ECONOMICS OF CONSUMER CITIES

Ph.DDOCTOR OF PHILOSOPH

Tools for the analysis of B-cell clonal diversity in immune repertoires

The lymphocytes of an organism harbor a diverse collection or repertoire of antigen receptors (antibodies (Ab) on B cells and T cell receptors (TCR) on T cells). This diversity of the adaptive immune repertoire is important for effective immune defense. In my thesis, I have developed computational methods to study the diversity and landscape of the immune repertoire using data from next-generation sequencing experiments and have applied these tools to the study human B cells in the blood and in different tissues. The first tool I developed was an efficient and accurate means of identifying the nearest germline-encoded variable (V) region gene in Ab and TCR rearrangements using anchoring sequences. I used this method to demonstrate that the length of the V gene sequence and its level of somatic mutation influenced the reliability of V gene assignment. Only with adequate V gene assignment can closely related sequences be reliably grouped together into clones (i.e. sequences from lymphocytes that likely derive from a common progenitor cell). With this method, I contributed to a computational pipeline that can process millions of antibody or TCR sequences in hours to days, unlike earlier methods. I used this method to identify and track expanded B-cell clones through the human body, creating an atlas of clonal connections between the blood, bone marrow, spleen, lung, mesenteric lymph node, jejunum, ileum and colon. To power the analysis to study clonal overlap between the tissues, I performed rarefaction analysis on biological replicates to determine how many replicates and how large the clones needed to be to have confidence in clones being present or absent in the different tissue sites. To quantify the level of overlap between two-tissue sites, I used the cosine statistic and showed that my analysis was robust to different measures of clones and clone size. To visualize the tissue distribution of large clones, I created line-circle plots, in which clones are displayed as lines with circles corresponding to the tissues. The sizes of the tissue circles were proportional to clone copy numbers and the colored wedges within the circles indicated the fraction of sequencing libraries that contained sequences from the clone. My analysis revealed the clones tended to partition into two large networks in the human body, one in blood-rich tissues such as the bone marrow, spleen and lung, and another network that was more separated from the blood, in the gastrointestinal tract. I also used methods to visualize and characterize the diversification within B-cell clones due to somatic hypermutation, including lineage tree analysis, the analysis of four-fold silent mutations (mutations that do not change the amino acid sequence), methods for studying intermingling of lineage tree branches (clumpiness) and the use of Bayesian modeling approaches. I used these methods to show that GI tract clones in the human body harbor higher levels of somatic hypermutation and that there is extensive sharing of sequence variants within individual clonal lineages between different sites within the GI tract. Finally, I studied selection of mutations within clonal lineages over time in patients with an autoimmune disease (Sjogren's syndrome) and showed that large clones that were resistant to B-cell depletion therapy were under negative selection. Together, my analysis tools provide a useful means to systematically and quantitatively characterize diversity at the population (repertoire) level and at the clonal level. These tools can be applied to future immune repertoire profiling to study immune responses to vaccines, cancer and infectious disease.Ph.D., Biomedical Science -- Drexel University, 201

α- and β-Amino C−H Functionalization through Cooperative Catalysis:

Thesis advisor: Masayuki WasaWhen a catalytic reaction is carried out between two reactants, usually only onereactant is activated by a single catalyst while the other component is pre-activated so that the sluggish reactivity was compensated. In order to broaden the substrate scope, the development of cooperative catalysts that can generate both electrophilic and nucleophilic species in situ represents a compelling research objective. This thesis is focused on the development of cooperative catalyst systems and their applications to α- and β-amino C−H bond functionalization. In the first chapter of this thesis, a brief summary of the present cooperative catalysts will be discussed. In the second chapter, the development of cooperative acid/acid catalysts for the α-alkynylation of N-alkylamines will be discussed. Typically, catalytic α-amino C−H alkynylation process is carried out under oxidative conditions, and enantioselective reactions are confined to tetrahydroisoquinoline derivatives. We disclose a strategy for the union of N-alkylamines and trimethylsilyl alkynes through cooperative actions of two Lewis acids, B(C 6 F 5 ) 3 and a Cu-based complex without the use of oxidants. We proposed that various propargylamines can be synthesized through the reaction between a L n Cu−alkynyl complex and an iminium ion that are generated in situ. Furthermore, the utility of this protocol was demonstrated by applications in late stage α-alkynylation of bioactive amines and stereoselective synthesis of propargylamines. In the third chapter of this thesis, catalytic and regioselective deuteration of β-amino C−H bonds in an array of N-alkylamine-based pharmaceutical compounds will be described. Isotopic labeling of β-amino C−H bond is promoted by the cooperative action of Lewis acidic B(C 6 F 5 ) 3 and Brønsted basic N-alkylamine, converting a bioactive amine first into an iminium ion and then the corresponding enamine. Meanwhile, the acid/base catalysts can also promote the dedeuteration of acetone-d 6 to afford a deuterated ammonium ion and a boron enolate. Ensuing deuteration of the enamine by deuterated ammonium ion followed by borohydride reduction leads to the formation of β-deuterated bioactive amines with up to 99% deuterium incorporation.Thesis (MS) — Boston College, 2020.Submitted to: Boston College. Graduate School of Arts and Sciences.Discipline: Chemistry

Mixup for Test-Time Training

Test-time training provides a new approach solving the problem of domain shift. In its framework, a test-time training phase is inserted between training phase and test phase. During test-time training phase, usually parts of the model are updated with test sample(s). Then the updated model will be used in the test phase. However, utilizing test samples for test-time training has some limitations. Firstly, it will lead to overfitting to the test-time procedure thus hurt the performance on the main task. Besides, updating part of the model without changing other parts will induce a mismatch problem. Thus it is hard to perform better on the main task. To relieve above problems, we propose to use mixup in test-time training (MixTTT) which controls the change of model's parameters as well as completing the test-time procedure. We theoretically show its contribution in alleviating the mismatch problem of updated part and static part for the main task as a specific regularization effect for test-time training. MixTTT can be used as an add-on module in general test-time training based methods to further improve their performance. Experimental results show the effectiveness of our method.Comment: 11 page

FD-Align: Feature Discrimination Alignment for Fine-tuning Pre-Trained Models in Few-Shot Learning

Due to the limited availability of data, existing few-shot learning methods trained from scratch fail to achieve satisfactory performance. In contrast, large-scale pre-trained models such as CLIP demonstrate remarkable few-shot and zero-shot capabilities. To enhance the performance of pre-trained models for downstream tasks, fine-tuning the model on downstream data is frequently necessary. However, fine-tuning the pre-trained model leads to a decrease in its generalizability in the presence of distribution shift, while the limited number of samples in few-shot learning makes the model highly susceptible to overfitting. Consequently, existing methods for fine-tuning few-shot learning primarily focus on fine-tuning the model's classification head or introducing additional structure. In this paper, we introduce a fine-tuning approach termed Feature Discrimination Alignment (FD-Align). Our method aims to bolster the model's generalizability by preserving the consistency of spurious features across the fine-tuning process. Extensive experimental results validate the efficacy of our approach for both ID and OOD tasks. Once fine-tuned, the model can seamlessly integrate with existing methods, leading to performance improvements. Our code can be found in https://github.com/skingorz/FD-Align.Comment: Accepted by NeurIPS 202

A high infectious simian adenovirus type 23 vector based vaccine efficiently protects common marmosets against Zika virus infection.

Zika virus (ZIKV) has spread in many countries or territories causing severe neurologic complications with potential fatal outcomes. The small primate common marmosets are susceptible to ZIKV, mimicking key features of human infection. Here, a novel simian adenovirus type 23 vector-based vaccine expressing ZIKV pre-membrane-envelope proteins (Sad23L-prM-E) was produced in high infectious titer. Due to determination of immunogenicity in mice, a single-dose of 3×108 PFU Sad23L-prM-E vaccine was intramuscularly inoculated to marmosets. This vaccine raised antibody titers of 104.07 E-specific and 103.13 neutralizing antibody (NAb), as well as robust specific IFN-γ secreting T-cell response (1,219 SFCs/106 cells) to E peptides. The vaccinated marmosets, upon challenge with a high dose of ZIKV (105 PFU) six weeks post prime immunization, reduced viremia by more than 100 folds, and the low level of detectable viral RNA (103.66) and T-cell response (>726 SFCs/106 PBMCs) were acquired 1-2 weeks post exposure to ZIKV, while non-vaccinated control marmosets developed long-term high titer of ZIKV (105.73 copies/ml) (P<0.05). No significant pathological lesions were observed in marmoset tissues. Sad23L-prM-E vaccine was detectable in spleen, liver and PBMCs at least 4 months post challenge. In conclusion, a prime immunization with Sad23L-prM-E vaccine was able to protect marmosets against ZIKV infection when exposed to a high dose of ZIKV. This Sad23L-prM-E vaccine is a promising vaccine candidate for prevention of ZIKV infection in humans

Marmoset Viral Hepatic Inflammation Induced by Hepatitis C Virus Core Protein via IL-32.

Common marmosets infected with GB virus-B (GBV-B) chimeras containing hepatitis C virus (HCV) core and envelope proteins (CE1E2p7) developed more severe hepatitis than those infected with HCV envelope proteins (E1E2p7), suggesting that HCV core protein might be involved in the pathogenesis of viral hepatitis. The potential role of HCV core in hepatic inflammation was investigated. Six individual cDNA libraries of liver tissues from HCV CE1E2p7 or E1E2p7 chimera-infected marmosets (three animals per group) were constructed and sequenced. By differential expression gene analysis, 30 of 632 mRNA transcripts were correlated with the immune system process, which might be associated with hepatitis. A protein-protein interaction network was constituted by STRING database based on these 30 differentially expressed genes (DEGs), showing that IL-32 might play a central regulatory role in HCV core-related hepatitis. To investigate the effect of HCV core protein on IL-32 production, HCV core expressing and mock constructs were transfected into Huh7 cells. IL-32 mRNA and secretion protein were detected at significantly higher levels in cells expressing HCV core protein than in those without HCV core expression (P < 0.01 and P < 0.001, respectively). By KEGG enrichment analysis and using the specific signaling pathway inhibitor LY294002 for inhibition of PI3K, IL-32 expression was significantly reduced (P < 0.001). In conclusion, HCV core protein induces an increase of IL-32 expression via the PI3K pathway in hepatic cells, which played a major role in development of HCV-related severe hepatitis