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The identification and understanding of early drivers in malignancy is crucial to revert preleukemic events and prevent leukemic relapse. Del(20q) is one of the most common primary cytogenetic abnormalities found in preleukemic malignancies from myeloproliferative neoplasms to myelodysplastic syndrome (MDS). Previous studies have identified a “common retained region” within 20q11.21 that is often amplified in a subset of MDS patients. PLAGL2 is one of the 4 genes identified to be within the minimally conserved amplified region. Indeed, in previously published datasets of MDS hematopoietic stem and progenitor cells (HSPCs) transcriptome, PLAGL2 is significantly elevated in del(20q) patients compared to healthy controls. However, we have found that its level is also higher in HSPCs of cytogenetically normal MDS patients with low blasts. Given these findings, we sought to define the role of PLAGL2 as a potential early driver of myeloid malignancies. Results In healthy cord blood (CB) HSPCs, PLAGL2 overexpression enhanced proliferation ex vivo, better maintained stemness and decreased apoptosis. Colony formation assays also identified increased output of the erythroid lineage. Xenotransplanted CB CD34+ HSPCs overexpressing PLAGL2 exhibited increased engraftment competitiveness and led to splenomegaly with signs of hypercellularity after 20 weeks, features consistent with clinical observations of hematological malignancy. Grafts derived from PLAGL2 overexpressing cells reproducibly maintained a significantly larger CD34+ HSPC compartment. Intriguingly we also identified that ~50% of PLAGL2-overexpressing grafts exhibited a significant erythroid (CD71+/CD235a+) component where none was observed in the control group. This unique finding of aberrant erythropoiesis is reminiscent of clinical observations in patients with 20q11.21 amplification, where a high proportion of erythroblasts in the marrow and in some cases progression to erythroleukemia was noted. To evaluate the progression of PLAGL2-overexpressing grafts, further secondary transplantations were carried out and showed the persistence of only immature erythroid progenitors (CD71+/CD235a-) coupled with a near complete absence of lymphopoiesis in the same grafts. Together, our data strongly suggests ectopic levels of PLAGL2 can independently drive the expansion of human HSPCs and enforce features of myeloid malignancy. To uncover the molecular mechanism underlying PLAGL2 function, we performed RNA-seq and CUT&RUN in human CB CD34+ HSPCs overexpressing PLAGL2. Gene set enrichment analysis of the transcriptome and over-representation analysis of bound genes both identified signatures consistent with LSCs. We compared these findings with identically-derived omics profiles of HSPCs overexpressing PLAG1, a closely related family member that our lab has identified as a potent expander of HSCs ex vivo but not capable of promoting malignant features. We found a strong common feature in the downregulation of ribosomal components and translation machinery, then functionally validated reduced protein synthesis in PLAGL2 overexpressing HSPCs through OP-Puro assays. We have shown dampened mRNA translation to be one of the mechanisms by which PLAG1 enhances stemness and survival of HSCs, one that potentially extends to PLAGL2 as well. However, we also identified discordant signatures, notably PLAGL2's unique capacity to reduce mitochondrial translation, a pathway associated with ineffective erythropoiesis and MDS and one potential pathway by which PLAGL2 can enforce malignant phenotypes. Finally, to investigate the potential of PLAGL2 as a therapeutic target in MDS and AML, we performed shRNA knockdown in MDSL, a human MDS cell line, and primary human AML. In vitro competitive assays with MDSL showed steady dropout of PLAGL2 depleted cells. Similarly, depletion of PLAGL2 in primary AML was also able to attenuate colony formation and engraftment in vivo, highlighting the therapeutic potential of PLAGL2 inhibition throughout myeloid malignancies. Conclusion We have identified PLAGL2's potential as an early independent driver of myeloid malignancy and aberrant erythroid differentiation. An understanding of PLAGL2 and its downstream mechanisms will not only further our understanding on the development of early myeloid malignancies but also potentially provide another avenue to treat or prevent leukemia before it manifests. / Thesis / Doctor of Philosophy (PhD)

As the IT industry marks a paradigm shift from the traditional system design model to System-On-Chip (SOC) design, the design of custom hardware, embedded processors and associated software have become very tightly coupled. Any change in the implementation of one of the components affects the design of other components and, in turn, the performance of the system. This has led to an integrated design approach known as hardware/software co-design and co-verification. The conventional techniques for co-design favor partitioning the system into hardware and software components at an early stage of the design and then iteratively refining it until a good solution is found. This method is expensive and time consuming. A more modern approach is to model the whole system and rigorously test and refine it before the partitioning is done. The key to this method is the ability to model and simulate the entire system. The advent of new System Level Modeling Languages (SLML), like SystemC, has made this possible. This research proposes a strategy to automate the process of partitioning a system model after it has been simulated and verified. The partitioning idea is based on systems modeled using Process Model Graphs (PmG). It is possible to extract a PmG directly from a SLML like SystemC. The PmG is then annotated with additional attributes like IO delay and rate of activation. A complexity heuristic is generated from this information, which is then used by a greedy algorithm to partition the graph into different architectures. Further, a command line tool has been developed that can process textually represented PmGs and partition them based on this approach. / Master of Science

Arguably, from the programmer's perspective, the programming model is the most important characteristic of any computer system. Perhaps this explains why, after many decades of research, architects and programmers alike continue to debate the appropriate programming model for parallel computers. Though thousands of programming models have been developed, standards such as PVM and MPI have made send-and-receive based message-passing the most popular programming model for distributed memory architectures. This thesis explores modifying the Single-Chip Message-Passing (SCMP) architecture to more efficiently support send-and-receive based message-passing. The proposed system is compared, for performance and programmability, to the active messaging programming model currently used by SCMP. SCMP offers a unique platform for send-and-receive based message-passing. The SCMP design incorporates multiple multi-threaded processors, memory, and a network onto a single chip. This integration reduces the penalties of thread switching, memory access, and inter-process communication typically seen on more traditional distributed memory parallel machines. The mechanisms proposed in this thesis to support send-and-receive based message-passing on SCMP attempt to preserve and exploit these features as much as possible. / Master of Science

With machine learning workloads currently at very large scales, models are distributed across large compute systems. On distributed systems, the performance of these models are limited by the bandwidth limitations of chip-to-chip communication. To relieve this bottleneck, spiking neural networks (SNNs) can be utilized to reduce inter-chip communication traffic utilizing inherit network sparsity. However, in comparison to traditional artificial neural networks (ANNs), SNNs can have significant degradation in performance with increased network scale and complexity. This research proposes a hybrid neural network accelerator that uses the best of both spiking and non-spiking layers by allocating a majority of resources to nonspiking layers on the interior of the chip while bandwidth-limited areas (e.g., I/O pads, or chip separation boundaries) employ spike-based data traffic. By limiting the overall use of spiking layers within the network, we realize the energy savings of SNNs without the a degradation in accuracy which comes with large spike-based models. We present a scalable chiplet architecture and show how hybrid data is managed with both spike and non-spiking data communication. We also demonstrate how the asynchronous spike-based model is integrated efficiently with the synchronous artificial-based deep learning workloads. We demonstrate that our hybrid architecture offers significant improvements in performance, accuracy, and energy consumption in comparison to SNNs and ANNs. With up to a 1.34× increase in energy efficiency and 1.56× decrease in single inference latency, the versatility of the architecture is demonstrated by its validation across multiple datasets, encompassing both language processing and computer vision tasks.

In the following work, nanoparticle quantum dot (QD) fluorophores have been exploited to measure biologically relevant analytes via a miniaturized sensor ensemble to provide key diagnostic and prognostic information in a rapid, yet sensitive manner—data essential for effective treatment of many diseases including HIV/AIDS and cancer. At the heart of this “nano-bio-chip” (NBC) sensor is a modular chemical/cellular processing unit consisting of either a polycarbonate membrane filter for cell-based assays, or an agarose bead array for detection of biomarkers in serum or saliva. Two applications of the NBC sensor system are described herein, both exhibiting excellent correlation to reference methods ((R² above 0.94), with analysis times under 30 minutes and sample volumes below 50 [mu]L. First, the NBC sensor was employed for the sequestration and enumeration of T lymphocytes, cells specifically targeted by HIV, from whole blood samples. Several different conjugation methods linking QDs to recognition biomolecules were extensively characterized by biological and optical methods, with a thiol-linked secondary antibody labeling scheme yielding intense, specific signal. Using this technique, the photostability of QDs was exploited, as was the ability to simultaneously visualize different color QDs via a single light pathway, effectively reducing optical requirements by half. Further, T-cell counts were observed well below the 200/[mu]L discriminator between HIV and AIDS and across the common testing region, demonstrating the first reported example of cell counting via QDs in an enclosed, disposable device. Next, multiplexed bead-based detection of cancer protein biomarkers CEA, Her-2/Neu, and CA125 in serum and saliva was examined using a sandwich immunoassay with detecting antibodies covalently bound to QDs. This nano-based signal was amplified 30 times versus molecular fluorophores and cross talk in multiplexed experiments was less than 5%. In addition, molecular-level tuning of recognition elements (size, concentration) and agarose porosity resulted in NBC limits of detection two orders of magnitude lower than ELISA, values competitive with the most sensitive methods yet reported (0.021 ng/mL CEA). Taken together, these efforts serve to establish the valuable role of QDs in miniaturized diagnostic devices with potential for delivering biomedical information rapidly, reliably, and robustly. / text

In order to carry out the complicated computation inside the high performance computing (HPC) systems, tens to hundreds of parallel processor chips and physical wires are required to be integrated inside the multi-chip package module (MCM). The physical wires considered as the electrical interconnects between the processor chips, however, have the challenges on placements and routings because of the unequal progress between the semiconductor and I/O size reductions. The primary goal of the research is to overcome package design challenges - providing a hybrid computing architecture with implemented 60 GHz antennas as the high efficient wireless interconnect which could generate over 10 Gbps bandwidth on the data transmissions. The dissertation is divided into three major parts. In the first part, two different performance metrics, power loss required to be recovered (PRE) and wireless link budget, on evaluating the antenna's system performance within the chip to chip wireless interconnect are introduced to address the design challenges and define the design goals. The second part contains the design concept, fabrication procedure and measurements of implemented 60 GHz broadband antenna in the application of multi-chip data transmissions. The developed antenna utilizes the periodically-patched artificial magnetic conductor (AMC) structure associated with the ground-shielded conductor in order to enhance the antenna's impedance matching bandwidth. The validation presents that over 10 GHz -10 dB S11 bandwidth which indicates the antenna's operating bandwidth and the horizontal data transmission capability which is required by planar type chip to chip interconnect can be achieved with the design concept. In order to reduce both PRE and wireless link budget numbers, a 60 GHz two-element array in the multi-chip communication is developed in the third part. The third section includes the combined-field analysis, the design concepts on two-element array and feeding circuitry. The simulation results agree with the predicted field analysis and demonstrate the 5dBi gain enhancement in the horizontal direction over a single 60 GHz AMC antenna to further reduce both PRE and wireless link budget numbers.

En 2010, Haurie et al. ont identifié deux isoformes différentes de la Pol III humaine : Pol IIIα, quin’est présente que dans les cellules souches embryonnaires et dans celles tumorales, et Pol IIIβ,exprimée constitutivement.L’expression ectopique de Pol IIIα affecte profondément l’équilibre de cellules différentiées, jusqu’àen induire la transformation oncogénique. Ceci n’est pas observé dans le cas de l’expression ectopiquede Pol IIIβ.A fin de définir les causes moléculaires de la transformation cellulaire induite par Pol IIIα, nousavons décidé d’étudier son transcriptome en comparaison avec celui de Pol IIIβ, à la recherche desgènes qui pourraient soutenir l’oncogenèse observée. Dans notre étude, nous avons adopté latechnique du ChIP-Seq, une approche innovatrice et puissante qui permet de cartographier tous lessites de liaison d’une protéine sur le génome. Elle comporte la purification de la protéine d’intérêtcomplexe avec ces cibles génomiques et le séquençage de ces dernières de manière massive.Le premier but de cette thèse a été représenté par la mise a point d’un protocole de ChIP-Seqoptimisé spécifiquement pour Pol IIIα et Pol IIIβ. Une fois obtenue une préparation de chromatine dequalité adéquate aux standards du ChIP-Seq, nous avons effectué les séquençages massifs etcartographié sur le génome tous les loci contactés par les deux isoformes de polymérase.De cette manière, nous avons identifié 1287 loci occupés par Pol IIIα et 1281 par Pol IIIβ. Les deuxpolymérases se co-localisent sur la plupart de ces sites, mais montrent aussi une occupationpréférentielle et spécifique sur une fraction de loci Pol III. Cette disproportion pourrait contribuer auphénomène cellulaire observé par Haurie et al.Le manuscrit de thèse détaille les résultats de cette analyse et les conclusions qui en dérivent.Nous y avons ajouté une synthèse des travaux réalisés antérieurement et concertants la caractérisationdes promoteurs de gène qui codent pour les snoARN chez la levure S.cerevisiae, positionnée en toutefin de document. / In eukaryotes, transcription is carried out by DNA-dependent RNA polymerases I, II and III (or I-V inplants). These RNA polymerases are specialized in the transcription of specific groups of genes.Human RNA polymerase III (Pol III) transcribes small noncoding RNAs involved in the regulation oftranscription (7SK RNA), RNA processing (U6 RNA, RNAse P, RNAse MRP), translation (tRNAs,5S RNA) or other cellular processes (vault RNAs [multidrug resistance], adenoviral RNAs [VA-I,VA-II], Epstein-Barr virus RNAs [EBER1, EBER2]). It has furthermore been reported that somemicroRNAs of viral or cellular origin may also be transcribed by Pol III.Interestingly, increased Pol III transcription levels accompany or cause cell transformation. Themechanisms underlying this phenomenon are still largely unknown. Recently, two distinct isoforms ofhuman Pol III have been discovered (Haurie et al., 2010). RPC32β-containing Pol IIIβ is ubiquitouslyexpressed and essential for growth and survival of human cells. In contrast, RPC32α-containing PolIIIα is dispensable for cell survival and its expression is restricted to undifferentiated embryonic stem(ES) cells and to tumor cells.The distinct effects of Pol IIIα and Pol III β on cell growth and transformation may be explained bythe transcription of isoform-specific target genes. To identify such isoform-specific target genes, wespecifically targeted RPC32α and of RPC32β subunits in chromatin immunoprecipitation (ChIP)experiments and analyzed the co-precipitated DNA sequences by high throughput sequencing (ChIPseq.).Genome wide localization of RPC32α and RPC32β subunits of Pol III revealed the presence of bothsubunits on many of the known Pol III-transcribed genes, suggesting redundant activities of bothisoforms of Pol III in transcription of these genes. We also found that some of the genes known to betranscribed by Pol III are only occupied by either RPC32α or by RPC32β, suggesting that these genesare exclusively transcribed by Pol IIIα or by Pol IIIβ, respectively.RPC32α and RPC32β ChIP-seq. results furthermore led to the identification of novel Pol III candidategenes in HeLa cells. Moreover, we found high levels of Pol IIIα or Pol III β at some of the annotatedtRNA pseudogenes, implicating that these genes may be transcribed. The functions of RNAstranscribed from novel putative Pol III genes or from tRNA pseudogenes remain to be determined.

Este trabalho descreve o desenvolvimento de um processo de fabricação de mini e microdispositivos fluídicos baseado na utilização de um equipamento de usinagem a laser de CO2 para criação de relevos sobre base de poli(metacrilato de metila) e na selagem térmica contra igual material. Inicialmente, o equipamento laser foi detalhadamente caracterizado, o que possibilitou elaborar métodos para a construção de microcanais de forma mais eficiente e com menores chances de defeitos. Tipicamente, os canais apresentaram seção transversal triangular em torno de 200 µm de largura e 100 µm de profundidade, sendo possível, no entanto, criar canais com outras características. A etapa de selagem entre a tampa e a base que apresentou melhores resultados consiste em pressurização acima de 6 kgf·cm-2 e aquecimento a 110 ºC durante 45 minutos, seguido de resfriamento por 1 h. Os microcanais selados por esta técnica, resistiram a pressões superiores a 3,5·kgf·cm-2. O processo desenvolvido se mostrou adequado para a criação de protótipos, sendo também suas principais características: (1) a facilidade de incorporação de regiões de grandes dimensões (como reservatórios) em conjunto com os microcanais, (2) número reduzido de etapas de produção e (3) boa uniformidade química da parede interna dos canais, o que é particularmente interessante para microdispositivos aplicados à Química Analítica / A microfabrication process based machining using CO2 laser on poly(methyl methacrylate) and thermal sealing is described. Initially, the laser equipment was characterized in detail, which allowed developing strategies for the construction of microchannels more efficiently and less failure-prone. Typically, the channels had a triangular cross section around 200 µm in width and 100 µm in depth. It is possible, however, create channels with other features. The sealing step that showed better results consists in to pressurize at 6 kgf·cm-2 and heating at 110 °C during 45 minutes, followed by natural cooling for 1 h. The microchannels sealed by using this procedure resisted pressures above 3.5 kgf·cm-2. The process proved to be adequate for prototyping and also has other main features: (1) easiness of incorporation of large regions (such as reservoirs) together with the microchannel; (2) reduced number of manufacturing steps and (3) good chemical uniformity of the inner wall of the channel, which is particularly interesting for microdevices applied to Analytical Chemistry.

The Nelore breed represents the vast majority of Brazilian Zebuine cattle (Bos taurus indicus). The great adaptability of the Nelore breed to Brazilian tropical climate, however, is not associated with meat tenderness (MT). It is known that MT is influenced by several environmental factors, but also genetic composition. In the first chapter, we report a genome-wide analysis of copy number variation (CNV) inferred from Illumina® Bovine High Density SNP-chip data for a Nelore population of 723 males including 30 sires. We detected >2600 CNV regions (CNVRs) representing ≈6.5% of the Bos taurus genome. The CNVR size was 65 kb on average, ranging from 5 kb to 4.3 Mb. A total of 1155 CNVRs (43.6%) overlapped 2750 genes. They are enriched for important functions such as immune response, olfactory reception and processes involving guanosine triphosphate (GTP). The GTP processes have known influence in skeletal muscle physiology and morphology. Quantitative trait loci for MT, partly specific for Nelore, overlapped a substantial fraction of CNVRs and two CNVRs were found proximal to glutathione metabolism genes that are associated with MT as well. Comparing our results with previous studies revealed an overlap in ≈1400 CNVRs (>50%). We selected 9 CNVRs that overlapped regions associated with MT and we validated them in all 30 sires by qPCR. There was identified many genomic regions of structural variation in Nelore with important implications on the MT phenotype. In the second chapter, a total of 34 animals of the population were subjected to transcriptome analysis and meat tenderness (MT) phenotyping. We identified 170 CNV fragments (CNVFs) residing in 20 CNVRs, which occurred in different frequencies between animals with tougher and softer meat genetic potential. A considerable fraction of the identified CNVFs affected gene expression of the MT genes, which play important roles in glycogen metabolism, connective tissue turnover, membrane transporters and glutathione pathways. We also detected that several CNVRs substantially influenced the expression of overlapped and nearby genes, where the increase or decrease of copy number correlated well with the change in gene expression. Among them are two CNVRs at chromosomes 12 and 23, which are in the vicinity of previously described QTLs for MT in Nelore breed. Several CNVFs, which are more frequent in animals with genetic potential for softer or tougher MT, showed significant differences in gene expression. Those regions are linked to important biological functions with highly relevant influences on MT and skeletal muscle physiology. / A raça Nelore é predominante no rebanho zebuíno brasileiro (Bos taurus indicus). A grande adaptabilidade da raça Nelore ao clima tropical brasileiro, no entanto, não está associada à maciez de carne (MT). Sabe-se que MT é influenciada por vários fatores ambientais e pela composição genética. Foi realizada uma análise de todo o genoma para inferir Variação no Número de Cópias de Segmentos Genômicos (Copy Number Variation - CNV) a partir de dados oriundos de chip de SNP (Illumina® Bovine High Density), para uma população de 723 machos Nelore, incluindo 30 ancentrais da população. Foram detectadas >2600 regiões de CNV (CNVRs) representando ≈6.5% do genoma bovino. O tamanho médio do CNVR foi de 65 kb, variando de 5 kb até 43 Mb. Um total de 1155 CNVRs (43.6%) obtiveram sobreposição com 2750 genes. Estes genes foram enriquecidos para as funções importantes, tais como resposta imunológica, recepção olfativa e processos que envolvem o trifosfato de guanosina (GTP). As vias metabólicas do GTP conhecidamente influenciam a fisiologia e a morfologia do músculo esquelético. Loci de características quantitativas (QTLs) para MT, alguns específicos para Nelore, sobrepuseram uma fração substancial das CNVRs encontradas. Dois CNVRs foram encontrados em região proximal à genes do metabolismo da glutationa os quais também são associados com MT. Comparando os resultados com estudos anteriores ≈1400 CNVRs (>50%) foram sobrepostos. Nove CNVRs em regiões associadas com MT foram validados nos 30 ancentrais por qPCR. Em conclusão, foram identificadas regiões genômicas de variação estrutural no Nelore, com potenciais implicações sobre o fenótipo MT. No segundo capítulo, um total de 34 animais da população foi submetido à análise do transcriptoma e análise de potencial genético para MT. Foram identificados 170 fragmentos de CNV (CNVFs) mapeados em 20 CNVRs, os quais mostraram frequências significativamente diferentes entre animais com potencial genético para carne mais dura ou mais macia. Uma fração considerável dos CNVFs identificados afetaram a expressão gênica de genes MT (anteriormente descritos como associados à MT ou fisiologia do músculo esquelético), os quais desempenham um papel importante no metabolismo de glicogênio, volume do tecido conjuntivo, transportadores de membrana e vias metabólicas da glutationa. Um número considerável de CNVRs foram associados à expressão de genes sobrepostos e nas proximidades, onde o aumento ou diminuição do número de cópias foi associado com a mudança na expressão gênica. Dois CNVRs associados foram mapeados para os cromossomo 12 e 23, estando próximos a QTLs anteriormente descritos para MT na raça Nelore. Vários CNVFs, entre animais com potencial genético para carne mais macia ou dura, mostraram diferenças significativas na expressão gênica. Essas regiões estão ligadas a importantes funções biológicas com influências altamente relevantes para MT e para a fisiologia do músculo esquelético.