Sickle cell disease (SCD) is a severe hemoglobin disorder caused by co-inheritance of a single mutation in the β-globin gene of adult hemoglobin (HbA; α2β2). This alteration leads to the formation of sickle hemoglobin (HbS; α2βS2) and deformed, sickle-shaped red blood cells (RBCs). Sickle RBCs obstruct small blood vessels resulting in anemia, excruciating pain crises, organ damage, and stroke. For the millions of people affected by this disease, life expectancy is only 40-60 years of age. The only cure for SCD is hematopoietic stem cell (HSC, CD34+) transplantation, which requires a human leukocyte antigen (HLA)-matched donor. However, this option runs the risk of complications associated with graft versus host disease and infection. Before birth, individuals with SCD do well because their RBCs are filled with γ-globin containing fetal hemoglobin (HbF; α2γ2), which inhibits the formation of HbS. In fact, some SCD patients who co-inherit mutations that allow for high-level expression of HbF into adulthood are asymptomatic. This suggests that genetic modification of the patient’s own HSCs to permit HbF production would be a viable therapeutic alternative to HSC transplantation. Our work has focused on the use of lentiviral vectors to introduce an exogenous γ-globin gene or shRNA sequences designed to knockdown repressors of γ-globin, such as the zinc-finger transcription factor, BCL11A, to prevent silencing of the endogenous γ-globin genes allowing for persistent expression of HbF. Despite significant progress using both approaches, we have been unable to increase the level of HbF > 30%; a curative threshold for SCD patients who continue to produce HbF into adulthood. The goal of my project was to combine these approaches into a single lentiviral vector to achieve co-regulated, erythroid-specific expression and augmented levels of HbF. I successfully modified the insulated, erythroid-specific γ-globin vector (termed V5m3-400) to include microRNA (miR)-adapted shRNAs (or shmiRs) targeting BCL11A (based on miR-30 and miR-E architectures) in the first and second noncoding introns of the γ-globin genomic sequences. Inclusion of shmiRs had no appreciable effect on integrity of the integrated provirus or vector titer. Vector performance was initially tested using human K562 erythroleukemia cells expressing a flag-tagged version of BCL11A. In this cell line, BCL11A knockdown was significantly improved using miR-E-shRNAs due to a dramatic increase (up to 350-fold) in processing of mature shRNA sequences. The miR-E vectors also provided high-level expression of γ-globin. Erythroid-specific expression of the γ-globin transgene and BCL11A knockdown was confirmed in maturing erythroid cells derived from transduced CD34+ cells of a healthy donor resulting in a 50% increase in HbF levels compared with cells transduced with V5m3-400 as a control. While encouraging, I was unable to discriminate HbF derived from the vector-encoded versus endogenous γ-globin genes. To address this, I introduced a single base change in exon 2 of the γ-globin gene encoded by V5m3-400 such that threonine replaces isoleucine at amino acid 75 (I75T). This variant was successfully distinguished from endogenous γ-globin gene products by reverse phase high performance liquid chromatography (HPLC) in culture-differentiated erythroid cells. Based on these findings, I created compound γ-globin/shmiR-E vectors that include the I75T substitution (I75Tγ-globin/shmiR-E). Future studies will focus on testing this novel vector design in erythroid cells derived from transduced CD34+ cells of healthy donors and patients with SCD. I anticipate that this compound vector has the potential to maximize γ-globin expression and promote levels of HbF that are unlikely to be safely and effectively achieved by conventional globin gene addition or shRNA knockdown approaches alone.

Made available in DSpace on 2013-01-07T15:55:06Z (GMT). No. of bitstreams: 2 license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) 15.pdf: 2584815 bytes, checksum: 6349ee7c691ec4772382bd325a63ae85 (MD5) Previous issue date: 2007 / Analisa os estudos médicos brasileiros sobre a anemia falciforme publicados nas décadas de 1930 e 1940. Esta dissertação orienta-se pela compreensão da relação entre sangue, doença e raça no pensamento médico brasileiro dos anos de 1930 e 1940, quando a anemia falciforme era considerada uma enfermidade que se observava principalmente, pela presença de hemácias falciformes no sangue e por uma variedade de sintomas clínicos, sobretudo pela anemia. Como a freqüência desta doença era maior nos negros do que nos brancos, a anemia falciforme era qualificada geralmente como uma doença racial.

Made available in DSpace on 2014-06-11T19:26:05Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-02-28Bitstream added on 2014-06-13T19:53:58Z : No. of bitstreams: 1 torres_ls_me_sjrp.pdf: 665668 bytes, checksum: 4aaf2ab7e9956b74cfe43d4216ad0030 (MD5) / A anemia falciforme (AF) afeta milhões de pessoas em todo o mundo e está associada a altas taxas de morbidade e mortalidade. Apresenta uma série de manifestações fenotípicas, que são influenciadas por fatores genéticos e ambientais, resultando em fenótipos diversificados, e um tratamento bastante eficaz tem sido o uso de hidroxiureia (HU), que ameniza os sintomas e a necessidade de transfusão sanguínea e hospitalização. Estudos de associação de genomas já demonstraram que polimorfismos genéticos podem desempenhar influência no perfil clínico dos pacientes, assim como na resposta destes à medicação. Os polimofismos -308G/A no gene TNFA e -509C/T no gene TGFB aumentam a produção das suas respectivas citocinas que atuam principalmente em vias inflamatórias e são fortes candidatos a estarem envolvidos na ocorrência de episódios vaso-oclusivos característicos da doença. Dessa forma, o objetivo do trabalho foi verificar a frequência desses polimorfismos em portadores da AF, com e sem o uso de HU, e possível relação com a gravidade das manifestações clínicas da doença. Foram obtidas 588 amostras de sangue periférico de pacientes com doença falciforme em acompanhamento no HEMORIO. A partir destas, foram separados, aleatoriamente, 240 pacientes com AF, cujo genótipo foi confirmado por procedimentos laboratoriais clássicos e moleculares. Estes foram genotipados para os polimorfismos -308G/A (TNFA) e -509C/T (TGFB) por PCR-RFLP. Os dados hematológicos e clínicos parciais de 118, dos 240 pacientes, foram obtidos por questionário e consulta aos prontuários médicos e banco de dados. A frequência do polimorfismo -308G/A foi de 0,83 em homozigose e 17,92% em heterozigose. Para o polimorfismo -509C/T, foi de 6,25% e 48,33%, respectivamente. Não foi observada associação entre o polimorfismo -308G/A e as manifestações clínicas... (Resumo completo, clicar acesso eletrônico aba / Sickle cell anemia (SCA) affects millions of people worldwide and is associated with high morbidity and mortality. This affection shows various phenotypic manifestations, which are influenced by genetic and environmental factors, resulting in many phenotypes, and the most effective treatment has been the use of hydroxyurea (HU), which improves the symptoms and the requirement for blood transfusion and hospitalization. Genome association studies have shown that genetic polymorphisms may play role on the clinical profile of patients, as well as in response to these medications. The -308G/A and -509C/T polymorphisms, in TNFA and TGFB genes respectively, increase production of their cytokines, which act on inflammatory pathways and are strong candidates to be involved in the occurrence of vaso-occlusive episodes. The aim of this study was to determine the frequency of these polymorphisms in patients with AF, with and without HU utilization, and possible relationship with the severity of clinical manifestations of disease. We obtained 588 peripheral blood samples of patients with sickle cell disease at HEMORIO. From these, were separated at random 240 patients with AF, whose genotype was confirmed by classical and molecular laboratory procedures. They were genotyped for polymorphisms-308G/A (TNFA) and-509C/T (TGFB) by PCR-RFLP. The hematological and clinical data of 118 of the 240 patients, were obtained by questionnaire and medical records and database. The frequency of polymorphism -308G/A was 0.83% in homozygous and 17.92% in heterozygous. For the polymorphism -509C/T, was 6,25% and 48.33% respectively. No association between polymorphism -308G/A and clinical manifestations in patients was found. Concerning the polymorphism-509C/T, the mutant allele (T) proved to be a risk... (Complete abstract click electronic access below)

La drépanocytose est la première maladie génétique en France et notamment en Guadeloupe. Il s’agit d’une maladie du sang qui est due à une mutation ponctuelle au niveau du gène de la β-globine, laquelle entre dans la composition de l’hémoglobine. Ainsi les drépanocytaires ont une hémoglobine anormale appelée « HbS », contrairement à l’hémoglobine normale « HbA ». En condition de faible oxygénation, l’HbS polymérise et forme des fibres à l’intérieur des érythrocytes. Ces fibres rigidifient et fragilisent les globules rouges. Par conséquent ils peuvent bloquer la circulation à cause de leur faible déformabilité, et causer des crises vaso-occlusives douloureuses, complication caractéristique de la drépanocytose. Ce modèle physiopathologique classique a été complété par les résultats plus récents montrant l’importance du rôle des leucocytes dans l’établissement de ces obstructions. Par ailleurs, les globules rouges des drépanocytaires sont plus prompts à l’hémolyse en raison de leur fragilité. En raison de l’hémolyse exacerbée, l’hémoglobine se retrouve dans le plasma et diminue la biodisponibilité du principal vasodilatateur, le monoxyde d’azote. De plus, les globules rouges rigides et déformés entrainent activation de l’endothélium. Il en résulte dans la drépanocytose, un contexte pro-inflammatoire et pro-adhérent mais aussi pro-coagulant.Ce contexte est propice à l’activation des cellules sanguines et notamment à celle des plaquettes et des érythrocytes qui par bourgeonnement de leur membrane, émettent alors en grandes quantités, des vésicules sub-micrométriques appelées microparticules, ou MP. En l’absence de traitement curatif applicable à tous les patients drépanocytaires, nous avons décidé d’étudier le profil mais aussi le rôle des MP de patients drépanocytaires dans le but de mieux comprendre cette maladie et dans l’espoir de peut-être découvrir une nouvelle piste diagnostique ou thérapeutique.Nous avons donc montré que les patients SC ont des concentrations sanguines en MP plus importantes que les sujets AA, mais moindres que les patients SS. Etonnamment les MP SC, qu’elles soient d’origine érythrocytaire ou plaquettaire, ont aussi plus de phosphatidylsérine (PS) à leur surface que les MP SS. Ce phospholipide étant impliqué dans l’activation de la cascade de la coagulation, il serait intéressant d’évaluer l’intensité de cette activation par des MP SS ou SC. On pourrait aussi comparer ces intensités à celle induite par des MP de patients SS sous hydroxyurée. En effet nous avons aussi montré que 2 ans après avoir commencé ce traitement, les MP érythrocytaires des patients ont une taille plus importante et une exposition de la PS diminuée drastiquement.Les MP étant physiologiquement dans le sang, elles peuvent entrer en contact avec les cellules sanguines mais aussi avec l’endothélium vasculaire. Etant donné l’importance des changements que connaît cet endothélium chez les drépanocytaires (pro-adhérent, pro-inflammatoire et pro-coagulant), nous nous sommes ensuite focalisés sur l’impact des MP de drépanocytaires sur les cellules endothéliales. Ces dernières provenaient de la moelle osseuse humaine, territoire fréquemment affecté par les vaso-occlusions. Il ressort de ces travaux que les MP de patients SS et SC induisent, par rapport aux MP de sujets AA, une surexpression dose-dépendante de gènes impliqués dans l’adhérence (ICAM-1, VCAM-1, E-sélectine), dans l’inflammation (IL-6, IL-1β et CD40-I) et dans la coagulation (TF). Au niveau protéique, ICAM-1 est aussi surexprimé. En effet les MP SS induisent dès 4 heures d’incubation, une augmentation de la densité moyenne d’ICAM-1 membranaire, ainsi qu’une augmentation de la proportion de cellules exprimant cette protéine. ICAM-1 étant impliquée dans l’adhérence des leucocytes à l’endothélium (roulement, adhérence ferme et même transmigration). / Sicle cell disease (SCD) is the first genetic disease in France and more specifically in Guadeloupe. It is a blood disorder due to a point mutation in the β-globin gene. The corresponding peptide chain being a part of hemoglobin, SCD patients have an abnormal hemoglobin called “HbS”, contrary to the normal one, so called “HbA”. In hypoxic conditions, HbS forms polymers inside red blood cells (RBCs), thereby making them rigid but also fragile. Consequently, RBCs can stop blood flow due to their low deformability, and so cause a painful vaso-occlusive crisis, which is a complication characterizing SCD. This pathophysiological model has been modified by recent results showing the involvement of leukocytes in the establishing of these occlusions. Besides, sickle RBCs are more prone to hemolysis owing to their being fragile. Due to this exacerbated hemolysis, hemoglobin is released in the plasma and diminishes the bioavailability of the main vasodilator, nitrite monoxide. Moreover, rigid sickled RBCs entail endothelium activation, which results in a pro-inflammatory, a pro-adhesive and a pro-coagulant context. This latter favors blood cells activation, among which are platelets and erythrocytes that bud high quantities of submicrometric membrane vesicles called microparticles, or MPs. In the absence of curative treatment for all patients, we decided to study the profile but also the role of MPs from SCD patients to better understand this disease and hoping to find a new diagnostic or therapeutic pathway. We showed that SC patients have lower MP levels than SS patients, but higher MP levels than AA subjects. Surprisingly, we have observed that SC MPs, whether they derive from RBCs or platelets (PLTs), have higher densities of exposed phosphatidylserine (PS) than SS MPs. Since this phospholipid is involved in the activation of the coagulation cascade, it would be interesting to evaluate the intensity of this activation by SS or SC MPs. One could also compare these intensities to the one induced by MPs from SS patients under hydrocarbamide. Indeed we also showed that 2 years after the beginning of this treatment, erythrocyte-derived MP are larger and expose PS much less.As MPs are physiologically in the blood, they can interact with blood cells but also with the vascular endothelium. Given the known changes of this endothelium in SCD (pro-adhesive, pro-inflammatory and pro-coagluant), we then focused on the impact of SCD MPs on endothelial cells (ECs). These cells came from human bone marrow, a territory frequently affected by vaso-occlusions. These experiments showed that SS and SC MPs, induce, compared to AA MPs, a dose-dependent overexpression of genes involved in adherence (ICAM-1, VCAM-1, E-selectin), in inflammation (IL-6, IL-1β and CD40-I) and in coagulation (TF). At the protein level, ICAM-1 is also overexpressed. Indeed SS MPs induce within 4 hours of incubation, an increase of the mean membrane density of ICAM-1, but also an increased proportion of cells bearing this protein. As ICAM-1 is involved in leukocytes adherence to the endothelium (rolling, firm adhesion and even transmigration), SS MPs may, by triggering ICAM-1 overexpression at the endothelium surface, allow their adherence to the endothelium, thereby promoting RBC adherence and so the occlusion of the vessel and the occurring of a VOC. It would be interesting to determine which type of MP cause the overexpression of ICAM-1 and to evaluate if it is sufficient to increase in vitro adherence of leukocytes to ECs stimulated with MPs. This would allow to evaluate how important MPs are when considering the fight against sickle cell disease.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / A anemia falciforme (AF) Ã uma das alteraÃÃes genÃticas mais comuns em nosso paÃs. Os haplÃtipos da &#61538;s-globina estÃo associados com a heterogeneidade clÃnica apresentada pela doenÃa. Os portadores de AF sÃo submetidos a elevados nÃveis de estresse oxidativo. A hidroxiurÃia (HU) tem sido utilizada no tratamento por sua aÃÃo de elevar os nÃveis de hemoglobina fetal e aÃÃo antioxidante. O estudo teve como objetivo investigar o impacto do uso da HU e dos haplÃtipos no perfil oxidativo nos pacientes com AF. Foram analisadas amostras de 64 pacientes acompanhados no Hospital UniversitÃrio Walter CantÃdio - HUWC e do grupo controle de 20 indivÃduos sem hemoglobinopatias. Os pacientes foram estratificados em grupos: uso de HU > 1 ano, uso de HU &#8804; 1 ano e sem uso de HU (SHU). A confirmaÃÃo do diagnÃstico e a anÃlise dos haplÃtipos foram realizadas atravÃs de estudo molecular. As dosagens do nitrito (NO2-), malonaldeÃdo (MDA), glutationa peroxidase (GSH-Px), catalase (CAT), glutationas total (GSSG+GSH), oxidada (GSSG) e reduzida (GSH) e da relaÃÃo GSSG/GSH (glutationas oxidada/reduzida) foram realizadas por espectrofotometria. Os nÃveis mÃdios de NO2-, e MDA mostraram-se mais elevados no grupo SHU, sendo significante apenas para o MDA (p < 0,05). O grupo SHU apresentou atividade mÃdia das enzimas CAT (p = 0,031) e GSH-Px (p = 0,036) inferiores aos demais grupos e maior relaÃÃo GSSG/GSH (p < 0,05). A avaliaÃÃo do estresse oxidativo em relaÃÃo aos haplÃtipos demonstrou que na populaÃÃo sem uso de HU os nÃveis de NO2- e de MDA foram semelhantes entre os grupos, com um aumento nÃo significante da GSH-Px no grupo Bantu/n em relaÃÃo ao Benin/n e do Ãndice GSSG/GSH no grupo Benin/n em relaÃÃo ao Bantu/n. Na populaÃÃo em uso de HU verificou-se um aumento nÃo significante dos nÃveis de NO2- no grupo Bantu/n em relaÃÃo ao Benin/n com resultados semelhantes de MDA entre os grupos e um aumento significante da GSH-Px (p<0,03) no grupo Benin/n em relaÃÃo ao Bantu/n. Os resultados da CAT foram semelhantes entre os grupos. A relaÃÃo GSSG/GSH foi maior, porÃm nÃo significante no grupo Bantu/n em relaÃÃo ao Benin/n. Os resultados do presente estudo reforÃam a hipÃtese de que os pacientes com AF apresentam um estado hiperoxidativo com nÃveis elevados dos produtos do estresse oxidativo e diminuÃdos do perfil antioxidante e que a hidroxiurÃia teve um impacto sobre o perfil oxidativo. No entanto, em relaÃÃo ao impacto dos haplÃtipos no estresse oxidativo, estudos posteriores com uma maior amostragem devem ser recomendados para confirmar nossos resultados, considerando que o grupo de pacientes nÃo tratados com HU foi menor que o grupo de pacientes em uso de HU, o que pode ter interferido na anÃlise estatÃstica dos resultados. / The sickle cell anemia is one of the most common genetic disorders in our country. The &#61538;-globin haplotypes are associated with the clinical heterogeneity of the disease. Individuals with sickle cell disease are subjected to high levels of oxidative stress. Hydroxyurea (HU) has been used as treatment and it increases the levels of fetal hemoglobin having an action antioxidant. The study aimed to investigate the impact of the use of HU and haplotypes in the oxidative status in patients with sickle cell anemia. Samples from 64 patients treated at the University Hospital Walter CantÃdio - HUWC and the control group of 20 individuals without hemoglobinopathies. Patients were stratified into groups: the first one using HU > 1 year, the second using HU &#8804; 1 year and the third using of HU (SHU). Confirmation of the diagnosis and analysis of haplotypes were performed by molecular study. The measurements of nitrite (NO2-), malonaldehyde (MDA), glutathione peroxidase (GSH-Px), catalase (CAT), glutathione total (GSSG+GSH), oxidized (GSSG) and reduced (GSH) and the ratio GSSG/GSH (glutathione oxidized/reduced) were performed by spectrophotometry. Mean levels of NO2- and MDA were shown to be higher in SHU group, being significant only for MDA (p < 0,05). The assessment of oxidative stress in relation to haplotypes showed that the population without the use of HU levels of NO2- and MDA were similar between groups, with an insignificant increase of GSH-Px in the Bantu/n compared with Benin/n and index GSSG/GSH in the Benin/n compared with Bantu/n. In the population using the HU there was a nonsignificant increase in the levels of NO2- in the Bantu/n compared with Benin/n with similar results between groups of MDA and a significant increase in GSH-Px (p <0.03) in the Benin/n compared with Bantu/n. The results of CAT were similar between groups. The ratio of GSSG/GSH was higher but not significant in the Bantu/n compared to Benin/n. The results of this study support the hypothesis that patients with the sickle cell anemia have a state hiperoxidativo products with high levels of oxidative stress and decreased antioxidant status and that of hydroxyurea had an impact on oxidative stress. However, regarding the impact of the haplotypes in oxidative stress, further studies with a larger sample should be recommended to confirm our results, considering that the group of patients not treated with HU was lower than the group of patients using HU, the which may have interfered in the statistical analysis of results.

Orientador: Helena Zerlotti Wolf Grotto / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-08T13:21:42Z (GMT). No. of bitstreams: 1 Noronha_JoseFernandodeAlmeida_D.pdf: 5060365 bytes, checksum: 9a433693a12ebc4fc3a72a0639d93c73 (MD5) Previous issue date: 2007 / Resumo: Introdução: Plaquetas reticuladas (PRs) são plaquetas jovens recentemente liberadas pela medula óssea para a circulação e que possuem alto conteúdo de RNA em seu citoplasma. As PRs são descritas como plaquetas grandes, densas e mais ativas no processo de formação do trombo hemostático. A participação das plaquetas ativadas nos fenÃ'menos vaso-oclusivos que acometem pacientes com síndrome falciformes (SF) já está documentada, mas a avaliação do papel das PRs não foi ainda descrito. Assim, avaliamos o número e a atividade das PRs, correlacionando-os com os níveis séricos de P-selectina solúvel (sCD62p), interleucinaâ?¿6 (IL-6), interleucina-3 (IL-3) e trombopoietina (TPO) em sangue periférico de pacientes em diferentes estágios clínicos das SF. Casuística e Métodos: Foram estudados 89 pacientes adultos, sendo 38 em fase â?¿estávelâ??, 27 em crise hemolítica (CH), 27 em crise vaso-oclusiva (CVO) e 30 indivíduos saudáveis (grupo controle - GC). Os parâmetros plaquetários, incluindo a quantificação de macroplaquetas foram determinados em analisador hematológico. Através da técnica da citometria de fluxo com o uso de anticorpos monoclonais e thiazole orange (TO), realizamos as identificações e quantificações das plaquetas ativadas (anti CD41a + anti CD62p), PRs (anti CD41a + TO) PRs ativadas (TO + anti CD62p). As dosagens de sCD62p, IL-6, IL-3 e TPO foram realizadas pela técnica de ELISA. Resultados: O número de macroplaquetas foi significativamente superior nas 3 fases clínicas das SF em relação ao GC. O número de plaquetas ativadas, tanto reticuladas como maduras foi superior em todas as fases clínicas quando comparadas ao GC. Os valores de PRs mostraram-se mais elevados no grupo de pacientes em CVO do que na fase â?¿estávelâ?? e na CH. Em todos os grupos as PRs apresentaram um maior grau de ativação quando comparadas às plaquetas maduras. Os pacientes em CVO apresentaram maiores níveis de sCD62p em relação ao GC. Nas diferentes fases das SF observamos níveis séricos elevados de IL-6, IL-3 e TPO, embora não tenha sido observada correlação entre essas determinações com os números absolutos de PRs, Plaquetas ativadas (CD62p+) e PRs ativadas (TO+/CD62p+). Conclusões: Os resultados sugerem a contribuição das PRs na trombogênese das Síndromes Falciformes. Níveis elevados das interleucinas provavelmente indicam a participação das mesmas no processo inflamatório que acompanha os fenÃ'menos de vaso-oclusão, mas aparentemente esses moduladores inflamatórios não exercem efeito sobre a trombopoiese em pacientes com SF / Abstract: Introduction: Reticulated Platelets (RPs) are youngest platelets released recently from bone marrow to the blood and are characterized by the high citoplasmatic RNA content. RPs are described as higher size, denser and more active in the formation of thrombus than mature platelets. The participation of activated platelets in vaso-occlusive process in sickle cell disease patients have been documented, but the evaluation of the RPs role has not been established at the moment. We evaluated the number and activity of RPs and correlated them with serum soluble P-selectin (sCD62p), Interleukin-6 (IL-6), Interleukin 3 (IL-3) and thrombopoetin (TPO) levels in patients with sickle cell diseases in different clinical expressions. Casuistic and Methods: Eighty-nine adult patients were studied: 38 in steady-state, 27 in hemolytic crisis (HC), 27 in vascular-occlusive crisis (CVO) and 30 healthy individuals (control groupâ?¿CG). Platelet parameters including the percentage of larger platelets were obtained by an automatic hematological analyzer. Monoclonal antibodies, thiazole orange (TO) dye and flow cytometric technique were used to identify and to quantify activated platelets (anti CD41a+ and anti CD62p+), RPs (anti CD41a+ and TO+) and activated RPs (TO+ and anti CD62p+). Soluble CD62p, IL-3, IL-6 and TPO determinations were measured by ELISA tests. Results: The number of macroplatelets was significantly higher in steady-state, CVO and HC groups than in CG. The number of activated mature platelets and activated RPs was higher in all stages of the disease when compared with CG. PRs values were more elevated in group of patients with CVO than in HC and steady-state. The degree of activation was higher in PRs than in mature platelets independently on sickle cell disease phase. CVO patients showed higher serum levels of sCD62p than CG. IL-6, IL-3 and TPO serum levels were increased in sickle cell disease, but there was not a correlation between those determinations and parameters related to platelets. Conclusions: Our results suggest that PRs contribute to the thrombogenesis process in sickle cell disease. Increased serum levels of interleukins probably indicate the participation of PRs in inflammatory process which is associated to vascular-occlusive phenomenon, but apparently those inflammatory mediators do not have an effect on thrombopoiese in sickle cell disease patients / Doutorado / Ciencias Biomedicas / Doutor em Ciências Médicas

Orientador: Nicola Amanda Conran Zorzetto / Dissertação (mestrado) - Universidade Estadual de Campinas. Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-15T08:53:01Z (GMT). No. of bitstreams: 1 Miguel_LedianaIagalo_M.pdf: 1386194 bytes, checksum: b81f5aa45e96b255588df48747c1397c (MD5) Previous issue date: 2010 / Resumo: A adesão anormal das células brancas e vermelhas ao endotélio, que desencadeia numa diminuição do fluxo de sangue na microcirculação, é um dos principais fatores envolvidos na iniciação da vaso-oclusão em pacientes falciformes (AF). O estado inflamatório crônico, característico nos pacientes com AF, eleva a circulação de citocinas, as quais podem contribuir significativamente para a ativação e adesão das células vermelhas e brancas ao endotélio. O óxido nítrico (NO) e a via de sinalização dependente em NO têm importante efeito inibidor nas propriedades adesivas de leucócitos. Drogas que aumentem a biodisponibilidade de NO ou que atuem na via de sinalização NO-GMPc podem ser benéficas no tratamento de alguns aspectos da AF. Já é de conhecimento que pacientes com AF apresentam níveis elevados de algumas citocinas presentes no plasma, assim sendo, este estudo teve como objetivo avaliar os efeitos in vitro das citocinas nas propriedades adesivas de neutrófilos e células vermelhas de indivíduos controles e pacientes com AF. Adicionalmente, foram determinados os efeitos de BAY 73-6691, um inibidor da enzima hidrolizante de GMPc, fosfodiesterase 9A (PDE9A) e BAY 41-2272, um ativador de guanilato ciclase, na ausência ou presença da estimulação pelas citocinas na adesão dessas células. Os neutrófilos e as células vermelhas de indivíduos controles e pacientes com AF foram isolados de sangue periférico. A adesão das células à fibronectina foi determinada utilizando o ensaio de adesão estático na presença ou ausência das citocinas IL-8 (10-500ng/ml), TNF-alpha (10-100ng/ml) e GM-CSF (0,1-10ng/ml) e/ou na presença/ausência de BAY 73-6691 (60µM), BAY 41-2272 (60nM) ou DMSO como veículo (0.2%v/v). Como previamente demonstrado, os neutrófilos de pacientes com AF (neutrófilos AF) possuem uma maior capacidade de aderir à FN do que os neutrófilos de indivíduos controle (neutrófilos CON). A estimulação das células in vitro com as três citocinas aumentaram significativamente as adesões à FN dos neutrófilos CON e aumentou ainda mais a adesão dos neutrófilos AF. A incubação de ambos os neutrófilos, CON e AF, com BAY 73-6691, mas não BAY 41-2272, reduziu significativamente as propriedades adesivas à FN; esse evento foi acompanhado por uma diminuição da expressão das moléculas de adesão, L-selectina e CD11b (subunidade Mac-1) na superfície de neutrófilos AF. Além do mais, nas concentrações utilizadas, BAY 73-6691, mas não o BAY 41-2272, diminui significativamente a adesão de neutrófilos CON e AF após estimulação com IL-8, TNF-a e GM-CSF. No entanto, esse evento não foi acompanhado por alterações na expressão da moléculas de adesão na superfície de neutrófilos AF quando estimulados com IL-8. As células vermelhas de indivíduos AF também apresentaram uma maior capacidade de se aderir à FN quando comparadas às células de indivíduos controles. No entanto, ao contrário dos neutrófilos, na presença de IL-8 (10-500ng/ml) e TNF-a (0.1-1µg/ml), não houve alteração das propriedades adesivas dessas células tanto de indivíduos controles quanto das células de pacientes com AF. Além disso, BAY 73-6691 e BAY 41-2272, não alteraram a adesão basal tanto das células vermelhas de controles quanto pacientes com AF. Os principais mediadores inflamatórios, utilizados em concentrações fisiologicamente relevantes, foram capazes de aumentar as propriedades adesivas de neutrófilos, mas não das células vermelhas, de indivíduos controles e AF. Portanto, sugerimos que as citocinas inflamatórias circulantes podem desempenhar um papel na indução das propriedades adesivas dos neutrófilos em pacientes falciformes; em contrapartida, outros fatores além do estímulo inflamatório, podem ser mais importante para induzir a adesão das células vermelhas de pacientes AF. Dados sugerem que agentes que aumentam os níveis de GMPc intracelular podem ser úteis para reduzir as propriedades adesivas de neutrófilos AF, mesmo na presença de um estado inflamatório. PDE9A é altamente expressa pelas células hematopoiéticas e a inibição desta enzima, com conseqüente elevação de GMPc, pode representar um alvo terapêutico para drogas que são tecido/célula específicas, necessitando de mais estudos in vivo e in vitro para a terapêutica de AF / Abstract: The adhesion of both red and white cells to the vessel walls of the microcirculation initiates vaso-occlusion in sickle cell disease (SCD). The chronic inflammatory nature of SCD leads to elevation of circulating cytokines in patients, which may contribute significantly to the activation of red and white cells and their consequent adhesion. Nitric oxide (NO) and the NO-dependent signaling pathway have important inhibitory effects on cellular adhesive properties. Drugs that enhance NO bioavailability or NO-cGMP-dependent signaling may hold potential for treatment of various aspects of SCD. It is known that levels of certain cytokines are augmented in the plasma of SCD individuals; therefore, this study aimed to observe the effect of cytokines, on the in vitro adhesive properties of neutrophils (neu) and red blood cells (RBC) from healthy control (CON) and steady-state SCD (SCD) individuals. Furthermore, the effects of BAY 73-6691, an inhibitor of the cGMP-hydrolyzing enzyme, phosphodiesterase 9A (PDE9A) and BAY 41-2272, a guanylate cylase activator, on non-stimulated and cytokine-stimulated cell adhesion were determined. Neutrophils and red blood cells (RBC) were isolated from the peripheral blood of CON and SCD individuals. Cell adhesion to immobilized fibronectin was assessed using static adhesion assays in the presence or absence of the cytokines, IL-8 (10-500ng/ml), TNF-alpha (10-100ng/ml) and GM-CSF (0,1-10ng/ml) and/or in the presence/absence of BAY 73-6691 (10-60µM), BAY 41-2272 (60nM) or DMSO vehicle (0.2%v/v). As previously demonstrated, SCDneu have a greater capacity to adhere to FN than CONneu. Stimulation of cells in vitro with all three cytokines significantly augmented both CONneu adhesion to FN and further increased SCDneu adhesion. The incubation of both CONneu and SCDneu with BAY 73-6691, but not BAY 41-2272, significantly reduced their adhesions to FN; this was accompanied by a decrease in the expressions of the L-selectin and CD11b (Mac-1-subunit) adhesion molecules on the SCAneu surface. Furthermore, BAY 73-6691, but essentially not BAY 41-2272, significantly inhibited CONneu and SCDneu adhesion stimulated by IL-8, TNF-alpha and GM-CSF. However, this was not accompanied by alterations in adhesion molecule presentation on IL-8-stimulated SCAneu. As previously reported, SCD RBC have a greater capacity to adhere to FN, in vitro, compared to CON RBC. However, in contrast to neutrophils, cytokines IL-8 (10-500ng/ml) and TNF-alpha (0.1-1µg/ml) did not alter the capacities of neither CON RBC nor SCD RBC to adhere to FN. Furthermore, BAY 73-6691 and BAY 41-2272 did not affect either basal CON RBC or SCD RBC adhesion. Key SCD inflammatory mediators were found, at physiologically relevant concentrations, to augment the adhesive properties of neutrophils from control and SCD individuals. Circulating inflammatory cytokines may play a role in the induction of leukocyte adhesive properties in SCD; in contrast factors other than inflammatory stimuli may be more important for induction of SCD RBC adhesion. Data suggest that elevation of intracellular cGMP may be an important approach for reducing SCD leukocyte adhesive properties, even in an inflammatory environment. PDE9A is highly expressed in hematopoietic cells and inhibition of this enzyme, with consequent augmentation of cGMP, may represent a tissue/cell-specific therapeutic drug target worthy of further in vitro and in vivo studies as a therapy for SCD / Mestrado / Ciencias Biomedicas / Mestre em Ciências Médicas

Orientador: Nicola Amanda Conran Zorzetto, Fernando Ferreira Costa / Texto em português e inglês / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-24T19:18:22Z (GMT). No. of bitstreams: 1 Garrido_VanessaTonin_D.pdf: 6610754 bytes, checksum: 9b799d90821835bae6ac5d806b1d2f1c (MD5) Previous issue date: 2014 / Resumo: A anemia falciforme (AF) é uma hemoglobinopatia hereditária resultante de uma mutação no gene que codifica a subunidade ?-globina, levando à produção da hemoglobina S (HbS) nos eritrócitos. Com a polimerização da HbS, durante a desoxigenação, ocorre a deformação e fragilização das células vermelhas, resultando em anemia hemolítica e eventos vaso-oclusivos. As crises vaso-oclusivas são a principal causa de morbidade nos pacientes com anemia falciforme e as plaquetas parecem ter um papel importante nesse processo, pois uma vez ativadas elas secretam e expressam mediadores que induzem uma resposta inflamatória tanto em leucócitos como em células endoteliais. A proposta deste trabalho foi investigar a produção e expressão dos mediadores inflamatórios derivados de plaquetas, LIGHT e CD40L, em controles (indivíduos saudáveis; CON), pacientes com anemia falciforme (AF) e pacientes com anemia falciforme em terapia com hidroxiureia (AFHU). Também avaliamos o envolvimento das plaquetas e seus mediadores na ativação de leucócitos e células endoteliais. Os níveis plasmáticos de ambas citocinas foram significativamente maiores em indivíduos AF e AFHU do que nos indivíduos controle e, curiosamente, apesar da hidroxiureia ser capaz de diminuir a concentração plasmática de algumas citocinas inflamatórias, a terapia com essa droga não foi associada com qualquer alteração nos níveis de LIGHT ou CD40L. Foi observada uma correlação expressiva da concentração de LIGHT com níveis plasmáticos de CD40L, IL-8, ICAM-1, Trombospondina-1 e TNF-?, enquanto que a concentração plasmática de CD40L correlacionou-se com os níveis de TNF-? e principalmente com Trombospondina-1, indicando que tanto LIGHT como CD40L podem estar participando ou então refletindo a inflamação crônica presente na anemia falciforme. A expressão proteica de LIGHT foi significativamente maior na superfície de plaquetas de indivíduos AF e AFHU em comparação com plaquetas CON e apresentou uma correlação com marcadores de ativação plaquetária. A secreção de LIGHT pelas plaquetas foi determinada por ELISA e concentrações significativas dessa citocina puderam ser detectadas no sobrenadante de plaquetas CON e AF, sugerindo que essas células podem ser uma fonte importante de LIGHT na circulação. Apesar da expressão de CD40L não ter sido detectada na superfície das plaquetas de pacientes e controles, as plaquetas de pacientes AF secretaram uma quantidade maior de CD40L em comparação aos controles e foi observada uma correlação significativa entre a liberação de LIGHT e CD40L em plaquetas de pacientes AF, indicando que pode existir uma associação na secreção dessas duas citocinas. A expressão dos receptores de LIGHT (HVEM e LT?R) e de CD40L (CD40) foi avaliada por citometria de fluxo em plaquetas, neutrófilos, linfócitos e monócitos. Foi observado que o receptor HVEM estava mais expresso em plaquetas e linfócitos de pacientes com anemia falciforme, enquanto que a expressão do receptor CD40 estava elevada nas plaquetas, nos neutrófilos, nos linfócitos e nos monócitos de pacientes, comparando com o grupo controle. Esses dados mostram que a via de sinalização de LIGHT e CD40L pode estar alterada na anemia falciforme, contribuindo com a ativação dos leucócitos. Quando avaliamos a participação das plaquetas na ativação dos leucócitos, observamos que as plaquetas de indivíduos com anemia falciforme foram eficientes em aumentar a expressão do marcador de ativação, CD69, nos linfócitos e também em induzir o fenótipo pró-inflamatório nos monócitos. Enquanto que a co-cultura de HUVECs com plaquetas demonstrou que as plaquetas de pacientes com anemia falciforme possuem uma capacidade maior de induzir a expressão de ICAM-1 em células endoteliais do que as plaquetas de indivíduos controle. Na presença de anticorpos anti-CD40L observamos uma redução drástica no aumento da expressão de ICAM-1 pelas plaquetas e apesar dessa expressão também ter sido reduzida na presença de anticorpos anti-LIGHT, esses resultados não foram estatísticamente significativos. Interessantemente, altas concentrações plasmáticas de LIGHT estavam associadas com a elevada velocidade de regurgitação tricúspide, um indicativo de hipertensão pulmonar na anemia falciforme e uma associação significativa também foi encontrada entre níveis elevados de CD40L e pacientes com histórico de Síndrome Torácica Aguda. Essas evidências sugerem que LIGHT e CD40L parecem estar contribuindo com a ativação dos leucócitos e do endotélio, exercendo um papel importante na fisiopatogenia da anemia falciforme e aparentemente nas manifestações clínicas desta doença. Os resultados encontrados neste estudo evidenciam a importância que as plaquetas e seus mediadores inflamatórios, LIGHT e CD40L, podem ter na propagação da inflamação vascular presente na anemia falciforme, se tornando possíveis alvos para novas abordagens terapêuticas / Abstract: Sickle cell disease results from a single amino acid substitution in the gene encoding the ?-globin subunit, leading to hemoglobin S production in red blood cells. Polymerization of deoxygenated sickle hemoglobin leads to decreased deformability of red blood cells, resulting in hemolytic anemia and vaso-occlusive events. Platelets appear to play an important role in the vaso-occlusive process, as following their activation they express and secrete mediators that induce an inflammatory response in endothelial cells and leukocytes. The purpose of this study was to investigate the production and expression of LIGHT and CD40L on platelets, the presence of this protein in the plasma of controls (healthy subjects; CON), sickle cell anemia patients (AF) and sickle cell anemia patients on hydroxyurea therapy (AFHU). In addition, this study evaluated the involvement of platelets and their mediators, LIGHT and CD40L, in the activation of leukocytes and endothelial cells. Plasma levels of both cytokines were significantly higher in AF and AFHU individuals than in control individuals and interestingly, HU therapy was not associated with a reduction in these levels. A significant correlation was observed between levels of LIGHT with plasma levels of CD40L, IL-8, ICAM-1, Thrombospondin-1 and TNF-?, whereas the plasma concentration of CD40L correlated with levels of TNF-? and especially with plasma Thrombospondin-1. LIGHT expression was significantly higher on the surface of platelets from AF and AFHU subjects, compared with CON individuals and this expression demonstrated a correlation with markers of platelet activation. LIGHT secretion was determined by ELISA and significant concentrations of this cytokine could be detected in the supernatant of platelets from CON and AF individuals, indicating that platelets may be an important source of LIGHT. Although CD40L expression was not detected on the platelet surface in patients or controls, sickle platelets secreted an increased amount of CD40L, compared to controls. A significant correlation was observed between CD40L and LIGHT release in sickle cell patients, indicating that the production of these two proteins may be tightly coupled. The expression of LIGHT (HVEM and LT?R) and CD40L (CD40) receptors was evaluated by flow cytometry on the surface of platelets, neutrophils, lymphocytes and monocytes. An increased HVEM receptor expression was observed on the platelets and lymphocytes of sickle cell patients, whereas the expression of the CD40 receptor was elevated on platelets, neutrophils, lymphocytes and monocytes from sickle cell patients, compared to control subjects. Evaluating the contribution of platelets to leukocyte activation, we observed that platelets from sickle cell anemia individuals increased the expression of the activation marker, CD69, on lymphocytes and also induced a pro-inflammatory phenotype on monocytes. Co-culture of HUVEC with platelets demonstrated that sickle cell platelets have an increased ability to induce ICAM-1 expression on endothelial cells than platelets from control subjects. Furthermore, in the presence of anti-CD40L antibodies, a drastic reduction was observed in this increase. Although the expression of endothelial ICAM -1 was also reduced in the presence of anti-LIGHT antibodies, these results were not statistically significant. Interestingly, high plasma concentrations of LIGHT were associated with elevated tricuspid regurgitant velocity, indicative of pulmonary hypertension in sickle cell anemia. A significant association was also found between high levels of CD40L and patients with a lifetime history of acute chest syndrome. LIGHT and CD40L appear to contribute to leukocyte and endothelial activation, playing an important role in the pathophysiology of sickle cell anemia and apparently in the clinical manifestations of this disease. These results highlight the important role that platelets and their inflammatory mediators may play in the vascular inflammation that is known to occur in sickle cell anemia / Doutorado / Fisiopatologia Médica / Doutora em Ciências

Actuellement, l’Hydroxyurée est le seul traitement médicamenteux qui permet d’améliorer la qualité de vie et l’espérance de vie des patients drépanocytaires. Ses effets sont essentiellement liés à l’augmentation de l’HbF par l’inhibition de la polymérisation de l’hémoglobine S qui constitue la base physiopathologique de la drépanocytose. Cependant d’autres paramètres de réponse contribuent à l’amélioration clinique, principalement les atteintes chroniques, et ce indépendamment de l’augmentation du %HbF. Le phénotype hémolytique est associé aux globules rouges denses (DRBC) qui constituent une sous population de globules rouges présentant une densité supérieure à 1,11. L’amélioration des manifestations chroniques est associée à une diminution du %DRBC qui semble être une cible thérapeutique de l’Hydroxyurée. Par son efficacité de plus en plus démontrée, l’Hydroxyurée pourrait être le traitement de recours dans le traitement de fond de la drépanocytose, cependant son mode d’action reste encore partiellement élucidé.Ce travail a été réalisé dans le but de mieux comprendre le mode d’action de l’Hydroxyurée.La première étude consiste à déterminer les facteurs prédictifs de la diminution des DRBC sous Hydroxyurée. Après 6 mois de traitement, une diminution significative du %DRBC associée à une amélioration des paramètres d’hémolyses a été observée. Cet effet est indépendant de l’augmentation du %HbF. La diminution du %DRBC semble être plus constante que l’augmentation du %HbF sous Hydroxyurée.La deuxième étude a pour but de mettre au point une nouvelle approche de mesure de la teneur en HbF par globule rouge. L’outil de quantification actuellement utilisé donne une mesure du pourcentage moyen de l’HbF. Or la teneur en HbF par globule rouge peut varier pour un même %HbF conduisant à une variabilité de réponse clinique et biologique. Dans l’hypothèse d’un seuil d’HbF inhibant la polymérisation de l’hémoglobine S, une méthode précise de mesure de la teneur en HbF par globule rouge a été développée. Dans le cadre d’une preuve de concept, une étude de la distribution de l’HbF a été effectuée au cours du traitement par Hydroxyurée. Le suivi longitudinal montre une distribution hétérogène de l’HbF avant le début du traitement par Hydroxyurée. La distribution de l’HbF devient homogène après 6 mois sur l’ensemble des globules rouges avec une diminution rapide du nombre de globules rouges ne contenant pas d’HbF au profit des autres populations globulaires.La troisième étude a pour but d’optimiser le traitement par Hydroxyurée. En effet les atteintes rénales associées à la drépanocytose sont de plus en plus fréquentes et constituent un facteur de mortalité. Elles peuvent influencer la pharmacocinétique de l’Hydroxyurée car l’excrétion rénale constitue une voie d’élimination de l’Hydroxyurée. Nous avons d’abord développé unetechnique de mesure simple et fiable de dosage de l’Hydroxyurée. La pharmacocinétique de l’Hydroxyurée a été comparée entre les patients drépanocytaires présentant une fonction rénale normale avec ceux présentant une insuffisance rénale modérée et une hyper filtration glomérulaire. Une diminution de l’élimination de l’Hydroxyurée associée à une augmentation des concentrations plasmatiques ont été observées chez les insuffisants rénaux. La pharmacocinétique de l’Hydroxyurée semble être similaire chez les patients normo-rénaux et hyperfiltrants.Ce travail a apporté des éléments de réponse que nous jugeons important quant à l’action de l’Hydroxyurée sur les DRBC impliqués dans la vasculopathie chronique et sur la distribution de l’HbF. Nous avons également apporté une nouvelle technique de mesure de l’Hydroxyurée et montré qu’une adaptation de dose est nécessaire chez les patients ayant une insuffisance rénale. / Hydroxyurea, the only sickle-cell–disease approved drug, has proven its efficacy in ameliorating patient quality of life and life expectancy. The classical biological response of Hydroxyurea therapy is increased fetal hemoglobin (HbF) which inhibits deoxy hemoglobin S polymerization. However, other parameters also support clinical benefits of Hydroxyurea especially for chronic organ failures which are related to hemolysis. Sickle-cell–disease hemolytic phenotype is associated with dense red blood cells (DRBC) defined as having a density >1,11. Improvement of chronic clinical complications is associated with a marked decrease in %DRBC emphasizing its role as a therapeutic target for Hydroxyurea treatment. Despite the known benefits in both acute and chronic sickle-cell–disease manifestations, the mechanism action of Hydroxyurea has not been fully elucidated.This thesis was undertaken in order to investigate the mechanism of action of Hydroxyurea in sickle-cell–disease treatment.The first aim is to determine biological parameters predictive of %DRBC decrease under Hydroxyurea. A significant decrease in %DRBC after 6 months of Hydroxyurea therapy was observed. This biological response was associated with a decrease in hemolysis. However no correlation with %HbF increase was detected; the decrease in %DRBC under Hydroxyurea was more constant than the increase in %HbF.The second aim of this study was to develop a new approach for HbF determination in individual red blood cells. Indeed, routine HbF quantification only provides a mean value. However clinical amelioration is not exclusively dependent on the average %HbF, since a wide distribution of HbF content per cell could indicate a HbF content threshold for inhibition of deoxy hemoglobin S polymerization. In this context, a simple and precise method was developed in order to assay HbF content per cell. The distribution of HbF during Hydroxyurea therapy was analyzed as proof of concept. The longitudinal monitoring showed an uneven distribution of HbF before Hydroxyurea and a normal distribution in the whole red blood cells after 6 months of therapy. A rapid decrease in red blood cells not containing HbF in favor of cells expressing a high quantity of HbF was observed.The third objective consisted of Hydroxyurea therapy optimization. Chronic kidney disease is a frequent complication during sickle-cell–disease. Since Hydroxyurea is cleared from plasma partially by renal excretion, impaired renal function could potentially affect Hydroxyurea pharmacokinetics. A new simple Hydroxyurea dosage method was performed in this context.Hydroxyurea pharmacokinetic parameters were compared among sickle-cell–disease patients with normal renal function, moderate renal insufficiency and renal hyper filtration. A decrease in Hydroxyurea elimination associated with a marked plasma concentration was observed inrenally impaired patients. No significant difference was observed between renal hyper filtration and normal patients.These results provide answers about Hydroxyurea effect on DRBC which are involved in chronic vasculopathy and on HbF distribution. Hydroxyurea dosage method underpins the need for dose adjustment in renally impaired patients.

Ischemic stroke is defined as a blockage or reduced flow of blood to select areas of brain tissue due to either plaque formation or buildup of blood clots in the small blood vessels. A characteristic of sickle cell anemic patients is the potential for them to experience a similar type of blockage due to the sticky nature of the sickled red blood cells as well as defective oxygen delivery to the brain. Because of this similarity, sickle cell anemia may represent a good animal research model for therapeutic intervention based on stroke models. In recent studies, Granulocyte-Colony Stimulating Factor (GCSF), has been shown to exhibit a robust range of neuroprotective properties against neurological disorders including ischemic stroke through preservation of the endoplasmic reticulum (ER) by modulating various ER stress pathways. Through cognitive deficit analysis in the form of behavioral and locomotor experiments in addition to in situ biomarker analysis by way of western blotting and immunohistochemistry, we found that G-CSF gene therapy exhibited neurogenic and neuroprotective effects in ischemic mouse models and could possibly serve as a good therapy for other diseases that share similar pathology to stroke. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2021. / FAU Electronic Theses and Dissertations Collection