Type 2 diabetes (T2D) is characterized by impaired beta cell function. The generation of various types of cellular stresses, including oxidative stress and ER stress, and the induction of cellular senescence can contribute to beta cell dysfunction. Recent studies have demonstrated associations between petrochemical exposure and beta cell dysfunction, particularly through induction of cellular stress. One class of compounds, commonly found in crude oil, are sulphur-containing heterocyclic aromatic compounds (S-HACs). S-HACs have been previously demonstrated to induce cellular stress in mammalian cells. This thesis aims to determine if S-HACs can induce cellular stress in beta cells and, consequently, impair beta cell function, particularly insulin production. Rat pancreatic beta cells, INS-1Es, were treated with two commonly occurring S-HACs, BNT(2,3D) and DBT, at doses which reflect non-occupational exposure levels. Upon treatment, various functional assays and qPCR experiments were performed for examining glucose uptake, ROS production, cellular senescence, ER stress and intracellular insulin production. It was observed that both BNT(2,3D) and DBT significantly increased glucose uptake and ROS production in the beta cells and upregulated the mRNA expression of various ER stress markers. In addition, BNT(2,3D) also induced cellular senescence, likely through a p53-independent pathway. This suggests that S-HACs may induce oxidative stress and ER stress in exposed beta cells, and some S-HACs may cause irreversible cell cycle arrest in response to these cellular stresses. However, intracellular insulin content in the INS-1Es was not altered by exposure to either S-HAC, suggesting that S-HACs may not impair insulin production. Nevertheless, the significant accumulation of ROS in S-HAC-exposed beta cells and the subsequent induction of cellular senescence by some S-HACs may alter other important beta cell functions, including mitochondrial function and insulin secretion, which could lead to the development of T2D; suggesting the potential for S-HACs to be novel beta cell toxicants. / Thesis / Master of Science (MSc)

Cellular stress is central to the understanding of pathological mechanisms and the development of new therapeutic strategies and serves as a biomarker for disease progression in neurodegeneration, diabetes, cancer, cardiovascular and other chronic diseases. The common cellular stress assay (CSA) based on Seahorse technology in peripheral blood mononuclear cells (PBMCs) shows inconsistent results, which prevents its use as a biomarker for the progression of chronic diseases. Therefore, the aim of this study was to investigate potential factors that affect the CSA in PBMCs. We measured the CSA parameters in PBMCs from study participants and compared the results according to the potential factors, namely, the PBMC isolation method, age, seasonal variation and the gender of the study participants. PBMCs were isolated by OptiPrep® and RobosepTM-S methods. PBMCs isolated with the OptiPrep method showed much higher extracellular acidification and higher respiration compared to Robosep-isolated cells. Moreover, OptiPrep-isolated cells showed a higher number of outliers for the proton production rate (PPR) and a high respiratory quotient, indicating impurities with other cells, such as platelets, and technical inconsistencies. PBMCs from older individuals showed higher maximal respiration, spare capacity and extracellular acidification than younger participants. Additionally, in winter, maximal respiration and spare capacity decreased. From spring until early autumn, spare capacity and maximal respiration continuously increased. Elderly males also showed higher basal respiration, spare capacity and extracellular acidification than females. In conclusion, the findings of this study clearly demonstrate that the results of CSA parameters measured in PBMCs are influenced by the PBMC isolation method, age, seasonal variation and gender. Therefore, we recommend that researchers and physicians properly interpret the results of CSA parameters in PBMCs by considering these factors. It is important to use separate CSA evaluation standards based on the isolation method, age, gender and season-dependent factors. To assess the cellular stress situation in PBMCs, both extracellular acidification and mitochondrial respiration should be taken into account. Further study of additional factors, such as mitochondrial mass, should be conducted to improve the measurement of CSA parameters for the assessment of the real mitochondrial fitness.

Le système lymphatique a pour rôle principal de drainer les fluides tissulaires et de participer à la surveillance immune. La lymphangiogenèse est principalement activée par deux facteurs : les vascular endothelial growth factors-C et -D (VEGF-C et VEGF-D). Ces facteurs participent à la progression métastatique à la fois en stimulant la prolifération des vaisseaux lymphatiques dans la tumeur et en périphérie et en provoquant la dilatation des vaisseaux collecteurs facilitant ainsi le passage des cellules tumorales de la tumeur primaire aux ganglions lymphatiques. Lors de son développement, une tumeur est soumise à de nombreux stress cellulaires tels que l'hypoxie, l'inflammation ou la déprivation en nutriments. Ma thèse a donc été consacrée à l'analyse des régulations moléculaires permettant une surexpression des facteurs lymphangiogéniques en condition de stress cellulaire. Lors d'un premier projet de recherche j'ai démontré que l'hypoxie réduit la transcription de VEGF-C ainsi que sa traduction coiffe-dépendante mais activait une traduction médiée par un site interne d'entrée du ribosome (IRES). De plus, cette activation de la synthèse de VEGF-C en hypoxie est indépendante de HIF-1a et stimule la lymphangiogenèse dans les tumeurs ainsi que dans les ganglions lymphatiques participant ainsi à la dissémination tumorale. Dans un second temps, j'ai démontré que le VEGF-D possédait lui aussi un IRES activé par un choc thermique. L'activité de l'IRES du VEGF-D est régulée par la localisation subcellulaire d'un facteur trans-activateur de l'IRES (ITAF) : la nucléoline. Cette protéine est exportée du noyau vers le cytoplasme lors d'un choc thermique. Ce processus est inhibé par un traitement avec un anti-inflammatoire non stéroïdien ce qui supprime l'activation de la traduction IRES dépendante du VEGF-D. Enfin lors d'un troisième projet, j'ai travaillé sur le lymphœdème secondaire, une pathologie caractérisée par une destruction du système lymphatique où la lymphangiogenèse doit être stimulée. J'ai évalué l'impact de l'hormonothérapie, principal traitement du cancer du sein, sur l'expression du VEGF-C et -D. J'ai démontré que l'estradiol stimulait l'expression de ces facteurs alors que l'hormonothérapie diminue leur synthèse et provoque une destruction de l'endothélium lymphatique. En conclusion, mes trois projets de thèse m'ont permis d'étudier les régulations moléculaires des deux facteurs lymphangiogéniques principaux dans des pathologies provoquées par une lymphangiogenèse excessive ou insuffisante. / Tumor lymphangiogenesis promotes lymph node metastasis using two mechanisms consisting of lymphatic vessels proliferation and dilatation to facilitate tumor spread. Lymphangiogenesis is mainly promoted by two growth factors: the vascular endothelial growth factor C and D. My PhD was focused on the molecular regulations inducing a tumoral over-expression of these two factors during stress. I demonstrated that hypoxia reduced VEGF-C transcription and cap-dependent translation while activating an Internal Ribosome Entry Site (IRES)-dependent mechanism of translation. This upregulation of VEGF-C in hypoxia is independent of HIF-1a; and stimulates lymphangiogenesis in tumors and lymph nodes and contribute to lymphatic metastasis. Then, I have discovered that VEGF-D had an IRES selectively activated by heat shock. VEGF-D IRES is regulated by a subcellular relocalization of an ITAF, the nucleolin. This molecular process is reversed by non-steroidal anti-inflammatory drugs (NSAID) that decrease VEGF-D IRES translation initiation by targeting the nucleolin. Finally, I worked on secondary lymphedema, a pathology that is characterized by a disruption of the lymphatic network where lymphangiogenesis thus need to be restored. In this condition, I have evaluated the impact of hormone therapy, the main treatment for breast cancer, on the regulation of lymphangiogenic factors. I found that estradiol stimulates the expression of VEGF-C and-D contrary to the hormone therapy that inhibits VEGF-C and -D and thus mediates a disruption of the lymphatic endothelium. Taken together, my PhD projects have allowed me to study the regulations of VEGF-C and -D in pathologies mediated either by an excessive or an insufficient lymphangiogenesis.

The ability to accurately predict danger and implement appropriate protective responses is critical for survival. Environmental fluctuations can cause damage at the cellular level, leading to the misfolding and aggregation of proteins. Such damage is toxic to cells: in age-related neurodegenerative diseases like ALS, Parkinson’s, Alzheimer’s and Huntington’s Diseases, the accumulation of damaged proteins in the brain ultimately leads to neuronal cell death and disease onset. To date, there is still no cure to combat the progressive degeneration and cell death seen in the brains of patients. Cells within an animal possess defense programs to minimize protein damage. One such defense mechanism is the activation of a program called the Heat Shock Response, which increases production of protective proteins known as heat shock proteins (HSPs). These HSPs act as molecular chaperones to assist with the clearing out of damaged proteins. This program is implemented by a conserved transcription factor, Heat Shock Factor 1 (HSF-1). However, in brains of patients with degenerative diseases, this protective mechanism, for reasons yet unknown, is not constantly activated. My thesis has involved the discovery of innate mechanisms that exist in organisms to activate this cellular protective mechanism against protein misfolding. My research, using the model organism Caenorhabditis elegans, has shown that the protective heat shock response in the cells of the animal can be triggered through neurohormonal signaling. The neurohormonal signaling that I am studying is one that is highly conserved across all organisms from plants to insects to mammals – serotonergic signaling. The stimulation of serotonergic signaling appears sufficient to activate the Heat Shock Response, even in the absence of real damage. In fact, the neuronal release of serotonin facilitates a pre-emptive upregulation of protective genes in the animal, which we have observed to be able to reduce the accumulation of damaged proteins in a C. elegans model of Huntington’s Disease. Additionally, I have seen that anticipating danger can enhance the animal’s stress response in a serotonin-dependent manner, thus facilitating better survival against a subsequent insult that can cause protein damage. Together, these studies present the novel possibility of protection against neurodegenerative disease via modulation of neurotransmission and/or neurosecretion. They also allow for understanding how sensory inputs are coupled to gene expression under stressful conditions. I hope to understand the mechanism by which animals adapt to changes in their environment by coordinating their sensory input with changes in behavior and gene expression.

Cadmium (Cd), is a non-essential metal with no known physiological function. It is known to alter redox state by disrupting the mitochondrial electron transport chain, as well as inactivating protein and non-protein thiols. It is thus believed that oxidative stress may comprise an important part of the mechanism of Cd toxicity. Accordingly, the initial cellular response to acute Cd exposure is defensive, where various anti-oxidant defence systems are triggered. One of the induced systems is the haem oxygenase-1 (HO-1). Its activation is mediated by the transcription factor Nrf2, which is the general regulator of cellular defence against oxidative stress. The protective effects of HO-1 are mediated, in part, through the generation of potent anti-oxidant bilirubin (BR) and its metabolites, which exploit the intrinsic antioxidant properties of these species at a cellular level. The oxidative metabolism of BR is an important route of detoxification in addition to glucuronidation. However, the major enzyme(s) involved in this oxidative degradation are not known. This thesis presents evidence for a major role of the hepatic cytochrome P450 2a5 (Cyp2a5) in BR degradation during Cd intoxication, where the BR levels are elevated following induction of HO-1. Treatment of DBA/2J male mice with CdCl2 induced both the Cyp2a5 and HO-1, and increased the microsomal BR degradation activity. By way of contrast, the total cytochrome P450 (CYP) content and the expression of Cyp1a2 were down-regulated by the treatment. The induction of the HO-1 and Cyp2a5 was significant at the mRNA, protein and enzyme activity levels. In each case, the up-regulation of the HO-1 preceded that of the Cyp2a5 with a 5-10 hr interval. In addition, BR totally inhibited the microsomal coumarin hydroxylase activity (a Cyp2a5-catalysed reaction) with an IC50 approximately equal to the substrate concentration. The MROD activity, catalysed mainly by the Cyp1a2, was inhibited up to 36% by BR. The microsomal BR degradation was inhibited by coumarin and by a monoclonal antibody against the Cyp2a5 by about 90%. In addition, 7-methoxyresorufin, a substrate for Cyp1a2, inhibited BR degradation activity by approximately 20%. A study using Nrf2 null mutant mice suggests that Cd-mediated induction of Cyp2a5 is dependent on the transcription factor Nrf2. Additionally, acute exposure to Cd activated localisation of Nrf2 from the cytoplasm to the nucleus. Furthermore, electrophoretic mobility shift assay (EMSA) analysis suggests that Cd induced sequence-specific binding of various species of the StRE-binding proteins on the 5’-flanking region of the Cyp2a5 gene. Collectively, these observations strongly suggest that BR may act as a substrate for the hepatic Cyp2a5, a major catalyst for BR degradation under conditions of substantial elevation of BR levels following induction of HO-1 by Cd. Secondly, the concurrent up-regulation of the HO-1 and Cyp2a5 during Cd-mediated injury implicates a coordinated regulation of two enzyme systems in the maintenance of balancing BR production and elimination. Finally, StRE-binding proteins, in particular Nrf2, may be involved in the regulation of the Cyp2a5 gene, which leads to the oxidation of BR. However, the respective roles of these factors in the regulation of the Cyp2a5 gene, as well as the coordinated regulation of ho-1 and Cyp2a5 genes remain an open question, requiring further investigations.

Die myotone Dystrophie ist eine autosomal dominant vererbte, multisystemische Erkrankung mit skelettmuskulärem Fokus und wird in zwei Untergruppen eingeteilt, die myotone Dystrophie Typ I (MD1), verursacht durch eine CTG-Trinukleotidexpansion im DMPK-Gen auf Chromosom 19, und die myotone Dystrophie Typ II (MD2), verursacht durch eine Tetranukleotidexpansion im CNBP-Gen auf Chromosom 3. Die Nukleotidexpansionen akkumulieren als mRNA intranukleär und tragen über die Beeinflussung des alternativen Splicings von über 30 Genen zum Erkrankungsmechanismus bei. Daneben akkumulieren die mRNA-Repeats im Zytoplasma und induzieren über die Repeat-assoziierte-non-AUG-Translation (RAN-Translation) die Akkumulation aberranter Proteine. Wenngleich die Patienten der MD2 einen milderen Krankheitsverlauf erleben, zeigen sie ein erhöhtes Auftreten von Autoimmunerkrankungen. In der Pathogenese der Autoimmunerkrankungen spielt die durch Interferon-Typ-I vermittelte Immunreaktion eine wichtige Rolle. In der AG Günther konnte eine erhöhte Expression interferonstimulierter Gene und die erhöhte Expression von Markern des Stresszustandes des endoplasmatischen Retikulums in Fibroblasten von MD2-Patienten nachgewiesen sowie die erhöhte Prävalenz von Autoimmunerkrankungen in MD2 gezeigt werden. Die Auswertung serologischer Parameter im Rahmen dieser Arbeit erlaubte eine klinische Charakterisierung der Patienten und ergab einen positiven Zusammenhang zwischen Höhe der Myoglobin-Konzentration im Serum und Länge der CCTG Repeats. Es wurde die Hypothese aufgestellt, dass die zytoplasmatischen RNA-Repeats über die RAN-Translation und Akkumulation aberranter Proteine zur Induktion von ER-Stress führen, welcher über die gemeinsame Kontaktflächen der Mitochondrien-assoziierten Membranen (MAMs) in die Mitochondrien übertragen wird. Im Rahmen des mitochondrialen Stresszustandes könnte es zu einer Freisetzung mitochondrialer DNA und zur Aktivierung des zytosolischen DNA-Rezeptors cGAS und einer nachfolgenden Expression von Interferon und Interferon-stimulierten Genen kommen, die für das vermehrte Auftreten der Autoimmunerkrankungen verantwortlich sind. Zur Überprüfung der Hypothese sollte nach Analyse der Proteinexpression der ER-Stress-Marker eIF2α und peIF2α eine Hemmung der ER-Stressreaktion vorgenommen werden. Die anschließende Untersuchung des mitochondrialen Stresszustandes und die Unterbrechung der MAMs sollte Aufschluss über die Rolle der Mitochondrien in der Pathogenese der Autoimmunität liefern. In dieser Arbeit konnte die erhöhte Expression des ER-Stressmarkers peIF2α nach Stressinduktion in Fibroblasten von MD2-Patienten gezeigt werden. Dies könnte auf eine Sensibilisierung des Signalweges im Rahmen eines chronischen Stresszustandes hinweisen. Der Versuch einer Behandlung der Fibroblasten mit Ursodesoxycholsäure (UDCA), einem chemischen Chaperon, mit Ziel der Suppression der ER-Stressreaktion erzielte keine Reduktion der Expression der ER-Stressmarker unter Nativniveau. Nach Stimulation des ER-Stresses konnte mit UDCA-Vorbehandlung dennoch die Reduktion auf Nativniveau erzielt werden. Zur Analyse des mitochondrialen Stresszustandes wurde eine Färbung mit Mitotracker-Farbstoffen und anschließender Durchflusszytometrie und eine Messung der reaktiven Sauerstoffspezies (ROS) vorgenommen, welche ein verringertes mitochondriales Membranpotential und eine erhöhte ROS-Expression in den MD2-Fibroblasten ergaben und mit einem mitochondrialen Stresszustand einhergehen könnten. Nach Unterbrechung der räumlichen Verbindung zwischen ER und Mitochondrium durch die Herunterregulation des gemeinsamen Strukturproteins PERK konnten Hinweise auf eine verringerte ROS-Expression in den MD2-Fibroblasten gefunden werden. Die Ergebnisse dieser Arbeit unterstützen die Hypothese der Funktion der mitochondrialen Stressreaktion in den Fibroblasten der MD2-Patienten, wenngleich die Autoimmunität-vermittelnden Mechanismen noch Gegenstand weiterführender Untersuchungen sein sollten.

DCs são células especializadas na apresentação de antígenos (Ags) para linfócitos T virgens e indução de respostas imunes primárias. Deficiências no processo de eliminação de células apoptóticas relacionam-se com desenvolvimento de doenças auto-imunes. Para avaliar o efeito de células apoptóticas sobre os processos de maturação e atividade funcional de DCs derivadas in vitro de monócitos aderentes de doadores saudáveis, células não aderentes obtidas após uma 2ª etapa de aderência por 3 dias foram submetidas a aquecimento e/ou irradiação. 48h após, células somente irradiadas (37i) apresentaram maior porcentagem de apoptose que células aquecidas e irradiadas (43i), sugerindo que o calor protege da apoptose induzida. Na cocultura com iDCs ou mDCs (iDC+TNF), iDC+37i apresentaram aumento de CD1a, correlacionado com altos níveis de IL-10 e inibição de autoestimulação linfocitária. mDC+43i apresentaram níveis de CD1a semelhantes a iDC, baixos níveis de IL-10, altos níveis de IL-12p70 e altos índices de auto e aloestimulação linfocitária. Concluímos que o fenótipo e função de DCs é modulado diferencialmente na presença de 43i, que induzem ativação in vitro, enquanto 37i induzem DCs com características moduladoras dependente de lipídios. / DCs are specialized in presenting Ags to naïve lymphocytes, inducing primary immune responses or immunological peripheral tolerance, through tissue turnover by scavenging dying cells. Works with human cells are scarcer and controversial about the latter. Then, the aim of this work was to investigate the effect of apoptotic cells (Nadhs) on maturation and function of human monocyte-derived DCs in vitro. The results suggest that heating Nadhs before irradiation (43i) seems to protect them from apoptosis induced by irradiation (37i). DCs were cocultured with 37i or 43i, simultaneously to TNF-a addition in the 5th day of culture. In the 7th day, there was an association between high levels of CD1a, IL-10, low levels of IL-12p70 and decreased allostimulation induced by iDC+37i. mDC+43i had high levels of IL-12p70, CD86 and proliferation index, associated with low levels in CD1a expression. We conclude that DCs phenotype and function are differentially modulated in the presence of 43i, which induce DCs activation, or 37i, which induce regulatory phenotype and function in DCs. We suggest that these protocols for DCs activation with 43i or 37i could be used, respectively, as models of in vitro auto-reactivity or homeostatic immunomodulation of DCs in vitro.

Os mecanismos pelos quais a produção in vitro de embriões (PIVE) bovinos gera embriões com excessivo acúmulo lipídico, com elevado estresse celular e com reduzida criotolerância ainda são desconhecidos. Também permanece desconhecido quando essas alterações acontecem, se acontecem desde a maturação oocitária e o papel que as células do cumulus possuem neste mecanismo. O objetivo do presente trabalho foi estudar e comparar o metabolismo lipídico e homeostase celular, além dos perfis de miRNAs, durante a maturação oocitária e o desenvolvimento embrionário inicial in vivo e in vitro em bovinos. Para isso, o trabalho foi dividido em 4 estudos. No Estudo 1, intitulado \"A maturação in vitro gera complexos cumulus-oócitos metabolicamente desregulados e estressados em bovinos\" foram analisadas as células do cumulus e oócitos de complexos cumulus-oócitos (COCs) imaturos e maturados in vivo e in vitro. Foram realizadas análises de quantificação de lipídeos, espécies reativas de oxigênio (EROs), glutationa reduzida (GSH), razão ATP/ADP assim como expressão de mRNAs e miRNAs relacionados com as vias de metabolismo e homeostase celular. A partir dos dados obtidos neste estudo, concluímos que a maturação in vitro (MIV) provoca aumento de lipídeos no COC, diminuição de GSH e da atividade mitocondrial nos oócitos, acompanhado por uma desregulação massiva das vias relacionadas a metabolismo e homeostase nas células do cumulus da MIV. No Estudo 2, intitulado \"A proteína ligadora de ácidos graxos 3 (Fatty Acid Binding Protein 3 - FABP3) e as projeções transzonais estão envolvidas no acúmulo lipídico durante a maturação in vitro em oócitos bovinos\" foi constatado aumento do conteúdo lipídico e da expressão da FABP3 nas células do cumulus da MIV, quando comparado ao sistema in vivo. Ainda, imunolocalizamos a FABP3 dentro das projeções transzonais (TZPs) e verificamos um aumento concomitante da FABP3 e dos lipídeos oocitários nas primeiras 9 horas da MIV. Mediante a remoção das TZPs às 9 horas da MIV e consequente diminuição lipídica observada no oócito, concluímos que existe um possível transporte de ácidos graxos a partir das células do cumulus até o oócito mediante FABP3 e TZPs. No Estudo 3, intitulado \"Alterações no metabolismo lipídico e homeostase celular entre embriões bovinos produzidos in vivo e in vitro\", verificamos que, mesmo utilizando um cultivo in vitro com baixa tensão de oxigênio e sem soro, os blastocistos da PIVE possuem maiores níveis de lipídeos e EROs que aqueles produzidos in vivo. Além disso, constatamos que essas alterações nos lipídeos durante a PIVE não estão acompanhadas de alterações de expressão, porém, existe um aumento na expressão de genes relacionados com estresse. No último estudo, \"O sistema in vitro altera o perfil de miRNAs durante a maturação oocitária e desenvolvimento embrionário inicial em bovino\", constatamos as diferenças no perfil de miRNAs e nas vias reguladas por estes nas células do cumulus e oócitos maturados in vivo e in vitro e em blastocistos produzidos in vivo e in vitro. Verificamos uma maior regulação por miRNAs durante a maturação nas células do cumulus comparado ao oócito. Além do mais, tanto no COC quanto nos blastocistos, os miRNAs mostraram regular vias importantes do metabolismo, comunicação celular e vias de sinalização importantes para a maturação e desenvolvimento embrionário inicial. Conjuntamente, este trabalho permitiu elucidar as diferenças que a PIVE provoca no metabolismo e homeostase celular e na expressão de miRNAs. / The mechanism through which in vitro embryo production (IVEP) generates embryos with higher lipid accumulation, overstressed and with reduced cryotolerance is unknown. It is also unknown when these alterations take place, if occurs since the oocyte maturation and the role of cumulus cells in this mechanism are also unknown. The aim of the present work was to study and compare the lipid metabolism and cellular homeostasis, as well as the miRNAs profile during oocyte maturation and early in vivo and in vitro embryo development in bovines. For that, the present work was divided in 4 studies. In Study 1, entitled \"in vitro maturation generates metabolically disrupted and overstressed cumulus-oocyte complexes in bovines\" cumulus cells and oocytes from immature and in vivo and in vitro matured cumulus-cells complexes (COC) were analyzed. Analysis of lipid stores, oxygen reactive species (ROS), reduced glutathione and ATP/ADP ratio quantification, as well as mRNAs and miRNAs involved with metabolism and cellular homeostasis pathways were performed. From the obtained data in this study, we concluded that in vitro maturation (IVM) leads to an increase of lipids in the COC, a decrease of oocyte GSH and mitochondrial activity, as well as a massive deregulation in metabolism and homeostasis related pathways in MIV cumuls cells. In Study 2, \"Fatty Acid Binding Protein 3 and transzonal projections are involved on lipid accumulation during in vitro maturation in bovine oocytes\" we observed an increase of lipid accumulation and FABP3 expression in MIV cumulus cells when compared to the in vivo system. Furthermore, we immunolocalized the FABP3 inside the transzonal projections (TZPs) and verified a concomitant increase of FABP3 and oocyte lipids on the first 9 hours of MIV. By removing the TZPs at 9 hours of MIV and by the consequent lipid decrease observed in the oocyte, we concluded that there is a possible traffic of fatty acids from the cumulus cells to the oocyte mediated by FABP3 and TZPs. In Study 3, entitled \"Alterations in lipid metabolism and cellular homeostasis between in vivo and in vitro produced bovine embryos\", we observed that, even though under serum free and low oxygen tension used during in vitro culture was used, IVP blastocysts had higher lipid and ROS levels than the counterparts produced in vivo. Moreover, we found that these lipid alterations during IVP are not followed by corresponding genes expression alterations, however, there is an increase of the expression of stress related genes. In the last study, \"in vitro system alters miRNAs profile during oocyte maturation and early embryo development in bovines\", we observed differences in miRNAs profiles and in pathways modulated by them in in vivo and in vitro matured cumulus cells and oocytes and in in vivo and in vitro produced blastocysts. We observed an intense miRNAs regulation during maturation in cumulus cells when compared to the oocyte. Furthermore, in both COC and blastocysts, miRNAs regulated important metabolism, cellular communication pathways, as well as important signaling pathways for maturation and early embryo development. Taken together, the findings of the present work allowed us to elucidate the differences caused by IVEP on cell metabolism and homeostasis as well as on miRNAs expression.

Dimethylsulphoniopropionate (DMSP) and dimethylsulphoxide (DMSO) (collectively DMSP/O) are produced by marine algae, including symbiotic algae within corals. These sulphur compounds are important not only in sulphur cycle dynamics but also in potentially mediating atmospheric conditions, alleviating the effects of climate change and contributing to reef health. Most research has focused on the production of DMSP and its major degradation product, the climatically active gas, dimethylsulphide (DMS) by Acropora corals in the Great Barrier Reef. However, mechanisms for the production and release of DMSP/O by different reef taxa is poorly understood. Recently the importance of mesophotic reefs as refugia for shallow water corals has been postulated, however their role in the marine sulphur cycle is unknown. This research aimed to improve our understanding of the contemporary and climate change induced seawater and tissue production of DMSP/O in a range of reef environments and taxa. This was achieved through a combination of laboratory and field - based studies, using modern and established techniques. An effect of both elevated temperature and OA on increased tissue and seawater concentrations of DMSP/O production is reported in field and laboratory studies. Contrasting effects of benthic cover on tissue DMSP/O distributions and seawater DMSP are also noted. The importance of the physical and hydrodynamic environment on biogeochemical connectivity both within a reef and between neighbouring reefs is also focussed on. Crucially, however, the novel tissue and seawater data from mesophotic sites suggests that deeper reefs could affect the biogeochemistry of their shallow water counterparts. The key finding from this work is that climate change will result in increased seawater DMSP concentrations via two mechanisms; through the increase of cellular production of DMSP/O in all reef taxa, and by increasing the biomass of prolific DMSP producers as reefs transition to a fleshy/macroalgal assemblage. Whilst this could potentially mediate the effects of climate change, it will probably also worsen overall reef health, lead to a restructuring of reef communities from the microbial level upwards and will have possibly permanent and deleterious effects on overall ecosystem function.

Histones are chromatin-associated proteins localized to the nucleus. However, extracellular histones are present in biofluids from healthy individuals and become elevated under disease conditions, such as neurodegeneration and cancer. Hence, extracellular histones may have important biological functions in healthy and diseased states, which are not understood. Histones have been reported in the proteomes of extracellular vesicles (EVs), including microvesicles and exosomes. The main aim of this thesis was to determine whether or not extracellular histones are secreted via EVs/exosomes. In an initial study (Paper I), I optimized methods for human embryonic kidney (HEK293) cell culture, transfection and protein detection using western blotting. In the main study (Paper II), I used oligodendrocyte cell lines (rat OLN-93 and mouse Oli-neu) to investigate the localization of histones to EVs. Western blotting of EVs purified from OLN-93 cell-conditioned media confirmed the presence of linker and core histones in them. Immunolocalization and transmission electron microscopy confirmed that histones are localized to EVs, as well as intraluminal vesicles (ILVs) within multivesicular bodies (MVBs). This suggests that histones are secreted via the MVB/exosome pathway. Localization of histones in EVs was investigated by biochemical/proteolytic degradation and purification followed by western blotting. Surprisingly, histones were associated with the membrane but not the luminal fraction. Overexpression of tagged histones in HEK293 cells confirmed their conserved, membrane localization. OLN-93 cell EVs contained both double stranded and single stranded DNA but nuclease and protease digestion showed that the association of histones and DNA with EVs was not interdependent. The abundance of histones in EVs was not affected by differentiation in Oli-neu cells. However, histone release was upregulated as an early response to cellular stress in OLN-93 cells and occurred before the release of markers of stress including heat shock proteins. Interestingly, a notable upregulation in secretion of small diameter (50-100 nm) EVs was observed following heat stress, suggesting that a sub-population of vesicles may be involved specifically in histone secretion in response to stress. Proteomic analyses identified the downregulation of endosomal sorting complex required for transport (ESCRT) as a possible mechanism underlying increased histone secretion. In Paper III, I developed methods to quantify extracellular histone proteins in human ascites samples from ovarian cancer patients.   In summary, we show for the first time that membrane-associated histones are secreted via the MVB/exosome pathway. We demonstrate a novel pathway for extracellular histone release that may have a role in both health and disease.