Drug delivery plays a crucial role in the chemotherapeutic treatment of cancerous solid tumours. A drug, no matter how potent, is only truly effective when it can be delivered to all targeted cells. In recent years it has been recognised that the poor response of tumours to chemotherapy is in part due to inadequate drug delivery. Numerous strategies have been developed to overcome this issue. Of particular interest to the present work is the application of ultrasound and cavitation, which has been shown to be capable of enhancing drug delivery in solid tumours. These enhancements are attributed to the acoustic cavitation of microbubbles and the effects cavitation induces in the surrounding tissue. To better understand how ultrasound and cavitation can enhance drug delivery, an instrument was developed that is capable of monitoring in real-time and in-situ the effect of ultrasound and cavitation on drugs and drug analogues within flow channel models. The developed instrument was used to investigate the effect of ultrasound and cavitation on drug-eluting beads used for chemoembolisation, the effects of drug loading on microbubble dynamics, the effects produced by different cavitation agents, and the performance of passive acoustic mapping as a means of cavitation monitoring. The findings of the above investigations include: more physiologically relevant characterisations of drug-eluting beads pharmacokinetics, the possibility of significant changes in microbubble dynamics due to drug loading, a lack of general correlation between detected cavitation activity and induced effects, and the potential of passive acoustic mapping for monitoring cavitation and ultrasound induced effects. These and other findings also demonstrate the utility of the developed instrument for studying the many facets and applications of ultrasound and cavitation mediated drug delivery.

Thesis advisor: Eranthie Weerapana / Over the past two decades there have been a variety of programmed cell death (PCD) pathways to emerge. Among these emerging PCD pathways ferroptosis is has been of especial interest as its iron-dependent reliance on the generation of reactive oxygen species (ROS) links this PCD pathway to some of the most pervasive pathologies including cancer and neurodegeneration. In order to broaden our understanding of ferroptosis we applied a number of proteomic based methods in effort to identify novel ferroptotic mediators. Before the application of proteomic methods, we developed complementary pharmacological and genetic ferroptosis models. With these models we identified ferroptosis-induced changes in protein abundance. Using these data, we generated CRISPR-Cas9-mediated knockouts of protein disulfide isomerase A1 (PDIA1) and cytosolic acetyl-CoA acetyltransferase (ACAT2) that were found to exhibit altered susceptibility to the induction of ferroptosis. With data on protein abundance changes we then profiled ferroptosis-induced changes in abundance corrected cysteine reactivity. Many proteins displayed significant changes in cysteine reactivity that will require further investigation in order to determine if they are drivers or a downstream consequence of ferroptosis. Finally, we used a cell surface biotinylation reagent together with proteomics in effort to identify potential cell surface markers of ferroptosis. This lead to the identification of multiple known cell membrane proteins with ferroptosis altered cell surface labeling. Future studies will seek to confirm the observed alterations with complementary methods. Together these studies illustrate the dynamic responses of the proteome to the induction of ferroptosis. / Thesis (PhD) — Boston College, 2024. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

A polycrystalline diamond was tested as a bearing material for a tilting pad thrust bearing to be used in an electric submersible pump, which elevates process fluids from the bottom of well bores. The goal of this study was to compare the PCD to a current best of technology, which is stainless steel with an engineering polymer.This study found that PCD can handle larger loads than current technology but is limited in size due to diamond sintering and manufacturing constraints. The maximum size is Ø75mm.

A polycrystalline diamond was tested as a bearing material for a tilting pad thrust bearing to be used in an electric submersible pump, which elevates process fluids from the bottom of well bores. The goal of this study was to compare the PCD to a current best of technology, which is stainless steel with an engineering polymer.This study found that PCD can handle larger loads than current technology but is limited in size due to diamond sintering and manufacturing constraints. The maximum size is Ø75mm.

Drosophila provide an excellent model organism in which to study cilia as there are only two ciliated cell types; the sensory neurons and sperm cells. The chordotonal neuron is one such ciliated cell and is required for hearing, proprioception and gravitaxis. Mechanical manipulation of the cilium that extends from the neuronal dendrite is required for signal transduction. Chordotonal neuronal differentiation is regulated by a transcription factor cascade. Atonal begins the cascade, which is then continued by RFX and Fd3F for ciliary genes (Cachero et al 2011, Newton et al 2012). Genes expressed in developing chordotonal neurons are downstream of these transcription factors and their characterisation can further elucidate how neuronal differentiation is regulated. Ciliary genes are highly enriched in developing chordotonal cells; uncharacterised genes enriched in these cells can therefore be considered candidate ciliary genes (Cachero et al 2011). A behavioural assay was conducted to identify further genes that could have a role in ciliary formation and function. Candidate genes were identified by combining enrichment data with previous genomic, proteomic and transcriptomic studies of cilia. A climbing assay of RNAi mediated knock down of these genes identified a number of candidates for future work. One gene found to be highly enriched in developing chordotonal neurons is CG11253. CG11253EY10866 P element insertion mutant flies show a mild uncoordinated phenotype in a climbing assay consistent with reduced chordotonal organ function. Male flies are also infertile due to a lack of motile sperm. CG11253 is expressed in motile ciliated cells and is conserved in organisms with motile cilia. CG11253 expression is also regulated by RFX and Fd3F, suggesting that it is involved in cilium motility. This was confirmed by electron microscopy, which showed disruption of axonemal dynein arm localisation in chordotonal cilia and sperm flagella. A CG11253::mVenus fusion protein was found to localise mainly to the cytoplasm and to a lesser extent the cilia of chordotonal neurons. Patients with symptoms consistent with Primary Ciliary Dyskinesia (PCD), a condition caused by cilium immotility, have subsequently been found to have point mutations in ZMYND10, the human homologue of CG11253. The identification of PCD patients with ZMYND10 mutations showed that investigating cilium motility in Drosophila chordotonal neurons could identify novel PCD genes. It was thought that investigating previously uncharacterised targets of Fd3F could identify novel genes involved in cilium motility and thus candidate PCD genes. CG31320 is a gene regulated by RFX and Fd3F and conserved in organisms with motile cilia. RNAi mediated knock down of CG31320 resulted in both a mild uncoordinated phenotype and male infertility due to a lack of motile sperm. Electron microscopy showed a complete loss of axonemal dynein arms in chordotonal neuron cilia. An mVenus fusion protein of CG6971, an inner dynein arm component, was also mislocalised from the cilia in CG3132027 deletion mutant larvae. This shows that CG31320 is required for the appropriate localisation of the axonemal dynein arms and thus cilium motility. This further showed that uncharacterised genes enriched in chordotonal neurons and regulated by Fd3F could be novel ciliary genes required for cilium motility. Our collaborators and Horani et al (2012) showed that the human homologue of CG31320 (HEATR2) is mutated in patients with PCD, further confirming that this method can be used to identify PCD genes. I have identified two factors required for cilium motility. Disruption of the axonemal dynein arms in both cases results in reduced coordination, and lack of fertility due to sperm immotility. Mutations in the human homologues of these genes have been found to result in PCD. This indicates that further PCD genes could be identified from genes enriched in Drosophila chordotonal neurons that are regulated by Fd3F.

Programmed Cell death (PCD) is a highly conserved and genetically controlled event that plays important roles in animal development, homeostasis and disease. Our first objective was to discover and characterize new genes involved in PCD. Since many PCD genes are conserved in Drosophila, and steroid-induced PCD of larval salivary glands (SGs) is transcriptionally regulated with features of both apoptosis and autophagy, we used this exceptionally well-suited in vivo system and performed Serial Analysis of Gene Expression (SAGE) in three pre-death stages. SAGE identified 1244 expressed transcripts, including genes involved in autophagy, apoptosis, immunity, cytoskeleton remodeling, and proteolysis. Of the 1244 transcripts, 463 transcripts belonged to knownlpredicted genes and were 5-fold differentially expressed prior to cell death. Next, we investigated the role of differentially expressed genes from SAGE, in cell death or cell survival, by RNA interference (RNAi ) in l(2)mbn haemocyte Drosophila cells. l(2)mbn cells undergo morphological changes in response to ecdysone treatment, and ultimately undergo PCD. We used cell viability, cell morphology, and apoptosis assays to identify the death-related genes and determined their ecdysone dependency and function in cell death regulation. Our RNAi screen identified six new pro-death related genes, including SH3PXJ and Soxl4, and 21 new pro-survival genes including SoxN. Identification of Soxl4 as pro-death and SoxN as pro-survival suggests that these Sox box proteins may have opposing roles in ecdysone-mediated cell death. Our final objective was to elucidate the function of CG409], a Drosophila homologue of human TNF-alpha induced proteins 8 (TNFAIP8) we identified from SAGE. We created loss-of-function and overexpression mutants of CG4091 to study gene function in vivo and employed immunoprecipitation and mass-spectrometry assays to identify proteins interacting with CG409] in vitro. We identified two proteins that are involved in n-fatty acid oxidation and several cytoskeletal proteins as interaction partners. Immunofluorescence based assays in vivo and in vitro revealed that CG409] is necessary for cytoskeletal remodeling. Further, defects in CG4091 expression affect cellular functions such as autophagy and lipid metabolism/trafficking that require an intact cytoskeleton. Together, our studies provided new insights into the molecular mechanisms involved in Drosophila SG cell death.

Programmed Cell death (PCD) is a highly conserved and genetically controlled event that plays important roles in animal development, homeostasis and disease. Our first objective was to discover and characterize new genes involved in PCD. Since many PCD genes are conserved in Drosophila, and steroid-induced PCD of larval salivary glands (SGs) is transcriptionally regulated with features of both apoptosis and autophagy, we used this exceptionally well-suited in vivo system and performed Serial Analysis of Gene Expression (SAGE) in three pre-death stages. SAGE identified 1244 expressed transcripts, including genes involved in autophagy, apoptosis, immunity, cytoskeleton remodeling, and proteolysis. Of the 1244 transcripts, 463 transcripts belonged to knownlpredicted genes and were 5-fold differentially expressed prior to cell death. Next, we investigated the role of differentially expressed genes from SAGE, in cell death or cell survival, by RNA interference (RNAi ) in l(2)mbn haemocyte Drosophila cells. l(2)mbn cells undergo morphological changes in response to ecdysone treatment, and ultimately undergo PCD. We used cell viability, cell morphology, and apoptosis assays to identify the death-related genes and determined their ecdysone dependency and function in cell death regulation. Our RNAi screen identified six new pro-death related genes, including SH3PXJ and Soxl4, and 21 new pro-survival genes including SoxN. Identification of Soxl4 as pro-death and SoxN as pro-survival suggests that these Sox box proteins may have opposing roles in ecdysone-mediated cell death. Our final objective was to elucidate the function of CG409], a Drosophila homologue of human TNF-alpha induced proteins 8 (TNFAIP8) we identified from SAGE. We created loss-of-function and overexpression mutants of CG4091 to study gene function in vivo and employed immunoprecipitation and mass-spectrometry assays to identify proteins interacting with CG409] in vitro. We identified two proteins that are involved in n-fatty acid oxidation and several cytoskeletal proteins as interaction partners. Immunofluorescence based assays in vivo and in vitro revealed that CG409] is necessary for cytoskeletal remodeling. Further, defects in CG4091 expression affect cellular functions such as autophagy and lipid metabolism/trafficking that require an intact cytoskeleton. Together, our studies provided new insights into the molecular mechanisms involved in Drosophila SG cell death.

Programmed Cell death (PCD) is a highly conserved and genetically controlled event that plays important roles in animal development, homeostasis and disease. Our first objective was to discover and characterize new genes involved in PCD. Since many PCD genes are conserved in Drosophila, and steroid-induced PCD of larval salivary glands (SGs) is transcriptionally regulated with features of both apoptosis and autophagy, we used this exceptionally well-suited in vivo system and performed Serial Analysis of Gene Expression (SAGE) in three pre-death stages. SAGE identified 1244 expressed transcripts, including genes involved in autophagy, apoptosis, immunity, cytoskeleton remodeling, and proteolysis. Of the 1244 transcripts, 463 transcripts belonged to knownlpredicted genes and were 5-fold differentially expressed prior to cell death. Next, we investigated the role of differentially expressed genes from SAGE, in cell death or cell survival, by RNA interference (RNAi ) in l(2)mbn haemocyte Drosophila cells. l(2)mbn cells undergo morphological changes in response to ecdysone treatment, and ultimately undergo PCD. We used cell viability, cell morphology, and apoptosis assays to identify the death-related genes and determined their ecdysone dependency and function in cell death regulation. Our RNAi screen identified six new pro-death related genes, including SH3PXJ and Soxl4, and 21 new pro-survival genes including SoxN. Identification of Soxl4 as pro-death and SoxN as pro-survival suggests that these Sox box proteins may have opposing roles in ecdysone-mediated cell death. Our final objective was to elucidate the function of CG409], a Drosophila homologue of human TNF-alpha induced proteins 8 (TNFAIP8) we identified from SAGE. We created loss-of-function and overexpression mutants of CG4091 to study gene function in vivo and employed immunoprecipitation and mass-spectrometry assays to identify proteins interacting with CG409] in vitro. We identified two proteins that are involved in n-fatty acid oxidation and several cytoskeletal proteins as interaction partners. Immunofluorescence based assays in vivo and in vitro revealed that CG409] is necessary for cytoskeletal remodeling. Further, defects in CG4091 expression affect cellular functions such as autophagy and lipid metabolism/trafficking that require an intact cytoskeleton. Together, our studies provided new insights into the molecular mechanisms involved in Drosophila SG cell death. / Medicine, Faculty of / Medical Genetics, Department of / Graduate

Programmed Cell Death (PCD) is an essential process utilised for the defence and development of all multicellular organisms. In plants however, relatively little is known about the genes involved with the regulation and execution of this process. In particular, even less is known about the molecular components which act high up in the PCD pathway. In this thesis, we carried out an investigation into the novel peptide-encoding gene KISS OF DEATH (KOD). In Arabidopsis, KOD was found to be involved with mediating the elimination of the suspensor, an organ which undergoes developmental PCD. Two mutant alleles of KOD showed reduced PCD in both the suspensor and during heat-shock induced PCD of root hairs. Over-expression of KOD in plant tissues was sufficient to cause death in leaves or whole seedlings and involved the activation of caspase-like proteolytic activity. KOD-induced PCD was found to require light in leaves and is also sensitive to the PCD suppressor genes AtBI-1 and P35. We suggest that KOD acts high up in the PCD cascade as its expression resulted in depolarisation of the mitochondrial membrane, which is an early step in plant PCD. KOD appears to be a plant-specific peptide that is sufficient to induce PCD in Arabidopsis in the absence of external triggers. Typical BLAST searches yielded no obvious homolog for KOD, therefore a bioinformatics screen of the Arabidopsis genome was carried out. This screen for small genes similar in size to KOD enabled us to detect 10 previously unidentified genes, one of which may represent a putative KOD homolog. In summary, KOD appears to be a novel pro-PCD component of the Arabidopsis cell death machinery and represents the first plant peptide to be involved with a form of developmental PCD.

Les travaux présentés dans cette thèse ont porté sur la specificité des réponses cellulaires de l’halophyte obligatoire Cakile maritima au choc salin et la régulation des événements précoces impliqués dans la mort cellulaire programmée et la survie en condition de salinité. Dans une première étape, nous avons montré que cette plante est aussi tolérante aux chocs salins répétés qu’au stress salin progressif. Cependant, on a observé de zones de mort cellulaires sur les feuilles âgées soumises à un choc salin sévère (400 mM NaCl). Pour mieux cerner la cascade d’événements impliqués dans ce processus de mort cellulaire, nous avons poursuivi nos expériences sur des suspensions cellulaires de C. maritima, dont nous avons-nous même optimisé les conditions d’obtention, et des suspensions cellulaires d’Arabidopsis thaliana (glycophyte modèle). Chez les deux espèces, nous avons observé une mort cellulaire programmée qui dépend de la durée et l’intensité du traitement salin appliqué, et qui met en jeu les mêmes événements cellulaires notamment la dépolarisation de la membrane plasmique due à l’entrée de Na+ par les NSCCs, un dysfonctionnement mitochondrial, une production d’anions superoxydes et une activation de protéines de type caspase. La tolérance de C. maritima au stress salin serait potentiellement due à une forte accumulation d’ascorbate qui permettrait à cette halophyte de mieux réduire les dommages générés par le stress oxydatif. C. maritima s’est aussi distinguée par une meilleure capacité de contrôler l’accumulation cytoplasmique de Na+, conduisant à la survie de ses cellules en condition de salinité. Cette étude sur la mort cellulaire induite par le NaCl chez les cellules en culture de C. maritima nous a aussi permis de mettre en évidence deux types de comportement dans cette population de cellules en culture : l’un lié à une dépolarisation soutenue en réponse au NaCl conduisant probablement à la mort de ces cellules, l’autre lié à une dépolarisation transitoire indiquant que l’influx de Na+ au travers des NSCC était régulé permettant probablement aux cellules présentant ce comportement de survivre en évitant l'accumulation excessive de Na+ dans le cytosol. Dans la dernière partie de ce travail, nous avons mis en évidence la capacité de C. maritima d’exclure Na+ via le système SOS. Ce résultat suggère l’existence d’une deuxième voie de signalisation induite parallèlement à celle conduisant à la mort cellulaire. Cette voie, impliquant une production rapide d’oxygène singulet, pourrait permettre un influx de Ca2+ dans le cytoplasme activant la protéine SOS3 et en cascade SOS2 et SOS1 et les H+-ATPases de la membrane plasmique permettant un efflux du Na+ via SOS1 hors des cellules. / AbstractThis work aimed at understanding the specificity of cellular responses of the obligate halophyte Cakile maritima to salt shock and regulation of early events involved in programmed cell death and survival under salinity conditions. In a first step, we have shown that this plant is tolerant upon both repetitive salt shocks and gradual salt application. However, we have observed a cell death zones on older leaves subjected to a severe shock saline (400 mM NaCl). To better understand the cascade of events involved in the cell death process, we continued our experiments on suspension culture of C. maritima, which we have optimized ourselves the conditions for establishment and suspension culture of Arabidopsis thaliana (glycophyte model). In both species, salinity induced programmed cell death that depends on the duration and the intensity of the applied salt treatment. Also, the same cellular events, including depolarization of the plasma membrane due to the Na+ influx by NSCCs, mitochondrial dysfunction, production of superoxide anions and activation of caspase-like proteins, occurs early in response to salt stress. C. maritima tolerance to salt stress is potentially due to a strong accumulation of ascorbate that would allow this halophyte to better reduce damage generated by oxidative stress. C. maritima is also distinguished by a better ability to control the cytoplasmic accumulation of Na+, leading to the survival of its cells under salinity conditions. This study on cell death induced by NaCl in cell culture of C. maritima also allowed us to identify two types of behavior in this population of cells in culture: one related to a sustained depolarization in response to NaCl probably leading to death of these cells, the other linked to a transient depolarization indicating that the Na+ influx through the NSCC was probably regulated allowing cells exhibiting this behavior to survive by avoiding excessive accumulation of Na+ in the cytosol. In the last part of this work, we have demonstrated the ability of C. maritima to exclude Na+ via the SOS system. This result suggests the existence of a second signaling pathway induced in parallel to that leading to cell death. This pathway, involving a rapid production of singlet oxygen, could allow a Ca2+ influx in the cytoplasm that acts as an elicitor for activation of SOS3 protein and SOS2-SOS1 cascade and H+- ATPases of the plasma membrane allowing Na+ efflux via SOS1 out of cells.