In this study we found that tumour cells can be radiosensitised by targeting the DNA damage response kinases, ATM and ATR. Furthermore, we highlight that Wee1 inhibitors, which are already under the clinical trials need to be further investigated in combination with radiation in the context of tumour hypoxia. In addition, we observed that induction of autophagy using STF-62247 can lead to radiosensitisation of VHL deficient RCC cells. Our studies with the rapamycin analogue temsirolimus, already in the clinic for the treatment of various cancers, can be a potential candidate as a radiosensitiser for RCC cells. Overall, these finding led us to investigate further whether autophagy inducing compounds, which are either in clinic or in clinical trials, can effect the response to radiation. From a panel of candidate drugs which are known to induce autophagy we identified an aminopeptidase inhibitor, CHR-2797. CHR-2797 induces autophagy in the oesophageal cancer cell lines FLO-1 and OE21. Although, our results with CHR-2797 demonstrate it as a potential radiosensitiser, the mechanism of its radiosensitisation needs to be established. Our results from CHR-2797-induced radiosensitisation, further led us to investigate if other aminopeptidase inhibitors have a role in radiosensitisation. Therefore, we selectively screened candidate aminopeptidase inhibitors and identified some promising effects on radiosensitivity.

The weak selectivity of chemotherapeutic drugs against tumors has sustained efforts to develop better chemotherapeutic agents that are more potent and selective at destroying tumor cell populations versus normal tissues. This project focuses on evaluating the cell killing effects of the microtubule inhibitor, stilbene 5c, against melanoma cancer. We utilized an in vitro murine melanoma model to study the effects of stilbene 5c on tumor proliferation and survival, as well as growth arrest and cell death. Our findings indicate that stilbene 5c promotes dose dependent cell death in melanomas with the induction of apoptosis and autophagy. The role of autophagy was further assessed using the pharmacological autophagy inhibitor, Bafilomycin A1. It was concluded that autophagy was partially cytoprotective as inhibition of autophagy was shown to induce extensive cell death through an increase in apoptosis. Residual surviving cells were shown to be in a state of growth arrest characterized to be senescence. These findings indicate that stilbene 5c could potentially be developed for the treatment of melanoma.

The standard of care for unresectable lung cancer is chemoradiation. However, therapeutic options are limited and patients are rarely cured. While Radiation therapy is effective at killing tumor cells or inhibiting their growth initially, development of resistance to treatments and recurrence of tumors are major issues. One of the major goals of Dr. Gewirtz’s laboratory has been to develop strategies to overcome the resistance and attenuate disease recurrence. One of these attempts involve employing vitamin D and its analogs in combination with radiation therapy. Our proposed studies were based on a previous finding where vitamin D and vitamin D analogs such as EB 1089, were shown to enhance the response to radiation in breast cancer through the promotion of autophagy. We extended these studies to non-small cell lung cancer (NSCLC) and were able to validate that 1,25-D3 (the hormonally active form of vitamin D) and EB 1089 does in fact sensitize A549 and H460 cells and prolonged the growth arrest induced by radiation alone and suppressed proliferative recovery, which translated to a significant reduction in clonogenic survival. In H838 or H358 NSCLC cells, which lack the vitamin D receptor or functional p53, respectively, 1,25-D3 failed to modify the extent of radiation-induced growth arrest or suppress proliferative recovery post irradiation. Sensitization to radiation in H1299 NSCLC cells was evident only when p53 was induced in otherwise p53 null H1299 NSCLC cells. Sensitization by 1,25-D3/ EB 1089 was not associated with increased DNA damage, decreased DNA repair or an increase in apoptosis, necrosis or senescence. Instead sensitization appeared to be a consequence of the conversion of the cytoprotective autophagy induced by radiation alone to a novel cytostatic form of autophagy by the combination of 1,25-D3 or EB 1089 with radiation. While both pharmacological and genetic suppression of autophagy or inhibition of AMPK phosphorylation sensitized the NSCLC cells to radiation alone, inhibition of the cytostatic autophagy induced by the combination treatment reversed sensitization. Evidence for selectivity was provided by lack of radiosensitization in normal human bronchial cells and cardiomyocytes. Taken together, these studies have identified a unique cytostatic function of autophagy that appears to be mediated by the vitamin D receptor, p53 and possibly AMPK in the promotion of an enhanced response to radiation by 1,25-D3 and EB 1089 in NSCLC.

Autophagy is the process of “cell self-eating” which has been implicated both in cell survival and cell death. Sphingosine kinase 1 (SphK1) regulates the intracellular balance between ceramide and sphingosine, bioactive lipids associated with cell death, and sphingosine-1-phosphate (S1P), whose actions are associated with survival and proliferation. Previous studies have implicated upregulation of SphK1 in the induction of autophagy. In this study, SK1-I, a SphK1 specific competitive inhibitor, induced autophagy in a concentration and time dependent manner in HCT116 colorectal carcinoma cells. This autophagic response was observed to be more intense in wild type p53 expressing HCT116 cells than in p53 null cells and ultimately led to non-apoptotic death in wild type and apoptotic death in p53 null cells. In agreement, cell death in wild type cells was not accompanied by cleavage of polyADP ribose polymerase, a hallmark of apoptosis. Knockdown of Beclin 1 demonstrated that it and its binding partners do not have a significant role in the induction of autophagy in response to SK1-I treatment. Similarly, mTORC1 signaling was not observed. In contrast, SK1-I markedly decreased Akt phosphorylation. However, this might not be the sole factor important for SK1-I induced autophagy, as pharmacological inhibition of Akt only led to a comparatively weak autophagic response. Indeed, phosphorylation of the endoplasmic reticulum (ER) stress marker eIF2 α, was greatly reduced, suggesting that an ER mediated mechanism also contributes to SK1-I induced autophagy. Thus, SK1-I induced autophagy was likely triggered by ER stress signaling and led to non-apoptotic cell death in the more highly autophagic wild type 53 expressing cells. These results suggest that an isotype specific SphK1 inhibitor might be a useful adjunct for the treatment of cancer or other diseases in which enhancement of cytotoxicity or autophagy is desirable.

Abstract The yeast Saccharomyces cerevisiae on solid media forms multicellular colonies. Cells within colonies undergo differentiation and metabolic diversification, including formation of two layers of cells called Upper and Lower cells. The metabolic activity of U and L cells is different. For instance a higher level of autophagy was observed in U cells. This thesis includes a literature review of molecular mechanisms of autophagy in yeasts. Yeast colonies, under starvation conditions, produce volatile ammonia signal. This signal allows them communicate over a distance. Studies, revealing cell differentiation within colonies and ammonia signalling among colonies, were performed in colonies of laboratory strains. Strains isolated from nature, so-called wild strains, form distinct structured colonies, termed fluffy. Yeast within fluffy colonies also form different cell types. However the situation seems to be more complex that within smooth colonies of laboratory strains. Strains were constructed during this study, which express marker proteins Icl2p, Pox1p, Mae1p, Pma1p, Pma2p, Ino1p, Met17p and Atg8p fused with fluorescent labels in order to study cell differentiation and other processes within fluffy colonies. Furthermore, a new system for ...

Doctor of Philosophy / Department of Entomology / Marcelo Ramalho-Ortigao / Kun Yan Zhu / Microbial communities residing within the midgut of insect vectors play a critical role in the response to various zoonotic and human pathogens, and can directly alter the development and survival of the insects. Sand flies are the primary vector of Leishmania, the causative pathogen of leishmaniasis, a neglected tropical disease. Sand flies acquire many microbes from the soil where immature stages develop until emergence as adults. Gram-negative Pantoea agglomerans and gram-positive Bacillus subtilis are two bacteria commonly associated with sand fly populations. Here, I demonstrated that an EGFP- and a GFP-expressing version of these two bacteria localize to different compartments of the midgut; a phenomenon that is achieved, in part, to pH differences found across the length of the gut. Additionally, P. agglomerans is able to selectively induce midgut epithelial apoptosis while B. subtilis does not. This is accompanied by differential immune and homeostasis responses to both bacteria highlighted by immune pathway suppression via the Poor Immune Response upon Knock-in (Pirk) gene. These effects may actually be representative of a broader type of response to bacterial infection that might be present across several insect species. Finally, I demonstrated that during metamorphosis the sand fly relies, at least in part, upon the activation of multiple genes from the autophagy pathway to aid in generating adult tissues. More specifically, I demonstrate, using microscopy, the presence of ATG6 in the cytoplasm of developing midgut epithelial cells of the sand fly pupae.

L’anémie est un symptôme fréquent chez les patients atteints de cancer. La libération de la cytokine pro-inflammatoire TNFα, un inhibiteur connu de l’érythropoïèse, en est l’une des causes. L’érythropoïèse est un processus nécessitant l’arrêt de la prolifération et l’autophagie. Les résultats précédents ont montré que le TNFα inhibe l’expression des marqueurs érythroïdes et module l’expression de facteurs de transcription (FT) hématopoïétiques. Notre objectif est d’étudier l’implication de la voie TNFα/sphingomyélinase (SMase)/céramide dans l’inhibition de l’érythropoïèse en utilisant des cellules souches hématopoïétiques CD34+ induites à se différencier par l’érythropoïétine recombinante (Epo). Par l’utilisation de céramides exogènes, de SMase bactérienne et d’inhibiteurs de SMases, nous montrons l’implication de la voie SMase/céramide dans l’inhibition de l’expression des marqueurs érythroïdes mais également dans l’induction de la différenciation myéloïde avec une augmentation de l’expression du CD11b. Cet effet sur la différenciation est corrélé à la modulation du réseau FT/miR impliquant GATA-1, GATA-2 et PU.1 et les miR-144, 451, 155, 146a et 223. De plus, l’analyse par microscopie électronique à transmission, l’absence de formation de punctae GFP-LC3 et l’accumulation de SQSTM1/p62 montrent que le TNFα et les céramides inhibent l’autophagie induite par l’Epo. L’analyse des protéines impliquées dans la régulation de l’autophagie montre que le TNFα et les céramides activent mTOR. Son implication est confirmée par l’utilisation de rapamycine et l’inhibition de ULK1 et Atg13. De plus, le TNFα et les céramides inhibent l’expression de bécline 1 et de la formation du complexe Atg5-Atg12. Ces résultats démontrent que la voie TNFα/SMase/céramide joue un rôle dans l’homéostasie hématopoïétique par l’inhibition de l’érythropoïèse au profit de la myélopoïèse, en impactant les réseaux de régulation FT/miR et le processus d’autophagie / Anemia is a common symptom in cancer patients. It can be caused by the release of pro-inflammatory cytokines such as TNFα, a known inhibitor of erythropoiesis. Erythropoiesis involves proliferation arrest and autophagy. Our previous studies showed that TNFα inhibits the expression of erythroid markers as well as hematopoietic transcription factors (TF) expression. The aim is to study the involvement of TNFα/sphingomyelinase (SMase)/ceramide pathway in erythropoiesis inhibition using recombinant erythropoietin (Epo)-induced CD34+ hematopoietic stem cells. Using exogenous ceramides, a bacterial SMase and sphingomyelinase inhibitors, we show the involvement of SMase/ceramide pathway in the inhibition of erythroid markers as well as the induction of myeloid differentiation as shown by the increase in CD11b expression. This effect is correlated to the modulation of the TF/miR network involving GATA-1, GATA-2 and PU.1 as well as miR-144, 451, 155, 146a and 223. We show that TNFα and ceramides inhibit Epo-induced autophagy through transmission electron microscopy analysis, the absence of GFP-LC3 punctae formation and SQSTM1/p62 accumulation. Analysis of proteins involved in autophagy regulation showed that TNFα and ceramides activate mTOR, which is confirmed using rapamycin as well as the inhibition of ULK1 and Atg13. Moreover, TNFα and ceramides inhibit Beclin 1 expression and Atg5-Atg12 complex formation. These results demonstrate the role of TNFα/SMase/ceramide pathway in hematopoietic homeostasis through an erythropoiesis-myelopoiesis switch resulting from perturbation of TF/miR network and autophagy

Os éteres de difenilas polibromados (PBDEs) são largamente usados como retardantes de chama e têm sido detectados em diversas amostras biológicas, devido às suas características físico-químicas e bioacumuladoras, as quais fazem com que estes compostos tenham uma elevada persistência no meio ambiente. A exposição a estes compostos pode ser nociva ao homem e aos organismos vivos, uma vez que muitos estudos têm relatado que este composto possui atividade citotóxica. Desta forma, no presente estudo, investigamos o mecanismo de ação dos BDE-47, BDE-99, BDE-154 e BDE-209 sobre o processo autofágico de células HepG2, pois além de ser um processo de reciclagem de moléculas intracelular, a autofagia atua ainda na citoproteção celular. Foram realizados ensaios de imunocitoquímica para detecção de alterações no processo autofágico, no qual foi avaliado, sob microscopia de fluorescência, o padrão de distribuição das proteínas LC3-I e LC3-II; paralelamente, foi realizada a técnica de western blotting para a quantificação de três proteínas associadas ao processo de autofagia - LC3, Beclin-1 e p62. Em nossas análises de imunocitoquímica pode-se observar que os quatro PBDEs em estudo foram capazes desencadear o processo de autofagia, sendo que a magnitude do efeito tóxico foi dependente do tempo de exposição (24 e 48 horas), da concentração utilizada (0,1; 1; 5; 25 ?mol/L) e variável de acordo com o congênere em questão, sendo os BDE-47 e BDE-99 os que apresentaram maior potencial de ativação da autofagia. Além disso, esta indução de autofagia ainda pode ser detectada nos ensaios de western blotting, pois os PBDEs alteraram os níveis de LC3-II, Beclin-1 e p62, havendo um padrão de toxicidade semelhante ao visualizado nos ensaios de imunoistoquímica. Adicionalmente, devido ao fato dos PBDEs poderem se acumular no meio ambiente, principalmente em organismos aquáticos, uma análise dos efeitos destes compostos sobre um modelo ecotoxicológico foi realizada. O zebrafish (D. rerio) foi utilizado como organismoteste, a fim de avaliar a toxicidade destes contaminantes ambientais sobre seus estágios embrionários e larvais. Foram analisados endpoints de letalidade, subletalidade e teratogenicidade após a exposição aos PBDEs em estudo. Sendo que os compostos BDE-47, BDE-99 e BDE-209 (na presença de luz) apresentaram alterações em pelo menos um dos indicadores avaliados, sendo que o parâmetro mais afetado foi a inflação das bexigas natatórias, indicando uma possível teratogenicidade causada por estes compostos. Por outro lado, os BDE-154 e BDE- 209 (na ausência de luz) não causaram malformações no organismo-teste. Diante dos resultados obtidos, pode-se concluir que estes compostos podem causar alterações na homeostase celular desencadeando o processo autofágico, sendo que se absorvidos por organismos, podem afetar o desenvolvimento de espécies como o zebrafish. / Polybrominated diphenyl ethers (PBDEs) are widely used as flame retardants and they have been detected in several biological samples due to their physicochemical and bioaccumulating characteristics, which make these compounds to have a high persistence in the environment. The exposure to these compounds can be harmful to man and other living organisms, since many studies have reported that this compound has cytotoxic activity. Thus, in the present study, we investigated the mechanism of action of the BDE-47, the BDE-99, the BDE-154 and the BDE-209 on the autophagic process of HepG2 cells, because beyond being an intracellular molecule recycling process, autophagy acts also in the cell cytoprotection. Immunocytochemistry assays were performed to detect changes in the autophagic process, in which the distribution pattern of the LC3-I and LC3-II proteins was evaluated under fluorescence microscopy. In parallel, the western blotting technique was used to quantify three proteins associated with the autophagy process - LC3, Beclin and p62. In our analyzes of immunocytochemistry it can be observed that the four PBDEs in the study were able to trigger the autophagy process, and the magnitude of the toxic effect was dependent on the exposure time (24 and 48 hours), on the used concentration (0.1; 1; 5; 25 ?mol/L), and it varies according to the congener. The BDE-47 and BDE-99 are the ones with the highest potential for autophagy activation. In addition, this autophagy induction can still be detected in the western blotting assays, because the PBDEs altered the levels of LC3-II, Beclin-1 and p62, with a similar pattern of toxicity as seen in the immunohistochemical assays. In addition, due to the fact that PBDEs may accumulate in the environment, mainly in aquatic organisms, an analysis of the effects of these compounds on an ecotoxicological model was performed. The Zebrafish (D. rerio) was used as a test organism in order to evaluate the toxicity of these environmental contaminants on their embryonic and larval stages. In this study, lethality, sublethality and teratogenicity endpoints were analyzed after exposure to the PBDEs. The BDE-47, BDE-99 and BDE-209 compounds (in the presence of light) presented alterations in at least one of the indicators evaluated, and the most affected parameter was the swim bladder inflation, indicating a possible teratogenicity caused by these compounds. On the other hand, the BDE-154 and the BDE-209 (in the absence of light) did not cause malformations in the test organism. Considering the results, it can be concluded that these compounds are able to cause changes in cellular homeostasis triggering the autophagic process, and whether absorbed by organisms, they can affect the development of species such as zebrafish.

A autofagia é o processo de degradação de estruturas celulares através do seu direcionamento ao lisossomo. As proteínas TRIMs reconhecem as -cargas? autofágicas e reúnem o complexo de nucleação do fagóforo, contudo se desconhece a função de cada domínio e a importância da atividade de E3 ligase para a sua atividade. A proteína TRIM49 clonada e expressa em E. coli ou em células humanas HEK293T não apresentou atividade de E3 ubiquitina ligase in vitro e reduziu os níveis totais de ubiquitinação in vivo, indicando que não é um E3 ubiquitina ligase. Células desafiadas com Htt74Q apresentaram menores níveis de citotoxicidade quando co-transfectadas com TRIM49 selvagem, mas não com os mutantes do domínio RING ou SPRY, indicando os dois domínios são necessários para sua atividade celular. A proteína selvagem se colocaliza com o marcador autofágico LC3, após o bloqueio da autofagia com bafilomicina A1. Os resultados indicam que a TRIM49 pode atuar na degradação intracelular de proteínas, por um mecanismo não dependente de atividade de E3 ligase. / Autophagy is the process of degradation of intracellular proteins through their directioning to the lysosome. TRIM proteins can directely recognize autophagic cargo and also act as a hub for the phagophore nucleation complex, however the function of each domain and the role of the E3 ligase activity in this process is unknown. The TRIM49 protein cloned and expressed in E. coli or in human cells HEK23T showed no ubiquitin E3 ligase activity in vitro and cells transfected with the wild type protein showed lower levels of polyubiquitinated proteins, indicating that TRIM49 is not a bona fide E3 ubiquitin ligase. Cells challenged with Htt74Q presented lower cytotoxicity levels when cotransfected with wild type TRIM49, when compared with the RING domain mutant or with the truncated protein lacking the SPRY domain, indicating that both domains are required for its cellular activity. The wild type protein colocalizes with the autophagic marker LC3 after treatment with the autophagy inhibitor bafilomycin A1. Taken together, these results indicate that the TRIM49 protein plays a role in protein degradation independently of a E3 ligase activity.

L'autophagie est une voie majeure de dégradation et de recyclage des constituants cytoplasmiques. C'est également un mécanisme de l'immunité innée et adaptative. Au cours de l'infection par le VIH-1, l'autophagie est déclenchée dans les lymphocytes T CD4 non infectés après contact avec les glycoprotéines d'enveloppe virale (Env). Cette autophagie participe alors à l'induction de l'apoptose dans ces cellules, mécanisme connu pour être responsable de la disparition des lymphocytes T CD4 au cours de l'infection, et donc de l'évolution vers la phase SIDA. Nos travaux ont permis de montrer que l'activité fusogénique de gp41 est responsable de l'induction d'autophagie et de la mort des lymphocytes T CD4 non infectés après leur contact avec Env. De façon tout à fait surprenante, il apparaît que l'autophagie est bloquée dans les lymphocytes T CD4 productivement infectés par le VIH-1 (souches X4 ou R5). Par contre, les macrophages, autre population cellulaire ciblée par ce virus, ne présentent aucun signe d'autophagie en réponse à la fixation de Env (souches X4 ou R5), alors qu'elle est induite dans les macrophages infectés par ces mêmes virus. Enfin, les derniers résultats nous ont permis de mettre en évidence une phase d'autophagie plus précoce, intervenant dans les deux heures suivant le contact avec Env. L'étude des acteurs impliqués dans l'induction de cette première phase a montré qu'elle était indépendante de la fusion membranaire induite par gp41, ainsi que de la liaison de Env aux récepteurs CD4 et CXCR4. Cette première phase d'autophagie est également présente dans les premières étapes d'infection des lymphocytes T CD4 par du virus libre, et est nécessaire à la réplication du VIH-1. Le VIH-1 adopte donc une stratégie d'exploitation du mécanisme autophagique afin d'optimiser sa réplication, mais également la propagation de l'infection. / Autophagy is a major catabolic pathway involved in the degradation and recycling of cytoplasmic components. It is also a mechanism of adaptive and innate immunity. During HIV-1 infection, autophagy is induced in uninfected CD4 T lymphocytes after contact with cells expressing HIV-1 envelope glycoproteins (Env, gp120/gp41). This process is a pre-requisite to their apoptosis. Thus, autophagy is implicated in the depletion of CD4 T lymphocytes during HIV-1 infection, mechanism leading to AIDS. The results obtained during my PhD have shown that the fusogenic function of gp41 is responsible for the induction of autophagy in the uninfected CD4 T lymphocytes. Surprisingly, this process is blocked in productively HIV-1-infected CD4 T lymphocytes (X4 or R5 strains). In contrast, uninfected macrophages, a preserved cell population during HIV-1 infection, do not undergo X4 or R5 Env-mediated autophagy. However, autophagosomes are present in infected macrophages. Our last results demonstrated that an early autophagic phase is triggered in target CD4 T cells 2h after the contact with Env-expressing cells. Neither the fusogenic function of gp41, nor gp120 binding to CD4 and CXCR4 is implicated in the induction of this early autophagic phase. Interestingly, this process seems necessary to establish an efficient HIV-1 replication. Our results suggest that HIV-1 adopts a strategy to use the autophagic pathway to its own profit and pointes this process as a key for the HIV-1-associated physiopathology.