Apparatus has been developed for the irradiation of V79-379A Chinese hamster lung fibroblast cells with 3.6 MeV protons from the Van de Graaff accelerator at the National Accelerator Centre in Faure. The original intention of this work was to investigate and measure a possible dose rate effect on the survival of V79 cells, in the dose range from zero to 25 Gy, at dose rates of about 3 Gy/s and 300 Gy/s. The survival curves initially obtained were anomalous in that they showed abnormally high levels of survival and a tendency to remain at a constant survival level for doses above 10 Gy. Systematic attempts to correct this observed anomaly, involved the following; apparatus improvements were made, a means of measuring the beam profile was devised, the current measuring device and the dosimetry were improved and a possible dose rate effect on intracellular oxygen was investigated. After these improvements, the anomalous effect was much reduced, but not entirely eliminated. The final results showed no significant difference between the survival of cells irradiated at dose rates of about 3 Gy/s and 300 Gy/s; qualitative differences were however noticeable. After correction for the effect of a non-uniform beam profile, the survival curves were significantly different to published work. This difference suggested a possible dose rate effect between dose rates of about 0.1 Gy/s and dose rates above 3 Gy/s.

Many genes are activated to influence the self-destruction programme of the cell. This programme entails synchronised instigation and implementation of numerous subprograms. The arrival of gene targeting aided in the determining of the functions of novel genes. Such genes may have been sequenced, but not functionally characterised. The fulfillment of this requirement through gene targeting technology has swiftly developed. The mode by which DWNN operate in organisms in which it is thought to be covalently linked to some other proteins, which have a definite role in apoptosis, is not yet unraveled. This study attempted the functional characterisation of DWNN in light to the hypothesis that it may be involved in Cytotoxic T lymphocyte killing and apoptosis.

Many genes are activated to influence the self-destruction programme of the cell. This programme entails synchronised instigation and implementation of numerous subprograms. The arrival of gene targeting aided in the determining of the functions of novel genes. Such genes may have been sequenced, but not functionally characterised. The fulfillment of this requirement through gene targeting technology has swiftly developed. The mode by which DWNN operate in organisms in which it is thought to be covalently linked to some other proteins, which have a definite role in apoptosis, is not yet unraveled. This study attempted the functional characterisation of DWNN in light to the hypothesis that it may be involved in Cytotoxic T lymphocyte killing and apoptosis.

Thesis (M.S.)--University of Wisconsin--Madison, 1968. / eContent provider-neutral record in process. Description based on print version record.

Urinary tract infections are generally preceded by transfer of organisms from the distal urethra to the bladder (20, 148). However, although urinary infections are predominantly due to pure cultures of Escherichia coli, the distal urethra contains a mixed flora in which E. coli is relatively uncommon and anaerobes predominate (73, 103). This discrepancy between the bladder and distal urethral flora may be due to differential adhesion or differential growth rates. In this dissertation I have tested the hypothesis that differential growth rates of urethral organisms in urine explains the predominance of E. coli as a pathogen. These experiments showed that the balance between bacterial growth and washout may have a pivotal role in the pathogenesis of infection and perhaps therefore in treatment. A model of the human bladder used for the pathogenesis studies was then used to study the activity of mecillinam and ampicillin under conditions simulating human urinary infection. The model proved realistic especially for synergy studies where shortcomings in conventional in vitro methods are a cause for concern. The following topics were studied. 1. Urine was chosen as a test medium for definitive experiments because growth rates of organisms other than E. coli were different in broth and in urine. A method for sterilizing urine in bulk was developed which did not affect growth supporting properties. 2. E. coli was shown to grow faster and to have a shorter lag period than almost all other organisms when studied in shake culture. 3. A continuous culture model of the human urinary bladder was employed for differential growth studies of organisms in sterilized human urine. This model reproduced many of the characteristics of the human lower urinary tract and enabled study of the balance between bacterial growth and the tendency of urine to wash organisms out of the tract. 4. Mixed cultures of approximately equal numbers of E. coli and a second potential urinary' pathogen were introduced into the bladder model and quantitative cultures performed at intervals up to 24 h. In 15 experiments E. coli eventually dominated the second pathogen which was sometimes undetectable at 24 h. Similar changes in bacterial populations seen in infected patients indicate that differential growth rates may be an important determinant of the pathogenicity of E. coli. 5. The use of the bladder model was then extended to investigations of antibiotic activity under realistic conditions. The value of the model for synergy studies with ampicillin and mecillinam was assessed by parallel conventional in vitro tests and an animal infection protection test*. The bladder model gave similar results to mammalian studies and appeared to be far superior to conventional methods. This model may be valuable in the initial assessment of new urinary antibiotics. 6. A representative array of organisms for the above study was selected following a survey of resistance patterns of 2000 clinical isolates of Enterobacteriaceae. An incidental by-product of this survey was the establishment of a breakpoint for mecillinam susceptibility in the Kirby-Bauer antibiotic disk test. 7. Work on the effect of mecillinam and/or ampicillin upon bacterial viability was extended to investigations of the relative contribution of permeability barriers and 3-lactamases to antibiotic susceptibility. Unlike ampicillin, mecillinam resistance of 77 clinical isolates of bacteria appeared to be independent of intracellular 3-lactamase levels, suggesting that the barrier effect may be more pronounced in bacterial resistance to mecillinam than to ampicillin. Kinetic studies using urine as a growth medium, and in particular the use of a bladder model have provided a unifying explanation of many features of both the pathogenesis and treatment of urinary infections. * Carried out by Dr. R.C. Cleeland. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

This document only includes an excerpt of the corresponding thesis or dissertation. To request a digital scan of the full text, please contact the Ruth Lilly Medical Library's Interlibrary Loan Department (rlmlill@iu.edu).

Organelles sequester biological phenomena within the cell, and allow an additional layer of complexity to life. The presence and maintenance of these organelles is crucial for cellular function. Two of the most expansive and complex organelles are the mitochondria and endoplasmic reticulum. These organelles contribute energy, protein folding and secretion, lipids, calcium regulation, and various other metabolites to the biology of the cell. Importantly, these organelles accumulate damage and cannot be derived de novo, therefore must be inherited and maintained in a functioning state. The study of these organelle quality control processes serves as the basis for my thesis. We use the budding yeast as a model organism to uncover conserved pathways affecting organelle, and ultimately cellular homeostasis. In yeast we find mitochondrial inheritance is critical for cell survival. Furthermore, not only is inheritance critical, but inheritance of a certain threshold of functional mitochondria appears critical in maintaining normal lifespan in yeast, identifying mitochondria as an aging determinant. By examining mutants that negatively affect mitochondrial inheritance in yeast, we established a role for phosphatidylcholine biosynthesis in organelle maintenance and inheritance. Glycerophospholipid biosynthesis plays a clear role not only in mitochondrial inheritance but also in that of the endoplasmic reticulum. We use insights gained from yeast to guide research into a human disease caused by similar glycerophospholipid biosynthetic deficiency.

Thesis (Ph.D.)--Michigan State University. Dept. of Pathobiology and Diagnostic Investigation, 2008. / Title from PDF t.p. (viewed on July 10, 2009) Includes bibliographical references (p. 157-174). Also issued in print.

The cytopathology of herpes simplex virus (HSV) in H.Ep.2 and BHK-21 cells was studied using the techniques of light microscopy, immunofluorescence, electron microscopy, autoradiography and cytogenetics. Both cell types supported rapid growth cycles of HSV resulting in the production of maximum titres after 22 - 24 hours of infection. Cultures treated with 10 µg/ml ara-C or 100 µg/ml IDU at the time of infection showed a 99% decrease in infectious virus production. HSV-infected H.Ep.2 and BHK-21 cells revealed typical virus-induced inclusion bodies and a generalized disorganization of the nucleus and cytoplasm. Syncytia formation was not observed but after 24 hours of infection, nearly 100% of the cells were rounded and often detached from the glass surface. Addition of 10 µg/ml ara-C or 100 µg/ml IDU failed to prevent virus cytopathology but did cause a characteristic cytoplasmic disruption and rounding of uninfected cells. Virus-infected cells also revealed at least four separate immuno-fluorescent elements after exposure to hyperimmune serum prepared in guinea pigs. These elements included small nuclear granules, amorphous nuclear masses, diffuse cytoplasmic antigens, and intense surface fluorescence. The nuclear antigens and cytoplasmic fluorescence appeared after treatment with ara-C or IDU but the surface fluorescence was not produced in the presence of the anti-viral agents. Herpes simplex virus developed in the nucleus of infected H.Ep.2 and BHK-21 cells. The virions were enveloped at the inner lamella of the nuclear membrane and after passing into the cytoplasm, were released from the cells by a process of reverse phagocytosis. Ara-C and IDU allowed the synthesis of certain viral antigens and the development of nuclear cytopathology but completely prevented the formation of infectious HSV particles. Both drugs caused a marked distortion of the mitochondria and endoplasmic reticulum in uninfected cells. DNA synthesis in HSV-infected cells, as measured by ³H-thymidine incorporation, was almost completely inhibited by 4 hours of infection. This early inhibition of cellular DNA synthesis was followed by an immediate increase in ³H-thymidine uptake corresponding to the synthesis of viral DNA. Both cell types showed a brief stimulation of mitosis prior to the complete inhibition observed after 20 hours of infection. Cellular and viral DNA synthesis and mitosis appeared to be inhibited in virus-infected and uninfected cells treated with ara-C or IDU. Infection with HSV resulted in severe chromosomal damage to H.Ep.2 and BHK-21 cells. Chromosomal abnormalities included chromatid gaps and breaks, enhanced secondary constrictions, fragmentation, erosion, and endoreduplication, and were dependent on virus dose and time of infection. The capacity of the virus to induce chromosomal aberrations in cultured cells was UV-inactivated approximately five times less rapidly than the infectious property. Ara-C acted synergistically with the virus to produce a large number of cells with multiple chromosome breaks and also caused a significant number of abnormalities in uninfected cells. In contrast, IDU treatment resulted in few aberrations over and above those produced by HSV and little damage in uninfected cells. It was concluded that HSV was capable of producing severe morphological and genetic alterations in cultured human and hamster cells. The antiviral agents ara-C and IDU were able to completely inhibit virus multiplication but were unable to prevent any of the virus-induced cytopathic effects in vitro. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

Some of the adverse effects associated with abstinence from chronic cocaine usage are similar to the psychiatric symptoms of affective illness. Some of these symptoms include dysphoria, agitation, anxiety and alterations in sleep and appetite (Gawin and Kleber, 1986, 1988). Since in the treatment of affective illness these symptoms are responsive to medications which regulate specific receptors in the serotonergic system, I studied the influence chronic cocaine exposure and abstinence has on the expression and behavioral responses of the 5-HT2A receptor. First, the specific binding parameters of [3H]ketanserin were assessed in the hippocampus and frontal cortex of rat brain tissue. These studies revealed relatively high affinity binding in both areas. The dissociation constants averaged 1.40 +/− 0.05 nM for the frontal cortex and 2.88 +/− 0.22 nM for the hippocampus. The density of the binding sites for the frontal cortex and hippocampus averaged 567 +/− 35 and 233 +/− 53 f moles/mg protein respectively. Additional receptor binding experiments conducted at various time points during and following cocaine exposure via subcutaneously implanted cocaine- or vehicle-containing Silastic capsules revealed no treatment-related differences in receptor density. Since previous studies have reported that various stressors (chronic forced swim test, restraint stress, or food restriction) may increase either the level of 5-HT in various brain areas, 5-HT2A receptor density, or behavioral responses to 5-HT2A receptor agonists. I also examined the interaction between the stress of minor surgery (associated with the capsule implantation), food restriction, and chronic cocaine treatment on head-twitch responses (HTRs) to the 5-HT2A agonist, 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI), in rats. Circulating cocaine concentrations were 116 +/− 11 ng/ml (low dose) and 330 +/− 75 ng/ml (high dose) at the end of the 3-day treatment period. Food restriction occurred through pair-feeding of control animals to high-dose cocaine-treated subjects that showed a transient drug-induced anorexia. Untreated rats subjected to surgery and food restriction exhibited a significantly elevated HTR at 3 days post-surgery compared to untreated animals given the same dose of DOI. This enhanced sensitivity to DOI subsequently declined in a time-dependent manner. High- but not low-dose cocaine treatment unexpectedly attenuated the stress-induced increase in DOI-elicited HTRs. Since the 5HT2A binding data revealed no treatment-related differences in receptor density, the result suggest that these alterations in behavioral responsiveness may reflect changes in 5-HT2A signal transduction mechanisms.