A lesão de isquemia e reperfusão (IRI) continua a ser um problema clínico e o estresse do retículo endoplasmático (ERS) parece ser um importante mediador desse processo. A presença da heme oxigenase-1 (HO-1) ou do monóxido de carbono (CO), parece proteger da IRI. O objetivo do nosso trabalho foi avaliar a papel da HO-1 e CO na IRI renal. A indução da HO-1 em camundongos promoveu uma proteção na IRI renal, com melhora da função renal, menos inflamação e atenuação do ERS. Ao avaliarmos o papel do CO, verificamos que há também uma proteção, mediada por p38, vias purinérgicas, estabilização de HIF-1a e eritropoietina. Há ainda uma melhora do metabolismo energético celular após o tratamento com CO. Enfim, podemos concluir que, na presença da HO-1 ou do CO, há uma melhora da lesão isquêmica, através de uma maior ativação de vias citoprotetoras, com atenuação do ERS, redução da inflamação e consequente melhora da função renal. / Ischemia-reperfusion injury (IRI) remains a clinical problem and endoplasmic reticulum stress (ERS) seems to be an important mediator of this process. The presence of heme oxygenase-1 (HO-1) or carbon monoxide (CO) appears to protect from IRI. The aim of our study was to evaluate the role of HO-1 and CO in renal IRI. The induction of HO-1 in mice promoted protection in renal IRI with improved renal function, less inflammation and attenuation of ERS. When evaluating the role of CO, we found that there is also a protection mediated by p38, purinergic signaling, HIF-1a stabilization and erythropoietin. There is still an improvement of cellular energy metabolism after treatment with CO. Finally, we conclude that, in the presence of HO-1 or CO, there is an improvement of the ischemic lesion, through greater activation of cytoprotective pathways, with reduced ERS, reducing inflammation and consequent improvement in renal function.

Between May 1985 and December 1986, a cohort of 1,502 pregnant women was recruited at two government clinics in Kosovo (formerly a province of Yugoslavia) for a study of the relationship between environmental lead (Pb) exposure and birth outcomes. Subsequently, a representative group of 541 of their children were selected for long-term follow up. The children were followed longitudinally at six-month intervals for 12 years to examine the effects of environmental Pb exposure on a variety of health outcomes including cognitive and motor function, anemia, endocrine function and growth. This work produced numerous landmark publications (Popovac et al, 1982; Graziano et al., 1990, 1991, 2004; Murphy et al., 1990; Factor-Litvak et al, 1993, 1996, 1998, 1999; Wasserman et al,. 1992, 1994, 1997, 1998, 2000) that contributed to the modification of environmental policies to reduce Pb exposure worldwide. The long-term study ultimately linked environmental Pb exposure from the Trepca mining and smelting operations in Mitrovica to adverse effects on intelligence, motor function, blood pressure, renal, endocrine and hematological functioning. Follow up rates over time were excellent in that 70% of the total cohort was evaluated at 6 years of age, and 65% were evaluated at 12 years of age, at which point the study was - until now - concluded. For the present study, we located 101 members of the original study cohort and requested their participation in a follow-up study in which participants were evaluated to assess their current blood lead (BPb) levels and health outcomes as follows: a) blood pressure; b) biomarkers of endothelial cell function that are associated with cardiovascular disease; c) and measurements of erythropoietin, a glycoprotein hormone produced in the kidney that regulates the production of red blood cells in the bone marrow. The participants, whose environmental exposure history is very well documented from 12 weeks of gestation through 12 years of age, were between 25-26 years of age during the follow-up study. We found a statistically significant association between BPb and systolic blood pressure (sBP), and a marginally significant association between BPb and diastolic blood pressure (dBP), which is consistent with a multitude of studies and meta-analyses referenced in this dissertation. These results provide further evidence that recent circulating dose, as estimated by BPb, or as estimated by lifetime cumulative exposure, is associated with slight increase in sBP. Furthermore, we detected a suggestive relationship between BPb and levels of circulating serum intercellular adhesion molecules (sICAM-1) and serum intravascular adhesion molecules (sVCAM-1), possibly a mechanism by which Pb may lead to increased BP. These findings support the hypothesis that the exposure to Pb either prenatally or in early adulthood, may lead to increased BP and increased circulating levels of sICAM-1 and sVCAM-1 later in life. Lastly, the results regarding the serum erythropoietin (EPO) production presented here resemble the findings reported in this cohort at 4.5 and 6.5 years of age and in contrast with the findings in this cohort when the study participants were 9.5 and 12 years of age (Graziano et al., 2004). In addition, they also contrast the findings reported in the anemic mothers of this study cohort (Graziano et al., 1991) where serum-EPO levels were lower in those with higher BPb levels.

Alzheimer’s disease (AD) is a devastating neurodegenerative condition in which a patient’s cognitive functioning, memory, and physical health progressively deteriorate. In order to treat physiological deterioration in AD, a neuroprotective recombinant human- erythropoietin (EPO) fusion protein was used. In addition to its ability to target amyloid beta (Aβ) aggregation, EPO has been shown to reduce inflammation, oxidative stress and synaptic loss. Recombinant human-erythropoietin (EPO) was combined with a chimeric transferrin receptor (TfR) monoclonal antibody (cTfRMAb) to form a fusion protein (cTfRMAb-EPO) that is able to cross the blood-brain barrier (BBB) by binding to the TfR expressed on the luminal side of the BBB. Thirty eight male APPswePSEN1dE9 (APP/PS1) mice were separated into four treatment groups (wildtype (WT) treated with saline, APP/PS1 treated with saline (TG), APP/PS1 treated with cTfRMAb-EPO (cTfRMAb-EPO), and APP/PS1 treated with rHu-EPO alone (rhu-EPO)) and were subcutaneously injected with their respective treatments twice a week for six weeks. Recognition memory and locomotive behavior were tested through the novel object recognition (NOR) task and open field (OF) test when the mice were 8 months old and again at 11 months old (after 8 weeks of treatment) to determine treatment effects. Both behavioral tests demonstrated a clear age effect in mice between 8- and 11-months old. In the NOR task, no significant differences in recognition memory were observed in TG, cTfRMAb-EPO, or rHu-EPO groups. Lastly, the OF test demonstrated no significant behavioral differences among treatment groups.

[Truncated abstract] Clinical therapeutic agents to directly inhibit ischaemic neuronal death are presently unavailable. One approach to developing therapeutics is based upon the identification of proteins up-regulated by 'preconditioning', a natural adaptive response utilised by the neural cells to counter damaging insults, such as ischaemia. Thus, my project aimed to firstly identify proteins differentially expressed following erythropoietin (EPO) mediated neuronal preconditioning and secondly to assess whether any of these proteins possessed neuroprotective activity using in vitro ischaemia like models. To achieve the first aim, it was shown that in vitro neuronal EPO preconditioning could: (i) induce cell signal changes in neuronal cultures, (ii) protect neurons against in vitro ischaemia and (iii) induce differential protein expression. Overall, 40 differentially expressed proteins were identified in cortical neuronal cultures following EPO preconditioning. In order to investigate the neuroprotective or neurodamaging activity of proteins induced by EPO preconditioning I developed an adenoviral expression system for use in neuronal cultures. To this end, I assessed the suitability of four promoters (cytomegalovirus [CMV], rous sarcoma virus [RSV], human synapsin 1 [hSYN1], rat synapsin 1 [rSYN1]) previously used to express proteins in neuronal cultures and demonstrated the superiority of the RSV promoter for this purpose. ... Finally, in order to validate this adenoviral expression system, I over-expressed the anti-apoptotic protein Bcl-XL in neuronal cultures and subsequently confirmed its neuroprotective activity in the in vitro ischaemia and oxidative stress models used in my project. Using this adenoviral vector system and the in vitro oxidative stress model I assessed a number of proteins up-regulated by EPO preconditioning. The results of this preliminary study indicated that cyclophilin A (CyPA), peroxiredoxin 2 (PRDX2) and superoxide dismutase 1 (SOD1) over-expression were neuroprotective. It was subsequently verified that adenoviral mediated over-expression of CyPA and PRDX2, v but not SOD1 in cortical neuronal cultures could protect neurons from in vitro ischaemia. I also confirmed that CyPA mRNA increased in the rat hippocampus in response to 3 minutes of global cerebral ischaemia. Interestingly, an increase in CyPA, PRDX2 or SOD1 protein was not observed in the same experimental paradigm. To investigate CyPA's mode of action I confirmed that cultured neurons, but not astrocytes, express the CyPA receptor, CD147. It was also demonstrated that administration of exogenous CyPA protein to neuronal cultures could protect neurons against oxidative and ischaemic injury. I further demonstrated that exogenous administration of CyPA induces a rapid and transient activation of the extracellular signal-regulated kinase (ERK) 1/2 pathway in neuronal cultures. From this observation, I have proposed that the extracellular mediated neuroprotective activity of CyPA occurs via CD147 receptor signalling and activation of ERK1/2 pro-survival pathways. Based on the findings reported in this thesis, the neuroprotective activities of PRDX2 and CyPA warrant further investigation as targets for the development of new therapies to treat cerebral ischaemia.

<p>Cardiovascular disease (CVD) is the major cause of death in patients with chronic renal failure (CRF). Endothelial function and oxidative stress (OS) have previously been shown to be important in the pathogenesis of CVD. In this thesis, the endothelium-dependent vasodilatation (EDV) and OS were investigated in the patients with CRF. Also the influence of L-arginine, erythropoietin and diclofenac on EDV were evaluated in patients with CRF. </p><p>Patients with CRF were found to be characterized by a defect EDV even after correction for traditional cardiovascular risk factors. This impairment was related to the degree of renal failure. </p><p>Measurement of OS markers in CRF patients demonstrated that these patients were in a state of OS compared to healthy controls. The most informative indices to evaluate the degree of OS in CRF were: oxidized glutathione (GSSG) level, ratio between oxidized and reduced glutathione (GSSG/GSH ratio), lag phase of lipoprotein fraction (LPF) and baseline diene conjugation level of LPF. </p><p>Simultaneously investigated OS markers and EDV demonstrated a relationship between OS and EDV in patients with CRF. EDV was positively correlated with total antioxidative activity, reduced glutathione (GSH) and lag phase of LDL. </p><p>Local infusion of L-arginine as a substrate for nitric oxide synthesis and diclofenac as an inhibitor of cyclooxygenase-derived vasoconstrictive agents augmented EDV in patients CRF. In contrast, the erythopoietin treatment (both acute and long-term) impaired EDV in CRF patients. </p><p>In conclusion, patients with CRF have increased levels of OS markers and impaired endothelial vasodilatory function. These factors may be important with respect to the high morbidity and mortality of CVD found in patients with CRF. One possible mechanism to reduce CVD in patients with CRF is to improve endothelial function and eliminate OS. Locally administrated L-arginine and diclofenae improved EDV but erythropoietin administration impaired EDV in patients with CRF. </p>

Cardiovascular disease (CVD) is the major cause of death in patients with chronic renal failure (CRF). Endothelial function and oxidative stress (OS) have previously been shown to be important in the pathogenesis of CVD. In this thesis, the endothelium-dependent vasodilatation (EDV) and OS were investigated in the patients with CRF. Also the influence of L-arginine, erythropoietin and diclofenac on EDV were evaluated in patients with CRF. Patients with CRF were found to be characterized by a defect EDV even after correction for traditional cardiovascular risk factors. This impairment was related to the degree of renal failure. Measurement of OS markers in CRF patients demonstrated that these patients were in a state of OS compared to healthy controls. The most informative indices to evaluate the degree of OS in CRF were: oxidized glutathione (GSSG) level, ratio between oxidized and reduced glutathione (GSSG/GSH ratio), lag phase of lipoprotein fraction (LPF) and baseline diene conjugation level of LPF. Simultaneously investigated OS markers and EDV demonstrated a relationship between OS and EDV in patients with CRF. EDV was positively correlated with total antioxidative activity, reduced glutathione (GSH) and lag phase of LDL. Local infusion of L-arginine as a substrate for nitric oxide synthesis and diclofenac as an inhibitor of cyclooxygenase-derived vasoconstrictive agents augmented EDV in patients CRF. In contrast, the erythopoietin treatment (both acute and long-term) impaired EDV in CRF patients. In conclusion, patients with CRF have increased levels of OS markers and impaired endothelial vasodilatory function. These factors may be important with respect to the high morbidity and mortality of CVD found in patients with CRF. One possible mechanism to reduce CVD in patients with CRF is to improve endothelial function and eliminate OS. Locally administrated L-arginine and diclofenae improved EDV but erythropoietin administration impaired EDV in patients with CRF.

Photodynamic therapy (PDT) derives its tumour selectivity from preferential photosensitizer accumulation and short light penetration in tissue. However, additional strategies are needed to improve the therapeutic index of PDT in oncological applications where light is delivered interstitially to large volumes (e.g. prostate), or when adjacent normal tissue is extremely sensitive (e.g. brain). Much research to improve PDT's selectivity is directed towards developing targeted photosensitizers. Here, I present two alternative strategies to improve PDT's selectivity, without compromising its efficacy. For interstitial delivery, I investigated whether customizable cylindrical diffusers can be used to deliver light doses that conform better to target geometries, specifically the prostate. Additionally, I examined whether the neuroprotectant erythropoietin, used as an adjuvant to PDT for brain tumours, can reduce the sensitivity of normal tissue, thereby improving treatment selectivity. To determine if tailored diffusers constitute an improvement over conventional ones, I introduce a novel optimization algorithm for treatment planning. I also analyze the sensitivity of the resulting plans to changes in the optical properties and diffuser placement. These results are contextualized by a mathematical formalism to characterize the light dose distributions arising from tailored diffusers. In parallel, I investigate the neuroprotective effects of erythropoietin in PDT of primary cortical neurons in culture and normal rat brain in vivo. I show that the most important parameter determining prostate coverage is the number of diffusers employed. Moreover, while tailored diffusers do offer an improvement over conventional ones, the improvement is likely masked by perturbations introduced by the uncertainties of light delivery. Although these results largely discard the use of tailored diffusers in prostate PDT, significant insight has been gained into PDT treatment planning, and tailored diffusers may still be advantageous in more complicated geometries. Additionally, I show that erythropoietin does not improve survival of PDT-treated neurons PDT, nor reduces the volume of necrosis in vivo, for the ranges of conditions and doses studied. To our knowledge, this is the first time this strategy has been tested in brain PDT and deserves to be investigated further, by using later time-points, functional outcomes, and other neuroprotectants.

In this study, it was aimed to investigate the effects of different co-carbon sources on therapeutically important glycoprotein, recombinant human erythropoietin (rHuEPO) production by Pichia pastoris by designing feeding strategies which were applied in the production phase of the bioprocess. During the experiments, the cell growth, sorbitol, mannitol, and methanol consumptions, recombinant human EPO production, alcohol oxidase activity, total protease concentrations and the by-products organic acid concentrations were analyzed. In this context, firstly, laboratory scale air filtered shake bioreactor experiments were performed by P. pastoris Mut+ strain to investigate the effects of mannitol and sorbitol. 50 gL-1 initial concentration of co-substrates was found more affordable and appropriate for cell concentration and recombinant protein production. Thereafter, six pilot scale bioreactor operations were designed and performed. In the first designed strategy (named as SSM strategy), batch-wise 50 g L-1 sorbitol was fed at t=0 h of the production phase and then sorbitol concentration was kept constant at 50 g L-1 by fed-batch feeding with a pre-determined specific growth rate of &mu / Srb0=0.025 h-1 within t=0-15 h of the production phase together with fed-batch methanol feeding with a pre-determined specific growth rate of &mu / M0=0.03 h-1. In the following bioreactor experiments co-substrate mannitol was fed to the system with different feeding strategies together with fed-batch methanol feeding with a pre-determined specific growth rate of &mu / M0=0.03 h-1. In the second strategy (MM), only 40 g L-1 mannitol was added to the system at t=0 h of the production phase. In the third strategy (MMM), after adding 50 g L-1 mannitol at t=0 h, mannitol concentration was kept constant at 50 g L-1 by fed-batch feeding with a pre-determined specific growth rate of &mu / Man0=0.11 h-1 within t=0-9 h of the production phase when the same cell concentration was attained in SSM strategy. In the fourth one (MLM), limiting amount of mannitol, 3 g L-1, was added at t=0 h and then mannitol concentration was kept constant at 3 g L-1 by fed-batch feeding with a pre-determined specific growth rate of &mu / Man0=0.005 h-1 within t=0-10 h of the production phase. After these strategies, several pulses, batch-wise, mannitol feeding strategies were performed. In the fifth strategy (MPM), besides 50 g L-1 initial mannitol feeding at t=0 h, adding second batch-wise mannitol at t=6 h, and third one at t=12 h were applied. In the last strategy (MPMG), four 50 g L-1 pulse feeding of mannitol were performed at t=0 h, 7 h, 14 h, and 24 h, containing glycerol, with an initial concentration in the fermentation medium being 8 g L-1. The highest extracellular rHuEPO production was achieved in the fifth strategy MPM as CrHuEPO=645 mg L-1 at t=9 h while the highest cell concentration was achieved in the first strategy SSM as Cx=109 gL-1 at t=48 h. The overall cell and product yields on total substrate were calculated as YX/St=0.22 g g-1 and YP/St=2.23 mg g-1 in the highest rHuEPO production case.

Photodynamic therapy (PDT) derives its tumour selectivity from preferential photosensitizer accumulation and short light penetration in tissue. However, additional strategies are needed to improve the therapeutic index of PDT in oncological applications where light is delivered interstitially to large volumes (e.g. prostate), or when adjacent normal tissue is extremely sensitive (e.g. brain). Much research to improve PDT's selectivity is directed towards developing targeted photosensitizers. Here, I present two alternative strategies to improve PDT's selectivity, without compromising its efficacy. For interstitial delivery, I investigated whether customizable cylindrical diffusers can be used to deliver light doses that conform better to target geometries, specifically the prostate. Additionally, I examined whether the neuroprotectant erythropoietin, used as an adjuvant to PDT for brain tumours, can reduce the sensitivity of normal tissue, thereby improving treatment selectivity. To determine if tailored diffusers constitute an improvement over conventional ones, I introduce a novel optimization algorithm for treatment planning. I also analyze the sensitivity of the resulting plans to changes in the optical properties and diffuser placement. These results are contextualized by a mathematical formalism to characterize the light dose distributions arising from tailored diffusers. In parallel, I investigate the neuroprotective effects of erythropoietin in PDT of primary cortical neurons in culture and normal rat brain in vivo. I show that the most important parameter determining prostate coverage is the number of diffusers employed. Moreover, while tailored diffusers do offer an improvement over conventional ones, the improvement is likely masked by perturbations introduced by the uncertainties of light delivery. Although these results largely discard the use of tailored diffusers in prostate PDT, significant insight has been gained into PDT treatment planning, and tailored diffusers may still be advantageous in more complicated geometries. Additionally, I show that erythropoietin does not improve survival of PDT-treated neurons PDT, nor reduces the volume of necrosis in vivo, for the ranges of conditions and doses studied. To our knowledge, this is the first time this strategy has been tested in brain PDT and deserves to be investigated further, by using later time-points, functional outcomes, and other neuroprotectants.

El objetivo principal de esta tesis fue el de realizar una validación y caracterización de las posibles variables que pueden afectar la metodología empleada para identificar las isoformas presentes en algunas de las especies de EPO (uEPO, rEPO alfa, rEPO beta y el NESP) tras su identificación mediante la técnica de IEF e inmunodetección