ca2+]i by fluorescence videomicroscopy, b) the photorelease of second messengers IP3 and Ca2+from their photolabile carriers, c) the loading of specific drugs by electroporation into defined zones of glial cultures, and d) identification of cell types by immunocytochemistry. The results of the Specific Aim 1 demonstrated the following: Mechanically-induced intercellular Ca2+ waves reequired PLC activation, the subsequent production of IP3 within the stimulated cell, and release of Ca2+ from intracellular calcium stores. Propagation of Ca2+waves depended on the presence of gap junctions. The release of Ca2+ via IP3 receptor/channels (IP3Rs) was necessary for Ca2+ wave propagation. In contrast, release of Ca2+ from ryanodine receptor/channels (RyRs) occurred in the mechanically-stimulated cell as well as in cells propagating a Ca2+ wave, but was not required for Ca2+ wave initiation and propagation. The propagation of Ca2+ waves through cells that contained heparin to block IP3Rs, or additional [Ca2+]i buffers, demonstrated that the regeneration of IP3 in the non-stimulated cells was not necessary for the propagation of the Ca2+ wave. Ca2+ waves were not mediated by extracellular signals, since Ca2+ waves were not affected by the extracellular perfusion or the inhibition of G proteins. Ca2+ was found to be a poor propagating signal of Ca2+ waves, since intercellular Ca2+ diffusion was not detected during Ca2+ wave propagation. These results are consistent with the hypothesis that Ca2+ waves propagate by diffusion of IP3molecules between neighboring cells via GIs. The [Ca2+]i increase in the stimulated cell occurred due to a Ca2+ influx from extracellular environment, and a release of Ca2+ from intracellular Ca2+ stores, and appeared to contribute to the activation of PLC and the generation of IP3. Ca2+ influx however, was not a necessary event in Ca2+ wave initiation or propagation, because Ca2+ waves occurred in the absence of extracellular Ca2+. By contrast, a [Ca2+]i increase in the absence of [IP3]i increase did not generate intercellular Ca2+waves. The results of the Specific Aim 2 demonstrated the following: An intercellular Ca2+ wave induced intracellular Ca2+ oscillations in a zone of cells at a specific distance from the stimulated cell. The initiation, frequency and duration of Ca2+ oscillations depended on the cells' distance from the Ca2+ wave origin, and not on the cell type or the magnitude of the Ca2+ wave. Modulation of the [IP3]i achieved by acetylcholine (ACh), a neurotransmitter that initiates IP3 production, or by intracellular photorelease of IP3 altered the oscillatory activity of individual cells and shifted the zone of oscillating cells away from the stimulated cell. Ca2+ oscillations spread through individual cells as an intracellular Ca2+ wave that was initiated from a specific site within the cell, independent of the orientation of the initial intercellular Ca2+ wave. These results are consistent with the hypothesis that an intercellular Ca2+ wave initiates Ca2+ oscillations by establishing a specific gradient of oscillation-promoting [IP3]i within the glial syncytium. The findings of this study support the hypothesis that intercellular diffusion of IP3 is the dominant mechanism of Ca2+ wave propagation and initiation of Ca2+ wave-induced Ca2+ oscillations. The significance of these results is that the glial syncytium may utilize specific intracellular Ca2+ oscillations to decode the position and strength of stimuli that induce intercellular Ca2+ waves, and thus integrate and coordinate multicellular functions of glia in the CNS.