Adult hemoglobin (HbA) is made up of two pairs of globin chains. Some rare mutations of the globin chains can result in high affinity towards hemoglobin molecule thus changing the equilibrium of normal oxygen loading in lungs and the delivery of same to the tissues. Because of change in the affinity to the oxygen these mutations can result in erythrocytosis (polycythemia). Here we discuss a case of Familial Polycythemia likely due to novel hemoglobin variant. 42-year Caucasian male presents to the clinic with high hemoglobin for several years but otherwise denies any symptoms of headache, vision changes, chest pain. He had history of multiple phlebotomies. His past medical history is significant for Polycythemia, PICC line associated clot,Type2 Diabetes, Hypertension, Epidural abscess. Social history is significant for smokeless tobacco but otherwise non-smoker, non-alcoholic, not an IVDA and doesn’t use testosterone. Family history is significant for his sister, her two 14year old daughters and multiple other family members with elevated hemoglobin and undergo phlebotomies, maternal grandfather died of cancer in his 30s. Physical examination is only significant for BMI of 32.46 otherwise no skin discoloration or cyanosis. Laboratory data WBC 9.4 with normal differential, hemoglobin 18.2, hematocrit 55.4, platelet count 180,000, peripheral blood smear was within normal limits, negative for JAK-2, normal erythropoietin, negative for hemochromatosis, Oxygen dissociation p50 of 19 which is low indicating left shifted dissociation curve, hemoglobin electrophoresis HbA: 61.2%, HbA2: 3%, HbF: 0%, Beta variant: 35.8%. In order to further characterize beta variant Bi-directional sequence analysis for Molecular alterations was performed and the following alterations were detected Gene: HBB, DNA change: Codon 39, heterozygous CAG>CCG Protein change: P.G1n39Pro. [glutamine (Q) to proline (P)]. HGVS: c.119A>C, p.Q40P, Classification: Likely deleterious variant. (GenBank accession number NM_000518.4). This is a previously unreported beta chain hemoglobin variant present. This hemoglobin variant is named as Hemoglobin Hyden based on the place where this is found in Hyden, Kentucky. There have been four variants reported at codon 40 of the beta globin gene, which is an external contact site between beta globin and alpha-2 globin. One variant, Hb vassa, is associated with mild hemolytic anemia. The three other variants (Hb Alabama, Hb Tianshui and Hb San Bruno) are not associated with clinical or hematological abnormalities. In our opinion p.Q40P is likely a cause of erythrocytosis. In order to further establish the causality it may be beneficial to test first degree relative to in this family in order to determine whether the p.Q40P alteration tracks with disease and is not present in unaffected individuals. Hemoglobin threshold for phelebotomy to lower the risk of thrombosis and cardiovascular events is yet to be defined.

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Due to the pandemic, research concerning SARS-CoV-2 became of the utmost importance. In this research, we aimed to find and synthesize a library of peptide epitopes that carry functional properties of the ACE-2 receptor binding to the virus protein for the purpose of creating a therapeutic treatment (i.e. viral inhibition). In order to do this, we used MST to determine binding affinity. After that, we validated the binding properties of our peptide epitopes and applied them as SARSCoV-2 antibody indicators using ELISA. We, then, functionalized gold nanoparticles with the peptide epitopes to assess its utility as a potential SARS-CoV-2 competitive inhibitor. From the set of peptides in the library, P25 showed the most functional properties in both MST and serological ELISA, while P1 successfully conjugated to the gold nanoparticles in different forms (PEG-P1, linker-P1, and mutated P1). Finally, P1 was validated to have antibody binding through sandwich ELISA. In the future, these findings can be applied to inhibit viral activity through drug delivery.

A decreased concentration of pepsinogen A in serum was found to be a marker of gastric cancer, similarly as a low ratio of pepsinogen A to pepsinogen C. In the present study we have compared properties of immunoglobulin fraction isolated from the egg yolks after immunization of laying hens with pepsinogen isolated from porcine gastric mucosa with those of present in rabbit antiserum obtained after the animal immunization with the same antigen. The characteristics of chicken antibodies against porcine pepsinogen and the comparison with rabbit antibodies raised against the same antigen was carried out using the following methods: ELISA, affinity chromatography on immobilzed antigen and bovine serum albumin, SDS and native electrophoresis and MALDI-TOF MS/MS. While the rabbit specific antibodies interacted with the used antigen and only slightly with bovine serum albumin and there was a diference between pre-immune IgG and specific IgG, in the case of chicken antibodies IgY it did not work. No diference was observed between ELISA tests performed with pre-immune serum and the serum after immunization with porcine pepsinogen and a high interaction of IgY with bovine serum albumin in pre-immune serum and specific IgY after the immunization were detected. Similar results were obtained in experiments with...

Alzheimers sjukdom (AD) är den vanligaste formen av demens och påverkar miljontals människor världen över. Förekomst av senil plack och tangles in hjärnan hos personer med AD har varit ett känt faktum sedan början av 1900-talet. Flera studier gjorda under de senaste åren har påvisat en kopplling mellan AD och Diabetes, då försämrad insulin-signalering och nedbrytning av glukos påverkar flera av de molekylära förändringar som uppvisas i AD. I det här projektet använde vi oss av immunofluorescence för att studera uttryck och lokalisering av två transkriptionsfaktorer involverade i nedbrytning av glukos; NPAS3 och RFX3, i hjärnbarken hos patienter med AD, Lewykroppsdemens (DLB) hälsosamma kontroller. Intensiteten på infärgningen av NPAS3 och RFX3 visade inte mängden protein och kvantifierades med algoritmer skrivna i ImageJ. Infärgningen av NPAS3 och RFX3 visade inte på någon signifikant skilland mellan de tre kohorterna och för att förstå mer om deras roll i nedbrytningen av glukos och om det kan påverka AD, måste både infärgnings- och kvantifieringsprocesserna använda i detta projekt optimeras. / Alzheimer's disease (AD) is the most common form of senile dementia. affecting millions o people worldwide. Beta amyloid plaques and neurofibrillary tangles are known to be part of AD pathology since the early nineteen hundreds. In recent years evidence showing that impairments in glucose metabolism can initiate plaque- and tangle formation, as well as several of the other degenerative mechanisms taking place in the AD brain, suggests a potential connection between Alzheimer's disease and Diabetes. Here we used immunofluorescence to study expression of two transcription factors involved in regulation of glucose metabolism; NPAS3 and RFX3. Expression of NPAS3 and RFX3 was investigated in temporal cortex from patients with AD, Dementia with Lewy bodies (DLB) and non-demented age matched controls. The intensity of the immunofluorescent stainings was assumed to be proportional to the amount of stained protein and was quantified in ImageJ. This explorative study did not reveal a significant difference between groups and staining- an quantification procedures would have to be optimized in order to get a clearer understanding about the role of NPAS3 and RFX3 in glucose metabolism, and if that could affect the progression of AD.

Is specificity an important issue regarding affinity reagents? What about the validation of affinity reagents today, is it good enough? This depends on the application and the producer of the reagent. Validation should be the most important marketing argument that can be found.Today there is a continuous growth of both the number of affinity reagents that are produced and the different types of affinity reagents that are developed. In proteomics they become more and more important in exploring the human proteome. Therefore, validated affinity reagents should be on top of every proteomic researcher’s list. How should this be accomplished?Better international agreements on how affinity reagents should be tested to be regarded as functional reagents are needed. One of the most important issues is the specificity of the affinity reagent. An international standard for which specific validation that is needed for different kinds of applications would be very useful.In this thesis, it is shown that the protein microarray platform that was established within the HPA project at KTH is a very good tool to determine the specificity of different affinity binders.In the first study, the production of mono-specific antibodies for tissue profiling in the Human Protein Atlas (HPA) project is presented. The section describing the use of protein microarrays for validation of the antibodies is relevant for this thesis. The implementation of protein microarrays in the HPA workflow was an important addition, because a deeper insight of the specificity of all the antibodies produced were now available.In a second study, bead based arrays were compared to planar protein microarrays used in the HPA project. In this study, 100 different bead identities were coupled with 100 different antigens and mixed together to generate an array. The correlation between the two types of assays was very high and the conclusion was that the methods can be used as backup to each other.A third study was a part of an international initiative to produce renewable affinity binders against proteins containing SH2 domain. Here, the HPA protein microarrays were modified to analyze different types of reagents produced at six laboratories around the world. Monoclonal antibodies, single chain fragment and fibronectin scaffolds were tested as well as mono-specific antibodies. It was shown to be possible to adapt protein microarrays used in the HPA project to validate other kinds of affinity reagents. / QC 20111117 / Development and applications of protein microarrays / The Swedish Human Proteome Resource (HPR) program

Preterm birth (PTB) is defined as birth before the 37th week of pregnancy and affects 15 million infants per year. Presently, there is no clinical test to determine PTB risk. A 3D printed microfluidic device is being developed as a clinical test for PTB risk via detection of a panel of biomarkers. A significant step is extraction of the PTB biomarkers from blood serum. In this work, I developed 3D printed microfluidic devices in which monoliths can be polymerized. Using the monolith as a solid support to attach antibody, I show that ferritin, one of the PTB biomarkers, can be selectively extracted from human blood serum. This is the first study where a monolith has been formed in a 3D printed microfluidic device and used to perform an immunoaffinity extraction. This work is an important step in developing a clinical test for PTB risk. The realization of this work also demonstrates that 3D printing can be used to fabricate functional microfluidic devices.

Companies compete for employees based on their perceived employer brand value. This study investigates what influences a strong or weak employer brand has on the preference for total rewards. The results should assist remuneration agents in appropriately leveraging the company’s employer brand value, as a factor, when compiling a total rewards package for potential employees. A questionnaire was developed, asking participants to indicate their preferences relating to total rewards in the context of their current employer, with regard to stronger and weaker employer brands. Results of the study indicate that potential employees would require financial reward increases in the range of 15% - 30% in order to change employment, irrespective of whether it is perceived as a strong or weak employer brand. It was observed that a stronger employer brand could offer increases closer to the bottom of the range compared to weaker employer brands which would have to pay a premium to the top end of the range Employer brand value appears to increase the total reward costs for companies, irrespective of how the brand is perceived. It is however beneficial to be viewed as a strong employer brand, as the value of this perception translates to a smaller premium compared to weaker employer brands. / Dissertation (MBA)--University of Pretoria, 2010. / Gordon Institute of Business Science (GIBS) / unrestricted

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