Initial experiments evaluated the impact of hematocrit on a bird’s ability to adapt to stress and what physiological mechanisms occurred to maintain oxygen carrying capacity (OCC). A final experiment was conducted to obtain proteomic evaluation of protein expression in monocytes of unstressed broilers. In initial experiments, ACTH treatment was applied to hematocrit separated broilers. Experiments evaluated effects of ACTH on broilers with low (19 to 22%, Experiment 1; 18-21%, Experiment 3), high (25 to 28%, Experiment 1; 24 to 27%, Experiment 3) or non-selected hematocrit levels (Experiment 2 and 3). After 4 d of ACTH, all treated birds had significantly increased (P < 0.1) pCO2, HCO3-, and corticosterone levels, indicating as stress raises pCO2, HCO3- must rise to maintain acid base balance. Birds not selected for hematocrit had significant drops in pO2 when given ACTH. Broilers compensate for low OCC through release of red blood cells from storage sites, indicated by decreases in organ hemoglobin and increases in hematocrit and blood hemoglobin when birds are given ACTH. Accelerated red blood cell formation does not appear to occur, because erythropoietin decreases following administration of ACTH to non-selected birds. ACTH induced stress, increased hemoglobin and hematocrit only in birds with low or non-selected hematocrit, suggesting high hematocrit birds prior to stress have an adaptive advantage during stress. Higher hematocrit prior to stress apparently provides ample OCC during stress. Unselected birds appear to require initiation of an inflammatory response to adapt to stress which can be noted by increases in total white blood cell count, monocytes, and heterophils and decreases in lymphocytes. High hematocrit birds appear less susceptible to stress effects by maintaining leukocytes at a constant level, while in non-selected birds lymphocyte percents drop. Proteomics was conducted on avian monocytes to reveal proteins related to immune functions, 3229 proteins were identified, with 46 involved in immune functions of professional antigen presenting cells. This protein data provides a means of comparing monocytes of stressed and unstressed animals in the future. In conclusion, evaluated hematocrit is advantageous in adaptation to stress through maintenance of high OCC, acid base balance and immune cells.

No description available.

Lines of White Leghorn chickens selected > 20 generations for (HA) and against (LA) antibody response to SRBC injected i.v. from 41 to 51 days of age, are now known to have diverged in primary antibody response to SRBC. Experiments described in this dissertation were designed to further evaluate the immune competence of these lines as influenced by age, diet, and a disease agent. A crossing experiment was also conducted to further describe the mode of inheritance of such competence. Humoral immunocompetence was evaluated by primary, memory, and maternal antibody responses to SRBC. Primary antibody response, measured 5, 10, and 20 days after inoculation with SRBC was greater in HA than LA chicks inoculated at 14, 21, and 28 days of age. In chicks injected at 7 days of age, a higher frequency of responders was observed for HA than LA chicks suggesting an earlier onset of immunocompetence in the high than low antibody line. Immunological memory antibody responses (secondary and tertiary) was studied in parallel experiments on two groups of chicks hatched at a 14-day interval. Chicks in both hatches were from the same matings of parental Lines HA and LA. Memory responses were evident in chicks at 14 days of age. Antibody responses to a second and third inoculation with SRBC were similar for both lines suggesting that genetic factors that influence primary and memory responses are not the same. The responses of LA chicks to repeat inoculations with SRBC were anamnestic whereas those of HA chicks initially inoculated at 28 days of age were not anamnestic. This study did not establish any major influence of nutrient density on either primary or memory immune responses even though the higher nutrient density diet improved growth performance. Assays in chicks indicated that maternal antibodies were transferred earlier into eggs laid by HA hens than in those of LA hens ( 7 to 9 days vs 10 to 12 days after inoculation) regardless of dosage administered. Response patterns whether assessed in terms of frequency of detection or magnitude of response showed divergence between the lines. Chicks of parental, reciprocal F , F , and backcrosses of 1 2 mating combinations of Lines HA and LA were injected with SRBC at 36 days of age. Contrasts between parental lines for antibody titers measured 5 and 12 days later showed higher antibody titers in HA than LA chicks. Sex-linked effects were evident because reciprocal contrasts for F crosses, individual heterosis, and 1 maternal heterosis were sex dependent. Response to marble spleen disease virus ( MSDV) measured 6 days after inoculation of chicks from parental, reciprocal F1, F2, and backcross matings of the lines, indicated that the mode of inheritance of spleen weight differed after infection. In the infected chicks, parental contrasts for absolute and relative spleen weights showed greater resistance to MSDV in LA than HA chicks. No other genetic effect was consistently important after infection. / Ph. D.

For 36 generations, White Leghorn chickens were selected for high (HAS) or low (LAS) antibody response to sheep red blood cells. The focus of this thesis was to investigate correlated responses in reproductive soundness and egg quality resulting from that selection. Forty-five hens and 25 roosters from each antibody line were used. In hens, commencement and intensity of lay, and egg quality, were analyzed; in both sexes, length of fertility was considered. Hens and roosters were mated to an intercross line to avoid confounding selection with sex effects. The LAS line was more reproductively sound, commencing lay at a younger age (11.67 ± 3.53 d; P < 0.001), lighter body weight (-169.46 ± 40.20 g; P < 0.001) and with greater intensity (2.68 ± 0.25%; P = 0.001) than the HAS line. Additionally, the LAS line had a greater length of fertility (hens: 2.43 ± 0.55 d; P < 0.001; roosters: 3.11 ± 0.71 d; P < 0.001). In contrast to their poorer reproductive soundness, the HAS line had superior egg quality compared to the LAS line. Egg shape index (4.12 ± 0.55; P < 0.001) and albumen height, measured in both mm (0.27 ± 0.12 mm; P < 0.001) and Haugh units (1.89 ± 0.91; P = 0.04), were superior in HAS hens. Selection for increased antibody response appeared to compromise reproductive soundness, perhaps due to limitations in available resources. However, the selection did not compromise egg quality. / Master of Science

The acoustic microscope is an efficient non-invasive tool that can explore the acoustic properties and the related mechanical microstructure of a wide diversity of materials, including biomedical and biological samples, which are, nowadays, among the most intriguing targets for investigations. In the presented work, an acoustic microscope with vector contrast is used to image and characterize the acousto-mechanical properties of chitosan, an abundant natural derivative of chitin known to be a biodegradable, nontoxic and versatile biopolymer that suits many biomedical applications such as its usage in tissue engineering. The work also presents key measurements for the study of the acousto-mechanical properties that are subject to variations during the life span of red blood cells (RBCs). The characteristic signature of fixed cells from groups of three different ages, fractionated according to mass density, is obtained from the acoustic microscope images. The analysis of these data enabled the quantitative comparisons between the acousto-mechanical properties (velocity and attenuation of ultrasound propagating in the cells, mass density, and bulk modulus of compression). Comparison of the contrasts in the acoustic micrographs for the cells of the different age groups is exploited to generate a model that determines the age of the individual cells in a sample of red blood cells collected from a healthy person. The dependence of the parameters of the cells including density, velocity and attenuation of longitudinal polarized ultrasonic waves travelling in the cells on the age of the cell is also presented. The output signal in dependence on the thickness of the sample, the so called V(d), represented as polar graph was exploited as the method of analysis of the data extracted from the acoustic micrographs imaged with ultrasound of a center frequency of 1.2 GHz. This procedure allows for the extraction of the quantitative information from a single image in magnitude and phase contrast and allows for height profiling with so called super resolution, relating to resolution below the diffraction limit, based on the developed modeling, beside of other advantages concerning the acoustic characterization of biomedical and biological samples. This method and the applications are presented and discussed together with the developed or adapted modeling.

No description available.

Recent advances in the clinical diagnosis and treatment of diseases using cell transplantation have emphasized the urgent need to cryopreserve many types of cells. In transfusion medicine, red blood cell (RBC) transfusions are used to treat anemia and inherited blood disorders, replace blood lost during or after surgery and treat accident victims and mass casualty events. In regenerative medicine, mesenchymal stem cell (MSC) therapy offers promising treatment for tissue injury and immune disorders. Current cryoprotective agents (CPAs) utilized for RBCs and MSCs are 40% glycerol and 10% dimethyl sulfoxide (DMSO), respectively. Although glycerol is required for successful cryopreservation of RBCs, it must be removed from RBCs post-thaw using costly and time-consuming deglycerolization procedures to avoid intravascular hemolysis. Unfortunately, while DMSO prevents cell damage and increases post-thaw MSC viability and recovery, recent reports have suggested that MSCs cryopreserved in DMSO display compromised function post-thaw. As a result, improvements to the current cryopreservation protocols such as reducing post-thaw RBC processing times and improving MSC function post-thaw are necessary in order to meet the increasing demands of emerging cellular therapies. Ice recrystallization has been identified as a significant contributor to cellular injury and death during cryopreservation. Consequently, the ability to inhibit ice recrystallization is a very desirable property for an effective CPA, unlike the conventional CPAs such as DMSO and glycerol that function via a different mechanism and do not control or inhibit ice recrystallization. Over the past few years, our laboratory has reported several different classes of small molecules capable of inhibiting ice recrystallization such as lysine-based surfactants, non-ionic carbohydrate-based amphiphiles (alkyl and aryl aldonamides) and O-linked alkyl and aryl glycosides. The use of these small molecule ice recrystallization inhibitors (IRIs) as novel CPAs has become an important strategy to improve cell viability and function post-thaw. With the overall goal to identify highly effective inhibitors of ice recrystallization, the first part of this thesis examines the IRI activity of three diverse classes of small molecules including carbohydrate-based surfactants bearing an azobenzene moiety, fluorinated aryl glycosides and phosphate sugars. While the majority of the carbohydrate-based surfactants and fluorinated aryl glycosides were not effective inhibitors of ice recrystallization, this work revealed that monosaccharides possessing a phosphate group could be effective IRIs. Our laboratory has previously demonstrated that small molecule IRIs β-PMP-Glc and β-pBrPh-Glc can protect human RBCs from cellular injury during freezing using reduced concentrations of glycerol (15% w/v). This was significant as reducing the concentration of glycerol can drastically decrease deglycerolization times. Consequently, structure- function studies were conducted on β-PMP-Glc and β-pBrPh-Glc to elucidate key structural features that further enhance their IRI activity and may increase their cryoprotective ability. In particular, the influence of an azido moiety on the IRI activity of β-PMP-Glc and β-pBrPh-Glc was investigated and it was determined that the position of the azide substituent on the pyranose ring is crucial for effective inhibition of ice recrystallization. Furthermore, the presence of an azido group at C-3 was found to increase the IRI activity of β-PMP-Glc and β-pBrPh-Glc. Despite the discovery that β-PMP-Glc and β-pBrPh-Glc are beneficial additives for the freezing of RBCs, a significant amount of cellular damage occurred during deglycerolization, resulting in very low cell recoveries. Thus, IRI active azido aryl glucosides were explored for their cryopreservation potential in RBCs to determine whether they could function as effective additives that reduce cellular damage post-thaw and improve cell recovery. One of the most significant results of this thesis is the discovery that azido aryl glucosides can successfully cryopreserve RBCs in the presence of 15% glycerol with significantly improved cell recovery. This thesis also explores the use of small molecule IRIs to improve the cryopreservation of MSCs. In particular, the addition of an N-aryl-aldonamide (2FA) to the standard 10% DMSO solution was found to enhance the proliferative capacity of MSCs post-thaw. Lastly, the ability of small molecule IRIs to cross the cell membrane and behave as permeating CPAs was evaluated in two different cell models, RBCs and human umbilical vein endothelial cells (HUVECs). These studies demonstrated that small molecule IRIs are capable of permeating the cell membrane and controlling intracellular ice recrystallization.

A hematopoese nas serpentes inicia-se durante a embriogênese e através dos processos de alterações da vida fetal. A primeira atividade eritropoética é extraembrionária, a partir de células mesodérmicas do saco vitelínico e durante o desenvolvimento embrionário torna-se intraembrionário. Em serpentes recém-nascidas e adultas, o principal foco hematopoético ocorre na medula óssea. O objetivo deste estudo foi caracterizar os diferentes estágios de maturação das células sanguíneas da serpente O. guibei, com base em estudos de microscopia, reações citoquímicas e aspectos ultraestruturais. Fragmentos de vértebras de serpentes recém-nascidas e adultas (n=11) foram coletados para obtenção da medula óssea que foi fixada em formol cálcio ou Bouin e processadas para histologia de rotina. Cortes histológicos, imprint de medula óssea e esfregaços sanguíneos foram corados com Rosenfeld, hematoxilina e eosina ou azul de metileno. As reações citoquímicas realizadas foram ácido periódico de Schiff (PAS), azul de toluidina (AT), Sudan Black B (SBB), benzidina peroxidase (PA) e fosfatase ácida (FA). Para a microscopia electrónica de transmissão (TEM), a medula óssea foi fixada em paraformaldeído a 4% + glutaraldeído 2%, em tampão Tyrode, pós fixados em tetróxido de ósmio a 1% e embebidos em resina Epon 812. A maior parte das células progenitoras de células sanguíneas foram identificadas em focos hematopoiéticos ativos na medula óssea de vértebras e costelas. As linhagens azurofílicas e linfoides foram morfologicamente similares aos de outros répteis. A linhagem granulocítica foi classificada como mieloblasto, promielócito, mielócito e granulócito maduro. Mielócitos podem ser diferenciados em basófilos com grânulos grandes, redondos e eletrondensidade homogênia ou heterófilos, com grânulos de tamanhos e formas variadas na análise MET. TB e PAS foram positivos nos grânulos imaturos e maduros basófilos. Por outro lado, heterófilos e azurófilos mostraram reação fortemente positiva para lípidos de SBB e BP. FA foi encontrado nos azurófilos em várias fases de maturação. As diferentes fases de eritrócitos foram classificadas como: proeritroblasto, eritroblasto basófilo, eritroblasto policromático, proeritrócitos e eritrócitos maduros. Os trombócitos apresentaram positivadade para PAS. As características dos trombócitos maduros e imaturos foram definidas através da TEM apresentando corpos densos, grânulos alfa, microtúbulos e sistema canalicular aberto. Concluindo, a medula óssea das costelas e vértebras é um importante foco hematopoético nas serpentes O. guibei recém-nascidas e adultas. Os aspectos morfológicos, citoquímicos e ultraestruturais são úteis para identificar e caracterizar os diferentes estágios de maturação das células sanguíneas / Hematopoiesis in snakes begins during embryogenesis and the process changes through fetal life. The first erytropoietic activity is extraembryonic from mesoderm cells of the yolk sac and during the embryonic development it becomes intraembryonic. In newborn and adult snakes, the main site of hematopoiesis occurs in the bone marrow. The aim of this study was to characterize different stages of blood cells maturation of O. guibei snakes, based on microscopic studies including cytochemical stains and ultrastructural features. Fragments of vertebrae of newborn and adult snakes (n= 11) were collected to obtain bone marrow that was fixed in Bouin or formol calcium and processed routinely for histology. Tissue sections, imprint of bone marrow and blood smears were stained with Rosenfeld, hematoxylin and eosin or methylene blue. The cytochemical reactions performed were periodic acid-Schiff (PAS), toluidine blue (AT), sudan black B (SBB), benzidine peroxidase (BP) and acid phosphatase (FA). For transmission electron microscopy (MET), bone marrow was fixed in paraformaldehyde 4% + glutaraldehyde 2% in Tyrode buffer, postfixed in 1% osmium tetroxide and embedded in Epon 812 resin. Most of progenitors of blood cells were identified in the active hematopoietic focus in bone marrow of vertebrae and ribs. The azurophilic and lymphocytic series were morphologically similar to those of other reptiles. Granulocytic lineage was classified as myeloblast, promyelocyte, myelocyte and mature granulocytes. Myelocyte can be differentiated into basophils, with large, round and electrondensity homogeneous granules or heterophils, with varied size and shapes granules in the TEM analysis. AT and PAS were positive in the immature and mature basophils granules. On the other hand, heterophils and azurophils showed strong positive reaction for lipids staining of SBB and BP. FA was found on azurophils in various stages of maturation. The different stages of erythrocytes were classified as: proerythroblast, basophilic erythroblast, polychromatic erythroblast, proerythrocyte and mature erythrocytes. Thrombocytics cells showed PAS positive. The characteristics of mature and immature thrombocytes were defined using TEM identifying the dense bodies, alpha granules, microtubules and open canalicular system. Concluding, the rib or vertebral bone marrow is an important hematopoietic site in the newborn and adult O. guibei snakes. The morphologic, cytochemical and ultrastructural characteristics are useful to identify and characterize different stages of maturation of blood cells

Introdução: A Hemoterapia sempre ocupou espaço entre o científico e o místico. Os gregos reconheciam o sangue como sustentáculo da vida e no século XVII, com a descoberta da circulação sanguínea, por Willian Harvey, a possibilidade de transfusão foi postulada por estudiosos da época. O primeiro relato de transfusão foi o de um procedimento feito empregando a infusão de sangue de carneiro para paciente com tifo, que faleceu. No início do século XX, iniciou-se realmente uma fase de maior compreensão de riscos, e possíveis benefícios com a utilização de sangue, hemocomponentes e hemoderivados. Com o desenvolvimento tecnológico dessa área da saúde, a preocupação com a segurança tornou-se cada vez maior. Dentro desse contexto, o presente trabalho compila e discute a ação dos agrotóxicos, descrita na literatura, sobre o sangue e hemocomponentes a fim de averiguar o quanto se sabe sobre a segurança e eficácia da liberação da doação, a produção e a utilização de hemocomponentes e hemoderivados provenientes de doadores expostos a agrotóxicos. Metodologia: O presente trabalho compilou resultados e observações de cinquenta publicações cientificas sobre o efeito dos agrotóxicos no sangue, empregando a pergunta norteadora: \"O que sabemos para considerarmos segura a utilização de hemocomponentes e hemoderivados provenientes de doadores expostos a agrotóxicos?\". Conclusão: O trabalho relatou os principais agrotóxicos empregados no Brasil e no mundo, seus efeitos sobre seres humanos e hemocomponentes, principalmente hemácias e plaquetas. Os dados, dos trabalhos consultados durante essa pesquisa, indicam sobrevida diminuída de plaquetas e hemácias do sangue e que há metabólitos de agrotóxicos no plasma de indivíduos, potenciais doadores de sangue, expostos aos agrotóxicos. Surge portanto, a reflexão sobre a qualidade do hemocomponente obtido de doadores expostos aos agrotóxicos, e a necessidade de novos estudos / Introduction: Hemotherapy has always occupied a space between the scientific and mythical field. The Greeks acknowledged blood as the sinews of life and in the 17th century, with the discovery of the blood stream by Willian Harvey, the possibility of transfusion was postulated by experts at that time. The first report of transfusion was a procedure performed by employing the infusion of sheep blood into a patient with typhus who died. In the beginning of the 20th century, we began to truly understand the risks and possible benefits with the use of blood, blood components and derivatives. With the technological development in this health field, the concern with safety became even greater. In this context, this work compiles and discusses the action of pesticides, described in the literature, about blood and its components in order to ascertain how much it is known about the safety and effectiveness of the release to the donation, production, and use of blood components and derivatives from donors exposed to pesticides. Methodology: The present work assembled results and transcriptions from 50 scientific publications regarding the pesticides effect on peripheral blood components, guided by the hypothesis of how much we know to consider safe the use of blood components and derivatives from donors exposed to pesticides. Conclusion: The study listed the main pesticides used in Brazil and in the world, their effects in human beings and blood components, particularly in red blood cells and platelets. Data from the reference indicate the shortened survival of platelets and red blood cells and the presence of pesticides\' metabolites in the plasma of healthy individuals who are potential blood donors exposed to pesticides. It arises here the reflection on the quality of the blood components obtained to donors exposed to pesticides and need for novel studies

Esse estudo teve por objetivo avaliar a viabilidade e qualidade in vitro do concentrado de hemácias felino (CHF), colhido e armazenado em bolsas e produtos nacionais, remanufaturadas, pelo Laboratório de Hemoterapia do Serviço de Anestesia da Faculdade de Medicina Veterinária e Zootecnia da Universidade de São Paulo, de acordo com normas definidas por órgãos regulamentadores como a ANVISA. Foram analisados parâmetros bioquímicos e hematológicos de 24 unidades de CHF nos dias 0, 14 e 21 de armazenamento. Utilizou-se sistema fechado para colheita e armazenamento do sangue com solução de anticoagulante-preservativa CPDA-1. A avaliação consistiu na mensuração de porcentagem de hematócrito, hemoglobina total, hemoglobina extracelular, porcentagem de hemólise, concentrações de potássio, lactato, glicose, ATP, pH, bicarbonato, pressão de CO2 e O2, inspeção visual da bolsa e cultura microbiológica aeróbia e anaeróbia. Os resultados obtidos foram avaliados estatisticamente por meio de testes paramétricos, sendo que as determinações de hematócrito, hemoglobina total não apresentaram variação significante nos 21 dias de preservação, enquanto os de potássio, lactato, pO2 aumentaram gradativamente de forma significante. Os níveis de ATP, glicose, pH, bicarbonato e pCO2 reduziram de forma significante com o decorrer do tempo. Não houve alteração à inspeção visual das bolsas de sangue, nem crescimento de microorganismos nas culturas realizadas. Por meio destas avaliações constatouse que o sistema remanufaturado com produtos nacionais pode ser empregado com segurança para obtenção de sangue felino tendo-se em vista que se manteve estéril, com eficiente conservação do concentrado de hemácias felino em CPDA-1 até o 21º dia de armazenamento. / This study aimed to evaluate the feasibility and quality of in vitro feline packed red blood cells (CHF), harvested and stored in bags and domestic products, manufactured, Hematology Laboratory at the Department of Anesthesia, Faculty of Veterinary Medicine, University of São Paulo, according to standards set by regulatory bodies such as ANVISA. Were analyzed biochemical and hematological parameters 24 units of CHF on days 0, 14 and 21 of storage. We used a closed system for collection and storage of blood with preservative-anticoagulant solution CPDA-1. The evaluation consisted in measuring percentage of hematocrit, total hemoglobin, extracellular hemoglobin, percentage of hemolysis, potassium concentrations, lactate, glucose, ATP, pH, bicarbonate, CO2 and O2 pressure, visual inspection of the bag and aerobic and anaerobic microbiological culture . The results were statistically analyzed using parametric tests, and determinations of hematocrit, total hemoglobin showed no significant variation within 21 days of preservation, while potassium lactate, pO2 gradually increased significantly. ATP levels, glucose, pH, pCO2 and bicarbonate decreased significantly with time. There was no change to the visual inspection of blood bags, or growth of microorganisms in the cultures performed. Through these evaluations it was found that the system refilled with domestic products can be used safely for obtaining blood feline bearing in mind that remained sterile, efficient storage of red blood cells feline in CPDA-1 until the 21 th days of storage.