Gene expression signatures of human primary monocytes from healthy individuals and XLA patients using deep RNA sequencing analysis / Hoda Mirsafian

Monocytes are essential cells of the innate immune system. They play important roles in the initiation and declaration of inflammation, generally through release of inflammatory cytokines, ROS (Reactive Oxygen Species) during phagocytosis and the activation of adaptive immune system. In this thesis, the transcriptome of primary monocytes from 6 healthy subjects and 3 patients with X-linked agammaglobulinemia (XLA), one of the inherited form of Primary immunodeficiency diseases (PIDs), were sequenced using deep polyadenylated (Poly(A)+) paired-end RNA sequencing (RNA-Seq) technique. The gene expression profiling was conducted on both healthy and disease RNA-Seq datasets. Approximately 1.3 billion reads were generated from healthy subject’s RNA-Seq datasets. Using this datasets, the expression of 17,657 genes (including 11,644 protein-coding, 3,515 non-coding, and 2,498 pseudogenes) and 81,419 transcripts (including 70,457 annotated transcripts and 4,935 novel transcripts) were profiled from healthy subjects. The sequencing also generated approximately 477 million reads from XLA patients’ samples which lead to the profile of 17,510 genes (including 11,788 protein-coding, genes 3,681 non-coding genes, and 2,041 pseudogenes) and 62,367 transcripts (including 58,136 annotated and 4,231 novel transcripts). A comparative study was conducted on gene expression profiles of 3 healthy male and 3 healthy female subjects to look into possible gender differences in expression patterns of immune-related genes. The results revealed that the innate immune-related genes are not equally expressed in primary monocytes of healthy male and female which indicated the disparity in innate immune response based on gender. Furthermore, the RNA-Seq datasets of the 6 healthy subjects were integrated with public domain RNA-Seq datasets of human monocytes to construct the gene reference catalogue of primary monocytes from healthy state monocytes. The long non-coding RNAs (lncRNAs) expression patterns analysis in monocytes was also conducted using these datasets which led to identification of several novel long intergenic non-coding RNAs (lincRNAs) that have not been previously reported in monocytes. A comparative study was performed on gene expression profiles of XLA patients and healthy male subjects. The analysis detected several innate immune-related genes which are differentially expressed between XLA patients and healthy subjects, suggesting impaired immune function of monocytes and increased of susceptibility to apoptosis in monocytes of XLA patients. The results also showed the significant changes in lncRNAs expression patterns in primary monocytes of XLA patients compared to healthy subjects which may play roles in regulating the cell cycle and apoptosis in primary monocytes of XLA patients. The high-resolution genome-wide transcriptome expression profile of primary monocytes present in this study would provide a better understanding of monocytes characterization and function in healthy and XLA states. It also facilitates the detailed analyses of innate immune system abnormalities and novel pathomechanism concerning XLA

Molecular sequence and structural comparison of BACE1 and BACE2 / Hoda Mirsafian

Beta-Secretase is a transmembrane aspartic protease that cleaves the amyloid precursor protein (APP) to generate amyloid beta peptide (Aβ) that is believed to be responsible for the Alzheimer’s disease (AD). Beta-Secretaes include BACE1 and BACE2 which are close homologues, sharing 61.5% similarity and 45% identity at the amino acid level. BACE1 is responsible for generating amyloid plaques in Alzheimer’s disease through cleaving Aβ peptides from the amyloid precursor protein, however, the fragments of Aβ produced by BACE2-cleaved have not been observed in senile plaques in AD. The early onset of dementia in patients with Down’s syndrome is believed to be associated with BACE2, and it is believed to be greatly expressed in breast cancers. Although the structure of BACE1 and BACE2 are very similar, there are some important different between their sequences and structure which made their function different with each other. In this study, I compared the conservation pattern of BACE1 and BACE2 throughout species and identified their active site by using evolutionary trace method. By using this method we can rank the functional significance of amino acids in the protein structure and also imply some data about the protein sequence-structure-function relationship. The result of this study identified that, in BACE1 and BACE2 structures, most of the conserved residues are located in the ligand binding site which are the key functionally residues for their activities. The other few conserved residues are more or less randomly localized in the rest of the structure. Also it was observed that, the structure of BACE1 and BACE2 reveals differences in some active site residues which may be of interest for the design of selective BACE1 or BACE2 inhibitors

Amino Acid Sequence and Structural Comparison of BACE1 and BACE2 Using Evolutionary Trace Method

Beta-amyloid precursor protein cleavage enzyme 1 (BACE1) and beta-amyloid precursor protein cleavage enzyme 2 (BACE2), members of aspartyl protease family, are close homologues and have high similarity in their protein crystal structures. However, their enzymatic properties differ leading to disparate clinical consequences. In order to identify the residues that are responsible for such differences, we used evolutionary trace (ET) method to compare the amino acid conservation patterns of BACE1 and BACE2 in several mammalian species. We found that, in BACE1 and BACE2 structures, most of the ligand binding sites are conserved which indicate their enzymatic property of aspartyl protease family members. The other conserved residues are more or less randomly localized in other parts of the structures. Four group-specific residues were identified at the ligand binding site of BACE1 and BACE2. We postulated that these residues would be essential for selectivity of BACE1 and BACE2 biological functions and could be sites of interest for the design of selective inhibitors targeting either BACE1 or BACE2

A Comparative Analysis of Synonymous Codon Usage Bias Pattern in Human Albumin Superfamily

Synonymous codon usage bias is an inevitable phenomenon in organismic taxa across the three domains of life. Though the frequency of codon usage is not equal across species and within genome in the same species, the phenomenon is non random and is tissue-specific. Several factors such as GC content, nucleotide distribution, protein hydropathy, protein secondary structure, and translational selection are reported to contribute to codon usage preference. The synonymous codon usage patterns can be helpful in revealing the expression pattern of genes as well as the evolutionary relationship between the sequences. In this study, synonymous codon usage bias patterns were determined for the evolutionarily close proteins of albumin superfamily, namely, albumin, α-fetoprotein, afamin, and vitamin D-binding protein. Our study demonstrated that the genes of the four albumin superfamily members have low GC content and high values of effective number of codons (ENC) suggesting high expressivity of these genes and less bias in codon usage preferences. This study also provided evidence that the albumin superfamily members are not subjected to mutational selection pressure

Transcutaneous induction of stimulus timing dependent plasticity in dorsal cochlear nucleus

The cochlear nucleus (CN) is the first site of multisensory integration in the ascending auditory pathway. The principal output neurons of the dorsal cochlear nucleus (DCN), fusiform cells, receive somatosensory information relayed by the CN granule cells from the trigeminal and dorsal column pathways. Integration of somatosensory and auditory inputs results in long-term enhancement or suppression in a stimulus timing dependent manner. Here we demonstrate that stimulus timing dependent plasticity (STDP) can be induced in DCN fusiform cells using paired auditory and transcutaneous electrical stimulation of the face and neck to activate trigeminal and dorsal column pathways to the CN, respectively. Long-lasting changes in fusiform cell firing rates persisted for up to two hours after this bimodal stimulation, and followed Hebbian or anti-Hebbian rules, depending on tone duration, but not somatosensory stimulation location: 50 ms paired tones evoked predominantly Hebbian, while 10 ms paired tones evoked predominantly anti-Hebbian plasticity. The tone-duration dependent STDP was strongly correlated with first inter-spike intervals, implicating intrinsic cellular properties as determinants of STDP. This study demonstrates that transcutaneous stimulation with precise auditory-somatosensory timing parameters can non-invasively induce fusiform cell long-term modulation, which could be harnessed in the future to moderate tinnitus-related hyperactivity in DCN