The administration of antibodies blocking the immune checkpoint molecules programmed cell death protein 1 (PD-1) or programmed cell death 1 ligand 1 (PD-L1) has evolved as a very promising treatment option for cancer patients. PD-1/PD-L1 inhibition has significantly enhanced expansion, cytokine secretion, and cytotoxic activity of CD4+ and CD8+ T lymphocytes, resulting in enhanced antitumor responses. Anti-PD-1 or anti-PD-L1 therapy has induced tumor regression and improved clinical outcome in patients with different tumor entities, including melanoma, non-small-cell lung cancer, and renal cell carcinoma. These findings led to the approval of various anti-PD-1 or anti-PD-L1 antibodies for the treatment of tumor patients. However, the majority of patients have failed to respond to this treatment modality. Comprehensive immune monitoring of clinical trials led to the identification of potential biomarkers distinguishing between responders and non-responders, the discovery of modes of treatment resistance, and the design of improved immunotherapeutic strategies. In this review article, we summarize the evolving landscape of biomarkers for anti-PD-1 or anti-PD-L1 therapy.

Diversos tipos celulares nas mais variadas condições têm sido usados como doadores de núcleo para a TN. Ainda não está claro se o estado fisiológico destas células afeta o posterior desenvolvimento dos embriões. Neste trabalho, testou-se a hipóese que fibroblastos bovinos em processo de MCP podem ser reprogramados na transferência nuclear. Fibroblastos foram cultivados até atingirem 60% de confluência, sincronizados por restrição de soro durante 24h e em seguida a MCP foi analisada por citometria de fluxo com a ténica da Anexina V/Iodeto de propídeo. Células Anexina positivas (MCP) e Anexinanegativas (Vivas) foram separadas por citometria de fluxo e utilizadas para a TNS. Céulas não coradas e não separadas no citômetro serviram como controle (Controle). Os embriões reconstruídos foram avaliados quanto à fusão, clivagem (2º dia de cultivo), blastocisto (7º dia) e prenhez (D30, D60 e nascimento). O índice de MCP dos blastocistos obtidos foi determinado. Os resultados foram analisados pela ANOVA ou teste de X2 com nível de significância de 5%. Não houve efeito nas taxas de fusão (p>0,05). Embriões reconstruídos com células MCP tiveram menor taxa de clivagem e formação de blastocistos (72,7% e 18,8%, respectivamente) em comparação ao grupo reconstruído com células Vivas (83,4% e 34,7%, respectivamente; p<0,05), não diferindo dos embriões Controle (77,3% e 27,3%, respectivamente; p>0,05). O índice de MCP do grupo de embriões MCP foi similar aos índices dos embriões clonados a partir de células Vivas e Controle (p>0,05). Após a transferência para receptoras, os grupos MCP, Vivas e Controle não diferiram com relação à taxa de prenhez aos 30d (18,1%, 13,3% e 27,5%, respectivamente; p>0,05). Entretanto aos 60d, a perda gestacional no grupo MCP (25%) foi inferior a do grupo Vivas (100%) e Controle (62,5%). Somente um nascimento, do grupo MCP (4,5% dos embriões transferidos), foi obtido no experimento. Conclui-se que células em processo de MCP, podem ser reprogramadas quando utilizadas como doadoras de núcleo na técnica de transferência nuclear, podendo estabelecer gestações e nascimentos, entretanto houve um efeito prejudicial nas taxas de desenvolvimento embrionário até o estádio de blastocisto assim como houve aumento do índice de MCP nos embriões reconstruídos. / It is not clear if the physiological status of the cells can affect further embryonic development in NT. We hypothesized that adult bovine fibroblasts in PCD can be reprogrammed when used as nuclear donors for cloning. Fibroblasts were cultivated until 60% confluency, synchronized by serum starvation for 24 h and stained with Annexin V and Propidium iodide (PI) by flow citometry. Annexin positive cells (PCD cells) and Annexin negative cells (Live cells) were sorted and used for NT. Unsorted, unstained cells were used as control (Control cells). After reconstruction, fusion, cleavage (day 2 of culture), blastocyst (day 7) and pregnancy rates (day 30, 60 and birth) were recorded. Apoptotic index of the embryos was determined by TUNEL. Data were analyzed with ANOVA and Chi-square test with 5% of significance level. There was no effect on fusion rates (p>0.05). Embryos reconstructed with PCD cells had lower cleavage and blastocyst rates (72.7 and 18.8%, respectively) compared with embryos reconstructed with Live cells (83.4 and 34.7%, respectively; p<0.05). Apoptotic index in embryos produced from cells in PCD was similar compared to embryos produced from Live and Control cells (p>0.05). Pregnancy rates were similar between cloned groups on day 30 after embryo transfer (p>0.05). However it was observed a reduced pregnancy loss in PCD group on day 60 (25%) compared with Control (62.5%) and Live (100%) groups. Only one calf, from PCD cells (4.5% of the transferred embryos), has been obtained in this experiment. In conclusion, it was showed that cells in PCD process can be reprogrammed when used as nuclear donors after NT producing even live animals. However, a negative effect on embryonic development and an increase in the apoptotic index of these embryos was observed.

A transição materno-embrionária é um fenômeno complexo caracterizado pela iniciação da transcrição no embrião e a substituição do mRNA materno pelo mRNA embrionário. O mRNA e as proteínas estocadas no oócito são utilizados nas primeiras clivagens e, posteriormente, o embrião deve iniciar a transcrição dos genes necessários ao seu desenvolvimento, que ocorre no estádio de oito células em embriões bovinos. Nesta etapa, podem ser observados embriões competentes a continuar o desenvolvimento, enquanto embriões incompetentes sofrem bloqueio. Pelo fato do bloqueio ocorrer na fase de ativação do genoma embrionário, formulou-se a hipótese de que o bloqueio estaria associado a genes transcritos neste momento, contrariamente à hipótese mais aceita de bloqueio passivo. O objetivo deste trabalho foi de identificar, categorizar e avaliar transcritos diferencialmente expressos entre embriões bovinos de desenvolvimento rápido e lento, além de elucidar possíveis vias de sinalização de morte ou sobrevivência celular. Para isso, foi feita uma hibridação em membrana de macro arranjo contendo genes humanos relacionados a ciclo celular, hibridada com aRNA marcado radioativamente oriundo de embriões bovinos produzidos in vitro de acordo com sua velocidade de desenvolvimento, seguido por RT-PCR e análise de vias de sinalização para validação da hibridação. A média de similaridade entre estes genes humanos e bovinos de 89,3%. Pelas membranas de macro arranjos foram identificados 120 genes com modulação diferencial entre embriões lentos e rápidos, sendo 100 genes com regulação superior nos embriões lentos. Entre os genes com modulação positiva nos embriões rápidos, 40% foram primariamente identificados como ligantes a proteínas e 25% têm atividade catalítica, com resultados similares no grupo de genes com modulação positiva nos embriões lentos. Por um lado, as diferenças de transcrição entre embriões de desenvolvimento rápido e lento não foram confirmadas pelo RTPCR. Mas os genes diferencialmente modulados estão associados e constitutivamente presentes em algumas vias de sinalização para morte celular. Os resultados sugerem que a ativação do genoma embrionário é necessária para a sinalização de vias de sobrevivência ou morte celular programada. Assim, o bloqueio do desenvolvimento não é um processo passivo, mas sim um processo ativo de transcrição de genes, ativando tanto a cascata de sobrevivência quanto a cascata de morte em embriões com baixo potencial de desenvolvimento. / Maternal-zygotic transition is a complex phenomenon characterized by the initiation of transcription in the embryo and the transition of maternal mRNA with embryonic mRNA. It is believed that the mRNAs and proteins synthethized by the oocyte during its growth and final maturation allow the zygote to develop during the early stages of embryo development up to the 8 cell-stage, the moment when the bovine embryo acquires transcriptional competence. Competent embryos are able to develop until blastocyst, while incompetent embryos block. Since the blockage occurs during embryo genome activation, we developed the hypothesis that gene transcription in incompetent embryos is associated with the blockage, instead of passive blockage. The aim of this work was to identify, categorize and analyze gene expression differences between fast cleavage and slow cleavage embryos, and discover possible signaling pathways to cell death or cell survival. We used a macroarray membrane spotted with human genes related to cell cycle and hybridizated with a radioactive labeled aRNA from fast or slow in vitro produced embryos. Real-time PCR and signaling pathways analysis were designed for further validation of the array. The mean similarity between human and bovine genes was 89,3%. According to the array membranes, it was possible to identify 120 genes differentially expressed between slow and fast cleavage embryos. Hence, the majority of the genes were more expressed in slow embryos (100 genes versus 20 genes in fast group). Among genes more expressed at fast embryos, 40% were identified as protein binding and 25% have catalytic activity, with similar results in slow embryos. In one hand, differences between fast and slow embryos transcripts were not confirmed by real-time PCR analysis. But on the other hand, the differentially expressed genes are somehow related to and constitutively present in some recognized death pathways. Together, these results presented herein suggest that embryonic genome activation is necessary for survival or cell death signaling. Moreover, developmental block is not a passive pathway, but rather a very active transcriptional pathway, leading to activation of cell survival genes prior to genes related to death in slow-developing embryos.

Au cours de ce travail de thèse, les deux approches permettant la caractérisation de nouveaux agents antileishmaniens ont été suivies : d’une part identification de molécules de synthèse originales et actives, par des méthodes de criblage, et d’autre part recherche de nouvelles cibles thérapeutiques leishmaniennes, faisant appel à des outils de biologie moléculaire, et de protéomique.Dans une première partie consacrée à l’optimisation du criblage antileishmanien de molécules de synthèse, nous avons notamment mis au point et validé une nouvelle méthode applicable aux formes promastigotes. Cette méthode reposant sur le principe de la bioluminescence, s’est avérée précise, rapide, répétable, sensible, automatisable et applicable à des isolats cliniques. Nous nous sommes également intéressés à la recherche d’une nouvelle méthode de criblage sur les formes amastigotes intracellulaires remplissant ces mêmes critères.Dans une seconde partie consacrée au lien entre la mort cellulaire programmée et les cystéines peptidases (métacaspase et Raptor) chez Leishmania, nous avons mis en évidence un lien possible entre métacaspase et autophagie chez L. infantum. De plus, nous avons identifié, in vivo, plusieurs substrats protéiques potentiels de ces peptidases : HSP70, ARN-Hélicase ATP-Dépendante et Lmjf09.1010 pour la métacaspase ; NDPKb et la protéine associée à l’ADN du kinétoplaste pour Raptor. Ces différents résultats ont permis de proposer un modèle conciliant les rôles possibles des cystéine-Peptidases métacaspase et Raptor au cours de l’autophagie et de l’apoptose chez Leishmania, rôles potentiellement dus au clivage de substrats protéiques spécifiques. / During this thesis work, two approaches affording the characterization of new antileishmanial agents were followed: on the one hand, the identification of new original antileishmanial synthetic drugs, through screening assays and on the other hand, research of new parasitic therapeutic targets by using molecular biology and proteomic tools. In the first part, dedicated to the optimization of the antileishmanial screening of synthetic compounds, we set up and validated a new bioluminescence-Based screening method for studying anti-Promastigote compounds. This method appears accurate, rapid, repeatable, sensitive and is also transposable to automats and usable on clinical isolates. In parallel, we investigated new screening protocols aiming at improving the screening in intracellular amastigotes which could meet the same criteria. In the second part focusing on the link between programmed cell death and cystein peptidases (metacaspase and Raptor) in Leishmania, our study first highlighted a possible link between metacaspase and autophagy in L. infantum. Moreover, we identified in vivo several potential subtrates for both peptidases: HSP70, ATP-Dependent RNA-Helicase and Lmjf09.1010 for the metacaspase; NDPKb and kinetoplast-DNA-Associated-Protein for Raptor. These results allowed us to propose a model reconciling the possible roles of cystein peptidases metacaspase and Raptor during autophagy and apoptosis in Leishmania, roles potentially due to the cleavage of specific proteic subtrates.

Ovarian cancer is a deadly disease that kills an estimated 15,000 women annually in the United States. It is estimated that approximately 10% of ovarian cancers are due to familial inheritance. The most commonly mutated genes in familial ovarian cancer are BRCA1 and BRCA2. It has been reported that cells carrying the BRCA1 185delAG mutation undergo an enhanced caspase-3 mediated apoptotic response. Here, we report on the transfection of cDNA coding for the putative truncated protein product of the BRCA1 185delAG mutant gene into BRCA1 wild-type human immortalized ovarian surface epithelial (IOSE) cells and ovarian cancer cells. Cells transfected with the BRCA1 185delAG truncation protein (BRAt) showed increased levels of active caspase 3, increased cleavage of caspase 3 substrates, PARP and DFF45, and decreased XIAP and cIAP1 following staurosporine (STS) treatment. BRAt also reduced Akt phosphorylation and over expression of activated Akt in BRAt cells restored caspase-3 activity to that seen in wild type cells. Further, BRAt expression increased chemosensitivity in platinum resistant ovarian cancer cells. Similarly, maspin protein has been shown to sensitize breast carcinoma cells to STS-induced apoptosis. We provide the first evidence that BRAt is sufficient to induce maspin protein in IOSE cells. IOSE cell lines carrying the BRCA1 185delAG mutation showed higher maspin levels than wild-type BRCA1 IOSE cell lines. BRCA1 wild-type IOSE cells were transfected with BRAt protein and showed increased maspin mRNA levels and increased nuclear maspin protein levels as compared to control cells. Additionally, both heterozygous carriers of the BRCA1 185delAG mutation and cells transfected with BRAt protein show an increased ability to activate the maspin promoter as compared to control cells. The transcription factor AP1 is at least partially required for full activation of the maspin promoter in BRAt cells, as siRNA directed towards c-jun decreased activation of the full-length maspin promoter. Taken together, our data demonstrate that truncated proteins arising from BRCA1 185delAG mutation increase Akt-mediated apoptosis by increasing nuclear maspin expression, suggesting a possible mechanism by which ovarian cancer patients with germline BRCA1 mutations may respond better to chemotherapy.

Doctor of Philosophy / Rice (Oryza sativa L.) is very susceptible to water deficit at any time during its life cycle as a semi-aquatic cereal crop. However, the consequential damage is particularly severe if water deficit occurs during reproductive phases. The conspicuous injury often observed in rice plants exposed to water stress during meiosis of the pollen mother cell is the reduction of grain set, which is attributed to the decline of male fertility. In spite of much research on drought-induced male sterility in rice, the underlying mechanisms of the problem are poorly understood. This project was therefore conducted to investigate the molecular mechanisms of water deficit-induced pollen sterility in rice. In this study three consecutive days of water deficit treatment at -0.5 MPa osmotic potential during anther development effectively reduced the leaf water potential (leaf) and the number of viable pollen which later led to a decrease in grain set. Moreover, this thesis demonstrates that the immediate deleterious effects of water deficit to plant fertility could be estimated using a young microspore viability index, which showed a strong correlation with mature viable pollen and grain set. The present work has also illustrated that oxidative stress appears to be a plausible cause for the decline of male fertility and grain set. Water deficit has induced the excessive production of reactive oxygen species (ROS) above the redox balance, which in turn caused detrimental effects to cellular DNA and might result in programmed cell death (PCD) in the anthers. Moreover, ROS accumulation effectively influenced ATP synthesis leading to a decrease in the level of ATP in the anthers. Excessive ROS accumulation after drought could be the consequence of insufficient activity of the antioxidant system, which has been illustrated by qRT-PCR expression analysis of major antioxidant genes. Down-regulation of those genes would increase the incidence of oxidative damage. In contrast, stable or up-regulated expression of these genes resulted in less oxidative damage. Detailed investigations of sugar metabolism in anthers has provided supplemental data to develop a model of sugar unloading and transport within anther using in situ hybridisation to mRNA techniques. Analysis of sugar transportation within the cellular compartments of anther has unveiled the role of sugar metabolism on pollen sterility in rice. qRT-PCR assays of genes associated with the sugar metabolic pathway has demonstrated that the supply of both sucrose and hexoses from the anther walls to the locules was not restricted after water deficit stress. The results indicate that water deficit might not cause sugar starvation for developing microspores as previously thought, nor inhibit the initial steps of sugar utilisation such as glycolysis. This thesis has suggested new ideas regarding the role of rising sugar levels to cope with oxidative stress in anthers. Sugar accumulation might have provided protection against oxidant damage by strengthening the antioxidant system. However, the interplay between sugar and oxidative stress is not straightforward and needs to be further characterised. In-depth investigations on the interaction between sugar signalling and oxidative stress responses may help indentify the role of sugars in protecting anthers under water deficit. Although many studies on drought and chilling stresses in rice anthers have been performed, the causal mechanism of male sterility still remains to be elucidated. Findings presented in this thesis may contribute to understanding molecular mechanisms of male sterility in rice as a response to drought stress. A more detailed investigation of mitochondrial respiration in rice anthers is required to further examine this problem. Finally, this thesis suggests that signalling molecules such as 14-3-3 proteins and abscisic acid (ABA) might act upstream of ROS production and antioxidant defence in plants. Further work on these molecules might therefore further illustrate how they influence plant fertility under water shortage conditions.

The steroid hormone ecdysone plays vital roles during Drosophila development. Pulses of 20E during Drosophila life cycle function as temporal cues, signaling the onset of metamorphic processes, including the stage specific programmed cell death of larval tissues. Ecdysone is the critical developmental cue orchestrating the metamorphic reformation of CNS, resulting in the formation of adult-specific neural circuitry. Ecdysone signaling is transduced by a heterodimeric receptor complex formed between two nuclear receptors: EcR and Ultraspiracle (USP). There are 18 nuclear receptors known in Drosophila and EcR is the only receptor whose functions in neuronal PCD have been well recognized. Therefore, the current study is aimed to define the role of nuclear receptors in neuronal cell death mechanisms in Drosophila. Here, I examine the function of nuclear receptors in PCD of two groups of peptidergic neurons: vCrz and CCAP. EcR and USP receptor complex on activation results in the coordinated transcriptional regulation of a host of transcription factors regulating genes essential for PCD. USP plays a dual role in ecdysone response, as its function is necessary for both activation and repression of ecdysone primary response genes. I have developed a possible dominant-negative mutant USP (usp3), and expressed it in flies using the GAL4-UAS system to illustrate the role of USP in ecdysone mediated PCD of vCrz neurons. Targeted expression of usp3 in corazonin neurons results in a complete blockage of PCD pathway. Another interacting partner of USP, Drosophila Hormone Receptor 38, however shows no involvement in PCD of vCrz neurons. I have also designed an ecdysone sensor to monitor the developmental timing of EcR activation in vCrz neurons. Further, I investigate the survival factors required for preventing the untimely PCD of these two groups of neurons. The study reveals that DIAP1 is required for the survival of larval vCrz and CCAP neurons. Also, the nuclear receptor E75 is shown to be critical for preventing premature PCD of CCAP neurons.

The resistance of cancer cells to traditional chemotherapeutic agents is a major obstacle in the successful treatment of cancer. Cancer cells manipulate a variety of signaling pathways to enhance resistance to anticancer agents; such mechanisms include disrupting the DNA damage response and hyperactivating survival signaling pathways. In an attempt to better understand the molecular mechanisms that underlie resistance to chemotherapeutic agents, we investigated multiple processes regulated by the Rad9-Rad1-Hus1 (9-1-1) complex and Bif-1. The 9-1-1 complex plays an integral role in the response to DNA damage and regulates many downstream signaling pathways. Overexpression of members of this complex has been described in several types of cancer and was shown to correlate with tumorigenicity. In this study, we demonstrate that disruption of the 9-1-1 complex, through loss of Hus1, sensitizes cells to DNA damaging agents by upregulating BH3-only protein expression. Moreover, loss of Hus1 results in release of Rad9 into the cytosol, which enhances the interaction of Rad9 with Bcl-2 to potentiate the apoptotic response. We also provide evidence that disruption of the 9-1-1 complex sensitizes cells to caspase-independent cell death in response to DNA damage. Furthermore, we found that loss of Hus1 enhances DNA damage-induced autophagy. As autophagy has been implicated in caspase-independent cell death, these data suggest that the enhanced autophagy observed in Hus1-knockout cells may act as an alternate cell death mechanism. However, inhibition of autophagy, through knockdown of Atg7 or Bif-1, did not suppress, but rather promoted DNA damage-induced cell death in Hus1-deficient cells, suggesting that in apoptosis-competent cells autophagy may be induced as a cytoprotective mechanism. The aberrant activation of survival signals, such as enhanced EGFR signaling, is another mechanism that provides cancer cells with resistance to DNA damage. We found that knockdown of Bif-1 accelerated the co-localization of EGF with late endosomes/lysosomes thereby promoting EGFR degradation. Our results suggest that Bif-1 may enhance survival not only by inducing autophagy, but also by regulating EGFR degradation. Taken together, the results from our studies indicate that the 9-1-1 complex and Bif-1 may be potential targets for cancer therapy as they both regulate sensitivity to DNA damage.

The precise structure of the adult Drosophila eye results from a coordinated process of cell sorting, differentiation and selective cell death in the retinal epithelium. Mutations in the gene echinus cause supernumerary pigment cells due to insufficient cell death. This study reports the identification of echinus and the characterization of its role in Drosophila retinal development. Using a combination of deletion mapping, gene expression analysis and genomic sequencing, echinus was cloned and several alleles were sequenced. echinus encodes a ~180kDa protein containing an ubiquitin hydrolase domain at its N-terminus and a polyglutamine tract at its C-terminus. echinus is expressed in the retina during pupal development and mutants of echinus have decreased levels of apoptosis during several stages of retinal development. Defects in the cell sorting process that precedes cell death are also observed in echinus loss-of-function mutants and echinus overexpression can cause defects in ommatidial rotation and the morphology of cone cells. echinus is a positive regulator of DE-cadherin and Enabled accumulation in adherens junctions of retinal epithelial cells. Genetic interactions were observed between echinus and the genes wingless, enabled and expanded. An immunofluorescence assay in Drosophila S2 cell cultured demonstrated that Echinus localizes to intracellular vesicles that do not appear to be endocytic in nature, and the C-terminal region of Echinus was shown to be necessary for this association. A protein interaction screen using an immunoprecipitation and mass spectrometry approach identified interactions between Echinus and the vesicle coat protein Clathrin, the scaffolding protein RACK1 and the casein kinase I epsilon (Dco). Co-immunoprecipitation additionally identified an interaction between Echinus and Enabled. This work has revealed echinus to be an important regulator of cell sorting and adherens junction formation in the developing retina and has identified multiple interactions between echinus and enabled, a regulator of the actin cytoskeleton.

HSP27, the Drosophila homolog of mammalian HspB1, is a nuclear sHsp that is both stress induced and developmentally regulated with a conserved cyto-protective function. It is multiply phosphorylated in vivo through an unconfirmed mechanism at unidentified residues. The effect of phosphorylation on its localization, oligomerization, and function is also not well understood. Here we report a genetic investigation into the role of Hsp27 in Drosophila development, and a preliminary investigation into the effect of phosphorylation on HSP27 localization and function in Drosophila S2 cells. Through a proteomic screen, a pro-apoptotic role for Hsp27 in embryonic developmentally regulated programmed cell death was suggested and supported by RNAi experiments, but not replicated using Hsp27null mutant stocks. These stocks were complicated by the intriguing appearance of multiple background mutations. Specific developmental defects in transgenic lines overexpressing phospho-mutant isoforms were then investigated. These too were subject to multiple independent incidences of background genetic mutation, which we believe may be related to Hsp27 mis-expression. We also studied the endogenous expression and localization pattern of HSP27 in stressed and unstressed Drosophila S2 cells. We found evidence that wild-type protein localization is influenced by stress. Finally, we took a first step toward understanding how phosphorylation might regulate HSP27 localization by examining the effect of targeted mutations of serine residues (S58, S71, and S75) on the localization pattern of exogenous HSP27. By characterizing the expression of endogenous and overexpressed HSP27 in Drosophila cells, we provide a foundation for future investigation into the regulated localization and function of HSP27 that can be extended to address the regulatory mechanisms that govern the protective capacities and oligomeric properties of phosphorylated HSP27 in Drosophila.