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.

The cyanobacterial neurotoxin β-N-methylamino-L-alanine (BMAA), a non-protein amino acid, first attracted attention in correlation to reports of high incidence of the unusual neurological disease amyotrophic lateral sclerosis/Parkinsonism-dementia (ALS/PDC) among the people of Guam in the South Pacific Ocean. Experimental studies have revealed that BMAA causes neuronal cell death. The neurotoxin is suggested to act via excitotoxicity through interaction with glutamatergic receptors. More importantly, BMAA is suggested to misincorporate in the synthesis of proteins, and contribute to protein misfolding and/or deleterious aggregation, which are hallmarks of several neurodegenerative disorders. A selective uptake of BMAA in the rat neonatal hippocampus can interfere with brain development, causing learning and memory impairments in adult rats. The aim of the present study was to investigate the effects of BMAA in human neuroblastoma SH-SY5Y cells. These cells were exposed to BMAA (10 μM, 50 μM, 100 μM or 500 μM) for 72 hours, and the expression of five selected proteins, including heat shock protein-27 (HSP-27), lysosomal associated membrane protein-1 (LAMP-1), CCAAT-enhancer-binding protein homologous protein (CHOP), Golgi associated plant pathogenesis related protein-2 (GLIPR-2), and glucose regulated protein-78 (GRP-78). They were carried out with immunohistochemistry (IHC). Results revealed an increased expression of all selected proteins, which indicates an uptake and shows the effects of BMAA in the cell cultures. Taken together, BMAA caused cellular stress, including endoplasmic reticulum (ER) stress that is correlated with HSP-27, LAMP-1, CHOP, GLIPR-2, and GRP-78. Further studies are needed in order to support the results. The experiments require being repeated using the same biomarkers as well as a combination of them with other biomarkers to elucidate the effects of BMAA.

A raiva é uma zoonose quase sempre fatal, que causa milhares de mortes humanas por ano em todo mundo. Essa enfermidade manifesta-se nos mamíferos em duas formas clínicas: furiosa e paralítica. Distinções em relação à evolução, manifestações clínicas, lesões, distribuição e respectiva carga viral no Sistema Nervoso Central (SNC) podem estar relacionadas às características de neuroinvasividade e neuropatogenicidade das diferentes variantes do vírus da raiva (RABV). O objetivo deste projeto foi estabelecer um modelo para o estudo da patogênese da raiva em camundongos inoculados com vírus fixo CVS/31 e variantes de rua do RABV originárias de bovino (variante 3 compatível com isolados de morcegos hematófagos) e de canídeo silvestre (variante compatível com isolados de canídeo silvestre). Para estabelecer o modelo murino, 24 camundongos isogênicos da linhagem BALB/c, de três semanas de idade foram inoculados com 103 DLIC50/0,03mL por via intracerebral (IC) para confirmar a neurovirulência das diferentes variantes do RABV. De modo concomitante, 32 camundongos desta mesma linhagem e faixa etária, foram inoculados com 105 DLIC50/0,03mL pela via coxim plantar (CP) com o intuito de mimetizar a progressão da infecção natural. Para o estudo da patogênese do RABV foram analisados durante um período de trinta dias após a inoculação (DPI) em camundongos pela via CP os seguintes parâmetros: manifestações clínicas, alterações histopatológicas, distribuição antigênica viral pela técnica de imuno-histoquímica (IHQ) e distribuição de RNA viral pela técnica da reação em cadeia da polimerase em tempo real precedida pela transcrição reversa (RT-qPCR) - sistema SYBR Green em diversos segmentos do SNC. Todos os camundongos inoculados com as três amostras do RABV apresentaram sintomas compatíveis com a forma paralítica da doença, tais como: piloereção, perda de peso, postura arqueada, prostração e paresia dos membros posteriores. Apesar de não ser possível observar diferenças de neuropatogenicidade entre as variantes virais, detectaram-se diversidade de virulência entre essas estirpes, demonstrado pelas distinções de período de incubação e taxa de letalidade. Ao analisar os resultados, também foi possível observar diferenças entre a neurovirulência e a neuroinvasividade das diferentes variantes do RABV. Nos camundongos inoculados com CVS/31 pela via CP, no 6º DPI, observaram-se predomínio de manguitos perivasculares na medula espinhal e degeneração neuronal em córtex e intensa distribuição antigênica de RABV no tronco encefálico. Nos camundongos inoculados via CP com amostras do RABV originárias de bovino, no 8º DPI, apresentaram moderada distribuição de manguitos perivasculares na medula e córtex e intensa distribuição antigênica no tálamo. Já nos animais inoculados com a amostra de canídeo silvestre, no 9ºDPI, relataram-se moderada distribuição de manguitos perivasculares no tronco encefálico e moderada distribuição antigênica no córtex. Além disso, foi observado discrepâncias na comparação entre a intensidade e distribuição de marcações antigênicas pela IHQ e inferência semi-quantitativa de distribuição de RNA viral analisada pelo RT-qPCR-sistema SYBR Green no SNC de camundongos / Rabies is a zoonotic disease usually fatal, causing thousands of human deaths each year worldwide. This disease manifests itself in mammals in two clinical forms: furious and paralytic. Distinctions regarding the evolution, clinical manifestations, injuries, distribution and their viral load on the central nervous system (CNS) may be related to the characteristics of neuroinvasiveness and neuropathogenicity of different variants of rabies virus (RABV). The objective of this project was to establish a model to study the pathogenesis of rabies in mice inoculated with fixed virus CVS / 31 and RABV Street variants originating from bovine (variant 3 - compatible with isolates from vampire bats) and wild canid (variant supports isolated from wild canid). To establish the mouse model, 24 mice of the inbred strain BALB/c, three weeks old were inoculated with 103DLIC50/0,03mL intracerebrally (IC) to confirm the neurovirulence of the different variants of RABV. Concomitantly, 32 mice of the same lineage and age were inoculated with 105DLIC50/0,03mL via footpad (CP) in order to mimic the natural progression of the infection. To study the pathogenesis of RABV CP the following parameters were analyzed over a period of thirty days after inoculation (DPI) in mice via CP: clinical, histopathological changes, viral antigen distribution by the technique of immunohistochemistry (IHC) and distribution of RNA by real-time polymerase chain reaction technique preceded by reverse transcription (RT-qPCR) - SYBR Green system in various segments of the CNS. All mice inoculated with the three RABV samples showed symptoms consistent with the paralytic form of the disease, such as ruffled fur, weight loss, hunched, prostration and paresis of the hind limbs. Although it is not possible to observe neuropathogenicity differences between viral variants, the virulence diversity is detected between these strains demonstrated by distinctions incubation period and fatality rate. When analyzing the results, it was also possible to observe differences between neurovirulence and neuroinvasiveness of different variants of RABV. In mice inoculated with CVS/31 via CP on the 6th DPI, there were predominance of perivascular cuffing in spinal cord and neuronal degeneration in cortex and intense antigenic distribution of the RABV in the brainstem. Mice inoculated via CP with RABV samples originating from bovine on the 8thDPI had moderate distribution of perivascular cuffing in the medulla and cortex and intense antigenic distribution in the thalamus. The animals inoculated with the sample of wild canid in 9thDPI reported a moderate perivascular cuffing distribution in the brainstem and moderate antigenic distribution in the cortex. Additionally, discrepancies were observed when comparing the intensity and distribution of antigenic tags by IHC, semi-quantitative inference viral RNA distribution analyzed by RT-qPCR using SYBR Green system in the mouse CNS

A raiva é uma zoonose quase sempre fatal, que causa milhares de mortes humanas por ano em todo mundo. Essa enfermidade manifesta-se nos mamíferos em duas formas clínicas: furiosa e paralítica. Distinções em relação à evolução, manifestações clínicas, lesões, distribuição e respectiva carga viral no Sistema Nervoso Central (SNC) podem estar relacionadas às características de neuroinvasividade e neuropatogenicidade das diferentes variantes do vírus da raiva (RABV). O objetivo deste projeto foi estabelecer um modelo para o estudo da patogênese da raiva em camundongos inoculados com vírus fixo CVS/31 e variantes de rua do RABV originárias de bovino (variante 3 compatível com isolados de morcegos hematófagos) e de canídeo silvestre (variante compatível com isolados de canídeo silvestre). Para estabelecer o modelo murino, 24 camundongos isogênicos da linhagem BALB/c, de três semanas de idade foram inoculados com 103 DLIC50/0,03mL por via intracerebral (IC) para confirmar a neurovirulência das diferentes variantes do RABV. De modo concomitante, 32 camundongos desta mesma linhagem e faixa etária, foram inoculados com 105 DLIC50/0,03mL pela via coxim plantar (CP) com o intuito de mimetizar a progressão da infecção natural. Para o estudo da patogênese do RABV foram analisados durante um período de trinta dias após a inoculação (DPI) em camundongos pela via CP os seguintes parâmetros: manifestações clínicas, alterações histopatológicas, distribuição antigênica viral pela técnica de imuno-histoquímica (IHQ) e distribuição de RNA viral pela técnica da reação em cadeia da polimerase em tempo real precedida pela transcrição reversa (RT-qPCR) - sistema SYBR Green em diversos segmentos do SNC. Todos os camundongos inoculados com as três amostras do RABV apresentaram sintomas compatíveis com a forma paralítica da doença, tais como: piloereção, perda de peso, postura arqueada, prostração e paresia dos membros posteriores. Apesar de não ser possível observar diferenças de neuropatogenicidade entre as variantes virais, detectaram-se diversidade de virulência entre essas estirpes, demonstrado pelas distinções de período de incubação e taxa de letalidade. Ao analisar os resultados, também foi possível observar diferenças entre a neurovirulência e a neuroinvasividade das diferentes variantes do RABV. Nos camundongos inoculados com CVS/31 pela via CP, no 6º DPI, observaram-se predomínio de manguitos perivasculares na medula espinhal e degeneração neuronal em córtex e intensa distribuição antigênica de RABV no tronco encefálico. Nos camundongos inoculados via CP com amostras do RABV originárias de bovino, no 8º DPI, apresentaram moderada distribuição de manguitos perivasculares na medula e córtex e intensa distribuição antigênica no tálamo. Já nos animais inoculados com a amostra de canídeo silvestre, no 9ºDPI, relataram-se moderada distribuição de manguitos perivasculares no tronco encefálico e moderada distribuição antigênica no córtex. Além disso, foi observado discrepâncias na comparação entre a intensidade e distribuição de marcações antigênicas pela IHQ e inferência semi-quantitativa de distribuição de RNA viral analisada pelo RT-qPCR-sistema SYBR Green no SNC de camundongos / Rabies is a zoonotic disease usually fatal, causing thousands of human deaths each year worldwide. This disease manifests itself in mammals in two clinical forms: furious and paralytic. Distinctions regarding the evolution, clinical manifestations, injuries, distribution and their viral load on the central nervous system (CNS) may be related to the characteristics of neuroinvasiveness and neuropathogenicity of different variants of rabies virus (RABV). The objective of this project was to establish a model to study the pathogenesis of rabies in mice inoculated with fixed virus CVS / 31 and RABV Street variants originating from bovine (variant 3 - compatible with isolates from vampire bats) and wild canid (variant supports isolated from wild canid). To establish the mouse model, 24 mice of the inbred strain BALB/c, three weeks old were inoculated with 103DLIC50/0,03mL intracerebrally (IC) to confirm the neurovirulence of the different variants of RABV. Concomitantly, 32 mice of the same lineage and age were inoculated with 105DLIC50/0,03mL via footpad (CP) in order to mimic the natural progression of the infection. To study the pathogenesis of RABV CP the following parameters were analyzed over a period of thirty days after inoculation (DPI) in mice via CP: clinical, histopathological changes, viral antigen distribution by the technique of immunohistochemistry (IHC) and distribution of RNA by real-time polymerase chain reaction technique preceded by reverse transcription (RT-qPCR) - SYBR Green system in various segments of the CNS. All mice inoculated with the three RABV samples showed symptoms consistent with the paralytic form of the disease, such as ruffled fur, weight loss, hunched, prostration and paresis of the hind limbs. Although it is not possible to observe neuropathogenicity differences between viral variants, the virulence diversity is detected between these strains demonstrated by distinctions incubation period and fatality rate. When analyzing the results, it was also possible to observe differences between neurovirulence and neuroinvasiveness of different variants of RABV. In mice inoculated with CVS/31 via CP on the 6th DPI, there were predominance of perivascular cuffing in spinal cord and neuronal degeneration in cortex and intense antigenic distribution of the RABV in the brainstem. Mice inoculated via CP with RABV samples originating from bovine on the 8thDPI had moderate distribution of perivascular cuffing in the medulla and cortex and intense antigenic distribution in the thalamus. The animals inoculated with the sample of wild canid in 9thDPI reported a moderate perivascular cuffing distribution in the brainstem and moderate antigenic distribution in the cortex. Additionally, discrepancies were observed when comparing the intensity and distribution of antigenic tags by IHC, semi-quantitative inference viral RNA distribution analyzed by RT-qPCR using SYBR Green system in the mouse CNS

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