Human herpesvirus 6A (HHV-6A) has yet to be definitively linked to a specific disease. This is due in part to the ubiquitous nature of the virus. Humanized Rag2-/-γc-/- (Rag-hu) mice were tested to determine if these were a suitable animal model to study the virus. Both cell-free and cell-associated virus was used for infection and both were found to be efficient at infecting the mice. Viral DNA was found in the plasma and cellular blood fractions, bone marrow, lymph node, and thymus, indicating successful infection and propagation of the virus in vivo. The CD3+CD4- population was depleted, while the CD3-CD4+ was increased in infected animals. The CD3-CD4+CD8- and CD3+CD4+CD8- populations were depleted and the CD3+CD4+CD8+ population increased when analysis was gated upon CD4+ cells. The CD3-CD4+CD8+ population expanded and the CD3-CD4+CD8- population was reduced when analysis was gated on the CD3- population. Additional flow cytometry analysis revealed increases in CD4+CD8+ double positive cells in the peripheral blood of cell-free infected mice, which could indicate improper T cell selection and a premature departure of these cells from the thymus, possibly contributing to autoimmunity. Previous research has shown that HIV and HHV-6A may have a synergistic effect on one another and that HHV-6A may act as a cofactor in the progression to AIDS. After determining the Rag-hu mouse model was suitable for studying HHV-6A infection, a coinfection of HHV-6A and HIV-1 was performed. Coinfected mice had fewer thymocytes when compared with the HIV-1 only, mock-infected, and to a lesser extent HHV-6A only groups which could indicate increased cell death in the coinfected group as well as possible disruptions in migration of cells, either causing cells to be sequestered in the bone marrow and unable to migrate to the thymus, or causing premature egress of the cells in the thymus due in part to premature upregulation of CCR7, both of which would explain the smaller cellular populations found in the coinfected mouse thymi. Additional studies were performed to determine if a preferential targeting existed between HHV-6A and HIV-1 as these viruses are found simultaneously coinfecting the same cell. Preferential targeting was not observed by cell-associated migration assay, but increased migration of HHV-6A-infected cells was observed in a CCL21 dependent manner. These studies have provided useful information about HHV-6A and its relevance to HIV/AIDS as well as a possible mechanism of the involvement of HHV-6A in multiple sclerosis (MS) and other autoimmune diseases.

Vascular stiffening is defined as the reduced ability of the blood vessels to expand in response to an increase in blood pressure. Vascular stiffening is often not appreciated as a disease in and of itself but is important to recognize because it is considered a predictor of many cardiovascular disease states. Mechanisms of vascular stiffening remain largely unknown; however the immune system has been found to play major roles in cardiovascular disease and arterial remodeling. This dissertation therefore seeks to illustrate the role of the adaptive immune system in vascular stiffening. This has been done by modeling vascular stiffness in transgenic mice lacking an adaptive immune system as well as immunosuppression in normal mice using a novel method to stimulate regulatory T cells with a cytokine immune complex. We have found that inhibition of the immune system by the use of a genetic knockout (RAG 1 ⁻/⁻ mice) or suppression of an existing immune system with an IL-2/anti-IL-2 complex reduces the development of angiotensin II-induced vascular stiffening. This dissertation supports the role of the adaptive immune system, and particularly CD4⁺T cells, in the development of vascular stiffening as well as the protective roles of Tregs in the disease. It also highlights the use of the IL-2/anti-IL-2 complex as a new potential therapy for vascular stiffness. Therapeutics that suppress adaptive immune function may be beneficial in the treatment of vascular stiffening.

The lymphoid tissues consist of distinct cell subpopulations of B and T cell lineages and possess complex signaling pathways that are controlled by a myriad of molecular interactions. During the fine-tuned developmental process of the lymphoid system, inappropriate activation of oncogenes and loss of tumor suppressor gene activity can push lymphocytes into uncontrolled clonal expansion, causing several lymphoid malignancies. V(D)J recombination is one such essential process, important for the proper development of the mammalian immune system. However, mistakes in normal V(D)J recombination can lead to deletion of tumor suppressor genes or activation of proto-oncogenes. In the first part of the study, the physiological and pathological roles of DNA binding domain of RAG1 have been characterized. RAG (Recombination Activating Gene) complex consisting of RAG1 and RAG2, is a site specific endonuclease responsible for the generation of antigen receptor diversity. It cleaves a specific DNA sequence termed as recombination signal sequence (RSS), comprising of a conserved heptamer and nonamer. Recent studies have shown that RAGs can also act as a structure-specific nuclease by cleaving flaps, heterologous loops, bubbles, hairpins etc. Nonamer binding domain (NBD) of RAG1 plays a central role in the recognition of RSS during its sequence specific activity. To investigate its DNA binding properties, NBD of murine RAG1 was cloned, overexpressed and purified from E. coli. Electrophoretic mobility shift assays showed that NBD binds with high affinity to nonamer in the context of 12/23 RSS. However, it did not bind to heteroduplex DNA, irrespective of the sequence of the single-stranded region. Interestingly, when a nonamer was present next to a heteroduplex DNA, NBD exhibited robust binding. NBD binding was specific to thymines when single stranded DNA containing poly A, C, G and T were used. Biolayer interferometry studies showed that the observed poly T binding to NBD was robust with a binding constant of 0.45±0.16 µM. >23 nt was essential for NBD binding at homothymidine stretches. On a double-stranded DNA, NBD could bind to A:T stretches, but not G:C stretches or random sequences. Although NBD is indispensable for sequence-specific activity of RAGs, external supplementation of purified nonamer binding domain to NBD deleted cRAG1/cRAG2 did not restore the sequence specific activity, suggesting that the overall domain architecture of RAG1 is important for maintaining its properties. Therefore, we define the sequence requirements of NBD binding to double- and single-stranded DNA, which will have implications in generation of chromosomal rearrangement and genomic instability in lymphoid cells. Genetic alterations are one of the hallmarks of lymphoid malignancies. Many genes involved in chromosomal abnormalities are known to play central roles in the development of normal lymphocytes. In the second part of the study, molecular mechanism associated with fragility of the transcription factor, B cell leukemia 11B (BCL11B) that drives malignant transformation of T-cells has been studied. BCL11B is a zinc finger protein transcription factor with multiple functions. It plays a key role in both development and subsequent maintenance of T-cells. BCL11B gene alterations are implicated in a number of diseases including T-cell malignancies. It acts as a haplo-insufficient tumor suppressor and loss of BCL11B allele leads to susceptibility to mouse thymic lymphoma and human T-ALL. Recent studies reveal heterozygous BCL11B mutations and deletions across each of the major molecular subtypes of T-ALL (15% of patients). Most of the BCL11B missense mutations identified so far affected the residues within BCL11B zinc finger domains of the exon 4. However, mechanism of generation of such specific mutations leading to altered functions of BCL11B remains to be explored. In the present study, we address the potential mechanism of fragility of BCL11B gene during leukemia genesis. Firstly, we have evaluated different regions of BCL11B gene for presence of non-B DNA sequence motifs. Studies using non-B DB database reveal clustering of several non-B DNA forming motifs at the region spanning exon 4 of BCL11B gene. In order to biochemically evaluate the potential of non-B DNA structure formation, two different regions of exon 4 were PCR amplified and cloned. Using bisulfite modification assay we demonstrate that, single strandedness exists at both region I and II of BCL11B exon 4, when the region is present on a plasmid DNA. Bisulfite reactivity on chromosomal DNA confirmed existence of such altered DNA structures in the context of human genome. In vitro gel shift assays showed formation of both intra and intermolecular G-quadruplexes. Primer extension studies revealed that non-B DNA structures could block polymerization during replication on a plasmid, leading to DNA replication arrest. Extrachromosomal assays showed that non-B DNA structure motifs, in contrast to its mutants, blocked transcription leading to reduced expression of green fluorescent protein (GFP) within cells. Many non-B DNA-forming sequences have been mapped to regions of common chromosomal breakpoints in human tumors, known as “hotspots”, which are associated with leukemia, lymphomas and genomic disorders. Thus, alternative DNA conformations are believed to contribute to mutations, deletions and other genetic instability, leading to the deregulation of cancer-related genes in malignant diseases such as leukemia and lymphoma. Activation induced cytidine deaminase (AID), is an essential enzyme involved in antibody diversification of immunoglobulin genes. However, aberrant AID expression in B- cell and non-B cell background is reported in various cancers including leukemia and lymphoma. AID activity requires single stranded DNA (ssDNA) as a substrate. Since activation induced cytidine deaminase (AID) deaminates cytosines when present on a single stranded DNA and its expression is deregulated in many cancers, we investigated the role of AID in BCL11B gene mutagenesis. We observed substantial AID expression in many T-cell leukemic cell lines. Thus, we hypothesize that AID might be targeted to single stranded DNA present at BCL11B exon 4 due to formation of non-B DNA structures such as G-quadruplexes causing AID mediated deamination, further leading to nucleotide alterations and the mutational signature observed at BCL11B exon 4 resulting in T-ALL. Based on our findings, we propose that single strandedness resulted due to formation of non-B DNA structures such as G-quadruplex DNA, triplex DNA or cruciform DNA during physiological processes like DNA replication and transcription at exon 4 of BCL11B, can act as the target for AID. Thus, our findings uncover a new possible link between non-B DNA structure motifs and AID expression in causing mutations at BCL11B exon 4 which could lead to T cell leukemia genesis. BCL11B is a bifunctional transcriptional regulator that can act as a repressor and transactivator, and is known to differentially control the expression of specific genes in a context-dependent manner. In order to understand the transcriptional network involving BCL11B, it was cloned, overexpressed and purified from E. coli. To investigate the DNA binding properties of BCL11B protein, electrophoretic mobility shift assays were performed. Our results lead to identification of a specific sequence motif that is responsible for DNA binding. Competition experiments in presence of specific and nonspecific oligomers further confirmed the binding specificity. Thus, in the present study, we have characterized the binding properties of nonamer binding domain of RAG1, emphasizing its pathological relevance in causing genomic instability in lymphoid cells. The study may help in better understanding of RAG induced genomic instability in lymphoid tissues and role of aberrant AID expression in inducing mutations at BCL11B Zinc finger domain, leading to its deregulation and culminating into T-cell leukemia

Recombination Activating Genes (RAGs) orchestrate the process called V (D) J recombination, which enables the vertebrate adaptive immune system to specifically recognize millions of antigens. During this recombination process, V (variable), D (diversity) and J (joining) gene segments of antibody (B cell receptor) and TCR (T cell receptor) join by different possible combinations to generate antigen receptor diversity. This unique site specific recombination process is actuated by lymphoid specific proteins called RAG1 and RAG2 (RAGs or RAG complex). RAGs recognize a conserved sequence motif flanking the above subexons called Recombination Signal Sequence (RSS). There are two types of RSS known as 12-RSS and 23-RSS, where a conserved heptamer sequence and nonameric sequence is separated by 12 or 23 bp, respectively. RAGs specifically bind to RSS by RAG1 Nonamer Binding Domain (NBD) and generate nicks which are converted to DSBs via a hairpin intermediate and finally repaired by Non-Homologous DNA End Joining (NHEJ), a major DSB repair pathway in eukaryotes. Thus, RAGs act as a sequence specific endonuclease, and is unique to higher eukaryotes. Therefore, reduced or loss of RAG activity could result in immune deficiency syndromes like Omenn Syndrome (OS) and Severe Combined Immunodeficiency (SCID). Apart from acting as a sequence specific nuclease, RAGs have been shown to cleave on altered DNA structures like mismatches (bubbles), hairpins, flaps, gaps, triplexes and 3’ overhangs. This structure specific nuclease activity is implicated in causing genomic instability in B and T cells, particularly leading to generation of chromosomal translocations in certain lymphoid cancers. However, unlike the sequence- specific cleavage activity, this novel property of RAGs is poorly understood. Structure-specific nuclease activity of RAGs was characterized by using heteroduplex DNA substrate containing bubble region. RAG proteins were overexpressed and purified from human cell line and used for the assay. Results showed that RAGs cleave different bubble substrates with different efficiency. The role of DNA sequence at single-stranded region of heteroduplex DNA on RAG cleavage was investigated by synthesizing the substrate DNA having either adenineguanine/ thymine/ cytosine in the bubble sequence. Interestingly, efficient RAG cleavage was observed only when cytosines were present at single-stranded region, while thymine bubbles were cleaved with much lower efficiency. Adenine and guanine containing bubbles were not cleaved by RAGs. This was the first observation showing sequence specificity during structure-specific nuclease activity of RAGs. Besides, it was observed that RAG cleavage on bubbles with cytosines resulted in DSB formation, which is essential for generation of chromosomal translocations. Further, such specificity and cytosine preference was observed, even when RAGs acted on other altered DNA substrates like hairpin loops, 3’ overhangs and gaps. When the role of flanking duplex region on RAG cleavage was tested, only the 5’ duplex nucleotide was critical for RAG cleavage reaction and cytosine was the most preferred nucleotide. By systematic mutation of bubble region, it was observed that the two cytosines present at the double strand-single strand junction are critical for RAG cleavage. A single nucleotide bubble (mismatch) with cytosines was cleaved by RAGs with low but detectable efficiency. A bubble with at least 2 nt length possessing cytosine was cleaved with higher efficiency resulting in both single-stranded nicks and DSBs. Based on these studies, “C(d)C(s)C(s)” was proposed as a novel recognition motif for RAG cleavage, on altered DNA structures, where“d” and “s” represent double- and single-strand region, respectively. To be targeted by RAGs in vivo, the altered DNA substrates have to compete with RSS, the physiological substrate. It is not known whether such structures will be cleaved by RAGs, when present along with RSS. Besides, the regulation of the both structure and sequence specific nuclease activities are not studied. To address the above questions, RAG cleavage on bubble substrates was performed in presence of RSS either in cis or trans configuration. Results showed that both bubble substrates and RSS were cleaved with similar efficiency by RAGs. In fact, they can compete out each other in a concentration dependent manner. When kinetics of RSS and bubble cleavage were performed, RSS cleavage reaction was faster and saturated within 10-15 min, while bubbles cleavage started slow and went on increasing with time. This difference in kinetics persisted when both substrates were present together. This could be a regulatory mechanism for targeting RAGs to RSS sites and limiting bubble cleavage which can be deleterious to cells. HMGB1, a DNA binding protein which is shown to enhance RSS binding and synapsis, does not affect RAG action on bubble substrates. RAG postcleavage complex is formed during V(D)J recombination process where RAGs remain bound to cleaved RSS after cleavage, which limits further RAG action on other sites. Such cleavage complex was not detected on bubble substrates, which suggests that after cleavage RAGs were not associated to DSBs of bubble cleavage. Finally, the nonamer binding domain of RAG1 involved in RSS binding in V(D)J recombination, was found to be dispensable for the structure-specific nuclease activity and it appears that RAGs bind to bubble substrates using a different domain. In summary, in this study, the structure-specific nuclease activity of RAGs was characterized. A novel sequence motif that can regulate this activity of RAGs was identified. Though altered structures can be equally favored substrates as RSS, differences in reaction kinetics, cleavage complex formation and separate DNA binding domains regulate RAG cleavage, when it acts as a structure-specific nuclease. Thus, this study may help in the better understanding of RAG induced genomic instabilities in lymphoid tissues.

No description available.

his thesis researches the complex intersection of Data Protection and Intelligent Chatbots (IC)at Scania Group. Developing intelligent chatbots in a secure and GDPR compliant way is highlycomplicated and multifaceted task. The purpose of this research is to provide Scania withorganizational knowledge on how this can be achieved. This study utilizes the Action DesignResearch framework to develop an artifact which integrates technological solutions with dataprotection considerations. By conducting a literature review and semi-structured interviews withemployees at Scania, three potential solutions are identified evaluated: ChatGPT Enterprise, theSecured AI Knowledge Repository (SAIKR), and Techtalker. Each solution offers differentcapabilities and compliance strategies: ChatGPT Enterprise, while practical, relies on contractualassurances for GDPR compliance with data stored in the USA. SAIKR, on the other hand, offersmore control with data stored and encrypted in Sweden, allowing for the use of advancedprivacy-preserving techniques. Techtalker, which is hosted directly by Scania, provides enhancedsecurity measures tailored to specific technical use cases. Based on the artifact and conclusionsof this research, generalized design principles for developing intelligent chatbots within acorporate structure are formulated. These four design principles encourages the utilization ofRAG and LLMs, safe and legal data localization, strong contractual safeguards with third-partyproviders, and a comprehensive risk analysis with stringent security measures.

The developmental processes of lymphopoiesis generate mature B lymphocytes from hematopoietic stem cells through increasingly restricted intermediates. Networks of transcription factors regulate these cell fate choices and are composed of both ubiquitously expressed and B lineage-specific factors. E-protein transcription factors are encoded by the three genes E2A, E2-2 (SEF2-1), and HEB. The E2A gene is required for B cell development and encodes the alternatively spliced proteins E12 and E47. During B lymphocyte development, the cells have to pass several checkpoints verifying the functionality of their antigen receptors. Early in the development, the expression of a pre-B cell receptor (pre-BCR) with membrane-bound immunoglobulin (Ig) heavy chain protein associated with surrogate light chain (SLC) proteins is a critical checkpoint that monitors for functional Ig heavy chain rearrangement. Signaling from the pre-BCR induces survival and a limited clonal expansion. Here it is shown that pre-BCR signaling rapidly down-regulates the SLCs l5 and VpreB and also the co-receptor CD19. Ca2+ signaling and E2A were shown to be essential for this regulation. E2A mutated in its binding site for the Ca2+ sensor protein calmodulin (CaM), and thus with CaM-resistant DNA binding, makes l5, VpreB and CD19 expression resistant to the inhibition following pre-BCR stimulation. Thus, Ca2+ down-regulates SLC and CD19 gene expression upon pre-BCR stimulation through inhibition of E2A by Ca2+/CaM. A general negative feedback regulation of the pre-BCR proteins as well as many co-receptors and proteins in signal pathways from the receptor was also shown. After the ordered recombination of Ig heavy chain gene segments, also Ig light chain gene segments are recombined together to create antibody diversity. The recombinations are orchestrated by the recombination activating gene (RAG) enzymes, other enzymes that cleave/mutate/assemble DNA of the Ig loci, and the transcription factor Pax5. A key feature of the immune system is the concept that one lymphocyte has only one antigen specificity that can be selected for or against. This requires that only one of the alleles of genes for Ig chains is made functional. The mechanism of this allelic exclusion has however been an enigma. Here pre-BCR signaling was shown to down-regulate several components of the recombination machinery including RAG1 and RAG2 through CaM inhibition of E2A. Furthermore, E2A, Pax5 and the RAGs were shown to be in a complex bound to key sequences on the IgH gene before pre-BCR stimulation and instead bound to CaM after this stimulation. Thus, the recombination complex is directly released through CaM inhibition of E2A. Upon encountering antigens, B cells must adapt to produce a highly specific and potent antibody response. Somatic hypermutation (SH), which introduces point mutations in the variable regions of Ig genes, can increase the affinity for antigen, and antibody effector functions can be altered by class switch recombination (CSR), which changes the expressed constant region exons. Activation-induced cytidine deaminase (AID) is the mutagenic antibody diversification enzyme that is essential for both SH and CSR. The AID enzyme has to be tightly controlled as it is a powerful mutagen. BCR signaling, which signals that good antibody affinity has been reached, was shown to inhibit AID gene expression through CaM inhibition of E2A.  SH increases the antigen binding strength by many orders of magnitude. Each round of SH leads to one or a few mutations, followed by selection for increased affinity. Thus, BCR signaling has to enable selection for successive improvements in antibodies (Ab) over an extremely broad range of affinities. Here the BCR is shown to be subject to general negative feedback regulation of the receptor proteins as well as many co-receptors and proteins in signal pathways from the receptor. Thus, the BCR can down-regulate itself to enable sensitive detection of successive improvements in antigen affinity. Furthermore, the feedback inhibition of the BCR signalosome and most of its protein, and most other gene regulations by BCR stimulation, is through inhibition of E2A by Ca2+/CaM. Differentiation to Ab-secreting plasmablasts and plasma cells is antigen-driven. The interaction of antigen with the membrane-bound Ab of the BCR is critical in determining which clones enter the plasma cell response. Genome-wide analysis showed that differentiation of B cells to Ab-secreting cell is induced by BCR stimulation through very fast regulatory events, and induction of IRF-4 and down-regulation of Pax5, Bcl-6, MITF, Ets-1, Fli-1 and Spi-B gene expressions were identified as immediate early events. Ca2+ signaling through CaM inhibition of E2A was essential for these rapid down-regulations of immediate early genes after BCR stimulation in initiation of plasma cell differentiation.

BACKGROUND: The research deals with the effect of the Student Rag Community Service's (SRCS) Pick–A–Leader programme on adolescents in a disadvantaged community. The SRCS has been in existence for 30 years and the Student Representative Council of the North–West University: Potchefstroom Campus, has authorised them to promote voluntary ativities for charity in Potchefstroom and its surrounding areas. The SJGD annually presents various programmes to communities in Potchefstroom; however, these programmes have not thus far been formally evaluated. One of these projects is the Pick–A–Leader (PAL) programme which focuses on leadership– and skills development for adolescents in a disadvantaged community. This study concentrates on the evaluation of the PAL–programme. AIM: The primary aim of this study was to determine the effect of the PAL–programme on the adolescents in a disadvantaged community. METHOD: The nature of the investigation was exploratory and descriptive. Mixed methodology was applied in this study. A quasi–experimental design was used in the form of a comparative pre– and post–test design. Focus groups and a number of measuring instruments were used to gather data. Participants in the experimental groups consisted of 45 grade 11 learners from 3 schools in a disadvantaged community of Potchefstroom. Members of the comparative groups also numbered 45 and were grade 11 learners from the same 3 schools as those in the experimental groups. RESULT: It was concluded that the SRCS PAL–programme had an effect on the adolescents. / Thesis (M.A. (MW))--North-West University, Potchefstroom Campus, 2011.

BACKGROUND: The research deals with the effect of the Student Rag Community Service's (SRCS) Pick–A–Leader programme on adolescents in a disadvantaged community. The SRCS has been in existence for 30 years and the Student Representative Council of the North–West University: Potchefstroom Campus, has authorised them to promote voluntary ativities for charity in Potchefstroom and its surrounding areas. The SJGD annually presents various programmes to communities in Potchefstroom; however, these programmes have not thus far been formally evaluated. One of these projects is the Pick–A–Leader (PAL) programme which focuses on leadership– and skills development for adolescents in a disadvantaged community. This study concentrates on the evaluation of the PAL–programme. AIM: The primary aim of this study was to determine the effect of the PAL–programme on the adolescents in a disadvantaged community. METHOD: The nature of the investigation was exploratory and descriptive. Mixed methodology was applied in this study. A quasi–experimental design was used in the form of a comparative pre– and post–test design. Focus groups and a number of measuring instruments were used to gather data. Participants in the experimental groups consisted of 45 grade 11 learners from 3 schools in a disadvantaged community of Potchefstroom. Members of the comparative groups also numbered 45 and were grade 11 learners from the same 3 schools as those in the experimental groups. RESULT: It was concluded that the SRCS PAL–programme had an effect on the adolescents. / Thesis (M.A. (MW))--North-West University, Potchefstroom Campus, 2011.

Of key importance for metamodelling are appropriate modelling formalisms. Most metamodelling languages permit the development of metamodels that specify tree-structured models enriched with semantics like constraints, references and operations, which extend the models to graphs. However, often the semantics of these semantic constructs is not part of the metamodel, i.e., it is unspeci ed. Therefore, we propose to reuse well-known compiler construction techniques to specify metamodel semantics. To be more precise, we present the application of reference attribute grammars (RAGs) for metamodel semantics and analyse commonalities and differences. Our focus is to pave the way for such a combination, by exemplifying why and how the metamodelling and attribute grammar (AG) world can be combined and by investigating a concrete example - the combination of the Eclipse Modelling Framework (EMF) and JastAdd, an AG evaluator generator.