No description available.

Thesis (Ph. D.)--Institute of Paper Chemistry, 1942. / Includes bibliographical references (p. 106-107).

No description available.

abstract: V(D)J recombination is responsible for generating an enormous repertoire of immunoglobulins and T cell receptors, therefore it is a centerpiece to the formation of the adaptive immune system. The V(D)J recombination process proceeds through two steps, site-specific cleavage at RSS (Recombination Signal Sequence) site mediated by the RAG recombinase (RAG1/2) and the subsequent imprecise resolution of the DNA ends, which is carried out by the ubiquitous non-homologous end joining pathway (NHEJ). The V(D)J recombination reaction is obliged to be tightly controlled under all circumstances, as it involves generations of DNA double strand breaks, which are considered the most dangerous lesion to a cell. Multifaceted regulatory mechanisms have been evolved to create great diversity of the antigen receptor repertoire while ensuring genome stability. The RAG-mediated cleavage reaction is stringently regulated at both the pre-cleavage stage and the post-cleavage stage. Specifically, RAG1/2 first forms a pre-cleavage complex assembled at the boarder of RSS and coding flank, which ensures the appropriate DNA targeting. Subsequently, this complex initiates site-specific cleavage, generating two types of double stranded DNA breaks, hairpin-ended coding ends (HP-CEs) and blunt signal ends (SEs). After the cleavage, RAG1/2 proteins bind and retain the recombination ends to form post-cleavage complexes (PCC), which collaborates with the NHEJ machinery for appropriate transfer of recombination ends to NHEJ for proper end resolution. However, little is known about the molecular basis of this collaboration, partly attributed to the lack of sensitive assays to reveal the interaction of PCC with HP-CEs. Here, for the first time, by using two complementary fluorescence-based techniques, fluorescence anisotropy and fluorescence resonance energy transfer (FRET), I managed to monitor the RAG1/2-catalyzed cleavage reaction in real time, from the pre-cleavage to the post-cleavage stages. By examining the dynamic fluorescence changes during the RAG-mediated cleavage reactions, and by manipulating the reaction conditions, I was able to characterize some fundamental properties of RAG-DNA interactions before and after cleavage. Firstly, Mg2+, known as a physiological cofactor at the excision step, also promotes the HP-CEs retention in the RAG complex after cleavage. Secondly, the structure of pre-cleavage complex may affect the subsequent collaborations with NHEJ for end resolution. Thirdly, the non-core region of RAG2 may have differential influences on the PCC retention of HP-CEs and SEs. Furthermore, I also provide the first evidence of RAG1-mediated regulation of RAG2. Our study provides important insights into the multilayered regulatory mechanisms, in modulating recombination events in developing lymphocytes and paves the way for possible development of detection and diagnotic markers for defective recombination events that are often associated immunodeficiency and/or lymphoid malignancy. / Dissertation/Thesis / Ph.D. Molecular and Cellular Biology 2012

RAGs (Recombination Activating Genes) are responsible for generation of antigen receptor diversity in case of B-cells and T-cells, through the process of combinatorial joining of different V (variable), D (diversity) and J (joining) gene segments. Each of these segments are flanked by recombination signal sequences (RSS), which consist of a conserved heptamer and nonamer separated by a less conserved spacer of 12 or 23 bp. RAGs recognize and cleave at the 5’ end of heptamer, leading to the formation of hairpin coding ends and blunt signal ends. The coding ends are joined through the process of no homologous DNA end joining (NHEJ), leading to the rearrangement of variable region of antigen receptors. Apart from its physiological property, RAGs can also act as a structure-specific nuclease. Previously, it has been shown that inadvertent action of RAGs on cryptic RSS and non B-DNA structures can lead to the generation of genomic instability and cancer. A very coordinated expression of RAGs has been observed in pro- and pre-B cells of the lymphoid system, which overlaps with the window of productive rearrangement during V(D)J recombination. Besides, studies by us and others have shown that RAG cleavage at altered DNA structures and cryptic RSS leads to chromosomal translocations resulting into cancer. However, several questions related to regulation of RAG expression and its activity in lymphoid cells remains to be answered. Previous studies have suggested regulation of RAG expression at different levels, such as methylation, ubiquitination, phosphorylation and by coordinate action of various transcription factors. In the present study, we evaluate the potential role of miRNAs in the regulation of RAG expression and its function in lymphoid cells. miRNAs are small, single-stranded non-coding RNAs, which play an important role in the regulation of gene expression. They play a critical role in the regulation of different cellular functions. Although there are miRNAs identified to play critical role during development of immune system, several key questions such as its role in the regulation of RAGs is yet to be addressed. In the current study, we have used bioinformatics approach to extract potential miRNAs that bind to 3’UTR of RAG1 and RAG2. miRNA expression datasets were downloaded from NCBI SRA database and extensive evaluation was done using various bioinformatics tools such as Bowtie, Sam tools, Bam tools, Bed tools and R package. We screened the miRNA expression profile across different stages of B-cell development (pro, pre, immature and mature B-cells), which overlap with the narrow window of RAG expression. The shortlisted miRNAs were further analyzed using miRNA databases such as miRBase, Targetscan and EMBL. Results showed that 33 miRNAs were specific to RAG1, among that one (miRNA1) followed RAG expression profile in B-cells. Besides miRNA2, which is a novel miRNA, was selected only on the basis of RAGs expression profile in a stage specific manner and the complementarity of the seed sequence of miRNA2 to the 3’UTR of RAG1 was checked manually. Interestingly, we observed that RAG1 expression was significantly down regulated in the presence of these miRNAs. However, there was no significant difference in the levels of other genes analysed. Further, semi-quantitative RT-PCR analysis confirmed the endogenous processing of pre-miRNA into mature miRNA using the cellular machinery. Besides, enrichment of 3’UTR of seed region of these miRNAs, enhanced the expression level of RAG1. Importantly, the enhancement in RAG1 expression level was limited in case of mature B-cells, where RAG expression is normally not observed. Further, transfection of lymphoid cells with miRNA inhibitors, specific to the miRNAs under study, showed the enhancement in RAG1 expression in lymphoid cells. In addition to this, specificity of selected miRNAs was confirmed by performing 3’UTR reporter assays, where enhanced luciferase expression was observed in case of mutant 3’UTR, while it was minimal in case of wild type constructs. Endogenous expression levels of selected miRNAs were evaluated in both lymphoid and nonlymphoid normal tissues and cancer cells using RT-PCR. Interestingly, we observed inverse correlation of expression levels of miRNA and RAG expression in all the cells tested. Besides, miRNA expression levels were less in pre-B cells and T-cells, owing to the increased expression of RAGs. Apart from this, recombinogenic potential of candidate miRNAs was assessed using episomal based V(D)J recombination assays. Interestingly we observed significant decrease (2-4 fold) in the V(D)J recombination efficiency when miRNA1 or 2 constructs were transfected in Nalm6 cells, as compared to that of controls, where no miRNAs were used. However, in case of Reh cells upon transfection with miRNA1construct, the decrease in recombination potential was upto 9 fold. Hence, we identify two miRNAs that can play an important role in the regulation of RAG1 expression and its physiological activity. Further, studies are being carried out to confirm their role in the regulation of RAG1 during different developmental stages of lymphoid cells in mice. As stated above, in addition to the sequence-specific activity, RAG possesses structure-specific nuclease activity as well. It has been shown that RAGs can cleave different types of altered DNA structures. Studies from our laboratory showed that even when RAGs act as a structure-specific nuclease there is a sequence bias. Presence of cytosine and thymine at the single-stranded region of heteroduplex DNA is important for RAG nicking and double-strand break (DSB) formation. In addition, proximity of a nonamer to bubble structures can enhance RAG cleavage. However, the role of immediate flanking sequences in the RAG mediated cleavage at heteroduplex regions is not understood. We investigated the role of flanking double-stranded DNA sequences in the regulation of RAG cleavage on non-B DNA structures. We found that RAG binding and cleavage on heteroduplex DNA is dependent on the length of double-stranded flanking region. Besides, immediate flanking regions of the heteroduplex DNA affected the RAG binding and cleavage in a sequence dependent manner. Interestingly, we also observed that the cleavage efficiency of RAGs at heteroduplex region was influenced by the phasing of DNA. Thus, our results suggest that sequence, length and phase positions of the DNA can affect the efficiency of RAG cleavage when it acts as a structure-specific nuclease. These findings provide novel insights into regulation of the pathological action of RAGs. Previous studies have shown that in addition to formation of coding and signal joints during V(D)J recombination, nonstandard V(D)J recombination products known as hybrid joints and open-shut joints may be formed, particularly in certain aberrant conditions such as defective NHEJ machinery. Interestingly, the hybrid and open-shut joints closely resemble the transposition mechanisms associated with transposons oretroviruses. Studies have also shown that RAGs possess structural similarity with integrases in domain organization. Both the proteins have Zinc Finger Binding domain (ZFB) which helps in multimerization of the protein, a central catalytic core domain comprising three acidic amino acids D, D and E essential for enzymatic activity and C-terminal domain (CTD) responsible for nonspecific binding to the DNA. Previous studies from our laboratory showed that, Elvitegravir, an inhibitor of integrase could interfere with the biochemical functions of the RAGs in vitro. Specifically, it inhibited the RAG binding and cleavage at RSS, hairpin formation, post-cleavage complex formation involving 12RSS and 23RSS. Using the episomal assay system that mimics signal joints (pGG49) and coding joints (pGG51), we show that Elvitegravir can inhibit V(D)J recombination inside cells. Interestingly we observed 3-6 fold decrease in the recombination frequency in signal ends joining, when treated with increasing concentrations (100, 500 and 1000 nM) of Elvitegravir. A 5-8 fold decrease in coding joints formation was also observed upon treatment with the inhibitor. The presence of recombination was confirmed by restriction digestion followed by sequencing analysis. Further analysis of recombination junctions revealed extensive deletion before joining in the case of Elvitegravir treated samples. Insertions or substitutions near to the recombination junctions were also prominent in treated samples. In depth analysis of sequenced junctions showed the presence of sequence having the features to form hairpins both upstream and downstream to the RSS sequences and was the site of cleavage in cases were higher deletion was observed. The analyzed recombinants did not show any signal joints or coding joints formation in treated samples. This suggests that Elvitegravir affects the physiological function, the V(D)J recombination of RAGs inside the cells. Thus, in the present study, we show that RAGs can be regulated by specific miRNAs. We have identified two potential miRNAs, which can regulate the RAG expression as well as its function in different stages of B- and T-cell development. Further, we also identify a novel regulatory mechanism for the structure-specific activity of the RAG complex. In addition to this, we find that integrase inhibitor, Elvitegravir, affects V(D)J recombination within B-cells, indicating its potential deleterious impact in HIV patients, which needs to be further evaluated.

No description available.

Large Language Models (LLMs) have the potential to enhance learning among students. These tools can be used in chatbot systems allowing students to ask questions about course material, in particular when plugged with the so-called Retrieval Augmented Systems (RAGs). RAGs allow LLMs to access external knowledge, which improves tailored responses when used in a chatbot system. This thesis studies different RAGs through an experimentation approach where each RAG is constructed using different sets of parameters and tools, including small and large language models. We conclude by suggesting which of the RAGs best adapts to high school courses in Physics and undergraduate courses in Mathematics, such that the retrieval systems together with the LLMs are able to return the most relevant answers from provided course material. We conclude with two RAG-powered LLM with different configurations performing over 64% accuracy in physics and 66% in mathematics.

Context: In both commercial and open-source software development, newcomers often join the development process in the advanced stages of the software development lifecycle. Newcomers frequently face barriers impeding their ability to make early contributions, often caused by a lack of understanding. For this purpose, we have developed an LLM-based tool called SPAC-B that facilitates project-specific question-answering to aid newcomers' understanding of software projects. Objective: Investigate the LLM-based tool's ability to assist newcomers in understanding software projects by measuring its accuracy and conducting an experiment. Method: In this study, a case study is conducted to investigate the accuracy of the tool, measured in relevance, completeness, and correctness. Furthermore, an experiment is performed among software developers to test the tool's ability to help newcomers formulate better plans for open-source issues. Results: SPAC-B achieved an accuracy of 4.60 in relevance, 4.30 in completeness, and 4.28 in correctness on a scale from 1 to 5. It improved the combined mean score of the plans of the 10 participants in our experiments from 1.90 to 2.70, and 8 out of 10 participants found the tool helpful. Conclusions: SPAC-B has demonstrated high accuracy and helpfulness, but further research is needed to confirm if these results can be generalized to a larger population and other contexts of use.

Characterizing the uncertainty in rainfall using stochastic models has been a challenging area of research in the field of operational hydrology for about half a century. Simulated sequences drawn from such models find use in a variety of hydrological applications. Traditionally, parametric models are used for simulating rainfall. But the parametric models are not parsimonious and have uncertainties associated with identification of model form, normalizing transformation, and parameter estimation. None of the models in vogue have gained universal acceptability among practising engineers. This may either be due to lack of confidence in the existing models, or the inability to adopt models proposed in literature because of their complexity or both. In the present study, a new nonparametric Matched Block Bootstrap (MABB) model is proposed for stochastic simulation of rainfall at daily time scale. It is based on conditional matching of blocks formed from the historical rainfall data using a set of predictors (conditioning variables) proposed for matching the blocks. The efficiency of the developed model is demonstrated through application to rainfall data from India, Australia, and USA. The performance of MABB is compared with two non-parametric rainfall simulation models, k-NN and ROG-RAG, for a site in Melbourne, Australia. The results showed that MABB model is a feasible alternative to ROG-RAG and k-NN models for simulating daily rainfall sequences for hydrologic applications. Further it is found that MABB and ROG-RAG models outperform k-NN model. The proposed MABB model preserved the summary statistics of rainfall and fraction of wet days at daily, monthly, seasonal and annual scales. It could also provide reasonable performance in simulating spell statistics. The MABB is parsimonious and requires less computational effort than ROG-RAG model. It reproduces probability density function (marginal distribution) fairly well due to its data driven nature. Results obtained for sites in India and U.S.A. show that the model is robust and promising.

This thesis presents the design and implementation of a generative Artificial Intelligence (AI)-based decision-support interface applied to the domain of pub- lic transport leveraging both offline and logged data from both past records and real-time updates. The AI assistant system was developed leveraging pre- trained Large Language Models (LLMs) together with Retrieval Augmented Generation (RAG) and the Function Calling Application Programming Inter- face (API), provided by OpenAI, for automating the process of adding knowl- edge to the LLM. Challenges such as formatting and restructuring of data, data retrieval methodologies, accuracy and latency were considered. The result is an AI assistant which can have a conversation with users, answer questions re- garding departures, arrivals, specific vehicle trips, and other questions relevant within the domain of the dataset. The AI assistant system has also been devel- oped to provide client-side actions that integrate with the user interface, enabling interactive elements such as clickable links to trigger relevant actions based on the content provided Different LLMs, including GPT-3.5 and GPT-4 with different temperatures, were compared and evaluated with a pre-defined set of questions paired with a respective ground truth. By adopting a conversational approach, the project aims to streamline infor- mation extraction from extensive datasets, offering a more flexible and feedback- oriented alternative to manual search and filtering processes. This way, traffic managers adapt and operate more efficiently. The traffic managers will also re- main informed about small disturbances and can act accordingly faster and more efficient. The project was conducted at Gaia Systems AB, Norrköping, Sweden. The project primarily aims to enhance the workflow of traffic managers utiliz- ing Gaia’s existing software for public transport management within Östgöta- trafiken. / Denna avhandling presenterar designen och implementationen av en generativ Artificiell Intelligens (AI)-baserad beslutsstödsgränssnitt applicerad på området för kollektivtrafik, utnyttjande både offline och loggad data från både tidigare händelser och realtidsuppdateringar. AI-assistentsystemet utvecklades med hjälp av Large Language Models (LLM) tillsammans med Retrieval Augmented Generation (RAG) och Function Calling API, tillhandahållet av OpenAI, för att automatisera processen att lägga till kunskap till en LLM. Utmaningar som formatering och omstrukturering av data, datahämtningsmetoder, noggrannhet och latens beaktades. Resultatet är en AI-assistent som kan ha en konversation med användare, svara på frågor om avgångar, ankomster, specifika fordonsturer och andra frågor relevanta inom datamängdens område. AI-assistentsystemet har också utvecklats för att tillhandahålla Client Actions som integreras med användargränssnittet, vilket möjliggör interaktiva element som klickbara länkar för att utlösa relevanta åtgärder baserade på den tillhandahållna innehållet. Olika LLM, inklusive GPT-3.5 och GPT-4 med olika temperaturer, jämfördes och utvärderades med en fördefinierad uppsättning frågor parat med en respektive sanning. Genom att använda en konversationell metod syftar projektet till att effektivisera informationsutvinning från omfattande datamängder och erbjuder ett mer flexibelt och feedbackorienterat alternativ till manuella sök- och filtreringsprocesser. På detta sätt kan trafikledare anpassa sig och arbeta mer effektivt. Trafikledarna kommer också att hållas informerade om mindre störningar och kan agera snabbare och mer effektivt. Projektet genomfördes på Gaia Systems AB, Norrköping, Sverige. Projektet syftar främst till att förbättra arbetsflödet för trafikförvaltare som använder Gaia's befintlig programvara för kollektivtrafikhantering inom Östgötatrafiken.