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In President's Report

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DigitalDocument

In Honors Theses

Abstract

Programmed cell death by apoptosis is a vital feature of multicellular life. It is characterized by a common set of morphological and biochemical changes that include chromatin condensation, caspase activation, DNA cleavage, membrane blebbing, and cellular fragmentation. Apoptosis can be reversed by a process called anastasis, which has been previously documented as a contributing mechanism in cancer recurrence following chemotherapy. The goal of our current work was to determine whether anastasis represented a more generalized cellular process independent of cancer. We used cultured mouse HT2 T-lymphocytes as a model system, a non-cancerous cell line that is dependent on the cytokine Interleukin 2 (IL2) for survival. Apoptosis was induced in HT2 cells with three different methods: IL2 deprivation (starvation), treatment with staurosporine, or camptothecin. We have previously shown that apoptosis induced by IL2 deprivation for 24 hours was reversible following IL2 re-addition, as cells were observed to gradually resume their normal physiological processes within 72 hours of rescue. Here, HT2 cells were also treated with staurosporine and camptothecin, washed free of each inducer, recovered in IL2, and analyzed by flow cytometry at 12, 24, 48, and 72 hours following the removal of the chemical inducer. Cell viability and executioner caspase 3/7 activity were determined jointly using SYTOX Red vital dye and NucView 488 substrate, respectively, whereas cell cycle distribution was monitored post-fixation using propidium iodide to determine DNA content per cell. Our findings revealed that apoptosis in HT2 cells was reversible following camptothecin treatment and IL2 deprivation and that HT2 cells gradually regained normal cell cycle distribution during the 72-hour rescue window. However, cells induced with staurosporine could not be rescued from apoptosis. Interestingly, sustained caspase activity was observed throughout the rescue period for cells treated with camptothecin for 9 hours. Our findings suggest that anastasis may be a normal physiological process in animal cells independent of malignancy but dependent on the inducer of apoptosis.

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Thesis

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In Honors Theses

Abstract

Programmed cell death (PCD) is critical to the development and maintenance of all multicellular organisms. We study the colonial marine urochordate Botryllus schlosseri, which undergoes synchronized PCD throughout the entire generation of adult zooids during the takeover or death phase of its budding cycle (cyclic blastogenesis). The adult generation is then resorbed by circulating phagocytes and recycled, as a new asexual generation reaches functional maturity. Due to the cyclical and predictable nature of PCD in this clonally-modular animal, Botryllus is a great model organism to study the regulation of PCD via cysteinyl aspartases called “caspases”. It is well documented that caspases -8 and -9 play a role in the initiation phase of PCD, while caspases -3 and -7 are used in its execution. In this study, we investigated whether caspase expression was developmentally-regulated in Botryllus. An expressed sequence tag (EST) project was carried out over the past two years, and oligonucleotide primers were designed to amplify caspase orthologs from Botryllus. In the current study, caspase expression patterns were investigated using developmentally-staged, clonal replicates from two genotypically- identical colonies. Our findings indicate that expression of executioner caspases is developmentally-regulated during cyclic blastogenesis, with loss of steady-state expression levels during the resorption phase of takeover. However, substantial variation was found in caspase expression patterns between genotypically distinct colonies. Antibody localization and siRNA studies will be required to determine the functional involvement of specific caspases during takeover and resolve the apparent discrepancies uncovered in our study.

Type of Resource

Thesis

Rights Statement

In Honors Theses

Abstract

Programmed cell death (PCD) is an important part in the development and maintenance of homeostasis in multicellular animals. We utilized the colonial marine urochordate Botryllus Schlosseri as a model system to investigate this process because it is an organism whose adult generation (zooids) cyclically undergoes massive PCD in all of its organs every 5 days at 21oC. Specialized blood phagocytes have previously been shown to play a major role in the clearance of apoptotic cell corpses during this death phase. Here, using fluorescence stereomicroscopy and laser-scanning confocal microscopy, we investigated phagocyte localization and function during colony homeostasis in Botryllus colonies via intravascular microinjection of fluorescent bioparticles and liposomes containing the phagocidal drug clodronate. Our findings indicate that both bioparticles and liposomes were rapidly targeted and engulfed within phagocytic cells localized within ventral islands of adult zooids. Knockdown of phagocyte function using clodronate liposomes further revealed that phagocytes were important throughout the blastogenic cycle of this animal, but most notably during the PCD phase. Clodronate colonies showed abnormalities consistent with phagocyte disruption. Using co-microinjection of liposomes and bioParticles, phagocyte populations were also observed to engulf foreign particles with different levels of specificity. The stationary endothelial cells only engulfed bioParticles, while migratory blood phagocytes ingested both types of particles. Collectively, our observations indicate that blood phagocytes are critical effectors in colony regeneration and homeostasis. Finally, in order to characterize molecular markers expressed in Botryllus phagocytic cell populations, a 180bp segment of a putative scavenger receptor homolog was also successfully isolated and amplified.

Type of Resource

Thesis

Rights Statement

In Honors Theses

Abstract

Apoptosis is a highly organized intracellular death program in multicellular animals. In the vertebrate immune system, apoptosis plays a central role in preventing the emergence of autoreactive lymphocytes. In this study, we used multiple stimuli (staurosporine, camptothecin, and cytokine deprivation - interleukin-2 or IL-2) to initiate apoptosis in IL-2 dependent, mouse HT-2 T-lymphocytes. All three inducers triggered DNA laddering and phosphatidylserine externalization. Propidium iodide staining and flow cytometry were also used to determine whether apoptotic cells accumulated in a specific stage of the cell cycle, and whether the mode of induction affected cell cycle distribution. Our findings indicate that IL-2 deprivation induces HT-2 cells to accumulate in G1, while cells treated with staurosporine and camptothecin accumulated in G2/M and S phase, respectively. We also used immunopblotting detection to investigate P27Kip1 protein expression for each condition. P27Kip1 is a member of the Cip/Kip family of cyclin-dependent kinase inhibitors whose function is to enforce the G1 restriction point. We found that P27Kip1 levels were significantly increased within 12 hrs and 6 hrs following both IL-2 deprivation and campthotecin exposure, respectively. Across all time points in the staurosporine experiments, P27Kip1 steady state levels were decreased. These results suggest that the mode by which apoptosis is induced differentially affects cell cycle distribution.

Type of Resource

Thesis

Rights Statement

In Honors Theses

Abstract

The increasing prevalence of antibiotic-resistant bacteria is an emerging problem for humans. Clinical misuse, overuse in agricultural and food settings, and limited numbers of new antibiotics have accelerated the proliferation of antibiotic-resistant bacteria. To confront this threat, scientists must develop new therapeutics that kill these antibiotic-resistant bacteria. In this study, we used <em>Escherichia coli</em> to analyze antibiotic and bacteriophage susceptibility. <em>E. coli</em> is a common, mostly benign, enteric, gram-negative bacteria. We isolated three <em>E. coli</em> strains from the Hans Groot Kill, a stream that runs through Union College's campus. We sought to assess various <em>E. coli</em> strains' antibiotic resistance, susceptibility to bacteriophages, and underlying resistance mechanisms. We compared the environmental <em>E. coli</em> isolates to known lab strains of <em>E. coli</em>. Using T4 bacteriophage, a well characterized viral pathogen that carries out a lytic cycle in <em>E. coli</em> bacteria, we conducted modified plaque assays with the three environmental isolates and lab strains "B" and "C." The environmental isolates were found to be highly resistant to T4 phage, while the lab strains were susceptible. We also conducted Kirby-Bauer antimicrobial disk susceptibility tests, using various gram-negative targeted drugs. We found the lab strains to be highly susceptible to all of the antibiotics, while the environmental isolates showed intermediate resistance to some of the antibiotics. Surprisingly, both environmental and lab strains were encapsulated. These patterns of resistance to phages and antimicrobial drugs suggest that environmental <em>E. coli</em> strains must have some mechanism for resistance that could be exploited as a novel means of treating drug-resistant infections.

Type of Resource

Thesis

Rights Statement

In Honors Theses

Abstract

Apoptosis is the principal form of programmed cell death by which multicellular animals rid themselves of old cells in order to allow new cells to grow. The colonial ascidian Botryllus schlosseri is a model system for homeostatic cell death and regeneration. During its unique, weekly budding cycle known as blastogenesis, new buds are produced asexually and eventually replace the old adult zooid generation through resorption via circulating phagocytes. Using polyester wax-embedded tissue sections, we have shown that TUNEL-positive (terminal deoxynucleotidyl transferase nick end-labeling), apoptotic cells are observed within circulating phagocytes in all stages of the colony's death phase (takeover), including the final stage during which the primary bud undergoes a growth surge and becomes a functional adult zooid. Suggested explanations of these intriguing results yield three potential theories: (1) the growing primary bud is a site of cell corpse elimination for the dying generation, (2) the phagocytosed materials of the dying generation are recycled and reincorporated into the new adult generation, or (3) the apoptotic cells of the dying adult zooid generation release mitogenic factors and stimulate proliferation in neighboring cell populations. Our findings are consistent with the idea that dying cells are trophic in nature, promoting growth and differentiation during whole body regeneration.

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Thesis

Rights Statement