Haemosporidian parasites are the agents of malaria. Countless vertebrates are affected by haemosporidians each year. Haemosporidians have been shown to be evolving at rapid rates; leading to new species of haemosporidians being discovered and new host associations being made. Adaptive molecular evolution was detected in an important hemoglobin degradation gene, falcilysin. At multiple sites across multiple genes involved in important functions signatures of negative selection were detected. The signatures of selection across non-hemoglobin degradation genes were indicative of evolutionary conservation when compared to the more variable hemoglobin degradation genes. This is probably due to the important role the hemoglobin degradation genes play in haemosporidian metabolism. A survey of local passerines detected a parasite prevalence rate of 57%. This included three genera of haemosporidians detected across six lineages and two more distantly related sequences. Leucocytozoon was detected for the first time in Mississippi songbirds, indicating the importance of surveying for understanding haemosporidian evolution and range.

Sickle cell disease (SCD) is one of the most common hemoglobinopathies worldwide. It is caused by a homozygous mutation in codon 6 of the beta globin gene (HBB), which leads to polymerization of the variant hemoglobin and sickled red blood cells that obstruct blood vessels and reduce oxygen delivery to tissues. Patients with SCD have multiple clinical problems, including pain crises, anemia and organ damage. However, not all patients with SCD display all these clinical manifestations. One major factor for reduced occurrences of symptoms is fetal hemoglobin (HbF). HbF is the main hemoglobin in the fetus, and declines one year after birth to less than one percent of total hemoglobin. Nevertheless, there are individuals who continue to have high levels of HbF into adulthood, which is beneficial for an individual with SCD because HbF reduces the amount of sickle polymer in red blood cells. There are three major quantitative trait loci (QTL) associated with high HbF. However, these QTL account for 20-45% of HbF variance. Therefore, further investigation is required to fully understand how HbF is regulated. The HBS1L-MYB intergenic polymorphism (HMIP) on chromosome 6q23 is one of the major QTL associated with high HbF. This region is also known to regulate other erythroid-specific traits due to an enhancer element that promotes the expression of the downstream gene, MYB, which controls hemoglobin expression and erythroid proliferation and maturation. The presence of RNA polymerase II binding and a 50-bp transcript suggested that a long noncoding RNA (lncRNA) is transcribed from this region. LncRNAs are non-protein-coding transcripts greater than 200 nucleotides and are involved in gene regulation. Therefore, it was hypothesized that a lncRNA is transcribed from the enhancer of MYB and regulates hemoglobin expression. I characterized a novel lncRNA, 1283 bp in length that was differentially expressed among various tissue types, among erythroid progenitor cells with different hemoglobin makeup, and also during erythroid differentiation. Furthermore, knockdown of this lncRNA, named the HBS1L-MYB intergenic long noncoding RNA (HMI-LNCRNA), significantly increased HbF. Taken together, these observations suggest that HMI-LNCRNA can be a possible therapeutic target to increase HbF expression in patients with SCD and β-thalassemia. / 2018-05-01T00:00:00Z

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Hemoglobin, from the erythrocytes of adult and fetal pigs, was isolated and analyzed by carboxymethylcellulose column chromatography and electrophoresis on polyacetate. These techniques demonstrate the existence of four fetal components (F0, F1A, F1B, F1C) and two adult components (A0, A1) based on their elution pH values and electrophoretic mobilities. The shift from-fetal to adult forms is initiated sometime between eight weeks after birth and one year. The change is not completed until sometime after one year and the major fetal forms (F0, F1A) maintain a 15% level in a mature adult. This report is in contrast with the results of early work that indicated that pig hemoglobin was homogeneous in nature. It conforms to the work by other researchers on other animals by showing that pig hemoglobin is heterogeneous.

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.

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Investigations into the nitrogen fixing apparatus in cyanobacterium Nostoc commune revealed a gene encoding for a hemoprotein, known as cyanoglobin. The cyanoglobin gene was isolated and subcloned into Escherichia coli previously. The study presented here encompasses the optimization of growth conditions for the transformed F. coli, with subsequent induction of cyanoglobin synthesis. These conditions were applied to large-scale (24-1) fermentor culture, permitting purification of approximately 200 mg cyanoglobin. Structural analyses, including absorption spectroscopy and circular dichroism, are presented. These studies indicate that cyanoglobin is a cytoplasmic hemoglobin with properties quite unlike those of leghemoglobin a and sperm whale myoglobin, which are used as references of comparison. For example, the optical spectral properties of oxycyanoglobin are different from those of leghemoglobin α and sperm whale myoglobin. In addition, the met-form of cyanoglobin has characteristics of a low-spin hemoglobin, in contrast to the high-spin met-forms of sperm whale myoglobin and leghemoglobin α. Unusually, the met- form of cyanoglobin fails to coordinate the strong-field ligands, cyanide and azide, at pH 7 and pH 9. The Soret region circular dichroism (CD) spectrum of cyanoglobin is unlike that of sperm whale myoglobin, yet is very similar to leghemoglobin α, suggesting a similar heme environment in these two hemoproteins. Far-UV CD of cyanoglobin revealed alphahelical character comparable to that of sperm whale myoglobin and leghemoglobin α. Cyanoglobin is the first monomeric hemoglobin detected in a prokaryote, raising questions concerning a possible role of cyanoglobin in early globin gene evolution. / Master of Science

Várias linhagens de camundongos resultam de mutações genéticas pontuais espontâneas ou induzidas e estas alterações podem apresentar fenótipos relevantes semelhantes a doenças hereditárias humanas. Massironi et al. (2006) desenvolveram vários camundongos BALB/c mutantes com o agente mutagênico etil-nitroso-uréia (ENU) no Biotério de Experimentação do Departamento de Imunologia do Instituto de Ciências Biomédicas (ICB) da Universidade de São Paulo. Destes, o mutante chamado anêmico foi objeto de estudo desta pesquisa, que tem como objetivo a sua fenotipagem. A avaliação hematológica revelou moderada anemia com intensa policromasia e reticulocitose, acompanhada de anisocitose, macrocitose, hipocromia, inclusões intraeritrocíticas e corpúsculos de Heinz, do nascimento até 18 meses de idade. Apresentaram também hemoglobinúria, bilirrubinemia, hiperfosfatemia e populações eritrocíticas com diferentes resistências à lise osmótica. No estudo da hemoglobina, não foi possível distinguir Hb anormal pela eletroforese em acetato-celulose em pH alcalino, ou cadeia globínica diferente pela eletroforese de cadeias globínicas em pH alcalino, mas constatou-se precipitação da hemoglobina no teste de estabilidade térmica e no teste de isopropanol. Na necrópsia, observou-se intensa esplenomegalia, especialmente nas fêmeas de 12 a 18 meses de idade, discreta hepatomegalia, moderada cardiomegalia e icterícia subcutânea. No exame histopatológico, observou-se intenso aumento da hematopoiese e hemossiderose nos órgãos hematopoiéticos e nas células tubulares renais notou-se deposição de ferro intracitoplasmático e houve aumento da concentração de creatinina plasmática animais mais idosos. Estes dados permitem classificar o quadro como anemia hemolítica congênita regenerativa crônica, semelhante a quadros de anemia por hemoglobina instável, descritos para humanos. Massironi (dados não publicados) concluiu o mapeamento genético deste mutante, que revelou a troca de aminoácidos na posição 88 da cadeia beta da hemoglobina (leucina por prolina), devido à troca de T por C no gene da globina &beta; Hbb-b1 no cromossomo 7, que corresponde à mutação da hemoglobina de Santa Ana descrita em humanos. A Hb Santa Ana é uma hemoglobina instável rara, com descrição de casos no Brasil, EUA, França, Hungria e Japão e que resulta em importante anemia hemolítica congênita crônica com evidente esplenomegalia. Este camundongo BALB/c mutante anêmico apresenta fenótipo e genótipo muito similares ao quadro de hemoglobina instável de Santa Ana e, considerando-se a importância dos modelos animais para o avanço dos estudos de doenças humanas, este modelo seria o primeiro camundongo mutante descrito para o estudo desta doença. / Many strains of mice result from spontaneous or induced punctual genetic mutations and these changes may present relevant and similar phenotypes to inherited human diseases. Massironi et al. (2006) developed some BALB/c mice mutants with the mutagen ethyl-nitroso-urea (ENU) in the Biotério de Experimentação, Departamento de Imunologia, Instituto de Ciências Biomédicas (ICB) of Universidade de São Paulo. From these, the mutant called anemic is the object of this research, which aims to characterize its phenotype. The hematologic evaluation revealed moderate anemia with intense reticulocytosis and polychromasia, followed by anisocytosis, macrocytosis, hypochromia and intraerythrocytic inclusion and Heinz bodies, presented from birth to 18 months of age. They also presented hemoglobinuria, bilirrubinaemia, hyperfosfatemia, and erythrocytic populations with different resistance to osmotic lysis. In the hemoglobin study, it was not possible to distinguish an abnormal Hb by cellulose acetate electrophoresis at alkaline pH nor a different globin chain with globin chains electrophoresis at alkaline pH, but there was hemoglobin precipitation at heat stability test and isopropanol test. At necropsy, there was evident splenomegaly - especially within the 12 to 18 months old mutant female group, discrete hepatomegaly, moderate cardiomegaly, and subcutaneous jaundice. On histopathologic examination, there was dramatic increase in the hematopoiesis and hemosiderosis in hematopoietic organs and intracellular iron deposition on tubular renal cells with elevated plasma creatinin concentration in the oldest animals. These data indicate a congenital hemolytic regenerative anemia, similar to human unstable hemoglobin anemia. By genetic mapping concluded by Massironi (data not published), it has been detected an amino acid exchange (leucine to proline) at position 88 in the beta globin protein, because of a T to C change in &beta; globin gene Hbb-b1 on chromosome7, which corresponds to the Santa Ana hemoglobin mutation described in humans. Hb Santa Ana is a rare unstable hemoglobin reported in Brazil, USA, France, Hungary and Japan, that presents cronic congenital hemolytic anemia with evident splenomegaly. This BALB/c mutant mouse presents similar genotype and phenotype to the human Santa Ana unstable hemoglobin. Considering the importance of animal models for human diseases studies advance, this model would be the first mutant mouse described for study of Hb Santa Ana.