Machine learning has been utilized in bio-imaging in recent years, however as it is relatively new and evolving, some researchers who wish to utilize machine learning tools have limited access because of a lack of programming knowledge. In electron microscopy (EM), immunogold labeling is commonly used to identify the target proteins, however the manual annotation of the gold particles in the images is a time-consuming and laborious process. Conventional image processing tools could provide semi-automated annotation, but those require that users make manual adjustments for every step of the analysis. To create a new high-throughput image analysis tool for immuno-EM, I developed a deep learning pipeline that was designed to deliver a completely automated annotation of immunogold particles in EM images. The program was made accessible for users without prior programming experience and was also expanded to be used on different types of immuno-EM images. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2020. / FAU Electronic Theses and Dissertations Collection

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Chloroplasts of many species of hornworts (Anthocerotae) have a structure that resembles the pyrenoid of green algae but whether these two structures are homologous has not been determined. We utilized immunogold labelling on thin sections to determine the distribution of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO), the major protein of algal pyrenoids, in sixteen hornwort species with and without pyrenoids. Several species (Phaeoceros laevis, Anthoceros punctatus, A. formosae, A. laminiferus, Folioceros fuciformis, Folioceros sp., Dendroceros tubercularis, D. japonicus, D. validus, Notothylas orbicularis, N. temperata, and Spaerosporoceros adscendens) have uniplastidic (or primarily uniplastidic) cells with large prominent multiple pyrenoids. In all of these species, the labelling is found exclusively in the pyrenoid and, with the exception of the Folioceros, Dendroceros, and Notothylas species, the labelling is randomly distributed throughout the pyrenoid. In the exceptional species, the pyrenoids have prominent pyrenoglobuli or other inclusions that are unlabelled. In Megaceros flagellaris and M. longispirus, the cells are multiplastidic (with the exception of the apical cell and some epidermal cells) and the chloroplasts lack pyrenoids. Anthoceros fusiformis and Phaeoceros coriaceus have primarily uniplastidic cells but the chloroplasts lack pyrenoids; only an area of stroma in the center of the plastid devoid of starch, reminiscent of a pyrenoid, is found. In all of the species lacking pyrenoids, RuBisCo is found throughout the stroma, including the stromal spaces made by the so-called channel thylakoids. No preferential accumulation of RuBisCo is found in the pyrenoid-like region in A. fusiformis and P. coriaceus. These data indicate that 1) the hornwort pyrenoid is homologous to algal pyrenoids in the presence of RuBisCo; 2) that at least some of the RuBisCo in the pyrenoid must represent an active form of the enzyme; and 3) that, in the absence of pyrenoids, the RuBisCo is distributed throughout the stroma, as in higher plants.

Immunolocalization of proteins within the cell is a significant and powerful tool that improves understanding of cellular functions and processes, such as molecule secretion during immune responses. Immunogold electron microscopy (IEM) is an immunohistochemistry technique that uses gold-conjugated antibodies and electron microscopy (EM) to identify and localize antigens at the ultrastructural level. Here, we are trying to develop and optimize an IEM staining protocol that targets glomerular proteins of interest in Minimal Change Disease (MCD), and eliminates background staining, and preserves tissue morphology. Using this optimized protocol, we hope to learn more about the relationship between IgG and Nephrin in MCD. Kidney biopsies diagnosed with MCD, Membranous Nephropathy (MN), and Thin Basement Membrane Disease (TBMD) and previously embedded in paraffin blocks were retrieved from the tissue archive of the Renal Pathology Laboratory at Boston Medical Center. MN and TBMD were selected as positive controls for IgG and Nephrin staining protocols, respectively. Co-staining of IgG and Nephrin was performed after the protocols for each target were optimized. During protocol development, it was observed that section quality is significantly affected by the angle and sharpness of the knife, and the thickness of the section. Moreover, section quality highly impacted gold particle localization. Ultimately, co-staining of IgG and Nephrin was successful in MCD cases. However, further improvements are needed to optimize IgG and Nephrin staining, and in turn, our understanding of MCD.

PKCe plays a very important role in mediating the protection against myocardial ischemia and reperfusion injury induced by ischemic preconditioning (IPC) and pharmacologic preconditioning (PPC). The redistribution of PKCe was assessed by subcellular fractionation and western blotting in the Langendorff-perfused rabbit heart. Either 5min ischemia or 5min administration of adenosine A1 and/or A3 agonists, bradykinin, angiotensin II, and d1-opioid agonists resulted in PKCe redistribution from the cytosol to the mitochondria. This effect of IPC on PKCe redistribution was visible up to at least 30min of reperfusion, while that of PPC was lost by 10min of drug washout, indicative of the transient nature of PKCe redistribution. PKCe redistribution to mitochondria by IPC was also visualized using immunogold electron microscopy. Thus, IPC and PPC caused PKCe redistribution from the cytosol to the mitochondria, which was longer-lasting in IPC than in PPC.

PKCe plays a very important role in mediating the protection against myocardial ischemia and reperfusion injury induced by ischemic preconditioning (IPC) and pharmacologic preconditioning (PPC). The redistribution of PKCe was assessed by subcellular fractionation and western blotting in the Langendorff-perfused rabbit heart. Either 5min ischemia or 5min administration of adenosine A1 and/or A3 agonists, bradykinin, angiotensin II, and d1-opioid agonists resulted in PKCe redistribution from the cytosol to the mitochondria. This effect of IPC on PKCe redistribution was visible up to at least 30min of reperfusion, while that of PPC was lost by 10min of drug washout, indicative of the transient nature of PKCe redistribution. PKCe redistribution to mitochondria by IPC was also visualized using immunogold electron microscopy. Thus, IPC and PPC caused PKCe redistribution from the cytosol to the mitochondria, which was longer-lasting in IPC than in PPC.

This study forms part of an investigation into the possible relevance of the lysosomal proteinases, cathepsins B, H, Land D, in cancer cell invasion. In this study, the main technique adopted was the Tokuyasu "cryo" method, in which the tissues were fixed, frozen and sectioned and labelled using the relevant antibodies, which were detected with protein A gold probes. In order to implement the Tokuyasu technique, it was necessary to rebuild a knife maker, for the production of adequately sharp glass knives, and to modify a sputter-coater into a glow-discharger, for rendering carbon-coated grids hydrophilic, to promote adhesion of hydrated sections. This study was directed towards human tissues and peptide antibodies were investigated as a means of avoiding isolation of proteins from scarce human tissue, and as a means of obtaining antibodies that will target specific regions of proteins of interest. Peptide antibodies were also considered promising for studies of proteinase trafficking and as immunoinhibiting agents, potentially useful in cancer therapy. Various prediction programmes were investigated for their effectiveness in predicting whether a given peptide sequence will elicit antibodies that will react with the native protein. Successful prediction would increase the success rate of peptide antibody production and thus lower the cost. Leucocytes were studied as a model of an invasive cell, since they are more readily available than tumour cells and serve the purpose during the development of methods. In the course of these studies, an optimal protocol for the fixation of PMNs was developed, involving lateral fixation of cut sections, that should be useful for future studies on these cells. Elastase and cathepsins D and G were found on the surface of activated PMNs and could thus play a role in the invasive properties of these cells. Studies on MCF-10A "normal" breast epithelial cells and their ras-transformed Neo-T counterparts revealed that upon transformation, lysosomes shift from a perinuclear position, to a more peripheral position. None of the cathepsins studied was found on the cell surface of either the normal or ras-transfected cells, suggesting that surface distribution of these enzymes may not be a requirement for invasiveness. These studies suggest that immunocytochemical investigation of cells, in the process of invading through a barrier membrane, might be profitable in elucidating the role of proteinases in invasive cancer. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1993.

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Chlamydomonas reinhardtii possesses a single-cell-based CO2-concentrating mechanism (CCM). The CCM is an important element of algal photosynthesis, metabolism, growth and biomass production, which works by increasing the concentration of inorganic carbon (Ci) in the pyrenoid, a dense RuBisCO-packed structure within the chloroplast. This suppresses RuBisCO oxygenase activity and associated photorespiration. The enhanced efficiency of CO2 assimilation in the pyrenoid via CCM had been modelled theoretically as a requirement for successful CCM in higher plant systems. The ultimate aim of my research is to understand the biogenesis of the pyrenoid using a set of CCM mutants with pyrenoidal defects. Immunofluorescence methods and spot growth tests under different CO2 concentrations were performed on mutants with CCM defects generated by an insertional mutagenesis screen. Morphological and physiological characterisation of these mutants revealed differences in the pyrenoid morphology, the ability for RuBisCO to aggregate into the pyrenoid and the formation of thylakoidal tubule network associated with the pyrenoid. The thylakoid tubule network may be linked to the transport of inorganic carbon into the pyrenoid as part of the CCM. Further characterisation of one of the mutants gave rise to the hypothesis that the gene of interest, Cre11.g467712 (SAGA), is a multi-functional anchor protein related to the structural formation of the pyrenoid and may be another essential component of the pyrenoid.

<i>Escherichia coli </i> remains a significant cause of diarrhea worldwide and in recent years a relatively high number of E. coli carrying gene for AIDA-I (adhesin involved in diffuse adherence) has been isolated from cases of neonatal and post-weaning diarr<i>Escherichia coli</i> remains a significant cause of diarrhea worldwide and in recent years a relatively high number of <i>E. coli</i> carrying gene for AIDA-I (adhesin involved in diffuse adherence) has been isolated from cases of neonatal and post-weaning diarrhea in pigs. AIDA-I adhesin and its gene aidA were first identified and characterized in <i>E. coli</i> isolated from a human case of infantile diarrhea. Recent studies have demonstrated a significant degree of homology between the AIDA-I adhesin isolated from porcine neonatal diarrheagenic <i>E. coli</i> isolates and that from a human <i>E. coli</i> isolate; however, the role of AIDA-I adhesin in the pathogenesis of diarrhea and the clinical significance of the AIDA-I <i>E. coli</i> virotype are unknown in humans or in animals. <p>First, in order to evaluate the role of AIDA-I adhesin, colostrum deprived newborn pigs were infected with: i) a wild strain PD20 (AIDA-I+/STb+) <i>E. coli</i>; ii) a mutant strain PD20M (AIDA-I-/STb+), generated by partial deletion of the aidA gene from the wild strain, iii) a complemented strain PD20C (AIDA-I+/STb+), generated by reintroducing the full length aidA gene into PD20M strain, and iv) a nonpathogenic <i>E. coli</i> strain PD71 used as negative control. Pigs infected with wild type (PD20) and complemented (PD20C) strains developed diarrhea between 15-19 h and 27-31 h after oral inoculation, respectively, in contrast to pigs infected with strains PD20M or PD71 that did not developed diarrhea. Intestinal colonization was evaluated by histology, imunohistochemistry (IHC), transmission electron microscopy (TEM), including immunogold electron microscopy (IGEM), and showed heavy bacterial colonization with biofilm formation in the large intestine with AIDA-I+ strains (PD20 and PD20C), but not in pigs infected with AIDA-I- strains (PD20M and PD71). In vitro assays showed marked diffuse adherence to HeLa cells, enhanced bacterial autoaggregation and significant biofilm formation by AIDA-I+ strains, when compared to AIDA-I- strains.<p>Second, 110 F4 negative <i>E. coli</i> isolates from problematic cases of diarrhea in pigs were subjected to multiplex polymerase chain reaction (M-PCR) for detection of the genes encoding the virulence factors F4, F5, F6, F18, F41, AIDA-I, EAE, STa, STb, LT, EAST1 and Stx2e. In this study, the prevalence of aidA gene among the 110 isolates was 8.2%, and the aidA gene was shown to be associated most commonly with EAST1 and STb genes. The genes for the F4, F5, F6 and F41 fimbriae were absent in all the AIDA-I+ <i>E. coli</i> isolates. <p>The clinical significance of the AIDA-I+ <i>E. coli</i> was studied using clinical data available for 35 of the 110 <i>E. coli</i> isolates, originating from 18 cases of diarrhea. Among these 18 diarrhea cases, 3 cases (5 isolates) were found to have AIDA-I+ <i>E. coli</i> and these were significantly associated with diarrhea cases of post-weaning age group. Enterotoxigenic <i>E. coli</i> strains were isolated from the majority (72.5%) of 18 diarrhea cases and a high proportion (23.1%) of these ETEC cases carried AIDA-I+ <i>E. coli</i>. <p>In conclusion, AIDA-I adhesin appears to be a significant virulence factor for intestinal colonization and induction of biofilm formation. Further, experimental studies and clinical data suggest that the AIDA-I/STb virotype may be important in the pathogenesis of pre-weaning and post-weaning diarrhea in pigs. Our results suggest that AIDA-I may play a significant role in the development of diarrhea in pigs. .hea in pigs. AIDA-I adhesin and its gene aidA were first identified and characterized in E. coli isolated from a human case of infantile diarrhea. Recent studies have demonstrated a significant degree of homology between the AIDA-I adhesin isolated from porcine neonatal diarrheagenic E. coli isolates and that from a human E. coli isolate; however, the role of AIDA-I adhesin in the pathogenesis of diarrhea and the clinical significance of the AIDA-I E. coli virotype are unknown in humans or in animals. First, in order to evaluate the role of AIDA-I adhesin, colostrum deprived newborn pigs were infected with: i) a wild strain PD20 (AIDA-I+/STb+) E. coli; ii) a mutant strain PD20M (AIDA-I-/STb+), generated by partial deletion of the aidA gene from the wild strain, iii) a complemented strain PD20C (AIDA-I+/STb+), generated by reintroducing the full length aidA gene into PD20M strain, and iv) a nonpathogenic E. coli strain PD71 used as negative control. Pigs infected with wild type (PD20) and complemented (PD20C) strains developed diarrhea between 15-19 h and 27-31 h after oral inoculation, respectively, in contrast to pigs infected with strains PD20M or PD71 that did not developed diarrhea. Intestinal colonization was evaluated by histology, imunohistochemistry (IHC), transmission electron microscopy (TEM), including immunogold electron microscopy (IGEM), and showed heavy bacterial colonization with biofilm formation in the large intestine with AIDA-I+ strains (PD20 and PD20C), but not in pigs infected with AIDA-I- strains (PD20M and PD71). In vitro assays showed marked diffuse adherence to HeLa cells, enhanced bacterial autoaggregation and significant biofilm formation by AIDA-I+ strains, when compared to AIDA-I- strains. Second, 110 F4 negative E. coli isolates from problematic cases of diarrhea in pigs were subjected to multiplex polymerase chain reaction (M-PCR) for detection of the genes encoding the virulence factors F4, F5, F6, F18, F41, AIDA-I, EAE, STa, STb, LT, EAST1 and Stx2e. In this study, the prevalence of aidA gene among the 110 isolates was 8.2%, and the aidA gene was shown to be associated most commonly with EAST1 and STb genes. The genes for the F4, F5, F6 and F41 fimbriae were absent in all the AIDA-I+ E. coli isolates. The clinical significance of the AIDA-I+ E. coli was studied using clinical data available for 35 of the 110 E. coli isolates, originating from 18 cases of diarrhea. Among these 18 diarrhea cases, 3 cases (5 isolates) were found to have AIDA-I+ E. coli and these were significantly associated with diarrhea cases of post-weaning age group. Enterotoxigenic E. coli strains were isolated from the majority (72.5%) of 18 diarrhea cases and a high proportion (23.1%) of these ETEC cases carried AIDA-I+ E. coli. In conclusion, AIDA-I adhesin appears to be a significant virulence factor for intestinal colonization and induction of biofilm formation. Further, experimental studies and clinical data suggest that the AIDA-I/STb virotype may be important in the pathogenesis of pre-weaning and post-weaning diarrhea in pigs. Our results suggest that AIDA-I may play a significant role in the development of diarrhea in pigs.