FSGS (focal segmental glomerulosclerosis) is a pathologic pattern that affects the glomerulus of the kidneys and is associated with progression toward end-stage kidney disease. It is characterized by destruction of the glomerular filtration system and replacement by scar tissue, which leads to chronic renal failure. An increase in glomerular capillary pressure may be an important factor in glomerulosclerosis. The podocyte is a major structural component of the filtration barrier. Since podocytes have an important role in maintaining the structural and functional integrity of glomerular filtration, a chronic increase in glomerular filtration pressure could ultimately damage and compromise the podocyte filtration apparatus. The segmental nature of FSGS suggests that podocyte damage may only occur at specific regions in the glomerulus. This poses the question as to whether differences in podocyte function, assessed by gene expression, may be dependent on their location in the glomerulus, and these regional differences might be responsible for FSGS development. Consequently, in order to evaluate this hypothesis there is a need to selectively isolate podocytes from various regions in a glomerulus. Therefore, the objective of this study was to determine the feasibility of selectively isolating podocyte cells from other renal cells using laser capture microdissection (LCM), and analyzing gene expression in these isolated cells using quantitative real-time polymerase chain reaction (qRT-PCR). If feasible, this method could be used to examine isolated cells for unique podocyte gene expression patterns that vary regionally throughout the glomerulus, and identify potential molecular and cellular mechanisms responsible for the initiation and progression of FSGS. Such information could then be used to identify new and pharmacologically accessible molecular targets. The specific goals of this project were to: 1) determine the feasibility of employment of the LCM system in the Boston University Department of Pathology for the capture of isolated mouse podocytes. 2) determine optimal tissue preservation and preparation methods for laser capture and mRNA analysis of isolated podocytes; and 3) determine the minimum number of isolated podocytes required to analyze gene expression using qRT-PCR. The results show that 1) liquid nitrogen was the preferred method of tissue freezing; 2) the use of Histogene stain improved cell identification during laser capture; 3) the LCM instrument parameters required for selective podocyte capture were identified; and 4) RNA quality obtained from the LCM samples was suboptimal. These results indicate that the possibility of using LCM for regional podocyte isolation and gene expression analysis is quite promising, and further optimization of the technique will likely yield an important new method for the study of kidney disease pathogenesis.

Keloid disease (KD) is a cutaneous fibroproliferative tumour characterised by heterogeneity, locally aggressive invasion and therapeutic resistance. Clinical, histological and molecular differences between the keloid scar centre and margin as well as recent evidence of the importance of epithelial-mesenchymal interactions (EMI) in KD pathobiology contribute to the complexity and diversity of KD, which coupled with the lack of a validated animal model have hindered research and effective management. Despite significant progress in the field of KD research, reliance on conventional monolayer cell culture and whole tissue analysis methods have failed to fully reflect the natural architecture, pathology and complexity of KD in vivo. In order to address these challenges, a site-specific in situ approach was therefore employed here for the first time in KD research. The first aim of this work was to compare the value of this contemporary approach with traditional methods of tissue dissection. The second aim was to compare the genomic expression between well-defined, distinct keloid sites and normal skin (NS). The third aim was to develop and explore hypotheses arising from this site-specific gene expression profiling approach, so as to enhance understanding of KD pathobiology as a basis for improved diagnostic and therapeutic strategies in future KD management. The fourth aim was to probe these hypotheses with relevant functional in vitro studies. The current site-specific in situ approach was achieved through a combination of laser capture microdissection and whole genome microarray, allowing separation of epidermis from dermis for keloid centre, margin and extralesional sites compared with NS. This in situ approach yielded selective, accurate and sensitive data, exposing genes that were overlooked with alternative methods of dissection. Identification of significant upregulation of the aldo-keto reductase enzyme AKR1B10 in all three sites of the keloid epidermis (KE) in situ, implicated dysregulation of the retinoic acid (RA) pathway in KD pathogenesis. This hypothesis was supported by showing that induced AKR1B10 overexpression in NS keratinocytes reproduced the keloid RA pathway expression pattern. Moreover, co-transfection with a luciferase reporter plasmid revealed reduced RA response element activity. Paracrine signals released by AKR1B10-overexpressing keratinocytes into conditioned medium resulted in TGFβ1 and collagen upregulation in keloid fibroblasts, suggesting the disturbed RA metabolism exerts a pro-fibrotic effect through pathological EMI, thus further supporting the hypothesis of RA deficiency in KE. Gene expression profiling further revealed an upregulation of NRG1 and ErbB2 in keloid margin dermis. Exogenous NRG1 led to enhanced keloid fibroblast migration with increased Src and PTK2 expression, which were attenuated with ErbB2 siRNA studies. Together with the observed failure to recover this expression with NRG1 treatment, suggested the novel KD pathobiology hypothesis that NRG1/ErbB2/Src/PTK2 signaling plays a role in migration at the keloid margin. In addition to these hypotheses, LCM methodology with comprehensive analysis of the data permitted the development of additional novel working hypotheses that will inform future KD research, including inflammatory gene dysregulation and cancer-like stem cells that may contribute to the therapeutic resistance characteristic of KD.

Laser capture microdissection (LCM) is a versatile computer-assisted dissection method that permits collection of tissue samples with a remarkable level of anatomical resolution. LCM's application to the study of human brain pathology is growing, although it is still relatively underutilized, compared with other areas of research. The present study examined factors that affect the utility of LCM, as performed with an Arcturus Veritas, in the study of gene expression in the human brain using frozen tissue sections. LCM performance was ascertained by determining cell capture efficiency and the quality of RNA extracted from human brain tissue under varying conditions. Among these, the relative humidity of the laboratory where tissue sections are stained, handled, and submitted to LCM had a profound effect on the performance of the instrument and on the quality of RNA extracted from tissue sections. Low relative humidity in the laboratory, i.e., 6-23%, was conducive to little or no degradation of RNA extracted from tissue following staining and fixation and to high capture efficiency by the LCM instrument. LCM settings were optimized as described herein to permit the selective capture of astrocytes, oligodendrocytes, and noradrenergic neurons from tissue sections containing the human locus coeruleus, as determined by the gene expression of cell-specific markers. With due regard for specific limitations, LCM can be used to evaluate the molecular pathology of individual cell types in post-mortem human brain.

Infection with "Pneumocystis" causes a ≥ 99% depletion of plasma S-adenosylmethionine (AdoMet) levels in both "Pneumocystis" pneumonia (PcP) animal models and patients. AdoMet is a critical cellular metabolic intermediate, with a pivotal role as methyl donor in a myriad of biochemical processes and necessary for the synthesis of the essential polyamines spermidine and spermine. In the target tissue of "Pneumocystis", the lung, levels of AdoMet were previously shown to be depleted experimentally using nicotine. Here we show that chronic administration of nicotine in an animal model of PcP resulted in decreased lung AdoMet content. Since "Pneumocystis" is dependent on this metabolite, PcP burden was also relived. We hypothesized that the underlying mechanism behind nicotine-induced AdoMet depletion was an increased consumption of AdoMet through the polyamine pathway where the increased activity of N-1-spermidine/spermine acetyl transferase raises the catabolic / anabolic cycling of polyamines, a process that utilizes AdoMet. In a critical test of our hypothesis, we found that blockage of polyamine metabolism via inhibition of the polyamine biosynthetic enzyme ornithine decarboxylase (ODC) hinders the effect of nicotine on lung AdoMet levels. Further support is provided by metabolite analyses showing nicotine to cause a strong diversion of AdoMet toward polyamine synthesis and away from methylation reactions; these shifts are also reversed by inhibition of ODC. Because the nicotine effect on "Pneumocystis" is so striking, we considered the possibility of tissue specificity. Using laser capture microdissection (LCM), we collected samples of lung alveolar regions (site of infection) and respiratory epithelium for controls. We found nicotine to cause increased ODC activity in alveolar regions but not airway epithelium; we conclude that tissue specificity likely contributes to the effect of nicotine on "Pneumocystis" pneumonia. Our studies demonstrate the feasibility of pharmacological manipulation of the polyamine pathway in order to reduce AdoMet levels in the lung and prompted the assessment of compounds alternative to nicotine with the potential to achieve a comparable effect. In vitro evaluation of the polyamine analog DENSPM along with putrescine in type II alveolar cell lines, indicates that although such a combination has the potential to induce polyamine flux, an apparent competition for the same polyamine transport system impairs simultaneous uptake of both compounds at effective concentrations. In conclusion, we showed that chronic nicotine administration causes reduction of AdoMet levels in rat lung following 21 days of treatment, by a mechanism involving the induction of polyamine flux, which is responsible of increased AdoMet utilization for polyamine biosynthesis. According to LCM-based analysis, this effect seems to be confined to the alveolar regions of the lung. / Biochemistry

No / The global prevalence Type 2 diabetes mellitus (T2DM) is escalating at a rapid rate. Patients with T2DM suffer from a multitude of complications and one of these is impaired wound healing. This can lead to the development of non-healing sores or foot ulcers and ultimately to amputation. In healthy individuals, wound healing follows a controlled and overlapping sequence of events encompassing inflammation, proliferation, and remodelling. In T2DM, one or more of these steps becomes dysfunctional. Current models to study impaired wound healing in T2DM include in vitro scratch wound assays, skin equivalents, or animal models to examine molecular mechanisms underpinning wound healing and/or potential therapeutic options. However, these do not fully recapitulate the complex wound healing process in T2DM patients, and ex vivo human skin tests are problematic due to the ethics of taking punch biopsies from patients where it is known they will heal poorly. Here, a technique is described whereby expression profiles of the specific cells involved in the (dys)functional wound healing response in T2DM patients can be examined using surplus tissue discarded following amputation or elective cosmetic surgery. In this protocol samples of donated skin are collected, wounded, cultured ex vivo in the air liquid interface, fixed at different time points and sectioned. Specific cell types involved in wound healing (e.g., epidermal keratinocytes, dermal fibroblasts (papillary and reticular), the vasculature) are isolated using laser capture microdissection and differences in gene expression analyzed by sequencing or microarray, with genes of interest further validated by qPCR. This protocol can be used to identify inherent differences in gene expression between both poorly healing and intact skin, in patients with or without diabetes, using tissue ordinarily discarded following surgery. It will yield greater understanding of the molecular mechanisms contributing to T2DM chronic wounds and lower limb loss. / European Commission 7th Framework Programme for Research and Technical Development - Marie Curie Innovative Training Networks (ITN), Grant agreement no 607886. Aveda, Hair Innovation & Technology, USA

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) now affects 1 in 68 children in the United States. Disorders within this spectrum share hallmark deficits in verbal and nonverbal communication, repetitive behavior, and social interaction. The cause of ASD is still unknown. Even though hundreds of genetic abnormalities have been identified in ASD, these markers account for less than 1% of all ASD cases. Researchers continue to search for pathological markers common to all or most cases of ASD. The research presented in this dissertation used a novel combination of state-of-the-art methods to investigate brain pathology in ASD. Postmortem anterior cingulate cortex (ACC) from ASD and typically developing brain donors was obtained from 2 national brain banks. The ACC was chosen for study because of its documented role in influencing behaviors characteristically disrupted in ASD. An initial study revealed elevated glial fibrillary acidic protein (GFAP) in ACC white matter from ASD brain donors compared to typically developing control donors. Laser capture microdissection was then employed to isolate specific cell populations from the ACC from ASD and control brain donors. Captured cells were used to interrogate potential gene expression abnormalities that may underlie biological mechanisms that contribute behavioral abnormalities of ASD. The expression of 4 genes associated with synaptic function, NTRK2, GRM8, SLC1A1, and GRIP1, were found to be significantly lower in ACC pyramidal neurons from ASD donors when compared to control donors. These expression abnormalities were not observed in ACC glia. Given robust evidence of neuronal and glial pathology in the ACC in ASD, a novel method for whole transcriptome analysis in single cell populations was developed to permit an unbiased analysis of brain cellular pathology in ASD. A list of genes that were differentially expressed, comparing ASD to control donors, was produced for both white matter and pyramidal neuron samples. By examining the ASD brain at the level of its most basic component, the cell, methods were developed that should allow future research to identify common cellular-based pathology of the ASD brain. Such research will increase the likelihood of future development of novel pharmacotherapy for ASD patients.

The airway epithelium has many roles in innate immunity including detection of pathogens and transmitting danger signals to other cell types. However, its role as a primary defender against infection is not well recognized. We have investigated methods of augmenting antiviral immunity by application of agents that stimulate viral killing, either in the extracellular space or within the cytoplasm. A recently described property of airway epithelial cells is direct oxidative killing of bacteria through the coordination of Duox and lactoperoxidase enzymes. We have exploited this property by supplementing airway cells with the lactoperoxidase substrate iodide to prevent viral infection. A second method for enhancing antiviral defenses is to supply small interfering RNAs (siRNAs) targeting essential viral genes. We have optimized antiviral siRNAs targeting respiratory syncytial virus by designing them to specifically target positive sense viral RNAs. Finally, we have initiated a project to discover host defense genes that are expressed in either the submucosal glands surface epithelium of human airway. This information will enable a better characterization of the roles for these structures in host defense pathways, and may identify other targets for augmentation of antiviral immunity.

Prion diseases are fatal and incurable neurodegenerative diseases that share many pathological similarities to other neurodegenerative diseases such as Alzheimer’s or Parkinson’s disease. One of the earliest pathological signs commonly detected in all of these diseases is the dysfunction followed by loss of neuronal synapses, spines and eventually dendrites that collectively contribute to disruption of normal brain function. These pathologies tend to progressively accumulate within the brain tissue such that extensive damage typically precedes clinical symptom manifestation and ultimate death of neurons. Clearly, understanding the molecular processes responsible for these pathologies could uncover critical pathway(s) that are responsible for propagating this brain damage and could therefore be exploited for therapy development. However, molecular mechanisms implicated in this early pathology remain unidentified. To address this gap in knowledge, this thesis describes a transcriptional approach coupled with specific isolation of neuronal-enriched tissue which was used to help delineate cellular pathways involved in prion-induced neurodegeneration. Profiling cell bodies of CA1 hippocampal neurons known to be affected during early prion disease revealed temporal alteration in both gene and microRNA (gene regulators) expression throughout disease. On a gene expression level, changes in transcript expression during preclinical disease were reminiscent of an activity-dependent neuroprotective gene signature previously described in the literature. These neuroprotective genes were induced during preclinical disease, diminished as disease progressed and were abolished at clinical disease. In support of this process, upregulation of the phosphorylated form of the neuroprotective transcription factor CREB was detected during preclinical disease in these neurons. Furthermore, several genes known to be induced by CREB were also upregulated at preclinical disease in prion-infected mice. Interestingly, expression of numerous deregulated microRNAs paralleled the neuroprotective gene signature of which several are known to remodel neuronal spines and dendrites. To determine whether other preclinically induced microRNAs were also capable of remodeling neuronal structures, gain-of-function studies were performed in primary mouse hippocampal neurons for the uncharacterized miR-26a-5p. Neurons over-expressing miR-26a-5p had enhanced spine density and dendrite arborization, similar to other preclinically-induced microRNAs. Together, these data suggests that CA1 hippocampal neurons induce a neuroprotective transcriptional signature that is evident early in the course of disease within CA1 hippocampal neurons and is abolished by clinical disease. Reestablishment of key molecules that can induce this neuroprotective signature at a time when these genes begin to dissipate could prolong prion disease onset and delay clinical symptom manifestation. / October 2015

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.