treatments, and neo-antigens are the targets of immune system in cancer patients who
respond to the treatments. The cancer vaccine field is focused on using neo-antigens from
unique point mutations of genomic sequence in the cancer patient for making
personalized cancer vaccines. However, we choose a different path to find frameshift
neo-antigens at the mRNA level and develop broadly effective cancer vaccines based on
frameshift antigens.
In this dissertation, I have summarized and characterized all the potential frameshift
antigens from microsatellite regions in human, dog and mouse. A list of frameshift
antigens was validated by PCR in tumor samples and the mutation rate was calculated for
one candidate – SEC62. I develop a method to screen the antibody response against
frameshift antigens in human and dog cancer patients by using frameshift peptide arrays.
Frameshift antigens selected by positive antibody response in cancer patients or by MHC
predictions show protection in different mouse tumor models. A dog version of the
cancer vaccine based on frameshift antigens was developed and tested in a small safety
trial. The results demonstrate that the vaccine is safe and it can induce strong B and T cell
immune responses. Further, I built the human exon junction frameshift database which
includes all possible frameshift antigens from mis-splicing events in exon junctions, and I
develop a method to find potential frameshift antigens from large cancer
immunosignature dataset with these databases. In addition, I test the idea of ‘early cancer
diagnosis, early treatment’ in a transgenic mouse cancer model. The results show that
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early treatment gives significantly better protection than late treatment and the correct
time point for treatment is crucial to give the best clinical benefit. A model for early
treatment is developed with these results.
Frameshift neo-antigens from microsatellite regions and mis-splicing events are
abundant at mRNA level and they are better antigens than neo-antigens from point
mutations in the genomic sequences of cancer patients in terms of high immunogenicity,
low probability to cause autoimmune diseases and low cost to develop a broadly effective
vaccine. This dissertation demonstrates the feasibility of using frameshift antigens for
cancer vaccine development.
The unicellular microalga Haematococcus pluvialis has emerged as a promising biomass feedstock for the ketocarotenoid astaxanthin and neutral lipid triacylglycerol. Motile flagellates, resting palmella cells, and cysts are the major life cycle stages of H. pluvialis. Fast-growing motile cells are usually used to induce astaxanthin and triacylglycerol biosynthesis under stress conditions (high light or nutrient starvation); however, productivity of biomass and bioproducts are compromised due to the susceptibility of motile cells to stress. This study revealed that the Photosystem II (PSII) reaction center D1 protein, the manganese-stabilizing protein PsbO, and several major membrane glycerolipids (particularly for chloroplast membrane lipids monogalactosyldiacylglycerol and phosphatidylglycerol), decreased dramatically in motile cells under high light (HL). In contrast, palmella cells, which are transformed from motile cells after an extended period of time under favorable growth conditions, have developed multiple protective mechanisms - including reduction in chloroplast membrane lipids content, downplay of linear photosynthetic electron transport, and activating nonphotochemical quenching mechanisms - while accumulating triacylglycerol. Consequently, the membrane lipids and PSII proteins (D1 and PsbO) remained relatively stable in palmella cells subjected to HL. Introducing palmella instead of motile cells to stress conditions may greatly increase astaxanthin and lipid production in H. pluvialis culture.
The unicellular green microalga Desmodesmus sp. S1 can produce more than 50% total lipid of cell dry weight under high light and nitrogen-limitation conditions. After irradiation by heavy 12C6+ ion beam of 10, 30, 60, 90 or 120 Gy, followed by screening of resulting mutants on 24-well microplates, more than 500 mutants were obtained. One of those, named D90G-19, exhibited lipid productivity of 0.298 g L-1⋅d-1, 20.6% higher than wild type, likely owing to an improved maximum quantum efficiency (Fv/Fm) of photosynthesis under stress. This work demonstrated that heavy-ion irradiation combined with high-throughput screening is an effective means for trait improvement. The resulting mutant D90G-19 may be used for enhanced lipid production.
Bismuth drugs, despite being clinically used for decades, surprisingly remain in use and effective for the treatment of Helicobacter pylori infection, even for resistant strains when co-administrated with antibiotics. However, the molecular mechanisms underlying the clinically sustained susceptibility of H. pylori to bismuth drugs remain elusive. Herein, we report that integration of in-house metalloproteomics and quantitative proteomics allows comprehensive uncovering of the bismuth-associated proteomes, including 63 bismuth-binding and 119 bismuth-regulated proteins from Helicobacter pylori, with over 60% being annotated with catalytic functions. Through bioinformatics analysis in combination with bioassays, we demonstrated that bismuth drugs disrupted multiple essential pathways in the pathogen, including ROS defence and pH buffering, by binding and functional perturbation of a number of key enzymes. Moreover, we discovered that HpDnaK may serve as a new target of bismuth drugs to inhibit bacterium-host cell adhesion. The integrative approach we report, herein, provides a novel strategy to unveil the molecular mechanisms of antimicrobial metals against pathogens in general. This study sheds light on the design of new types of antimicrobial agents with multiple targets to tackle the current crisis of antimicrobial resistance.
Competing endogenous RNAs (ceRNAs) are RNA molecules that sequester shared microRNAs (miRNAs) thereby affecting the expression of other targets of the miRNAs. Whether genetic variants in ceRNA can affect its biological function and disease development is still an open question. Here we identified a large number of genetic variants that are associated with ceRNA's function using Geuvaids RNA-seq data for 462 individuals from the 1000 Genomes Project. We call these loci competing endogenous RNA expression quantitative trait loci or ‘cerQTL’, and found that a large number of them were unexplored in conventional eQTL mapping. We identified many cerQTLs that have undergone recent positive selection in different human populations, and showed that single nucleotide polymorphisms in gene 3΄UTRs at the miRNA seed binding regions can simultaneously regulate gene expression changes in both cis and trans by the ceRNA mechanism. We also discovered that cerQTLs are significantly enriched in traits/diseases associated variants reported from genome-wide association studies in the miRNA binding sites, suggesting that disease susceptibilities could be attributed to ceRNA regulation. Further in vitro functional experiments demonstrated that a cerQTL rs11540855 can regulate ceRNA function. These results provide a comprehensive catalog of functional non-coding regulatory variants that may be responsible for ceRNA crosstalk at the post-transcriptional level.