Дисертації з теми "Stress signalling pathway"

>Magister Scientiae - MSc
Abiotic stress, mainly in the form of extreme temperatures, drought and salinity has caused major crop losses worldwide, putting a severe strain on agriculture. Salinity severely limits plant growth and productivity and affects all aspects of the plant’s development including the most crucial stage; germination. This study investigated the effect of salt (NaCl) stress on Sorghum bicolor seedlings and the role of exogenously applied calcium (Ca2+) to ameliorate the effects of salt stress during germination. Sorghum seeds were germinated in the presence and absence of various NaCl (100, 200 and 300 mM) and Ca2+ (5, 15 and 35 mM) concentrations. Several assays including physiological (germination and growth assays), biochemical (osmolytes and oxidative stress markers), anatomical (epidermal and xylem layers) and expression profiles of key genes [antioxidant (SbSOD, SbAPX2 and SbCAT3), Salt Overly Sensitive (SbSOS1, 2 and 3) pathway enzymes and the vacuolar Na+/H+ exchanger antiporter2 (SbNHX2)] were investigated. Salt stress delayed germination and negatively affected growth as observed by the reduced root and shoot length and decreased fresh and dry weight. There was an increase in proline content and oxidative stress markers (H2O2 and MDA) under salt stress. Oxidative stress resulted in damage to the epidermal and xylem layers as observed on Scanning Electron Microscopy (SEM) images. Quantitative real-time polymerase chain reaction revealed that salt stress induced the expression of SbAPX2, SbCAT3 and SbSOS1 genes, whereas SbSOD4A, SbSOS2, SbSOS3 and SbNHX2 genes were not affected by salt. Exogenous application of Ca2+ counteracted the harmful effects of salt stress by improving germination efficiency, promoting seedling growth, reducing oxidative damage and the Na+/K+ ratio, indicating the protective effect. Ca2+ also effectively protected the epidermis and xylem layers from the severe damage caused by salt stress. In the presence of Ca2+ the expression of SbAPX2 and SbCAT3 was reduced except for the SbNHX2 gene, which increased by 65-fold compared to the control. The results obtained suggests that sorghum is able to respond to salt stress by inducing osmolytes, the antioxidant defence system as well as the SOS pathway. Furthermore, 5 mM Ca2+ was determined as the optimum Ca2+ concentration required to enhance sorghum’s tolerance to salt stress.

The T-box gene family has achieved great prominence in the field of developmental biology because its members have been demonstrated to play important roles in embryonic development and mutations within several T-box genes are associated with a number of human congenital diseases. Several lines of evidence have also implicated members of the T-box gene family in cell cycle regulation and in cancer. Importantly, the highly related T-box factors, Tbx2 and Tbx3, can suppress senescence through repressing the cyclin dependent kinase inhibitors, p21wAF11 c1P118011 and p19ARF. Both Tbx2 and Tbx3 have also been linked to several cancers primarily because their expression levels have been found to be deregulated in these cancers. However, despite the pivotal role that members of the T-box family play in a wide variety of biological processes, very little is known about the biochemical pathways that regulate their levels and transcriptional activity. In view of the detrimental consequences resulting from altered levels of T-box proteins, as seen both in developmental disorders and in certain cancers, the need to identify such pathways is important. The aim of this study was therefore to identify kinases that phosphorylate and regulate the levels and activity of Tbx2 with the view to understanding its role in cell cycle regulation and cancer. This study shows that the p38 stress mitogen-activated protein kinase, and the mitotic cyclin A/Cdk2 and cyclin B1/Cdk1, are direct regulators of Tbx2 both in vitro and in vivo. It is possible that Tbx2 and Tbx3 may contribute towards the oncogenic process through their anti-senescence function, especially since a dominant negative form of Tbx2 induces senescence in melanoma cells overexpressing Tbx2. However, very little is known about whether Tbx2 is indeed regulated during replicative- or stress-induced senescence. In this study, using a breast cancer cell line known to overexpress Tbx2, the Tbx2 protein is shown to be specifically phosphorylated by the p38 kinase in response to stress induced by ultraviolet irradiation. Using site-directed mutagenesis and in vitro kinase assays, serine residues 336, 623 and 675 in the Tbx2 protein were identified as p38 target sites. These sites are also shown to be phosphorylated in vivo. Importantly, western blotting, immunofluorescence and reporter assays reveal that this phosphorylation leads to increased Tbx2 protein levels, predominant nuclear localisation of the protein, and an increase in the ability of Tbx2 to repress the p21wAF11 c/P118011 promoter. These results show, for the first time, that the ability of Tbx2 to repress the p21 gene is enhanced in response to a stress-induced senescence pathway. This leads to a better understanding of the anti-senescence function of Tbx2. The ability of Tbx2 to function as an anti-senescence factor, as well as its altered regulation being associated with certain cancers, suggests that"its levels may need to be tightly regulated during the cell cycle. Indeed, the Tbx2 protein was previously shown to be regulated during the various phases of the cell cycle, peaking at G2. The changes in the Tbx2 protein levels did not match changes in Tbx2 mRNA levels, suggesting that the protein may be regulated by posttranslational modifications such as phosphorylation. This study shows that the phosphorylation status of Tbx2 is regulated during the cell cycle with levels of phosphorylation peaking in G2 and M, in mouse and human cells respectively. Phosphorylation was shown to be specifically mediated by the mitotic kinases as demonstrated in experiments when the mitotic kinase inhibitor, olomoucine, was included. This study provides data to suggest that Tbx2 may be regulated differently during the cell cycle in mouse and human cells. Using site-directed mutagenesis and in vitro kinase assays, Tbx2 was found to be specifically phosphorylated at serine residues 192 and 336 by cyclin A/Cdk2 and serine residues 336 and 342 by cyclin 81/Cdk1. These sites are also targets for phosphorylation in vivo since mutating them altered the phosphorylation status ofTbx2. Moreover, both cyclin A and 81 were shown to bind Tbx2 in vitro and in vivo and the minimal region required for binding was mapped to its DNA-binding domain. Importantly, immunofluorescence demonstrates that the Tbx2 protein localises specifically to the nucleus at G2; this translocation was shown to be blocked in the presence of olomoucine. Furthermore, western blot analyses and reporter assays showed that pseudo-phosphorylation by cyclin 81/ Cdk1, but not cyclin A/Cdk2, leads to increased Tbx2 protein levels and an increase in the ability of Tbx2 to repress the p21wAFtiCIPt!Sou promoter. These results disclose, for the first time, that phosphorylation by cyclin A/Cdk2 and cyclin 81/Cdk1 of the Tbx2 protein regulates its activity. This data provide additional evidence to support a role for Tbx2 in the G2 and/or M phase of the cell cycle.

Les virus du papillome humain (HPV) sont des virus à ADN double-brin encapsidés appartenant à la famille des Papillomaviridae ayant un tropisme distinct pour les épithéliums squameux de type muqueux ou cutanés. Jusqu'à présent, plus de 200 types de HPV ont été isolés et regroupés dans un arbre phylogénétique composé de 5 genres nommés alpha, beta, gamma, mu et nu. Parmi eux, les types HPV muqueux à haut risque appartenant au genre alpha ont été associés au cancer du col de l'utérus ainsi qu'à des sous-groupes de carcinomes ano-génitaux et de la tête et du cou. Ces virus sont responsables d'environ 5% de tous les cancers viro-induits. Les types bêta du HPV ont un tropisme pour la peau et pourraient être impliqués dans le développement du cancer de la peau non mélanique (NMSC), en association avec la lumière ultraviolette (UV). Ainsi, les modèles expérimentaux in vitro et in vivo ont démontré les propriétés de transformation des oncoprotéines E6 et E7 du type HPV bêta 38. De plus, des études sur le modèle de souris transgénique, où E6 et E7 du HPV38 sont exprimés au niveau de la couche basale non différenciée de l'épithélium sous le contrôle du promoteur du gène humain de la kératine (K14), ont montré une très forte susceptibilité de la peau à la carcinogenèse induite par les UV par rapport aux animaux de type sauvage. Tout aussi important que leur capacité à promouvoir la transformation cellulaire, les virus oncogènes ont développé différentes stratégies pour prendre le dessus sur le système immunitaire de l'hôte, favorisant ainsi l'établissement d'une infection persistante. Par conséquent, savoir si des virus oncogènes potentiels ont la capacité d'interférer avec la réponse immunitaire pourrait fournir des preuves supplémentaires de leur implication dans la cancérogenèse humaine. Ici, nous montrons que les oncoprotéines E6 et E7 de HPV38 suppriment l'expression de Tolllike 9 (TLR9), récepteur des ADN double-brins, en favorisant l'accumulation de ΔNp73α, un antagoniste de p53 et p73. Des expériences d'immunoprécipitation de la chromatine ont montré que ΔNp73α fait partie d'un complexe de régulation négative transcriptionnelle qui se lie à un élément de réponse NF-kappaB dans le promoteur TLR9. Fait intéressant, l'expression ectopique de TLR9 dans des cellules HPV38 E6E7 a entraîné une accumulation des inhibiteurs du cycle cellulaire p21WAF1/Cip1 et p27kip1, une réduction de l'activité kinase associée à CDK2 et l'inhibition de la prolifération cellulaire. Ensemble, ces données indiquent que TLR9 est impliqué dans d'autres événements, en plus de la réponse immunitaire innée. Par conséquent, nous avons constaté que le traitement des kératinocytes humains primaires (HPK) avec différents stress cellulaires, par exemple l'irradiation aux UV, la doxorubicine et le traitement H2O2, conduisent à une induction de la transcription de TLR9. Cet évènement induit par les UV est arbitré par le recrutement de plusieurs facteurs de transcription sur le promoteur TLR9, tels que p53, NF-kappaB p65 et c-Jun. L'expression de E6 et E7 de HPV38 affecte fortement le recrutement de ces facteurs de transcription sur le promoteur TLR9, avec comme conséquence l'affaiblissement de l'expression du gène TLR9. En résumé, nos données montrent que HPV38, de manière similaire à d'autres virus avec une activité oncogénique bien connue, peut inhiber 'expression de TLR9. Plus important encore, nous mettons en évidence une nouvelle fonction de TLR9 dans le contrôle de la réponse cellulaire aux stress et nous montrons que E6 et E7 de HPV38 sont capables d'interférer avec un tel mécanisme. Ces résultats confirment le rôle des types HPV bêta dans la carcinogenèse de la peau, en fournissant des informations supplémentaires sur leur contribution précise dans le processus multi-étapes de développement du cancer
The human papillomaviruses (HPV) consist of a group of capsid-enclosed double-stranded deoxyribonucleic acid (dsDNA) viruses from the Papillomaviridae family that display a distinct tropism for mucosal or cutaneous squamous epithelia. Until now, more than 200 types of HPV have been isolated and grouped into a phylogenetic tree composed of 5 genera (alpha, beta, gamma, mu and nu papillomaviruses). Among them, the mucosal high-risk HPV types that belong to the genus alpha have been associated with cervical cancer as well as a subset of anogenital and head and neck carcinomas. They are responsible for approximately 5% of all virus-induced cancers. Beta HPV types have a skin tropism and have been suggested to be involved, together with ultraviolet light (UV), in the development of non-melanoma skin cancer (NMSC). For instance, in vitro and in vivo experimental models highlight the transforming properties of beta HPV38 E6 and E7. Specifically, studies of transgenic mouse model, where HPV38 E6 and E7 are expressed in the undifferentiated basal layer of epithelia under the control of the Keratin 14 (K14) promoter, showed a very high susceptibility to UV-induced skin carcinogenesis in comparison to the wild-type animals. Equally important as their ability to promote cellular transformation, oncogenic viruses have different strategies to overtake the host immune system thus guaranteeing persistent infection. Therefore, understanding whether potential oncogenic viruses have the ability to interfere with the immune response could provide additional evidence relating to their involvement in human carcinogenesis. Here, we show that the E6 and E7 oncoproteins from HPV38 suppress the expression of the dsDNA innate immune sensor Toll-like receptor 9 (TLR9) by promoting the accumulation of ΔNp73α, an antagonist of p53 and p73. Chromatin immunoprecipitation experiments showed that ΔNp73α is part of a negative transcriptional regulatory complex that binds to a NF-κB responsive element within the TLR9 promoter. Interestingly, ectopic expression of TLR9 in HPV38 E6E7 cells resulted in an accumulation of the cell cycle inhibitors p21WAF/Cip1 and p27Kip1, reduction of CDK2-associated kinase activity and inhibition of cellular proliferation. Together these data indicate that TLR9 is involved in additional events, besides the innate immune response. Accordingly, we observed that the treatment of human primary keratinocytes (HPKs) with different cellular stresses, e.g. UV irradiation, doxorubicin and H2O2 treatment, results in TLR9 up-regulation. This UVinduced event is mediated by the recruitment of several transcription factors to the TLR9 promoter, such as p53, NF-kB p65 and c-Jun. The expression of HPV38 E6 and E7 strongly affect the recruitment of these transcription factors to the TLR9 promoter, with consequent impairment of TLR9 gene expression. In summary, our data show that HPV38, similarly to other viruses with well-known oncogenic activity, can down-regulate TLR9. Most importantly, we highlight a novel function of TLR9 in controlling the cellular response to stresses and we show that HPV38 E6 and E7 are able to interfere with such mechanism. These findings further support the role of beta HPV types in skin carcinogenesis, providing additional insight into their precise contribution to the multistep process of cancer development

The serious nature of MDD has intensified the need to identify and elucidate new neurobiological targets for antidepressant drug action. Depression presents with evidence for degenerative pathology that relates to disturbances in excitatory glutamatergic pathways, particularly the N-methyl-D-aspartate (NMDA) receptormediated release of the pleiotropic molecule, nitric oxide (NO), and cyclic guanosine monophosphate (cGMP). The contribution of the glutamate-NO/cGMP pathway may realize great importance as a fundamental substrate underlying the pathophysiology of major depression. In the next generation of antidepressant drugs, the nitric oxide pathway could playa dynamic role in addressing urgent therapeutic needs. In this study, we have used a genetic model of depression, the Flinders Sensitive Line (FSL) rat, to investigate the surrogate markers of the NO/cGMP pathway. The aim was to determine whether the depressive-like behaviour of the hypercholinergic FSL rat is accompanied by altered activation of the NO/cGMP pathway. To this end, the extent to which the FSL and Flinders Resistant Line (FRL) rats differ neurochemically with regard to basal hippocampal and frontal cortical NOS-activity, as well as nitric oxide (NO) and cGMP accumulation, were determined. Additionally, select behavioural assessments were performed to confirm the anxiogenic phenotype of the FSL strain. For neurochemical determinations a sensitive fluorometric reversed phase highperformance liquid chromatographic (HPLC) assay was developed to analyze total nitrite and nitrate in brain tissue. Nitrate was enzymatically converted to nitrite before derivatization with 2,3-diaminonaphthalene (DAN). The stable and highly fluorescent product, 2,3-naphthotriazole (NAT), was quantified. Secondly, the quantity of the amino acid L-citrulline was measured by HPLC with electrochemical detection after o-phthalaldehyde (OPA) derivatization. L-citrulline formation was used as an index for nNOS activity. Finally, a direct, competitive enzyme immunoassay kit was used to determine the downstream activity of the NO-pathway in brain tissue. FSL rats were compared to FRL rats with respect to sensitivity to serotonin 5-HT1A . receptor-mediated hypothermia under our lab-conditions. The Open Field Test (OFT) behavioural assessment was performed to compare FSL with FRL groups under baseline conditions according to their level of inherent anxiety. The parameters used to measure anxiety were number of line crosses (locomotor activity), time spent in middle blocks and social interaction time between pairs of rats. As an additional behavioural assessment, the Forced Swim Test (FST) was performed to assess behavioural restraint measured as time of immobility. Basal cGMP levels in the frontal cortex were found to be significantly less in FSL than in FRL rats, whereas the levels in the hippocampus did not differ significantly. No other significant differences with respect to NO and nNOS activity were apparent in either of the brain areas. The hypothermia test confirmed a significantly greater decrease in temperature in the FSL rat than the FRL rat. The FST did not confirm any differences in immobility time between the two rat strains. In the OFT, FSL rat groups exhibited behaviour that indicated significantly more anxiety than FRL rats. Under basal conditions, FSL rats do not present with significant changes in markers of the NO cascade in the hippocampus and frontal cortex compared to FRL controls, including NOS activity as well as NO accumUlation. However, cGMP levels were found to be significantly lower in the frontal cortex of FSL rats versus FRL rats, although not in the hippocampus. Since the FSL rat is known to be hypercholinergic, these data support an interaction between the NO/cGMP pathway and the cholinergIc system in the frontal cortex but not hippocampus of FSL animals. The mechanisms and implications of such a mutual involvement need further clarification. Further, this anatomical differentiation may have important implications for understanding the role of NO in the depressive-like behaviour of the FSL rat and, indeed, may reveal more on the neurobiology and treatment of depression. Through the performed behavioural assessments, the FSL and FRL rats were successfully separated with respect to their anxiety phenotype as well as their heightened response to serotonergic challenge, thus confirming a contribution of both the serotonergic and cholinergic systems to the depressogenic nature of these animals. As concluding remark can be said that under normal basal conditions markers of the NO/cGMP signalling cascade are not altered in FSL vs FRL rats, although cGMP levels are reduced in the frontal cortex of FSL rats, supportive of an NO-independent mechanism of cGMP regulation, possibly involving ACh.
Thesis (M.Sc. (Pharmacology)--North-West University, Potchefstroom Campus, 2009.

Les troubles neurodéveloppementaux (TND) touchent environ 10 % des enfants et constituent une source majeure d'invalidité tout au long de la vie. Caractérisés par un développement défectueux du cerveau et une grande variabilité du tableau clinique des patients, qui compromet le diagnostic et l'émergence de solutions thérapeutiques, ils représentent un coût humain, sociétal et économique important. L'objectif de ce projet est de mieux comprendre une caractéristique commune des TND - la dérégulation des voies de réponse au stress - qui constituerait une clef de lecture pour comprendre ces pathologies. L'intégration des processus déclenchés par le stress est régie par les facteurs de transcription du choc thermique (HSF), qui sont fortement dérégulés dans plusieurs TND. Cela a deux conséquences : une altération de la réponse au stress des cellules neurales qui entraîne des défauts dans le développement du cerveau. Nous avons participé a montré que ces HSF sont essentiels au bon développement du cerveau. Plus précisément, l'équipe a démontré que HSF2 joue un rôle clef dans la régulation de la prolifération des cellules progénitrices et la migration neuronale dans le cortex en modulant l'expression de gène impliqués dans l'adhésion cellulaire. La modulation pharmacologique de cette voie pourrait donc offrir de nouvelles possibilités thérapeutiques. Dans une première étude, les mécanismes sous-jacents à la dérégulation des HSF ont été étudiés dans les cellules de patients atteints du syndrome de Rubinstein-Taybi (RSTS), un TND rare d'origine génétique causé par des mutations dans les gènes CREBBP ou EP300. Notre étude a montré une diminution des niveaux protéiques de HSF2 dans les fibroblastes et dans les modèles neuraux (2D et 3D) dérivés à partir de cellules souches pluripotentes induites (iPSC) provenant de patients RSTS. Cette diminution des niveaux protéiques de HSF2 résultait d'un défaut d'acétylation par CBP ou EP300, conduisant à l'ubiquitination et à la dégradation par le protéasome. En conséquence, les cellules RSTS présentaient une altération de la réponse au stress et une réduction de l'expression de gènes essentiels au développement neural, en particulier la N-cadhérine. La restauration des niveaux de HSF2, soit par l'inhibition du protéasome, soit par des mutations imitant l'acétylation, a permis de rétablir à la fois la réponse au stress et l'expression des gènes du neurodéveloppement. Nous avons constaté que la perturbation de la voie CBP/EP300-HSF2-N-cadhérine est récapitulée dans les modèles neuraux RSTS, qui présentent des anomalies de prolifération liées à une altération de l'adhésion cellule-cellule, en particulier dans la voie de la N-cadhérine. Sur la base de ces résultats et en collaboration avec Ksilink, mon projet de thèse CIFRE vise à développer un modèle cellulaire de TND basé sur les patients RSTS. Ce modèle permettra d'explorer comment les perturbations de la voie HSF pourraient contribuer à divers TND. Pour atteindre cet objectif, j'ai d'abord généré un mutant HSF2 qui mime la forme acétylée de la protéine dans les iPSC dérivés de fibroblastes de patients RSTS. En utilisant ce modèle isogénique comme référence, j'ai développé et validé un modèle de culture neural bidimensionnel et identifié de nouvelles cibles et phénotypes dépendants de HSF2 via une approche multiparamétrique allant de la transcriptomique à haut débit à des analyses morphologiques des cellules. Cette approche a permis d'identifier le facteur pro neuronal, ASCL1, et un phénotype morphologique, la formation de rosettes, comme clefs de lecture pour l'analyse par imagerie à haut contenu. Sur la base de ces deux phénotypes, j'ai utilisé le modèle neural pour cribler une sélection de molécules à potentiel thérapeutique par imagerie à haut contenu. Ces travaux ouvriront la voie à de nouvelles approches thérapeutiques visant à moduler les voies de réponse au stress, offrant ainsi de nouvelles possibilités de traitement des TND
Neurodevelopmental disorders (NDD) affect around 10% of children and are a major source of lifelong disability. Characterised by defective brain development and great variability in the clinical picture of patients, which compromises diagnosis and the emergence of therapeutic solutions, they represent a significant human, societal and economic cost. The aim of this project is to gain a better understanding of a common feature of NDDs - the deregulation of stress response pathways - which could provide a readout to understanding these pathologies. The integration of processes triggered by stress is governed by heat shock transcription factors (HSFs), which are strongly deregulated in several NDDs. This has two consequences: an altered stress response in neural cells leading to defects in brain development. We have helped to show that these HSFs are essential for proper brain development. More specifically, the team demonstrated that HSF2 plays a key role in regulating the proliferation of progenitor cells and neuronal migration in the cortex by modulating the expression of genes involved in cell adhesion. Pharmacological modulation of this pathway could therefore offer new therapeutic possibilities. In a first study, the mechanisms underlying HSF deregulation were investigated in cells from patients with Rubinstein-Taybi syndrome (RSTS), a rare genetic NDD caused by mutations in the CREBBP or EP300 genes. Our study showed a decrease in HSF2 protein levels in fibroblasts and in neural models (2D and 3D) derived from induced pluripotent stem cells (iPSCs) from RSTS patients. This decrease in HSF2 protein levels resulted from a defect in acetylation by CBP or EP300, leading to ubiquitination and degradation by the proteasome. As a result, RSTS cells showed an altered stress response and reduced expression of genes essential for neural development, in particular N-cadherin. Restoration of HSF2 levels, either by proteasome inhibition or by acetylation-mimicking mutations, restored both the stress response and the expression of neurodevelopmental genes. We found that disruption of the CBP/EP300-HSF2-N-cadherin pathway is recapitulated in RSTS neural models, which display proliferation abnormalities linked to altered cell-cell adhesion, particularly in the N-cadherin pathway. On the basis of these results and in collaboration with Ksilink, my CIFRE thesis project aims to develop a cellular model of NDD based on RSTS patients. This model will enable us to explore how perturbations in the HSF pathway could contribute to various NDDs. To achieve this objective, I first generated an HSF2 mutant that mimics the acetylated form of the protein in iPSCs derived from RSTS patient fibroblasts. Using this isogenic model as a reference, I developed and validated a two-dimensional neural culture model and identified new HSF2-dependent targets and phenotypes using a multiparametric approach ranging from high-throughput transcriptomics to cell morphological analyses. This approach made it possible to identify the pro-neuronal factor, ASCL1, and a morphological phenotype, rosette formation, as key readouts for analysis by high-content imaging. On the basis of these two phenotypes, I used the neural model to screen a selection of molecules with therapeutic potential using high-content imaging. This work will pave the way for new therapeutic approaches aimed at modulating stress response pathways, thereby opening up new possibilities for the treatment of NDD

Peroxiredoxins are small ubiquitous cysteine-containing proteins that exhibit high reactivity to hydrogen peroxide. Apart from their role as antioxidants, detoxifying hydrogen peroxide to water, peroxiredoxins have been implicated in other cellular processes, such as protein folding and signalling. Using S. cerevisiae as a model organism, we utilised a variety of techniques to examine previously unexplored links between peroxiredoxins and mitochondrial function. Firstly, we characterised the role of Gpx3 in yeast mitochondria. Proteomic work revealed the presence of Gpx3 in the mitochondrial intermembrane space (IMS) and we characterised when, how and why Gpx3 can be found within the mitochondria. We showed that cells lacking Gpx3 have aberrant mitochondrial morphology and defective protein import capacity and inner membrane potential upon H2O2 stress. Gpx3 translocates to the IMS via a targeting sequence encoded from a non-AUG codon. This provides a novel and unique molecular mechanism that protects mitochondria from the exceptional oxidative stress which their activity imposes. Secondly, we focused on the role of Tsa1 upon protein aggregation-induced stress. Previous studies using the proline analogue AZC to cause protein misfolding revealed that protein aggregates are localised adjacent to mitochondria and mitochondrial ROS are generated in response. We questioned what effect this might have on mitochondrial function and we showed that upon AZC treatment there is a drop in respiratory rate, dependent on Tsa1. We questioned whether Tsa1, like other peroxiredoxins, is involved in regulating signalling cascades and we showed that cells that are lacking Tsa1 have alterations in the activity of the cAMP/PKA pathway. In parallel, we looked for differences both in the proteome and the transcriptome to understand what is the cause of the lethality of a tsa1 strain upon protein aggregation stress. We propose a mechanism where Tsa1 mediates a transcriptional response to protein misfolding stress via the activity of the heat shock transcription factor, Hsf1. Finally, we focused on the role of the mitochondrial peroxiredoxin Prx1. Under conditions where the mitochondrial matrix is oxidised, either genetically or by chemical addition, we showed than an apoptotic pathway is activated, dependent on the redox state of thioredoxin, Trx3. We showed that Trx3 can interact with Prx1 and loss of Prx1 also stops the induction of cell death. Analysis of the interactome of Trx3 unraveled the involvement of Bxl1/Ybh3, the yeast BH3 domain-containing protein and Aim9, a previously uncharacterised protein with kinase-like motifs, in the progression of cell death. The data presented in this thesis widens our understanding of the function of peroxiredoxins and their involvement in the regulation of cellular cascades that ensure correct mitochondrial function and responses to stress.

[Truncated abstract] Reactive oxygen species (ROS) have been implicated to play a major role in many pathological conditions including heart attack and stroke. Their ability to modulate the extracellular signal-regulated protein kinase (ERK) and c-Jun Nterminal kinase (JNK) signalling pathways, thereby influencing cellular response has been well-documented. Recent studies implicate a central role for mitochondria in ERK and JNK activation by ROS although the mechanisms remained unresolved. Using Jurkat T-lymphocyte as a cell model, this study demonstrated increased mitochondrial ROS production as a result of decreased mitochondrial complex activities mediated by hydrogen peroxide treatment. This is the first study to show that mitochondria are not essential for activating ERKs, however damaged mitochondria producing ROS can be expected to cause sustained ERK activation . . . This study revealed that JNK and its upstream kinases MKK4, MKK7 and ASK1 are associated with the mitochondria. Furthermore, findings from this study imply that JNK resides in the mitochondrial matrix. This study is the first to demonstrate that mitochondrial JNK can be activated in a cell-free environment by signals originating from the mitochondria. Experimental work using isolated mitochondria demonstrated that mitochondrial JNK can be activated by ROS generated from the mitochondria themselves. Flavin-containing proteins appear to be the main sources of mitochondrial-ROS which signal through redoxsensitive proteins to activate mitochondrial JNK.

Upon exposure to heat or cold, Arabidopsis thaliana seedlings undergo rapid transcriptional reprogramming of several hundreds of genes that promote stress tolerance. Despite extensive characterisation of the transcriptional responses to these stimuli, however, relatively little is known about the mechanisms by which temperature signals are perceived and transduced in plant cells. High or low seasonal temperatures have large impacts on crop productivity and are expected to intensify given current global climatic projections. It is therefore of agricultural importance to better understand temperature signalling pathways in plants in order to find solutions to this problem. In this thesis, a chemical genomics screen for molecules activating or repressing heat-inducible genes in A. thaliana was performed in collaboration with Syngenta and the biological targets of these chemicals were predicted based on structural similarities to compounds with known modes of action. Many molecules that affect the function of chloroplasts or mitochondria either activate or repress heat-responsive genes, thus implicating these organelles in the regulation of plant temperature responses. In addition, the translation inhibitor cycloheximide was identified as a repressor of heat-inducible genes and an activator of early cold-inducible genes. Diverse translation inhibitors trigger a cytosolic influx of calcium ions and several inhibitors of translation elongation were found to strongly activate cold-inducible gene expression in a calcium-dependent manner. Furthermore, it was demonstrated that cold shock causes rapid translation repression in A. thaliana seedlings and that the elongation factor LOS1 is involved in cold- or cycloheximide-induced gene expression, thus implicating translational machinery in the regulation of temperature signalling in plants. Finally, one of the chemicals identified in the screen, S01A463859Y, was found to improve heat resilience in A. thaliana and may therefore be of potential use in enhancing crop productivity during thermal stress.

Contemporary theories regard that systemic inflammatory response in acute pancreatitis is elicited by the secretion of the pro-inflammatory cytokines, leukocytes activation and their interaction with the activated vascular endothelium. There is increasing evidence of the role of acinar cell injury and oxidative stress injury as key events of localised pancreatic response in acute pancreatitis, which triggers the production of pro-inflammatory cytokines and subsequently the escalation of systemic inflammatory response and remote organ dysfunction. The aim of this thesis is to assess the role of anti-oxidant supplementation in the outcome of severe acute pancreatitis and to investigate the effect of a central inflammatory pathway inhibition in acute pancreatitis.

The main aim of this project was to study Candida albicans cell wall biosynthesis in response to stress. The role of the MAPK, Ca2+/calcineurin and cAMP/PKA signal transduction pathways in regulating the C. albicans cell wall stress response was investigated. A library of mutants lacking receptors, signalling elements and transcription factors were screened for alterations in their ability to respond to a range of cell wall stressing agents, including CaCl2, Calcofluor White and caspofungin. Pretreatment of wild-type cells with CaCl2 and CFW, activates the Ca2+/calcineurin and PKC pathways, leading to an increase in chitin content, and reduced susceptibility to caspofungin. Although elevation of cell wall chitin content often resulted in decreased sensitivity to caspofungin, I show here that some strains with increased chitin levels remained sensitive to caspofungin. The results show that elevation of chitin is a common property of a range of mutants that are affected in coordinating cell wall stress pathways, but that multiple mechanisms are likely to operate in maintaining the robustness of the C. albicans cell wall. Some of the mutant strains of the MAPK, Ca2+/calcineurin and cAMP signalling pathways showed evidence of paradoxical growth, whereby less inhibition was achieved by higher concentrations of antifungal drug. The role of chitin-related genes and stress signalling pathways in regulating C. albicans paradoxical growth was also investigated. Based on these results, more detailed analyses were performed to investigate the correlations between sensitivity and resistance to caspofungin, in relation to paradoxical growth. The MAPK-Mkc1 and the calcineurin pathways played major roles in the paradoxical growth effect. There was a proportional relationship between echinocandin concentration and the chitin content of the cell wall although the chitin content did not continue to be upregulated by the highest echinocandin concentration. Different echinocandins, carbon source, cell morphology and medium composition influenced the extent of paradoxical growth effect. The existence of paradoxical growth in resistant strains such as Fks1 also highlights association of paradoxical growth with resistance mechanisms.

Les conditions contraignantes provoquent la modification de l’état redox et la signalisation liée aux formes actives de l’oxygène (ROS), dont les concentrations sont régulées par des systèmes antioxydant complexes. On dénombre de plus en plus de processus qui sont affectés par la régulation redox, mais nous avons toujours des connaissances fragmentaires quant à l’importance des interactions centrales entre ROS et systèmes antioxydants pour la signalisation cellulaire chez les plantes. Cette étude a utilisé des approches de génétique classique et inverse chez l’espèce-modèle, Arabidopsis thaliana dans le but d’élucider les rôles des catalases et des systèmes NADPH-glutathion-ascorbate dans le métabolisme du H₂O₂ et la signalisation qui en dépend. Une analyse de mutants ADN-T pour les trois gènes codant la catalase a révélé que la mutation cat2, à la différence de cat1 et de cat3, a fortement affecté la croissance et le développement de la plante. Lorsqu’il était cultivé dans l’air, le mutant cat2 présentait une croissance réduite à la fois au niveau de la rosette et des racines, mais ces effets étaient absents lors de la culture des plantes sous un taux de CO₂ élevé, suggérant que la taille diminuée est causée, directement ou indirectement, par une capacité compromise de métaboliser le H₂O₂ produit par la photorespiration. Une étude de cat2 cultivé dans des photopériodes différentes a mis en évidence une forte influence de la période d’illumination sur la signalisation oxydative et ceci d’une manière qui est indépendante de l’intensité du stress. Lorsque cat2 est cultivé en jours longs, le stress oxydant induit la voie de l’acide salicylique (SA), provoquant des lésions visibles sur les feuilles. Cette réponse au stress oxydant est annulée dans un double mutant cat2 g6pd5, chez lequel l’expression d’une forme spécifique de la glucose-6-phosphate déshydrogénase (G6PDH) a également été inactivée. Une approche de génétique classique a permis d’identifier plusieurs gènes susceptibles d’être impliqués dans la régulation de la formation de lésions SA-dépendante dans ce double mutant. Afin d’explorer les rôles des monodéshydroascorbate réductases (MDHAR) spécifiques dans des conditions optimales et de stress, des mutants d’insertion pour plusieurs gènes codant la MDHAR ont été obtenus. Ces mutants présentaient un phénotype sauvage dans des conditions de culture optimales, mais à la suite de son introduction dans le fond cat2, l’un d’entre eux a fortement modifié l’induction de la voie SA par le stress oxydant. Pris dans leur ensemble, les résultats soulignent l’importance de CAT2 et permettent de dessiner un lien fonctionnel entre des G6PDH et MDHAR spécifiques dans les voies de signalisation oxydative chez Arabidopsis, lien qui pourrait s’expliquer par la production de NADPH par la G6PDH et son utilisation par la MDHAR
Stress conditions lead to modified redox states and signaling linked to reactive oxygen species (ROS), whose cellular concentrations are regulated by complex antioxidative systems. While the list of processes subject to redox regulation continues to grow, our understanding of the importance of the core interactions between ROS and plant antioxidative systems in cell signaling remains very fragmentary. This work used forward and reverse genetics to analyze the roles of catalases and the NADPH-glutathione-ascorbate systems in H₂O₂ metabolism and related signaling in the model species, Arabidopsis thaliana. An analysis of T-DNA mutants for the three catalase-encoding genes revealed that cat2, but not cat1 or cat3, substantially impacted plant growth and development. While the cat2 mutant showed decreased shoot and root size when grown in air, both these effects were annulled by growth at high CO₂, suggesting that they were caused, directly or indirectly, by compromised capacity to metabolize photorespiratory H₂O₂. An analysis conducted in cat2 rosettes following growth in different photoperiods revealed that oxidative signaling is strongly influenced by day length in a manner that is independent of stress intensity. When cat2 is grown in long days, oxidative stress induces the salicylic acid (SA) pathway, leading to visible lesions on the leaves. This response to oxidative stress is annulled in cat2 g6pd5, which has additionally lost the function of a specific glucose-6-phosphate dehydrogenase (G6PDH). A forward genetics approach identified several genes that may be involved in regulating SA-dependent lesion formation in this double mutant. To explore the roles of specific monodehydroascorbate reductases (MDHAR) in optimal and stress conditions, insertion mutants for several MDHAR-encoding genes were obtained. While these mutants showed a wild-type phenotype in optimal growth conditions, one of them markedly altered induction of the SA pathway by oxidative stress when introduced into the cat2 background. Together, the results underline the importance of CAT2 and point to functional coupling between specific NADPH-producing G6PDH and NADPH-requiring MDHAR in oxidative stress signaling pathways in Arabidopsis

BACKGROUND:FGF signalling regulates numerous aspects of early embryo development. During gastrulation in amniotes, epiblast cells undergo an epithelial to mesenchymal transition (EMT) in the primitive streak to form the mesoderm and endoderm. In mice lacking FGFR1, epiblast cells in the primitive streak fail to downregulate E-cadherin and undergo EMT, and cell migration is inhibited. This study investigated how FGF signalling regulates cell movement and gene expression in the primitive streak of chicken embryos.RESULTS:We find that pharmacological inhibition of FGFR activity blocks migration of cells through the primitive streak of chicken embryos without apparent alterations in the level or intracellular localization of E-cadherin. E-cadherin protein is localized to the periphery of epiblast, primitive streak and some mesodermal cells. FGFR inhibition leads to downregulation of a large number of regulatory genes in the preingression epiblast adjacent to the primitive streak, the primitive streak and the newly formed mesoderm. This includes members of the FGF, NOTCH, EPH, PDGF, and canonical and non-canonical WNT pathways, negative modulators of these pathways, and a large number of transcriptional regulatory genes. SNAI2 expression in the primitive streak and mesoderm is not altered by FGFR inhibition, but is downregulated only in the preingression epiblast region with no significant effect on E-cadherin. Furthermore, over expression of SNAIL has no discernable effect on E-cadherin protein levels or localization in epiblast, primitive streak or mesodermal cells. FGFR activity modulates distinct downstream pathways including RAS/MAPK and PI3K/AKT. Pharmacological inhibition of MEK or AKT indicate that these downstream effectors control discrete and overlapping groups of genes during gastrulation. FGFR activity regulates components of several pathways known to be required for cell migration through the streak or in the mesoderm, including RHOA, the non-canonical WNT pathway, PDGF signalling and the cell adhesion protein N-cadherin.CONCLUSIONS:In chicken embryos, FGF signalling regulates cell movement through the primitive streak by mechanisms that appear to be independent of changes in E-cadherin expression or protein localization. The positive and negative effects on large groups of genes by pharmacological inhibition of FGF signalling, including major signalling pathways and transcription factor families, indicates that the FGF pathway is a focal point of regulation during gastrulation in chicken.

Pairing of cancer genome and transcriptome data has revealed that heterozygous mutations aren’t always expressed in cells. The potential for point mutation or genomic rearrangement to alter tumour allelic expression has implications for understanding cellular heterogeneity and application of treatments. Mutation of SPOP, PTEN and IDH-1 was assessed in 51 primary prostate cancer cultures to establish allelic heterozygosity and ascertain whether oncogenic change to coding regions altered allelic expression. No mutations were detected in the three genes, although 18% of tested cultures had loss of heterozygosity in PTEN. The TMPRSS2-ERG fusion, present in half of all prostate cancers, is selectively expressed at an allelic level by cancer stem cells. Monoallelic expression didn’t correlate with TMPRSS2 promoter hypermethylation. Prostate cultures expressed fusion transcript, however epigenetic features of monoallelically expressed genes were not investigated in the epithelial subpopulations. Understanding of allelic chromatin states may inform treatment strategies that permit tumour suppressor expression or oncogenic protein repression. Inability to predict indolent or aggressive progression of organ-confined prostate cancers has created the problem of surgical overtreatment. Focal therapies targeting the tumour core are being met with increasing rates of recurrence, necessitating development of novel treatments. The anti-cancer properties of Low Temperature Plasma (LTP) are being explored in prostate models where it produces autophagy and necrosis through generation of reactive species. Initial gene expression response to LTP and the activation of upstream transcription factors were analysed. LTP activated Nrf2, AP-1 and Notch signalling in patient matched prostate normal and cancer cultures. The progenitor-containing cell fraction was more responsive to LTP than differentiated epithelial cells in both transcription of response genes and nuclear accumulation of active Notch1. When linked to cell-fate outcomes, these immediate molecular responses of prostate cancer to LTP could be used as hallmarks of resistance or treatment efficacy in patients.

The aim of this thesis was to examine role of Sirt1 in the Notch signalling pathway, using Drosophila as a model organism. Based on in vivo and in vitro studies, we conclude that Sirt1 plays a positive role in Notch signalling. In embryonic S2N cells, Sirt1 is responsible for the protection from metabolic stress-induced down-regulation of subset of E(Spl) genes. During development, Sirt1 is responsible for proper Notch-dependent specification of SOPs and wing development. Sirt1 can regulate the Notch signalling on multiple levels via deacetylation of various substrates involved in the Notch signalling revealed by our proteomic survey.

Redox homeostasis maintenance is important for proper organism and cell function, for while relatively low amount of reactive oxygen (and nitrogen) species contributes to the fine tuning of signal transduction, excessive concentration of ROS (oxidative stress) has demonstrably harmful effects and is tightly connected to many pathological states. Cells therefore evolved broad palette of antioxidant mechanisms that express striking level of conservation among different species. Large, intricate stress response signaling networks have been already described; nonetheless, novel molecules employed in stress-related signaling are still being discovered. Several studies recently suggested transcription factors CSL, proteins essential for regulation of metazoan development as effectors of Notch signaling, are also involved in response to oxidative stress. The fission yeast Schizosaccharomyces pombe, well established model of response to various stresses, comprises two paralogs of CSL proteins - Cbf11 and Cbf12. We have found cells depleted of cbf11 are highly resistant to hydrogen peroxide. This resistance appears to be caused by upregulation of important stress responsive genes including ctt1, gst2, pyp2, and atf1. Cbf11 is therefore negative regulator of these genes, which suppresses their expression...

Research Doctorate - Doctor of Philosophy (PhD)
The lateral hypothalamus (LH) is anatomically positioned to receive and send an array of afferent and efferent signals within the brain. This is important as distinct behaviours relating to homeostasis are coordinated and executed by LH cells and signalling pathways. The LH can control appetitive behaviour as well as motivation and reward-seeking, as well as being sensitive to stress signals. Importantly, the LH has a heterogenous composition, comprising of many diverse cell types. The functional importance of these intra-LH populations is incompletely understood. Further, neuroscience research is only recently beginning to unravel the complex nature of afferent signals which can help control output from the LH. Afferent projections from brains areas such as the paraventricular nucleus of the hypothalamus (PVN) and nucleus accumbens shell (NAcSh) can control stress and reward-related behaviour, respectively. These regions are well positioned anatomically and physiologically to send important communications to the LH in order to help maintain homeostatic functioning. This thesis firstly demonstrated that early life stress-induced motivational deficits have the capacity to be overcome by chemogenetic manipulation of the LH. Next, we saw that repeated optogenetic stimulation of the PVN can reduce motivational drive for natural rewards. Importantly, these changes could be recapitulated by isolating the PVN→LH pathway. Lastly, using fibre photometry we found that orexin neurons almost exclusively respond to approach behaviour to food pellets and that the activity of orexin neurons depended on metabolic state and the palatability or caloric value of the food. Additionally, we provide electrophysiological evidence that this behaviour may be mediated by an inhibitory projection from the NAcSh. Overall, this work demonstrates that hypothalamic and extra-hypothalamic brain regions can provide vital input to the LH in mediating stress and motivated behaviours.

Tese de mestrado, Biologia Molecular e Genética, Universidade de Lisboa, Faculdade de Ciências, 2018
As plantas, devido à sua incapacidade de locomoção, estão confinadas ao local onde germinaram sendo, por isso, vulneráveis a condições ambientais que restringem o seu crescimento e desenvolvimento. Temperaturas extremas, seca, inundações, elevada salinidade, exposição a metais pesados, lesões mecânicas, condições de luz inadequadas e infeção por patogéneos estão entre as maiores causas de perda de produtividade agrícola a nível mundial. Para fazer face a estas flutuações ambientais, as plantas desenvolveram, por um lado, estratégias adaptativas de sobrevivência de carácter específico, que lhes permitem responder a um tipo particular de stress e, por outro, mecanismos gerais responsáveis pelo ajuste metabólico e pela reprogramação da expressão de genes, permitindo, assim, rapidamente reparar os componentes celulares danificados e alocar nutrientes para os processos adequados, de forma a restaurar a homeostasia. Em condições normais, as plantas convertem a luz em energia química sob a forma de açúcares que são depois distribuídos pelos vários órgãos da planta permitindo o seu correto crescimento e desenvolvimento. Contudo, condições desfavoráveis com impacto deletério nos processos de fotossíntese e respiração resultam, frequentemente, na diminuição dos níveis de energia celular da planta e afetam a alocação de açúcares para os órgãos em crescimento, levando à ativação da proteína cinase SnRK1. Esta, por sua vez, para restabelecer a homeostasia, ativa processos catabólicos e inibe processos anabólicos através da fosforilação de diversas enzimas metabólicas e de uma extensa reprogramação do transcriptoma, permitindo, assim, a aclimatação e sobrevivência das plantas. A SnRK1 pertence a uma família altamente conservada de cinases e partilha semelhanças estruturais e funcionais com as proteínas ortólogas “AMP-activated protein kinase” (AMPK), nos mamíferos, e “Sucrose non-fermenting 1” (SNF1), nas leveduras, funcionando como um complexo heterotrimérico composto por uma subunidade catalítica α e duas subunidades regulatórias, β e γ. Em Arabidopsis, existem três diferentes isoformas de SnRK1α, apesar de apenas duas (codificadas pelos genes SnRK1α1/KIN10 e SnRK1α2/KIN11) serem expressas constitutivamente em todos os tecidos da planta, três diferentes isoformas de SnRK1β (codificadas pelos genes SnRK1β1, SnRK1β2 e SnRK1β3) e apenas uma isoforma da subunidade γ, SnRK1βγ. Não obstante a sua enorme importância a nível da resposta a diferentes tipos de stress, a via de sinalização da SnRK1 vai além do mero ajuste metabólico nessas condições, estando igualmente implicada na sinalização de açúcares, associada a vias de sinalização mediadas por várias hormonas vegetais e envolvida na modulação do crescimento e desenvolvimento das plantas. Contudo, apesar do seu papel central, são poucos os mecanismos descritos, até à data, capazes de explicar a reprogramação transcripcional desencadeada pela SnRK1. O aumento da expressão de determinados genes, por seu lado, é atribuído em grande parte a fatores de transcrição “basic leucine Zipper” (bZIP) bem estabelecidos enquanto efetores a jusante da via de sinalização da SnRK1. Por outro lado, quanto à repressão de genes, os mecanismos subjacentes continuam maioritariamente desconhecidos, embora os microRNAs (miRNAs) tenham sido implicados na repressão de alguns dos alvos de SnRK1, ainda que através de mecanismos desconhecidos. Os miRNAs correspondem a uma classe de pequenos RNAs endógenos não codificantes com 20-24 nucleótidos que regulam a expressão de genes pós-transcricionalmente, através da clivagem e/ou do bloqueio da tradução de transcritos complementares. Desta forma, os miRNAs têm sido extensivamente implicados tanto no crescimento e desenvolvimento das plantas como na resposta a fatores de stress biótico e abiótico. O trabalho desenvolvido nesta tese de mestrado teve como principal objetivo aumentar o conhecimento sobre os mecanismos envolvidos na comunicação entre a via de sinalização de SnRK1 e os miRNAs. Para isso, foram testadas duas hipóteses não mutuamente exclusivas: a) SnRK1 afeta a biogénese de miRNAs e b) SnRK1 afeta a atividade de miRNAs. A biogénese de miRNAs ocorre no núcleo, englobando várias processos interdependentes desempenhados por componentes organizados num complexo. Em linhas gerais, a RNA Polimerase II (Pol II) é recrutada para o gene MIR, promovendo a sua transcrição e dando origem a um miRNA primário (pri-miRNA) que é processado pela proteína “DICER-like 1” (DCL1) com o auxílio das proteínas “Hyponastic-leaves 1” (HYL1) e “Serrate” (SE), originando um percursor de miRNA (pré-miRNA). Este pré-miRNA é novamente processado pela DCL1, originando uma pequena cadeia dupla formada por miRNA/miRNA*, que constituem respectivamente a cadeia guia e a cadeia passageira. A extremidade 3’ do duplex miRNA/miRNA* é metilada pela metil-transferase “Hua-Enhancer 1” (HEN1). Ainda no núcleo, a cadeia passageira é normalmente alvo de degradação e a cadeia guia, que constitui o miRNA maduro, é reconhecida e incorporada no complexo “RNA-induced Silencing Complex” (RISC). Este complexo tem como principal efetor uma proteína da família “ARGONAUTE” (AGO), responsável pelo reconhecimento, já no citoplasma, de transcritos-alvo com sequência complementar à do miRNA, e pela sua subsequente clivagem ou bloqueio de tradução. Problemas na biogénese de miRNAs refletem-se, normalmente, num aumento de pri-miRNAs e numa acumulação deficiente de miRNAs maduros. Deste modo, para testar a primeira hipótese, comecei por analisar a acumulação de dois miRNAs específicos – miR156 e miR319 – em plantas Arabidopsis tipo silvestre e em mutantes com perda parcial de função de SnRK1. Os resultados mostraram que a inativação parcial de SnRK1 levou à redução dos níveis de expressão de ambos os miRNAs testados. Ainda que não se consiga apurar por agora se este é um efeito específico para os miRNAs testados ou se se trata de um mecanismo geral, a confirmar-se futuramente estes mesmos resultados para outros miRNAs, pode potencialmente significar que a atividade de SnRK1 é uma condição necessária para a correta acumulação de miRNAs. Para perceber se SnRK1 afeta a atividade de miRNAs, e com base em resultados preliminares do laboratório da Baena-González, procurei avaliar se SnRK1 poderia eventualmente estar a interagir com a proteína AGO1, o maior efector do complexo de silenciamento RISC. O correto reconhecimento dos miRNAs pela proteína AGO adequada e a sua subsequente incorporação no complexo RISC representa a etapa final da biogénese de miRNAs e é crítico para a sua interação com os respetivos transcritos-alvo. Em Arabidopsis, a família AGO é constituída por dez membros, mas os miRNAs são, na sua grande maioria, incorporados na AGO1. Assim, comecei por testar a interação física entre SnRK1 e AGO1 através de ensaios par-a-par de dois híbridos em levedura (“Yeast Two Hybrid” - Y2H) seguido de uma co-imunoprecipitação. Apesar de ter sido detetada uma interação positiva entre SnRK1α1 e AGO1 em levedura, através da co-imunoprecipitação não foi possível detetar interações entre as duas proteínas in planta em folhas maduras de roseta. Este resultado poderá ser talvez devido ao carácter transiente ou fraco da interação ou à sua ocorrência específica em determinados tecidos ou fases de desenvolvimento. No futuro, seria importante repetir esta experiência em tecidos ou condições em que a expressão de AGO1 seja mais elevada, eventualmente, otimizando ainda as condições da co-imunoprecipitação, para que se possa confiantemente descartar ou confirmar a ocorrência de interação física entre SnRK1 e AGO1 in planta. Paralelamente a esta abordagem bioquímica, procurei ainda perceber se existia alguma interação genética entre AGO1 e SnRK1. Para tal, mutantes ago1-27, deficientes no silenciamento pós-translacional de genes, foram cruzados com mutantes com ganho e perda de função de SnRK1α1. A interação genética foi avaliada com base na caracterização fenotípica dos processos de germinação e enverdecimento dos cotilédones na presença de elevados níveis de glucose ou ácido abscísico (ABA) e com base no tempo de floração em condições de dias longos. Relativamente à germinação e enverdecimento dos cotilédones, enquanto que, sob elevados níveis de glucose, a mutação ago1-27 parece potenciar a hipersensibilidade do sobreexpressor de SnRK1α1, sob elevadas concentrações de ABA é o sobreexpressor de SnRK1α1 que parece aumentar o fenótipo hipersensível do mutante ago1-27. Estes resultados sugerem uma possível regulação negativa de AGO1 sobre SnRK1 e vice-versa cuja intensidade e sentido podem variar consoante o tipo de stress. Curiosamente, a mutação snrk1α1-3 reverteu parcialmente o fenótipo de floração atrasado do mutante ago1-27, com o duplo mutante a florir em média dois dias antes do que as plantas ago1-27 e com menos folhas de roseta do que as plantas do tipo silvestre, reforçando ainda mais o potencial papel de AGO1 enquanto regulador negativo de SnRK1. Finalmente, para explorar a interação funcional entre SnRK1 e AGO1, desenvolvi importantes ferramentas genéticas baseadas numa linha repórter SUC::SUL que irão permitir, no futuro, testar até que ponto diferentes níveis de SnRK1 influenciam a atividade silenciadora de AGO1. No sistema repórter SUC::SUL, um RNA de cadeia dupla do gene SULPHUR, é expresso sob o controlo do promotor SUC2, específico das células de companhia do floema, dando origem a pequenos RNAs de interferência que são incorporados na AGO1, reprimindo, assim, o transcrito SUL (envolvido na biossíntese de clorofila) e causando a clorose das células silenciadas na vasculatura. Novas linhas de plantas homozigóticas para este repórter contendo diferentes mutações das subunidades catalíticas de SnRK1 foram geradas e estão prontas para ser usadas em ensaios futuros para os quais a configuração experimental foi também aqui otimizada. Em suma, os resultados apresentados nesta tese fornecem novas evidências que apontam para uma possível ação de SnRK1 em diferentes níveis de regulação sobre a via de sinalização dos miRNAs, nomeadamente na acumulação de miRNAs e na atividade de AGO1, para controlar o crescimento, desenvolvimento e respostas das plantas a stress. Estudos futuros serão necessários para confirmar a extensão e mecanismo de impacto de SnRK1 na biogénese de miRNAs e para dissecar em detalhe a interação com AGO1.
As sessile organisms, plants are constantly exposed to environmental stresses that limit photosynthesis and/or respiration, thereby compromising ATP production, growth, and ultimately survival. To cope with these conditions, plants have evolved mechanisms that promote stress and defence responses at the expense of growth until the environmental conditions become favourable again. A core component of stress signalling pathways is the evolutionarily conserved sucrose non-fermenting 1 (SNF1)-related protein kinase 1 (SnRK1), the plant ortholog of yeast SNF1 and mammalian AMP-activated protein kinase (AMPK). SnRK1 is activated in response to declining energy levels during stress, implementing a vast metabolic and transcriptional reprogramming that restores homeostasis and thereby promotes plant survival. How SnRK1 regulates gene expression, and in particular how it exerts gene repression is poorly understood, but several lines of evidence suggest the involvement of microRNAs (miRNAs) in this process. miRNAs are 20–24nt non-coding RNAs that regulate gene expression posttranscriptionally mainly through cleavage and/or translation repression of complementary mRNA targets. MiRNAs have been extensively implicated in plant growth and development, but also in responses to environmental stress. SnRK1 has been shown to repress particular targets in a miRNA-dependent manner, suggesting that SnRK1 and miRNAs signalling pathways may be interconnected, even though the mechanisms underlying this connection are currently unclear. The aim of this thesis was to explore the mechanisms underlying this crosstalk by testing two non-mutually exclusive hypotheses: a) SnRK1 affects miRNA biogenesis and b) SnRK1 affects miRNA activity. To test the first hypothesis, I compared the levels of two specific miRNAs, miR156 and miR319, between wild-type Arabidopsis plants and SnRK1 partial loss-of-function mutants. Results showed that the partial inactivation of SnRK1 is accompanied by reduced levels of both miRNAs, suggesting that SnRK1 may be required for miRNA accumulation. To address whether SnRK1 affects miRNA activity and based on preliminary results from the Baena-González Lab, I asked whether SnRK1 could interact with ARGONAUTE 1 (AGO1), the major effector of the RNA-induced silencing complex (RISC). MiRNA activity is dependent on the correct loading of mature miRNAs into appropriate AGO proteins. In Arabidopsis, there are ten members of the AGO family, but most miRNAs are loaded into AGO1. I started by testing for a physical interaction between SnRK1 and AGO1 using yeast-two-hybrid (Y2H) pairwise assays, followed by co-immunoprecipitation from plant extracts. Results showed that SnRK1α1 physically interacts with AGO1 in yeast; on the other hand, by co-immunoprecipitation I was unable to detect in planta interactions between SnRK1α1 and AGO1 in mature leaves. However, to confidently discard or confirm a physical interaction in planta, it would be important to repeat the co-immunoprecipitation in tissues or conditions with higher AGO1 expression and, eventually, to optimise further the co-immunoprecipitation conditions. In parallel, I asked whether there were genetic interactions between AGO1 and SnRK1. For that, ago1-27 mutants were crossed to SnRK1α1 loss- and gain-of-function mutants and their genetic interaction was evaluated using both a phenotypic characterisation of early seedling development under increasingly high concentrations of glucose or ABA and a flowering time assessment in long day conditions. Regarding seedling development, ago1-27 enhances the hypersensitivity of the SnRK1α1 overexpressor (OE) to stress derived from high concentrations of glucose, whilst under high concentrations of ABA, SnRK1α1OE enhances the hypersensitive phenotype of ago1-27, suggesting that AGO1 and SnRK1 may negatively regulate each other. Moreover, the SnRK1 pathway seems to predominate under high sugar stress whilst AGO1 seems to be more important for ABA responses. Interestingly, the snrk1α1-3 mutation partially reverted the delayed flowering phenotype of ago1-27, further reinforcing that AGO1 is a negative regulator of SnRK1. Finally, to explore the functional interaction between SnRK1 and AGO1 in the future, I developed important genetic tools based on a SUC::SUL reporter line that will allow to test whether changes in SnRK1 levels and activity affect AGO1-mediated silencing activity. The SUC::SUL reporter is based on the vasculature-specific silencing of a gene involved in chlorophyll biosynthesis (SULPHUR), through small interfering RNAs derived from its sense and antisense expression under the SUC2 promoter. New plant lines for different snrk1α mutants harbouring this reporter were generated and are ready to be used in future assays, for which the experimental setup was also optimised here. Taken together, the results presented in this thesis suggest that SnRK1 may indeed regulate the miRNA pathway at different levels, affecting miRNA accumulation and potentially AGO1 function to control growth, development and plant stress responses. Further work will be required to confirm the extent and mechanism by which SnRK1 impacts on miRNA biogenesis and/or stability and to dissect the details of its interaction with AGO1.

Diabetes mellitus is a metabolic disease and its incidence is reaching epidemic proportions. Classically it can be divided into two types: type 1 diabetes mellitus, characterized by an insulin-dependent state, and type 2 diabetes mellitus (T2DM), where there is a resistance to the action of the hormone. External factors associated with lifestyle, such as eating behaviors, in particular, the excessive consumption of high caloric diets in combination with other factors, such as sedentary lifestyle, are the main causes for the increased incidence of T2DM. The metabolic deregulation associated with T2DM leads to the emergence of other comorbidities, notably a deregulation of male fertility. The hypothalamus-pituitary-testicle axis, also known as a reproductive axis, is sensitive to the metabolic changes induced by T2DM. Recent studies have shown that endocrine and metabolic alterations associated with T2DM affect the physiology of reproductive organs, mainly their mitochondrial bioenergetics. Maintaining mitochondrial function in reproductive organs is imperative for the maintenance of the reproductive capacity of the individual. Thus, we aimed to study the impact of T2DM on the molecular pathways underlying the control of the testicular and epididymal mitochondrial function. For this, we used an animal model of T2DM, in which we evaluated the expression of key proteins involved in the regulation of mitochondrial biogenesis and in mitochondrial function. We measured the activity of the enzymes of the antioxidant defense system. We also evaluated the effects of T2DM on the number of mitochondrial DNA copies and on the expression of the levels of the mitochondrial respiratory chain complexes in both tissues. Finally, we evaluated the parameters of oxidative stress (OS), such as carbonylation and proteins nitration, as well as lipid peroxidation. Our results showed that T2DM decreased the expression of sirtuin 1 (SIRT1) and sirtuin 3 (SIRT3) in the testicular tissue. There was also a decrease in the expression of complexes III and V of the mitochondrial respiratory chain, but the content of mitochondrial DNA (mtDNA) remained unchanged in the testicular tissue. There were no alterations on the activities of the antioxidant enzymes, however, these results were accompanied by an increase in lipid peroxidation and nitrate of proteins. In the epididymal tissue, a decrease was observed on the expression of the key regulator of mitochondrial biogenesis, the peroxisome proliferator activated receptor ? co-activator 1 a (PGC-1a), as well as on the SIRT1 and SIRT3 expression levels. Although there were no changes in the mtDNA content, DMT2 has induced a significant decrease in the expression of complexes II, III and V in the epididymis. There were also decreases in the activities of the enzymes involved in the system of antioxidant defenses, which were accompanied by an increase of protein nitration. The results suggested that T2DM disrupted the expression of key regulators of the mitochondrial biogenesis of the reproductive organs, thereby compromising the molecular pathways involved in the regulation of the mitochondrial function and, consequently in the maintenance of the antioxidant defense system. In this way, it is essential to deepen the knowledge in mitochondrial bioenergetics in order to develop possible therapeutic approaches to attenuate the increased decline of male fertility, especially in developed countries where the prevalence of metabolic diseases is a major public health concern.
A diabetes mellitus é uma doença metabólica cuja incidência está a aumentar na população mundial. Classicamente pode ser divida em dois tipos: tipo 1 que se carateriza por um estado de insulinodependência, e o tipo 2 em que se verifica uma resistência à ação da hormona. Fatores externos associados ao estilo de vida, como os maus hábitos alimentares, em particular o consumo frequente de dietas altamente calóricas, em combinação com outros fatores como o sedentarismo, são as principais causas para o incremento de patologias como a diabetes mellitus tipo 2 (DMT2). A desregulação metabólica associada à DMT2 leva ao aparecimento de outras comorbidades, nomeadamente uma diminuição da fertilidade masculina. O eixo hipotálamo-hipófise-testículo, conhecido como eixo reprodutivo, é sensível às alterações metabólicas induzidas pela DMT2. Estudos recentes têm mostrado que alterações endócrinas e metabólicas associadas à DMT2 afetam a fisiologia dos órgãos reprodutivos, nomeadamente a bioenergética mitocondrial pelo que a manutenção da função mitocondrial nos órgãos reprodutivos é essencial. Assim, pretendemos estudar qual o impacto da DMT2 nas vias moleculares subjacentes à regulação da função mitocondrial testicular e epididimal. Para isso usou-se o modelo animal de DMT2 onde se avaliou a expressão de proteínas-chave envolvidas tanto regulação da biogénese mitocondrial, como na ativação do sistema de defesa antioxidante. Também avaliámos os efeitos da DMT2 no número de cópias de DNA mitocondrial (mtDNA) e na expressão dos níveis dos complexos da cadeia respiratória mitocondrial em ambos os tecidos. Avaliámos ainda os danos induzidos pelo stress oxidativo, como a carbonilação e nitração de proteínas, assim como a peroxidação lipídica. Os resultados demonstraram que a DMT2 diminui a expressão da sirtuína 1 (SIRT1) e sirtuína 3 (SIRT3) no tecido testicular. Verificou-se uma diminuição na expressão dos complexos III e V da cadeia respiratória mitocondrial. Não se verificaram alterações no conteúdo do mtDNA testicular e nas atividades das enzimas antioxidantes. No entanto, estes resultados foram acompanhados por um aumento da peroxidação lipídica e nitração de proteínas. Ao nível epididimal, observou-se uma diminuição na expressão do regulador-chave da biogénese mitocondrial, o coativador 1 a do receptor ? activado pelo proliferador de peroxissoma, assim como a expressão das SIRT1 e SIRT3. Embora não se tenham verificado alterações significativas no conteúdo do mtDNA, a DMT2 induziu uma diminuição significativa da expressão dos complexos II, III e V. Também se observou uma diminuição significativa na atividade das enzimas envolvidas no sistema de defesas antioxidantes, que culminou com o aumento da nitração de proteínas. Os resultados obtidos sugerem-nos que a DMT2 induz uma diminuição da expressão de reguladores-chave da biogénese mitocondrial dos órgãos reprodutivos, comprometendo assim as vias moleculares envolvidas na regulação da função mitocondrial e, consequentemente, na manutenção do sistema de defesas antioxidantes. Desta forma, torna-se essencial aprofundar os conhecimentos na bioenergética mitocondrial para que se possam desenvolver novas abordagens terapêuticas, de modo a atenuar o aumento da infertilidade masculina, principalmente nos países mais desenvolvidos onde a elevada prevalência das doenças metabólicas é considerado um problema de saúde pública.