Differently expressed genes e.g. in a disease may play a role in the etiology or progression of the disease. The traditional approach of finding differentially expressed genes is to compare the expression levels in the groups, and produce a list of differentially expressed candidate genes. With many pairwise comparisons, the risk of introducing type I and type II errors is high. One solution is to group together genes that are co-expressed into modules. Weighted gene coexpression network analysis (WGCNA) uses a topological overlap module approach and has been proved to find patterns that have been undetected by gene-to-gene comparison methods. qPCR has high sensitivity and specificity, and advances in technology has increased its throughput. The goal of the project was to construct WGCNA modules from qPCR data and evaluate the WGCNA method in five previously published qPCR data sets. There was little overlap between the differentially expressed genes found in the published articles and the candidates found by WGCNA. In three data sets WGCNA failed to produce any significant genes. In one of the data set significant genes were found where the original article failed. In one data set, 19 out of 60 genes that are top-ranked by the original authors were found in significant WGCNA modules. The biggest challenge with this type of comparison is to determine whether results that differ from the published studies are more or less biologically relevant. It is difficult to draw conclusions on whether the method is suitable for use for analysis of qPCR data based on this study.

Streptococcus sanguinis is a gram-positive, non-motile bacterium native to human mouths. It is the primary cause of endocarditis and is also responsible for tooth decay. Two-component systems (TCSs) are commonly found in bacteria. In response to environmental signals, TCSs may regulate the expression of virulence factor genes. Gene co-expression networks are exploratory tools used to analyze system-level gene functionality. A gene co-expression network consists of gene expression profiles represented as nodes and gene connections, which occur if two genes are significantly co-expressed. An adjacency function transforms the similarity matrix containing co-expression similarities into the adjacency matrix containing connection strengths. Gene modules were determined from the connection strengths, and various network connectivity measures were calculated. S. sanguinis gene expression profile data was loaded for 2272 genes and 14 samples with 3 replicates each. The soft thresholding power β=6 was chosen to maximize R2 while maintaining a high mean number of connections. Nine modules were found. Possible meta-modules were found to be: Module 1: Blue & Green, Module 2: Pink, Module 3: Yellow, Brown & Red, Module 4: Black, Module 5: Magenta & Turquoise. The absolute value of module membership was found to be highly positively correlated with intramodular connectivity. Each of the nine modules were examined. Two methods (intramodular connectivity and TOM-based connectivity followed by network mapping) for identifying candidate hub genes were performed. Most modules provided similar results between the two methods. Similar rankings between the two methods can be considered equivalent and both can be used to detect candidate hub genes. Gene ontology information was unavailable to help select a module of interest. This network analysis would help researchers create new research hypotheses and design experiments for validation of candidate hub genes in biologically important modules.

A sepse é uma das maiores causas de mortalidade em pacientes hospitalizados, e uma complicação comum, tanto em pacientes clínicos quanto de cirurgias, admitidos em hospitais por causas não infecciosas. A sepse é especialmente comum em pacientes mais velhos, sendo portanto esperado que sua incidência aumente com o envelhecimento da população, e apesar da sua maior taxa de mortalidade, a resposta imune em idosos durante o choque séptico é muito similar à dos pacientes mais jovens. O objetivo desse estudo foi de conduzir uma análise de expressão gênica dos neutrófilos da circulação, tanto de pacientes adultos como de pacientes idosos, observando tanto os mRNAs e as vias em que estão envolvidos, como o papel dos ncRNAs, para um melhor entendimento da resposta imune do indivíduo idoso a infecções severas. Os RNAs de 24 indivíduos, divididos igualmente entre idosos e adultos, e entre pacientes em choque séptico e controles, foram hibridizadas em microarranjos de DNA. Deste experimento foram encontrados genes cuja expressão pode ser utilizada para diferenciar a resposta imune entre adultos e idosos. Estes genes foram observados concentrados em algumas vias, entre elas fosforilação oxidativa, disfunção mitocondrial, sinalização do TGF-, entre outras. Além da análise usando os mRNAs, esse trabalho mostra fortes indicações de interações de mRNAs com RNAs não codificadores longos, dos quais a maioria não têm função conhecida. Para propor uma função aos RNAs não codificadores foi construída uma rede de coexpressão em que alguns RNAs de função desconhecida se mostraram fortemente ligados à genes das vias moleculares do ribossomo e da mitocôndria. Também foi observado que para os idosos a rede de coexpressão é menos centralizada, suportando a hipótese de que alterar a expressão de alguns genes chave pode ser o fator determinante para alterar a expressão gênica e um conjunto maior / Sepsis is one of the major causes of mortality in hospitalized patients, and a common complication, both in clinical patients and in surgeries, admitted to hospital for non-infectious causes. Sepsis is especially common in older patients, and is therefore expected that its incidence increases as the population ages, and despite its higher mortality rate, the immune response in the elderly during septic shock is very similar to that of younger patients. The objective of this study was to conduct a gene expression analysis of circulating neutrophils, both adults and elderly patients, noting both the mRNAs, the pathways in which those are involved, and the role of ncRNAs, for a better understanding of the immune response of the elderly to severe infections. The RNAs of 24 individuals, equally divided among the adults and the elderly, and among patients in septic shock and controls, were hybridized to DNA microarrays. From this experiment many genes whose expression can be used to differentiate the immune response in adults and the elderly were found. These genes were concentrated in some metabolic pathways, including oxidative phosphorylation, mitochondrial dysfunction, TGF- signaling, and others. Besides the analysis using mRNAs, this work shows strong indications of mRNAs interactions with non coding RNAs, most of which have no known function. To propose a role for noncoding RNAs a coexpression network was built in which some RNAs of unknown function showed strongly connected to genes of the molecular pathways of the ribosome and mitochondria. It was also noted that for the elderly, the coexpression network is less centralized, supporting the hypothesis that altering the expression of a few key genes can be a determining factor for altering the gene expression of a larger set

A sepse é uma das maiores causas de mortalidade em pacientes hospitalizados, e uma complicação comum, tanto em pacientes clínicos quanto de cirurgias, admitidos em hospitais por causas não infecciosas. A sepse é especialmente comum em pacientes mais velhos, sendo portanto esperado que sua incidência aumente com o envelhecimento da população, e apesar da sua maior taxa de mortalidade, a resposta imune em idosos durante o choque séptico é muito similar à dos pacientes mais jovens. O objetivo desse estudo foi de conduzir uma análise de expressão gênica dos neutrófilos da circulação, tanto de pacientes adultos como de pacientes idosos, observando tanto os mRNAs e as vias em que estão envolvidos, como o papel dos ncRNAs, para um melhor entendimento da resposta imune do indivíduo idoso a infecções severas. Os RNAs de 24 indivíduos, divididos igualmente entre idosos e adultos, e entre pacientes em choque séptico e controles, foram hibridizadas em microarranjos de DNA. Deste experimento foram encontrados genes cuja expressão pode ser utilizada para diferenciar a resposta imune entre adultos e idosos. Estes genes foram observados concentrados em algumas vias, entre elas fosforilação oxidativa, disfunção mitocondrial, sinalização do TGF-, entre outras. Além da análise usando os mRNAs, esse trabalho mostra fortes indicações de interações de mRNAs com RNAs não codificadores longos, dos quais a maioria não têm função conhecida. Para propor uma função aos RNAs não codificadores foi construída uma rede de coexpressão em que alguns RNAs de função desconhecida se mostraram fortemente ligados à genes das vias moleculares do ribossomo e da mitocôndria. Também foi observado que para os idosos a rede de coexpressão é menos centralizada, suportando a hipótese de que alterar a expressão de alguns genes chave pode ser o fator determinante para alterar a expressão gênica e um conjunto maior / Sepsis is one of the major causes of mortality in hospitalized patients, and a common complication, both in clinical patients and in surgeries, admitted to hospital for non-infectious causes. Sepsis is especially common in older patients, and is therefore expected that its incidence increases as the population ages, and despite its higher mortality rate, the immune response in the elderly during septic shock is very similar to that of younger patients. The objective of this study was to conduct a gene expression analysis of circulating neutrophils, both adults and elderly patients, noting both the mRNAs, the pathways in which those are involved, and the role of ncRNAs, for a better understanding of the immune response of the elderly to severe infections. The RNAs of 24 individuals, equally divided among the adults and the elderly, and among patients in septic shock and controls, were hybridized to DNA microarrays. From this experiment many genes whose expression can be used to differentiate the immune response in adults and the elderly were found. These genes were concentrated in some metabolic pathways, including oxidative phosphorylation, mitochondrial dysfunction, TGF- signaling, and others. Besides the analysis using mRNAs, this work shows strong indications of mRNAs interactions with non coding RNAs, most of which have no known function. To propose a role for noncoding RNAs a coexpression network was built in which some RNAs of unknown function showed strongly connected to genes of the molecular pathways of the ribosome and mitochondria. It was also noted that for the elderly, the coexpression network is less centralized, supporting the hypothesis that altering the expression of a few key genes can be a determining factor for altering the gene expression of a larger set

The widely collected and analyzed genetic data help in understanding the underlying mechanisms of heterogeneous diseases. Cellular components interact in a network fashion where genes are nodes and edges are the interactions. The failure in individual genes lead to dys-regulation of sub-groups of genes which causes a disease phenotype, and this dys-functional region is called a disease module. Disease module identification in complex diseases such as asthma and cancer is a huge challenge. Despite the development of numerous sophisticated methods there is a still no gold standard. In this study we apply different parameter settings to test the performance of a widely used method for disease module detection in multi-omics data called Weighted Gene Co-expression Network Analysis (WGCNA). A systematic approach is used to identify disease modules in asthma and arthritis diseases. The accuracy of obtained modules is validated by a pathway scoring algorithm (PASCAL) and GWAS SNP enrichment. Our results differ between the tested data sets and therefore we cannot conclude with recommendations for an optimal setting that could perform best for multiple data sets using this method.

Approximately 6.3 million fractures occur annually in the U.S. and almost 10% of these fractures fail to heal normally. These non-union fractures adversely affect the patients’ quality of life and are an economic burden, due to both treatment costs and lost or reduced employment. Much of the biological and molecular basis of fracture healing and non-unions remains poorly understood. Previous studies have shown that hypophosphatemia produces a rachitic state and diminishes the endochondral ossification of long bone regeneration. Our studies used phosphate deficiency to produce a rachitic animal model in which to study delayed fracture healing. The goal of this study is to investigate the effects hypophosphatemia have on the healing bone’s temporal mRNA expression profiles, its correlations with bone phenotypes, and the effects diet and genetic strain have on significant bone-healing genes. Three strains — A/J, C57BL/6J, C3H/HeJ (AJ, B6, and C3 respectively) — of skeletally mature male mice had stabilized fractures produced in the right femur. Hypophosphatemia was produced by feeding a group (Pi) of mice with a low phosphate diet starting two days before surgery until 14 days after surgery when the regular diet is reintroduced. The control group (Ctrl) was fed the regular diet throughout. At harvest time points (post-operative days; POD) 3, 5, 7, 10, 14, 18, 21, 28, and 35, RNA was extracted from the fracture callus and quantified via microarray analysis. From a different set of mice, the calluses were extracted on POD 14, 21, and 35, for phenotype measurements. Diet and strain significant genes were identified by ANOVA with 11,037 from a total of 21,187 genes. These 11,037 genes were evaluated by Weighted Gene Co-expression Network Analysis (WGCNA) to correlate transcriptomic data to phenotypic properties of the healing bone. Additionally, the genes were also analyzed using a custom polynomial clustering method in order to cluster genes together based on similar temporal expression profiles. WGCNA results showed that, out of the list of 11,037 genes, 10,620 genes (from Ctrl group) and 10,351 genes (from Pi group) respectively clustered either into a group positively correlated with or a group negatively correlated with bone structural properties. In terms of biological functions present in the two gene groups, the positively correlated group consists of immune-related functions while the negatively correlated group consists of bone, cartilage, and vasculature functions. There were a greater number of bone-healing functions in the Pi groups relative to the Ctrl groups; this finding is consistent with known accelerated bone healing in the Pi group after phosphate has been returned to the diet at POD 14. Polynomial clustering results showed specific temporal gene expression differences between strains. Some genes, such as HIF-1α, had the same temporal gene expression regardless of diet or strain and are likely to be unaffected by phosphate deficiency while also being conserved across genetic strains. Other genes, such as IHH, had AJ temporal gene expressions, distinct from the B6 and C3, displaying a diminished peak with no compensation after phosphate recovery. Lastly, few genes, such as BMP2 and RUNX2, showed no Pi diet effects in AJ and C3 but were affected by the Pi diet in B6. Genes such as these may be attributable to previous findings where B6 bones are structurally less mineralized compared to AJ and C3 at POD 35 and beyond. These studies provided a highly detailed understanding of the temporal changes in the transcriptome in relation to both bone healing and the underlying changes at the organ level (bone phenotype). Through the analysis of specific genes’ temporal expressions, our findings further defined the role of phosphate deficiency in impaired bone repair. Future directions are to find the central hub gene, genes that are interconnected with the greatest number of other genes, for each different temporal genetic expression motif. Once identified, those temporally-clustered motifs and their central hub genes will be correlated directly with bone phenotypes in order to understand how these temporal transcriptomic profiles are associated with biomechanical and structural bone properties in the healing bone.

O objetivo deste trabalho foi estudar o transcriptoma do músculo Longissimus dorsi (LD) de bovinos da raça Nelore associado as características de área de olho de lombo (AOL) e espessura de gordura subcutânea (EGS), a fim de encontrar genes diferencialmente expressos (GDE), conjuntos de genes co-expressos, vias metabólicas e processos biológicos que regulam essas características. Foram utilizados dados de 385 bovinos Nelore castrados para obtenção das medidas fenotípicas de AOL e EGS, mensuradas entre a 12ª e 13ª costelas do músculo LD. Estes animais foram separados em dois grupos extremos, alto e baixo, contendo seis animais cada, baseados nos valores genômicos estimados (GEBV), para AOL e para EGS. Estes conjuntos de 12 animais foram submetidos à análise de expressão diferencial afim de encontrar GDE entre os grupos. Em seguida, a análise de enriquecimento funcional a partir da lista de GDE foi executada por meio do DAVID (Database for Annotation, Visualization and Integrated Discovery) e da combinação do BiNGO (Biological Networks Gene Ontology) e REVIGO (Reduce + Visualise Gene Ontology). Para AOL, foram identificados 101 GDE entre os grupos de alto e baixo GEBV. Desses, 72 genes apresentaram-se down-regulated e 29 up-regulated no grupo baixo. Para EGS, foram encontrados 18 GDE, dos quais treze apresentaram-se up-regulated e cinco down-regulated no grupo baixo. Dentre as vias metabólicas e processos biológicos encontrados para essas características, destacam-se a via de sinalização MAPK (bta04010) e a via da endocitose (bta04144) - AOL - e, os processos de biossíntese de andrógenos (GO:0006702) e via de sinalização canônica Wnt (GO:0060070) - EGS. Para encontrar conjuntos de genes co-expressos associados aos tratamentos (AOL e EGS), foi utilizado o pacote WGCNA (Weighted Correlation Network Analysis) do R, com dados de contagens de transcritos de 43 animais. Foram identificados 37 módulos, dentre eles, os módulos Azul, Verde-escuro e Salmon apresentaram correlação significativa de 0,3 com a EGS (P<0,10). Os genes destes módulos foram selecionados para análise de enriquecimento funcional quando seus valores de filiação ao módulo foram superiores a 0,7. O módulo azul apresentou o maior número de genes co-expressos, com 953 genes submetidos à análise de enriquecimento funcional. Foram encontradas seis vias metabólicas e 101 termos do Gene Ontology para este módulo, conforme análise do DAVID, além de 108 processos biológicos identificados pelo BiNGO/REVIGO. Os resultados do presente estudo enfatizam a complexidade da regulação gênica do músculo LD de bovinos Nelore associado às características de AOL e EGS. Estes resultados nos auxiliam a compreender melhor os diversos processos moleculares envolvidos na deposição muscular e de gordura de cobertura, características de carcaça economicamente importantes para a produção da carne bovina. / The aim of this work was to study the Longissimus dorsi (LD) muscle transcriptome of Nellore cattle associated with ribeye area (REA) and backfat thickness (BFT) to find differentially expressed genes (DEG), coexpressed gene modules, metabolic pathways, and biological processes that regulate these traits. Data from 385 Nellore steers were used to obtain the phenotypic measures of REA and BFT, measured between the 12th and 13th ribs of the LD muscle. These animals were divided into two extreme groups, high and low, with six animals each, based on the estimated genomic breeding values (GEBV) for REA and for BFT. These sets of 12 animals were submitted to differentially expressed gene analysis to find DEG between the groups. Then, the functional enrichment analysis from the list of DEG was performed by DAVID (Database for Annotation, Visualization and Integrated Discovery) and the combination of BiNGO (Biological Networks Gene Ontology) and REVIGO (Reduce + Visualize Gene Ontology). For REA, 101 DEG were identified between the high and low GEBV groups. Of those, 72 genes were down-regulated and 29 up-regulated in the low group. For BFT, 18 DEG were found, of which thirteen were up-regulated and five were down-regulated in the low group. Among the metabolic pathways and biological processes found for these traits, the MAPK signaling pathway (bta04010) and the endocytosis pathway (bta04144) - for REA -, the androgen biosynthetic processes (GO: 0006702) and the canonical Wnt signaling pathway (GO: 0060070) - for EGS - can be highlighted. To find coexpressed gene modules associated with the traits (REA and BFT), we used the WGCNA (Weighted Correlation Network Analysis) package from R, with data from transcript counts of 43 animals. A total of 37 modules were founded. Among them, the Blue, Dark Green and Salmon modules had a significant correlation of 0.3 with BFT (P<0.10). The genes of these modules were selected for functional enrichment analysis when their module membership values were higher than 0.7. The blue module had the highest number of co-expressed genes, with 953 genes submitted to functional enrichment analysis. Six metabolic pathways and 101 Gene Oontology terms were founded for this module by DAVID analysis, in addition, 108 biological processes were identified by BiNGO/REVIGO. The results of the present study emphasize the complexity of gene regulation in the LD muscle of Nellore cattle associated with REA and BFT. These results can help us to better understand the different molecular processes involved in muscle and fat deposition, which are economically important carcass traits for beef production.

O objetivo deste trabalho foi estudar o transcriptoma do músculo Longissimus dorsi (LD) de bovinos da raça Nelore associado as características de área de olho de lombo (AOL) e espessura de gordura subcutânea (EGS), a fim de encontrar genes diferencialmente expressos (GDE), conjuntos de genes co-expressos, vias metabólicas e processos biológicos que regulam essas características. Foram utilizados dados de 385 bovinos Nelore castrados para obtenção das medidas fenotípicas de AOL e EGS, mensuradas entre a 12ª e 13ª costelas do músculo LD. Estes animais foram separados em dois grupos extremos, alto e baixo, contendo seis animais cada, baseados nos valores genômicos estimados (GEBV), para AOL e para EGS. Estes conjuntos de 12 animais foram submetidos à análise de expressão diferencial afim de encontrar GDE entre os grupos. Em seguida, a análise de enriquecimento funcional a partir da lista de GDE foi executada por meio do DAVID (Database for Annotation, Visualization and Integrated Discovery) e da combinação do BiNGO (Biological Networks Gene Ontology) e REVIGO (Reduce + Visualise Gene Ontology). Para AOL, foram identificados 101 GDE entre os grupos de alto e baixo GEBV. Desses, 72 genes apresentaram-se down-regulated e 29 up-regulated no grupo baixo. Para EGS, foram encontrados 18 GDE, dos quais treze apresentaram-se up-regulated e cinco down-regulated no grupo baixo. Dentre as vias metabólicas e processos biológicos encontrados para essas características, destacam-se a via de sinalização MAPK (bta04010) e a via da endocitose (bta04144) - AOL - e, os processos de biossíntese de andrógenos (GO:0006702) e via de sinalização canônica Wnt (GO:0060070) - EGS. Para encontrar conjuntos de genes co-expressos associados aos tratamentos (AOL e EGS), foi utilizado o pacote WGCNA (Weighted Correlation Network Analysis) do R, com dados de contagens de transcritos de 43 animais. Foram identificados 37 módulos, dentre eles, os módulos Azul, Verde-escuro e Salmon apresentaram correlação significativa de 0,3 com a EGS (P<0,10). Os genes destes módulos foram selecionados para análise de enriquecimento funcional quando seus valores de filiação ao módulo foram superiores a 0,7. O módulo azul apresentou o maior número de genes co-expressos, com 953 genes submetidos à análise de enriquecimento funcional. Foram encontradas seis vias metabólicas e 101 termos do Gene Ontology para este módulo, conforme análise do DAVID, além de 108 processos biológicos identificados pelo BiNGO/REVIGO. Os resultados do presente estudo enfatizam a complexidade da regulação gênica do músculo LD de bovinos Nelore associado às características de AOL e EGS. Estes resultados nos auxiliam a compreender melhor os diversos processos moleculares envolvidos na deposição muscular e de gordura de cobertura, características de carcaça economicamente importantes para a produção da carne bovina. / The aim of this work was to study the Longissimus dorsi (LD) muscle transcriptome of Nellore cattle associated with ribeye area (REA) and backfat thickness (BFT) to find differentially expressed genes (DEG), coexpressed gene modules, metabolic pathways, and biological processes that regulate these traits. Data from 385 Nellore steers were used to obtain the phenotypic measures of REA and BFT, measured between the 12th and 13th ribs of the LD muscle. These animals were divided into two extreme groups, high and low, with six animals each, based on the estimated genomic breeding values (GEBV) for REA and for BFT. These sets of 12 animals were submitted to differentially expressed gene analysis to find DEG between the groups. Then, the functional enrichment analysis from the list of DEG was performed by DAVID (Database for Annotation, Visualization and Integrated Discovery) and the combination of BiNGO (Biological Networks Gene Ontology) and REVIGO (Reduce + Visualize Gene Ontology). For REA, 101 DEG were identified between the high and low GEBV groups. Of those, 72 genes were down-regulated and 29 up-regulated in the low group. For BFT, 18 DEG were found, of which thirteen were up-regulated and five were down-regulated in the low group. Among the metabolic pathways and biological processes found for these traits, the MAPK signaling pathway (bta04010) and the endocytosis pathway (bta04144) - for REA -, the androgen biosynthetic processes (GO: 0006702) and the canonical Wnt signaling pathway (GO: 0060070) - for EGS - can be highlighted. To find coexpressed gene modules associated with the traits (REA and BFT), we used the WGCNA (Weighted Correlation Network Analysis) package from R, with data from transcript counts of 43 animals. A total of 37 modules were founded. Among them, the Blue, Dark Green and Salmon modules had a significant correlation of 0.3 with BFT (P<0.10). The genes of these modules were selected for functional enrichment analysis when their module membership values were higher than 0.7. The blue module had the highest number of co-expressed genes, with 953 genes submitted to functional enrichment analysis. Six metabolic pathways and 101 Gene Oontology terms were founded for this module by DAVID analysis, in addition, 108 biological processes were identified by BiNGO/REVIGO. The results of the present study emphasize the complexity of gene regulation in the LD muscle of Nellore cattle associated with REA and BFT. These results can help us to better understand the different molecular processes involved in muscle and fat deposition, which are economically important carcass traits for beef production.

Increased alcohol consumption over time is one of the characteristic symptoms of Alcohol Use Disorder (AUD). The molecular mechanisms underlying this escalation in intake is still the subject of study. However, the mesocortical and mesolimbic dopamine pathways, and the extended amygdala, because of their involvement in reward and reinforcement are believed to play key roles in these behavioral changes. Multiple gene expression studies have shown that alcohol affects the expression of thousands of genes in the brain. The studies discussed in this document use the systems biology technique of co-expression network analysis to attempt to find patterns within genome-wide expression data from two animal models of chronic, high-dose ethanol exposure. These analyses have identified time-dependent and brain-regions specific patterns of expression in C57Bl/6J mice after multiple exposures to intoxicating doses of ethanol and withdrawal. Specifically, they have identified the PFC and HPC as showing long-term ethanol regulation, and identified Let-7 family miRNAs as potential gene expression regulators of chronic ethanol response. Network analysis also indicates neurotransmitter release and neuroimmune response are very correlated to ethanol intake in chronically exposed mice. Examining gene expression response to chronic ethanol exposure across a variable genetic background revealed that, although gene expression response may show conserved patterns, underlying differences in gene expression influence by genetic background may be what truly underlies voluntary ethanol consumption. Finally, combined network analysis of gene expression in the prefrontal cortex (PFC) of mice and macaques following prolonged ethanol exposure demonstrated that neurotransmission, myelination, transcription, cellular respiration, and, possibly, neurovasculature are affected by chronic ethanol across species. Taken together, these studies generate several new hypothesis and areas of future research into the continued study of druggable targets for AUD.

If small isoprenoids, the starting component of carotenoids, can be efficiently excreted from thermophilic cyanobacteria, they could help satisfy the demand for sustainably produced hydrocarbons. This is the driving force behind wanting to understand the response and regulation of isoprenoid pathways to environmental stimuli in the thermophilic cyanobacterium, Thermosynechococcus elongatus, BP-1. The portion of the isoprenoid pathway studied here is the carotenoid pathway since these products are critical to adaptation and they encompass the largest pool of isoprenoid compounds in cyanobacteria. Although synthetic biology in cyanboacteria has improved in recent years, there are many undiscovered metabolic complexities that make large-scale commercial production challenging. To address this need, I quantify and report for the first time metabolic shifts within the carotenoid pathway of BP-1 due to combined effects of temperature, pH and blue light. I show that metabolism shifts from the dicyclic into the monocyclic carotenoid pathway in response to pH, and that decreasing temperature drives flux into the end products of both pathways. Also, I report that the productivity of an uncommon carotenoid, 2-hydroxymyxol 2’-fucoside (HMF), approached 500 μg/L-day in cultures grown at 45 °C, high light intensity, and pH 8. In order to further elucidate these responses, I analyzed 42 RNAseq samples taken over time of BP-1 induced by cold and heat stress and compared these results to metabolomics data. I showed that crtR and crtG, two central carotenogenesis genes, are transcriptionally controlled and used weighted gene co-expression network analysis (WGCNA) to determine eight separate co-expressed modules of biological significance. Among the co-regulated heat response and cold response genes there were three and five non-coding RNA, respectively, providing targets for future investigation. Using subtractive genomics and transcriptional data I narrowed the potential missing steps of the myxol pathway in cyanobacteria to seven unknown BP-1 genes, two of which were confirmed not to be involved in the missing step(s). Finally, by generating a ΔcrtG mutant and testing it under different environmental parameters, I showed that HMF does not protect against high pH or low temperature (despite up-regulation at these conditions), and that CrtG has a higher affinity for monocyclic than dicyclic carotenoids. / text