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.

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.

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.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / A maneira mais difundida na observaÃÃo de cÃlulas sanguineas (hemÃcias) à aquela que utiliza microscopia Ãtica convencional. Devido ao limite de resoluÃÃo dos instrumentos Ãticos, novas tÃcnicas de microscopia colocam-se como alternativas para o estudo de cÃlulas, tais como: a microscopia eletrÃnica (de varredura e transmissÃo) e as tÃcnicas de varredura por sonda. Inclui-se neste Ãltimo grupo a microscopia de forÃa atÃmica (AFM). Este trabalho discute as possibilidades de uso da Microscopia de ForÃa AtÃmica ($emph{Atomic Force Microscopy}$ - AFM) em ciÃncias da vida, para ser mais especÃfico, na caracterizaÃÃo de eritrÃcitos. Cinco experimentos envolvendo hemÃcias e AFM estÃo aqui descritos: diferenciaÃÃo entre os grupos sanguÃneos AB+ e O+; anÃlise do perfil da membrana eritrocitÃria de indivÃduos sadios e portadores de SMD; preparaÃÃo de cÃlulas vermelhas para anÃlise em microscopia de forÃa atÃmica; anÃlise volumÃtrica de cÃlulas vermelhas; e monitoramento do envelhecimento de um eritrÃcito ao ar usando o AFM. No primeiro experimento, a rugosidade das membranas celulares dos grupos AB+ e O+ mostraram-se diferentes. Jà no segundo experimento, depressÃes foram encontradas sobre a membrana de pacientes com SMD e indivÃduos sadios, contudo, aparentemente mais profundas no primeiro grupo. O terceiro estudo trouxe à tona a importÃncia da preparaÃÃo adequada dos eritrÃcitos para medidas especÃficas em AFM. A quarta experiÃncia comprovou a capacidade da tÃcnica AFM de fornecer informaÃÃo de volume, o que tambÃm foi utilizado no Ãltimo experimento para monitorar o envelhecimento de uma hemÃcia ao ar. / The optical microscopy is the most employed technique used for visualize red blood cells (RBCs). But, due to its resolution limit, it is necessary to use other complementary techniques to study the cells, such as: the scanning and transmission electron microscopy, and the scanning probing microscopy. The Atomic Force Microscopy (AFM) is included in the last group. This work refers to the possibilities of using AFM in life science, focusing on the erythrocytes characterization. Five experiments involving red blood cells and AFM were carried out: AB+ and O+ blood types differentiation; RBCs membrane study of donors and patients with MDS (Myelodysplastic Syndrome); suitable preparation of red blood cells for AFM analysis; volume study of erythrocytes; and finally aging process observation of RBC in air. The first experiment determined the cell membrane roughness for AB+ and O+ groups, which were different. For the second one, depressions were found on the cell surface of both MSD patients and healthy people. These "holes" were deeper in the first group. The third experiment showed the importance of sample (RBCs) preparation for each AFM specific analysis. The fourth experimental procedure showed the AFM technique capability for providing volume information, which was also used in the last experiment to monitor the aging process of RBCs in air.

O curto tempo de armazenamento dos hemocomponentes é um dos fatores que dificulta e limita a quantidade de sangue que pode ser efetivamente armazenada, o que é uma desvantagem na medicina veterinária, pois o acesso a doadores é restrito e a demanda é contínua e cada vez maior na prática de clínicas e hospitais veterinários. Durante o armazenamento do sangue em baixas temperaturas, seja sob a forma de sangue total ou concentrado de eritrócitos, há uma queda intensa de metabólitos importantes para a viabilidade e funcionalidade dos eritrócitos. O desenvolvimento de meios e soluções de preservação sanguínea possibilitou o armazenamento dos eritrócitos e, consequentemente, facilitou o trabalho dos bancos de sangue. Portanto, a busca por melhores formas e soluções para preservação capazes de evitar ou diminuir estes efeitos prejudiciais durante o seu armazenamento é contínua, para que ao final se obtenha uma melhor qualidade do sangue transfundido. O presente trabalho avaliou o concentrado de eritrócitos felino armazenado na solução de CPDA-1 e CPD/SAGM durante 35 dias. Os dados laboratoriais foram comparados entre grupos e ao longo do tempo. Neste experimento foram utilizadas 10 bolsas de concentrado de eritrócitos felino divididos em dois grupos de cinco para avaliação de cada um dos aditivos. Os parâmetros laboratoriais K+, Na+, Cl-, lactato, HCO3-, amônia, glicose e pH foram avaliados nos dias 1, 7, 14, 21, 28 e 35 após a coleta. Vários parâmetros (K+, lactato, HCO3, glicose e cloreto) demonstraram que a solução CPD/SAGM manteve o metabolismo energético do eritrócito mais estável. Com este trabalho, foi possível entender melhor as alterações metabólicas sofridas pelos eritrócitos felinos durante o armazenamento. Concluímos que, apesar da solução CPD/SAGM se mostrar mais eficaz in vitro, são necessários mais estudos com relação aos hemocomponentes em gatos e à sua viabilidade pós-transfusional. / The short shelf life of blood products is one factor that complicates and limits the amount of blood that can be effectively stored, and it is a disadvantage in veterinary practice, because the access to donors is restricted and the demand is continuous and increasing at veterinary clinics and hospitals. During blood storage at low temperatures, either as whole blood or as packed red cells, there is a significant decrease of metabolites that are important for the viability and functionality of erythrocytes. The development of blood preservation solutions has enabled the storage of red blood cells and improved the service at the blood banks. Therefore, the search for better ways and blood preservation solutions to avoid or reduce these harmful effects during the storage conditions is continuous, in order to obtain the best blood product to be transfused. This study evaluated 10 bags of feline erythrocyte concentrate divided into two groups, stored in CPDA-1 and CPD/SAGM solutions during 35 days. The laboratory data were compared between groups and over time. K+, Na+, Cl-, lactate, HCO3-, ammonia, glucose and pH were assessed on days 1, 7, 14, 21, 28, and 35 after collection. On various parameters (K+, Cl-, HCO3-, glucose and lactate) solution of CPD/SAGM kept the energy metabolism of red blood cells more stable. With these results we can better understand the biochemical changes of feline erythrocytes during storage. We conclude that, although the CPD/SAGM solution shown to be more effective, more studies are needed to improve knowledge of feline blood components and post-transfusion viability.

Cryopreservation is a promising approach to long-term biopreservation of living cells, tissues and organs. The use of cryoprotective agents (CPAs) in combination with extremely low temperatures is mandatory for optimum biopreservation. CPAs (e.g., glycerol, trehalose, dimethyl sulphoxide (DMSO)), however, are relatively cytotoxic and compromise biopreserved cell quality. This is usually resultant in oxidative damage, diminishing cell functionality and survival rate. The growing market of cell therapy medicinal products (CTMPs) demands effective cryopreservation with greater safety, of which the currently available CPAs are unable to provide. The present study was aimed at developing cryomedia formulation to enhance the cryopreservation of nucleated and anucleated mammalian cells. Here, eleven compounds of a polyol nature were selected and examined for their cryoprotective properties. These compounds are derived from plants and honey, thereby ensuring their safety for human consumption. The selection was based on their molecular structure and chemical properties. Here, the presented study is divided into three main phases: 1) Screening the compounds panel for cryo-additive effects on cells during and post-cryopreservation and optimising the dose response and time course for trehalose and glycerol with and without the novel compounds; 2) Assessing the influence of biophysical criteria on biospecimen cryopreservation (e.g., biosampling procedure, cell age, donor age); 3) Establishing the mechanisms of action underpinning the modulatory effect of novel CPAs on biological pathways during cryopreservation. For the stated purposes, red blood cells (RBCs) obtained from sheep and humans were used to screen the compounds for novel cryo-additive agents. Cryosurvival rate was employed as an indication of the compounds' cryoprotective performance. Cellular biochemical profiles, including lipid and protein oxidative damage as well as key redox enzymatic activities (e.g., lactate dehydrogenase (LDH), glutathione reductase (GR)) were measured. The study revealed that nigerose (Nig) and salidroside (Sal) were significantly effective in protecting cells during the freeze-thaw cycle and recovery phases. Both compounds promoted the activity of GR and reduced oxidative stress mirrored by diminished LDH activity. This was also reflected in the protein and lipid oxidation levels, which was limited to a comparable level with the cells' prior freezing. Further studies on human leukaemia (HL-60) were carried out to elucidate the molecular and biological pathways associated with cryodamage and the modulatory effects of adding novel CPAs. The proteome profile and the corresponding biological functions were evaluated and iii showed that Nig and Sal protected cells against cryodamage. The additive compounds (Nig and Sal) demonstrated a unique and overlapping modulation effect pattern. Nig was found to highly influence proteins engaged with metabolic and energetic pathways, whereas Sal greatly affected nuclear and DNA-binding proteins. The current study concluded that novel CPAs have high potency in protecting cells and each compound has a unique effect on the cellular proteome. These features can be applied to designing cryomedia formulae with higher protective efficiency for targeted applications in cell based therapy and biopharmaceutical industries.