Small intestinal neuroendocrine tumors (SI-NETs) originate from serotonin-producing enterochromaffin (EC) cells in the intestinal mucosa. Somatostatin analogs (SSAs) are mainly used to control hormonal secretion and tumor growth. However, the molecular mechanisms leading to the control of SI-NETs are unknown. Although microRNAs (miRNAs) are post transcriptional regulators deeply studied in many cancers, are not well-defined in SI-NETs. We adopted a two-pronged strategy to investigate SSAs and miRNAs: first, to provide novel insights into how SSAs control NET cells, and second, to identify an exclusive SI-NET miRNA expression, and investigate the biological functions of miRNA targets. To accomplish the first aim, we treated CNDT2.5 cells with octreotide for 16 months. Affymetrix microarray was performed to study gene variation of CNDT2.5 cells in the presence or absence of octreotide. The study revealed that octreotide induces six genes, ANXA1, ARHGAP18, EMP1, GDF15, TGFBR2 and TNFSF15. To accomplish the second aim, SI-NET tissue specimens were used to run genome-wide Affymetrix miRNA arrays. The expression of five miRNAs (miR-96, -182, -183, -196a and -200a) was significantly upregulated in laser capture microdissected (LCM) tumor cells versus LCM normal EC cells, whereas the expression of four miRNAs (miR-31, -129-5p, -133a and -215) was significantly downregulated in LCM tumor cells. We also detected nine tissue miRNAs in serum samples, showing that the expression of five miRNAs is significantly increased in SSA treated patients versus untreated patients. Conversely, SSAs do not change miRNA expression of four low expressed miRNAs. Silencing miR-196a expression was used to investigate functional activities in NET cells. This experimental approach showed that four miR-196a target genes, HOXA9, HOXB7, LRP4 and RSPO2, are significantly upregulated in silenced miR-196a NET cells. In conclusion, ANXA1, ARHGAP18, EMP1, GDF15, TGFBR2 and TNFSF15 genes might regulate cell growth and differentiation in NET cells, and play a role in an innovative octreotide signaling pathway. The global SI-NET miRNA profiling revealed that nine selected miRNAs might be involved in tumorigenesis, and play a potential role as novel markers for follow-up. Indeed, silencing miR-196a demonstrated that HOXA9, HOXB7, LRP4 and RSPO2 genes are upregulated at both transcriptional and translational levels.

Primary wool follicles are initiated in the skin of sheep foetuses at approximately day 50 of gestation as the result of complex reciprocal molecular interactions between the mesenchyme and overlying epithelium. The lifetime wool production potential and fibre diameter of the Merino sheep is dependent on the total number of follicles initiated in utero. Understanding the molecular events that surround primary wool follicle initiation may provide approaches to enhance or manipulate this process in order to maximise the profitability of wool production enterprises. In order to study the morphological and molecular changes occurring during early wool follicle development, a foetal skin series spanning primary follicle initiation was generated. Foetal skin was sampled from the shoulder, midside and rump of four foetuses at 8 time points between day 43 and day 68 of gestation. Histological characterisation of the shoulder skin samples revealed that primary epidermal placodes emerged at around day 53, dermal condensates were visible from day 57 and downgrowth of the follicle began at day 68. An equation relating age of the foetus (day of gestation post AI) and crown-rump length, specific to Merino foetuses, was developed for use in future studies of this nature. Molecular markers of fibroblast migration, epidermal and dermal stem cells and cell proliferation were selected to test the hypothesis that dermal condensates are initiated at discrete sites beneath the epidermis as a result of a combination of migration and arrangement of multipotent pre-papilla cells. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis of RAC1 and RHOa (migration markers), β1-integrin and alkaline phosphatase (stem cell markers), proliferative nuclear cell antigen and cyclinB1 (proliferation markers), patched-1, selected tumor necrosis factor (TNF) signalling molecules and eleven reference genes was conducted using midside and rump skin samples from each of four foetuses from the 8 time points. geNorm analysis of the reference and target genes revealed that the migration markers RAC1 and RHOa along with GAPDH were the most stably expressed genes in this sample series. Significant changes in mRNA expression were detected for β1-integrin, alkaline phosphatase, patched-1 and the TNF members EDA, EDAR, TROY and TRAF6. Many of these significant differences in expression coincided with key morphological events. Significant differences in expression were also detected between the midside and rump samples for numerous transcripts. Laser capture microdissection (LCM) was implemented for analysis of the target transcripts within particular structures of foetal sheep skin. Frozen tissue sectioning, staining, LCM, RNA extraction and cDNA synthesis were optimised for qRT-PCR analysis of endogenous controls and selected TNF transcripts. Several RNA extraction methods and reverse transcription approaches were trialled to ensure optimum extraction and reverse transcription efficiency for this tissue type. Exogenous mRNA transcripts were also incorporated prior to RNA extraction and reverse transcription to track reaction efficiency between samples. A comparison of different slide types revealed that laser pressure catapulting from membrane slides was an absolute requirement for foetal skin tissue studies. Follicle regions (including the epidermal placode and dermal condensate) and the adjacent non-follicle regions were laser captured from foetal skin, and the mRNA expression levels of patched-1 and selected TNF members was compared. Preliminary qRT-PCR analysis using this technique revealed that EDAR, TROY and PTCH1 mRNA levels were higher in the follicle regions than the non-follicle regions. The TNF signalling pathway appears to play an important role in primary wool follicle initiation and patterning at different sites on the body. Spatial differences in expression of some of these regulators may be involved in initiating different types of follicles. The molecular events surrounding primary wool follicle initiation also show a high degree of conservation between sheep, humans, and mice. Considering the high degree of DNA sequence conservation as well as the histological, signalling and cycling similarities between sheep and humans, sheep may represent a better model for the study of human hair follicle initiation and disease than the currently used mice and rat models. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1523639 / Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 2010

O eucalipto é uma das espécies arbóreas mais plantadas no mundo atualmente, principalmente devido ao seu uso como matéria prima para as indústrias de celulose, papel e madeireira. Eucalyptus globulus e seus híbridos possuem baixos teores de lignina e despertam grande interesse da indústria, já que essa característica facilita e diminui o custo do processo de extração da celulose. Entretanto, essa espécie é recalcitrante ao enraizamento adventício, o que dificulta a propagação vegetativa de suas mudas. Com o objetivo de melhor entender os mecanismos moleculares envolvidos na recalcitrância ao enraizamento em E. globulus, o presente estudo envolveu análises de parâmetros morfológicos, anatômicos e moleculares durante a rizogênese adventícia nesta espécie. A exposição à auxina exógena reverteu o fenótipo recalcitrante em E. globulus, aumentando significativamente a porcentagem de enraizamento. O câmbio vascular foi identificado como uma região de acúmulo de auxina e local de origem das raízes adventícias. Através de análises da expressão gênica em células do câmbio, observou-se que TOPLESS e IAA12, repressores da sinalização de auxina, e ARR1, envolvido na rota de sinalização de citocininas, parecem atuar como reguladores negativos do enraizamento adventício. A alta expressão destes genes em plantas controle foi significativamente diminuída com aplicação de auxina exógena. Comparativamente, em espécie de fácil enraizamento, E. grandis, a expressão destes genes se manteve em níveis mais baixos em ambas as condições de tratamento, e a concentração de ácido indol-3-acético endógeno em plantas controle mostrou-se mais elevada. Análises do padrão proteico durante o enraizamento em plantas de E. globulus tratadas ou não com auxina exógena identificaram proteínas envolvidas em diversos processos biológicos, principalmente estresse oxidativo e metabolismo energético. Diferenças interessantes foram identificadas ao comparar as diferentes condições e fases do enraizamento. Várias proteínas foram claramente relacionadas com o respectivo fenótipo apresentado pela planta em cada situação, principalmente considerando plantas controle. Os resultados aqui apresentados representam avanços relevantes no conhecimento sobre a rizogênese adventícia em plantas lenhosas, podendo ser utilizados como ferramentas no desenho de estratégias visando melhorar o enraizamento em genótipos recalcitrantes de valor para a indústria. / Eucalyptus is one of the most planted tree species in the world today, mainly due to its use as raw material for paper, cellulose and wood industries. Eucalyptus globulus and its hybrids have low lignin contents and are of great interest to industry, as this feature facilitates and reduces costs of the cellulose extraction process. However, this species is recalcitrant to adventitious rooting, making vegetative propagation by cuttings difficult. Aiming at a better understanding of the molecular mechanisms involved in rooting recalcitrance in E. globulus, this study analyzed changes in morphological, anatomical and molecular patterns during adventitious rooting in this species. Exogenous auxin exposure reversed the recalcitrant phenotype in E. globulus, significantly increasing rooting percentage. The vascular cambium was identified as a region of auxin accumulation and also the site from where adventitious roots originated. Gene expression analysis in cambium cells indicated that TOPLESS and IAA12, auxin signaling repressors, and ARR1, involved in cytokinin signaling pathway, appear to act as negative regulators of adventitious rooting. The high expression of those genes in control plants was significantly decreased by exogenous auxin treatment. Comparatively, in an easy-to-root species, E. grandis, the expression of these genes was significantlylower in both treatment conditions, and the concentration of endogenous indole- 3-acetic acid in control plants was higher. Analysis of the protein pattern during rooting in E. globulus plants treated or not with exogenous auxin allowed the identification of proteins involved in diverse biological processes, mainly oxidative stress and energy metabolism. Interesting differences were identified when comparing different rooting conditions or phases. Several proteins were clearly associated with the respective plant phenotype in each situation, particularly considering control plants. These results represent relevant advances in the knowledge about adventitious rooting in woody plants and can be used as tools in the design of strategies aiming at improving adventitious rooting in recalcitrant genotypes of industrial value.

O eucalipto é uma das espécies arbóreas mais plantadas no mundo atualmente, principalmente devido ao seu uso como matéria prima para as indústrias de celulose, papel e madeireira. Eucalyptus globulus e seus híbridos possuem baixos teores de lignina e despertam grande interesse da indústria, já que essa característica facilita e diminui o custo do processo de extração da celulose. Entretanto, essa espécie é recalcitrante ao enraizamento adventício, o que dificulta a propagação vegetativa de suas mudas. Com o objetivo de melhor entender os mecanismos moleculares envolvidos na recalcitrância ao enraizamento em E. globulus, o presente estudo envolveu análises de parâmetros morfológicos, anatômicos e moleculares durante a rizogênese adventícia nesta espécie. A exposição à auxina exógena reverteu o fenótipo recalcitrante em E. globulus, aumentando significativamente a porcentagem de enraizamento. O câmbio vascular foi identificado como uma região de acúmulo de auxina e local de origem das raízes adventícias. Através de análises da expressão gênica em células do câmbio, observou-se que TOPLESS e IAA12, repressores da sinalização de auxina, e ARR1, envolvido na rota de sinalização de citocininas, parecem atuar como reguladores negativos do enraizamento adventício. A alta expressão destes genes em plantas controle foi significativamente diminuída com aplicação de auxina exógena. Comparativamente, em espécie de fácil enraizamento, E. grandis, a expressão destes genes se manteve em níveis mais baixos em ambas as condições de tratamento, e a concentração de ácido indol-3-acético endógeno em plantas controle mostrou-se mais elevada. Análises do padrão proteico durante o enraizamento em plantas de E. globulus tratadas ou não com auxina exógena identificaram proteínas envolvidas em diversos processos biológicos, principalmente estresse oxidativo e metabolismo energético. Diferenças interessantes foram identificadas ao comparar as diferentes condições e fases do enraizamento. Várias proteínas foram claramente relacionadas com o respectivo fenótipo apresentado pela planta em cada situação, principalmente considerando plantas controle. Os resultados aqui apresentados representam avanços relevantes no conhecimento sobre a rizogênese adventícia em plantas lenhosas, podendo ser utilizados como ferramentas no desenho de estratégias visando melhorar o enraizamento em genótipos recalcitrantes de valor para a indústria. / Eucalyptus is one of the most planted tree species in the world today, mainly due to its use as raw material for paper, cellulose and wood industries. Eucalyptus globulus and its hybrids have low lignin contents and are of great interest to industry, as this feature facilitates and reduces costs of the cellulose extraction process. However, this species is recalcitrant to adventitious rooting, making vegetative propagation by cuttings difficult. Aiming at a better understanding of the molecular mechanisms involved in rooting recalcitrance in E. globulus, this study analyzed changes in morphological, anatomical and molecular patterns during adventitious rooting in this species. Exogenous auxin exposure reversed the recalcitrant phenotype in E. globulus, significantly increasing rooting percentage. The vascular cambium was identified as a region of auxin accumulation and also the site from where adventitious roots originated. Gene expression analysis in cambium cells indicated that TOPLESS and IAA12, auxin signaling repressors, and ARR1, involved in cytokinin signaling pathway, appear to act as negative regulators of adventitious rooting. The high expression of those genes in control plants was significantly decreased by exogenous auxin treatment. Comparatively, in an easy-to-root species, E. grandis, the expression of these genes was significantlylower in both treatment conditions, and the concentration of endogenous indole- 3-acetic acid in control plants was higher. Analysis of the protein pattern during rooting in E. globulus plants treated or not with exogenous auxin allowed the identification of proteins involved in diverse biological processes, mainly oxidative stress and energy metabolism. Interesting differences were identified when comparing different rooting conditions or phases. Several proteins were clearly associated with the respective plant phenotype in each situation, particularly considering control plants. These results represent relevant advances in the knowledge about adventitious rooting in woody plants and can be used as tools in the design of strategies aiming at improving adventitious rooting in recalcitrant genotypes of industrial value.

O eucalipto é uma das espécies arbóreas mais plantadas no mundo atualmente, principalmente devido ao seu uso como matéria prima para as indústrias de celulose, papel e madeireira. Eucalyptus globulus e seus híbridos possuem baixos teores de lignina e despertam grande interesse da indústria, já que essa característica facilita e diminui o custo do processo de extração da celulose. Entretanto, essa espécie é recalcitrante ao enraizamento adventício, o que dificulta a propagação vegetativa de suas mudas. Com o objetivo de melhor entender os mecanismos moleculares envolvidos na recalcitrância ao enraizamento em E. globulus, o presente estudo envolveu análises de parâmetros morfológicos, anatômicos e moleculares durante a rizogênese adventícia nesta espécie. A exposição à auxina exógena reverteu o fenótipo recalcitrante em E. globulus, aumentando significativamente a porcentagem de enraizamento. O câmbio vascular foi identificado como uma região de acúmulo de auxina e local de origem das raízes adventícias. Através de análises da expressão gênica em células do câmbio, observou-se que TOPLESS e IAA12, repressores da sinalização de auxina, e ARR1, envolvido na rota de sinalização de citocininas, parecem atuar como reguladores negativos do enraizamento adventício. A alta expressão destes genes em plantas controle foi significativamente diminuída com aplicação de auxina exógena. Comparativamente, em espécie de fácil enraizamento, E. grandis, a expressão destes genes se manteve em níveis mais baixos em ambas as condições de tratamento, e a concentração de ácido indol-3-acético endógeno em plantas controle mostrou-se mais elevada. Análises do padrão proteico durante o enraizamento em plantas de E. globulus tratadas ou não com auxina exógena identificaram proteínas envolvidas em diversos processos biológicos, principalmente estresse oxidativo e metabolismo energético. Diferenças interessantes foram identificadas ao comparar as diferentes condições e fases do enraizamento. Várias proteínas foram claramente relacionadas com o respectivo fenótipo apresentado pela planta em cada situação, principalmente considerando plantas controle. Os resultados aqui apresentados representam avanços relevantes no conhecimento sobre a rizogênese adventícia em plantas lenhosas, podendo ser utilizados como ferramentas no desenho de estratégias visando melhorar o enraizamento em genótipos recalcitrantes de valor para a indústria. / Eucalyptus is one of the most planted tree species in the world today, mainly due to its use as raw material for paper, cellulose and wood industries. Eucalyptus globulus and its hybrids have low lignin contents and are of great interest to industry, as this feature facilitates and reduces costs of the cellulose extraction process. However, this species is recalcitrant to adventitious rooting, making vegetative propagation by cuttings difficult. Aiming at a better understanding of the molecular mechanisms involved in rooting recalcitrance in E. globulus, this study analyzed changes in morphological, anatomical and molecular patterns during adventitious rooting in this species. Exogenous auxin exposure reversed the recalcitrant phenotype in E. globulus, significantly increasing rooting percentage. The vascular cambium was identified as a region of auxin accumulation and also the site from where adventitious roots originated. Gene expression analysis in cambium cells indicated that TOPLESS and IAA12, auxin signaling repressors, and ARR1, involved in cytokinin signaling pathway, appear to act as negative regulators of adventitious rooting. The high expression of those genes in control plants was significantly decreased by exogenous auxin treatment. Comparatively, in an easy-to-root species, E. grandis, the expression of these genes was significantlylower in both treatment conditions, and the concentration of endogenous indole- 3-acetic acid in control plants was higher. Analysis of the protein pattern during rooting in E. globulus plants treated or not with exogenous auxin allowed the identification of proteins involved in diverse biological processes, mainly oxidative stress and energy metabolism. Interesting differences were identified when comparing different rooting conditions or phases. Several proteins were clearly associated with the respective plant phenotype in each situation, particularly considering control plants. These results represent relevant advances in the knowledge about adventitious rooting in woody plants and can be used as tools in the design of strategies aiming at improving adventitious rooting in recalcitrant genotypes of industrial value.

Atualmente observa-se que a proporção de pessoas com 60 anos ou mais está crescendo mais rápido do que a de outras faixas etárias. Um dos resultados desta transição epidemiológica é o aumento das doenças cujo fator de risco é o envelhecimento, entre elas, a doença de Parkinson. Embora muitas regiões exibam os sinais neuropatológicos da doença de Parkinson, a degeneração dos neurônios, contendo neuromelanina, da substância negra é considerada como sendo uma característica importante, representando o critério cardinal para o diagnóstico. No entanto, ainda não está claro por que certas regiões do cérebro, como a substância negra, são vulneráveis em algumas doenças neurodegenerativas e alguns neurônios vizinhos, às vezes morfologicamente indistinguíveis, permanecem preservados. Análises moleculares de populações de neurônios podem conduzir a uma melhor compreensão sobre a fisiologia dos mesmos, bem como os mecanismos envolvidos nos processos de doença. Na era pós genômica, realizar análises proteômicas são de grande interesse científico, pois permitem avanços no conhecimento dos processos biológicos. A técnica de microdissecção e captura a laser tem sido uma ferramenta importante e cada vez mais utilizada para aquisição de populações puras de células a partir de secções histológicas, evitando que áreas não pertencentes ao tecido alvo sejam dissecadas. A união destes métodos pode contribuir de maneira relevante para o entendimento fisiopatológico dos neurônios contendo neuromelanina da substância negra. No entanto, para que a microdissecção e captura a laser e as análises proteômicas sejam eficazes, é imprescindível a aplicação de um protocolo bem estruturado. Dentro desse contexto, o presente trabalho tem como objetivo criar um protocolo de microdissecção a laser de neurônios contendo neuromelanina em indivíduos cognitivamente normais, para subsequente análise proteômica. Os casos utilizados neste estudo são provenientes do Banco de Encéfalos Humanos do Grupo de Estudos em Envelhecimento Cerebral. Para o desenvolvimento da nossa proposta, contamos com a colaboração do Centro de Proteômica Médica da Universidade de Bochum, Alemanha. O protocolo foi desenvolvido baseado em outros previamente descritos na literatura e otimizado de acordo com objetivos pretendidos. Analisamos o plano anatômico de amostragem do tecido, o método de congelamento, a espessura do corte para a microdissecção, a solução utilizada para a coleta do tecido durante a microdissecção e o método de digestão proteolítica para posteriores análises proteômicas. Através de ensaios comparativos, alcançamos os resultados desejados e os mesmos foram validados através de análises por espectrometria de massas. Consequentemente, também fomos capazes de reconhecer fatores técnicos que possivelmente impossibilitariam um efetivo estudo do proteoma / Currently the worldwide proportion of people aged 60 years and over is growing faster than any other age group. This strikingly epidemiological transition results in an increase of aging related diseases, including Parkinson\'s disease (PD). Although many brain areas exhibit the neuropathological signs of Parkinson\'s disease, the degeneration of neuromelanin containing cells in the substantia nigra is considered a hallmark feature, representing cardinal diagnostic criteria for PD. However, why certain brain regions -- such as the substantia nigra -- are vulnerable in some neurodegenerative diseases, while some neighboring morphologically indistinguishable neurons remain preserved, is still unclear. Molecular analysis of specific neuronal populations can lead us to a better understanding about the physiological role played by these neurons and mechanisms involved in disease\'s processes. In a post-genomic era, proteomic analyses are of great scientific interest since they allow a better understanding of the biological processes. The laser capture microdissection technique has also became an important tool in biological research, being increasingly used for acquisition of pure populations of cells from histological sections, preventing the dissection of areas outside the target tissue. The combination of these methods can significantly contribute to understand the pathophysiological role of the containing neuromelanin neurons of the substantia nigra. However, for an effective application of both techniques, laser capture microdissection and proteomic analysis, it is essential the application of an efficient protocol. In this context, this study aims to establish a protocol for laser microdissection of containing neuromelanin neurons in cognitively normal individuals for subsequent proteomic analyses. We selected cases from the Brain Bank of the Brazilian Aging Brain Study. A collaboration with the Medical Proteome Center, University of Bochum, Germany took part during the development of our proposal. Our protocol was developed based on previous published protocols and optimized according the intended aims. We analyzed anatomical planes for neuronal collection, freezing methods, thickness of tissue for microdissection sections, solution for tissue collection during laser microdissection and the proteolytic digestion methods. Through our comparative tests, we have achieved the desired results and validated them by mass spectrometry analyses. Consequently, we were also able to exclude technical factors that could possibly preclude one effective proteome analysis

Autism spectrum disorder (ASD) is a neurodevelopmental disorder affecting social behaviors. ASD affects 1 in 59 children with males affected more frequently. ASD is postulated to result from excitatory and inhibitory neurotransmission imbalances. Brain-derived neurotrophic factor (BDNF) signaling affects ASD by influencing synaptogenesis, plasticity, and survival. Studying early in-utero neuropathological changes within ASD requires the use of animal models. Expression of BDNF-associated genes were analyzed within laser capture microdissected pyramidal neurons from the anterior cingulate cortex of male and female BTBR and valproic acid mouse models. No expression differences were found in any gene comparing the three groups. Gender comparisons did identify differences in NTRK2 and EFNB2. Significant correlations of gene expression were identified for male NTRK2 with EFNB2 and GRIN1 and EFNB2 with GRIN1 and female BDNF with GRIN1 expressions (p

No description available.

Lung cancer is the leading cause of cancer deaths worldwide due to the late stage of diagnosis as most patients do not present symptoms until late-stage disease. The two most common forms of non-small cell lung cancer are adenocarcinoma followed by squamous cell carcinoma, which differ in location, appearance and molecular phenotypes and require different approaches for treatment. Prior to the development of invasive lung cancer, premalignant lesions, or regions of abnormal cellular architecture, are often present in the lung. Understanding the pathologic heterogeneity and cellular and molecular alterations in premalignant lesions that precede or are adjacent to invasive carcinomas may help identify the earliest changes in lung carcinogenesis that could be used to identify biomarkers of progression and targets for intervention. Previous work by our group profiling endobronchial biopsies by bulk mRNA sequencing identified gene expression alterations associated with high grade squamous premalignant lesion histology and progression towards advanced histology. Molecular profiling combined with careful pathologic assessment of premalignant lesions are important steps in uncovering the biological processes that are dysregulated in premalignant lesions that progress; however, lung tissue is often limited and fixed. Biopsies of lung tissue, obtained using forceps or needles, are an important diagnostic and prognostic clinical tool although, only tiny amounts of tissue may be left for research. As a result, it is important to work closely with pathologists to review clinical pathology samples and optimize methodologies to use the limited biospecimens available to study these early changes associated with the development of lung cancer. My thesis work focused on the detailed pathologic annotation of lung premalignant lesion biopsies and whole tumor samples and the development of methodologies to profile these small biospecimens to advance lung cancer interception research. Lung squamous cell carcinoma is thought to originate from bronchial premalignant lesions that progress through a series of histological grades to dysplasia (mild, moderate, and severe), carcinoma in situ, and invasive carcinoma. Endobronchial biopsies of these premalignant lesions have heterogeneous pathologic features, and it is not known which features are associated with progression to cancer. In Aim 1 of my thesis, in collaboration with thoracic pathologists, whole slide biopsy images were annotated to identify the histologic grades of surface epithelium, including the presence of angiogenic squamous dysplasia, and stromal features such as the presence of a fibrotic basal membrane under the epithelium. Analyzing these annotations across 284 biopsies identified a significant association between former smokers and the presence of a fibrotic basal membrane. Additionally, we found that when exposed to asbestos, approximately two thirds of patients developed angiogenic dysplasia, while those who were never exposed had a 50% chance of developing it. We also observed a reserve cell dysplasia-like pattern characterized by its nuclear uniformity through all layers, increased nucleus to cytoplasm ratio, hyperchromia, and presence of cilia. This type of dysplasia is not described in the lung to date but is well described in the cervix as a transitional subtype between reserve cell hyperplasia and squamous dysplasia. We quantified the percentage of each histologic grade present in the epithelium of each biopsy and found that there was a weak, but significant, negative correlation between the percentage of normal epithelium and reserve cell dysplasia-like epithelium. The results suggest that reserve cell dysplasia in the lung may be worthy of more study, as it is found to be only inversely correlated with normal epithelium. More studies must be done to elucidate its true role in the premalignant to malignant process. To complement the pathologic analysis of endobronchial biopsies in Aim 2, we examined 31 lung cancer resection cases where premalignant lesions were present in the tumor margins. Our goal was to profile the RNA and DNA of multiple regions within each case to identify molecular alterations associated with the transition from premalignant to tumor tissue. The 31 cases were stained with H&E and mIHC panels using markers corresponding to epithelial, immune, and stromal cell types. We annotated the H&E stains alone and together with the mIHC stains and found that we annotated significantly more unique regions when considering both data modalities. We captured the annotated regions using an optimized laser capture microdissection (LCM) protocol. The goal of Aim 3 was to first optimize the LCM and isolation protocols for limited FFPE lung samples. We accomplished this using a smaller group of eight samples, of which six were LCM’d, by comparing different DNA and RNA isolation kits to select the kit that offered the highest quality and greatest amount of isolated DNA and RNA. The LCM tissue from the 31 cases is currently undergoing RNA and DNA sequencing and we hope the data analysis will identify unique tissue microenvironments associated with premalignant lesion progression. These findings contribute to existing lung cancer and premalignancy research, which, as a field, aims to identify progressing lesions and treat patients at earlier stages to decrease mortality. My thesis work has focused on characterizing the pathologic features of lung squamous premalignant lung lesions and their associations with premalignant progression to cancer and other clinical covariates such as smoking status and carcinogenic exposures. To fully understand these pathologic features, I have developed methods to isolate high quality RNA and DNA from these limited biospecimens to allow for the identification of the molecular alterations underpinning the pathological changes.

Muscle fiber composition correlates with insulin resistance, and exercise training can increase slow-twitch (type I) fibers and, thereby, mitigate diabetes risk. Human skeletal muscle is made up of three distinct fiber types, but muscle contains many more isoforms of myosin heavy and light chains, which are coded by 15 and 11 different genes, respectively. Laser capture microdissection techniques allow assessment of mRNA and protein content in individual fibers. We found that specific human fiber types contain different mixtures of myosin heavy and light chains. Fast-twitch (type IIx) fibers consistently contained myosin heavy chains 1, 2, and 4 and myosin light chain 1. Type I fibers always contained myosin heavy chains 6 and 7 (MYH6 and MYH7) and myosin light chain 3 (MYL3), whereas MYH6, MYH7, and MYL3 were nearly absent from type IIx fibers. In contrast to cardiomyocytes, where MYH6 (also known as α-myosin heavy chain) is seen solely in fast-twitch cells, only slow-twitch fibers of skeletal muscle contained MYH6. Classical fast myosin heavy chains (MHC1, MHC2, and MHC4) were present in variable proportions in all fiber types, but significant MYH6 and MYH7 expression indicated slow-twitch phenotype, and the absence of these two isoforms determined a fast-twitch phenotype. The mixed myosin heavy and light chain content of type IIa fibers was consistent with its role as a transition between fast and slow phenotypes. These new observations suggest that the presence or absence of MYH6 and MYH7 proteins dictates the slow- or fast-twitch phenotype in skeletal muscle. The technical challenges of human skeletal muscle fiber type identification have evolved over the past three decades (8). The typical normal adult has roughly equal amounts of slow- and fast-twitch fibers, designated type I and II fibers. In addition, a variable portion of the type II fibers is mixed, containing both fast- and slow-twitch fiber markers, called type IIa fibers, whereas type II fibers that contain only the fast-twitch phenotype are designated type IIx in humans. Exercise training can cause modest shifts in fiber composition from one of these types to a contiguous type, with the relationship being type I to IIa to IIx or type IIx to IIa to I. The tail end of each myosin heavy chain is attached to the tail of another myosin heavy chain, and each of these forms a complex with two myosin light chains. Many heavy and light chain complexes are intertwined to form the thick filaments of each sarcomere. Thin filaments are composed of actin, troponin, and tropomyosin. The myosin heavy chains contain ATPase, which is essential for shortening of the contractile apparatus in the sarcomere, resulting in muscle-generated movement of a body part. The pH optimum of the ATPase has been classically the histochemical technique for identifying fast, slow, and mixed fibers. However, for more than a decade, monoclonal antibodies that correlated with the ATPase designation of fast, slow, and mixed fibers by bright-field or immunohistochemical methods have been used (2). The widely used fast and slow myosin monoclonal antibodies were obtained from mice immunized with only partially purified human skeletal muscle myosin antigens. More recently, antibodies that were raised against specific individual myosin heavy and light chain proteins became commercially available. The 15 human genes that code myosin heavy chains are designated MYH1, MYH2, MYH3, MYH4, MYH6, MYH7, MYH7B, MYH8, MYH9, MYH10, MYH11, MYH12, MYH13, MYH14, MYH15, and MYH16 (17). MYH9, MYH10, and MYH11 are expressed primarily in smooth muscle. At least eight separate genes that code myosin light chains, MYL1, MYL2, MYL3, MYL4, MYL5, MYL6, MYL6B, and MYLPF, have been identified, and at least three of these have a second isoform (3). Our initial investigation of the expression of myosin heavy and light chains using laser capture microdissection (LCM) to obtain specific fiber type samples from human vastus lateralis biopsies yielded some unexpected results. These observations led us to question which isoforms of myosin heavy and light chains are actually characteristic of “fast” or “slow” fibers in human skeletal muscle. We used immunoblots, mass spectroscopic (MS) proteomics, and next-generation sequencing of muscle homogenates and of LCM-generated samples of individual fiber types from normal control subjects and subjects with extremely different muscle fiber composition to approach this question by evaluating muscle specimens from subjects with diverse and extremely different fiber compositions. The hypothesis that drove these studies was that fibers of each type would have consistent myosin heavy and light chains that are characteristic of the fiber type. This is the first report that the abundance of different myosin heavy and light chains corresponds to different muscle fiber types.