Seminario 1 Introducción a la metodología científica BCM- Gabriel Scicolone

Understanding Levels of Organization in Matter

Introduction to the Talk

• The talk aims to help us understand that matter is organized at different levels, with various sciences studying these levels using specific methodologies.

Scientific Methodologies

• Scientists in cellular and molecular biology generate knowledge through experimental results, employing an inductive methodology where findings are generalized. This contrasts with the deductive approach used by healthcare teams, including doctors, who apply scientific knowledge to solve individual patient problems.

Objectives of the Discussion

• Key objectives include understanding how scientific knowledge is acquired through research methods that address hypotheses and questions posed by scientists. Additionally, it emphasizes the importance of experimental models in transferring results applicable to humans.

Inductive vs Deductive Methods

• The inductive method involves conducting experiments on a small number of subjects (e.g., mice) and generalizing findings to other species, including humans. Conversely, the deductive method allows healthcare professionals to resolve specific cases using general scientific knowledge. Understanding both perspectives is crucial for applying these methodologies effectively in practice.

Levels of Complexity in Matter

Hierarchical Structure of Matter

• Matter exhibits various complexity levels: atomic, molecular, cellular (where cells form tissues), and organ systems (where organs perform specific functions). This hierarchy culminates at the organism level, which can range from unicellular organisms to multicellular ones like humans. Understanding this structure aids in studying biological systems comprehensively.

Role of Different Sciences

• Various sciences utilize distinct instruments to gather information across these organizational levels; for instance, optical instruments measure length related to matter's organization and resolution capabilities. The human eye has a resolution limit allowing differentiation between points separated by 100 microns; this concept is fundamental when discussing observational limits in biological studies.

Resolution Limits and Optical Instruments

Understanding Resolution

Microscopy and Its Role in Biological Sciences

Understanding Microscopy Types

• Resolution Power: Electron microscopes have a higher resolution than the human eye, allowing differentiation of points as close as 0.2 nanometers, compared to optical microscopes which can differentiate down to 0.2 microns.

• Human Eye vs. Microscopes: The human eye can see details separated by up to 100 microns, while optical microscopes extend this capability to 0.2 microns.

Anatomy and Histology

• Anatomy Studies: Anatomy utilizes the resolving power of the human eye to study the organization of the human body, including systems and organs.

• Histology Focus: Histology is concerned with tissue structure and uses optical microscopy to examine how cells and extracellular matrix are organized within tissues.

Advancements in Cellular Biology

• Cellular Biology Initiation: The advent of electron microscopy marked the beginning of cellular biology, enabling detailed studies of cell components.

• Molecular Techniques: Advances in molecular biology techniques allow for macromolecular analysis, enhancing understanding of cell composition and function.

Comparative Study Methods

• Different Scientific Approaches: Various fields like anatomy (studying systems), histology (studying tissues), cellular biology (studying cells), and molecular biology (studying molecules) utilize different microscopy techniques for their specific needs.

• Chemical Component Identification: Molecular biology employs techniques that help identify chemical components within cells and tissues without altering their structure.

Practical Applications in Histology

• Staining Techniques: Common staining methods include hematoxylin-eosin for identifying tissue structures; these will be used throughout histological studies.

• Visualization Techniques: Different staining methods such as trichrome stain help visualize extracellular matrix components like collagen fibers effectively under a microscope.

Insights into Experimental Methodologies

• Microscopy Variants: Examples include differential interference contrast microscopy for viewing live cells without staining, revealing chromosomes during cell division.

• Electron Microscopy Details: Transmission electron microscopy provides insights into organelles like nuclei and mitochondria through high-resolution imaging techniques using heavy metals for contrast enhancement.

Scientific Inquiry Methods

• Inductive vs. Deductive Reasoning: Scientists use inductive reasoning to generalize knowledge from observations while medical professionals apply deductive reasoning based on established knowledge to solve specific problems.

Understanding Scientific Knowledge Dissemination

Generalization of Statistical Results

• Statistical results can be generalized to all individuals within a population, highlighting the conservation of cellular and molecular mechanisms throughout evolution. This allows knowledge gained from one species to be applicable to humans.

Publication Process in Scientific Research

• Scientific knowledge is disseminated through published research papers in scientific journals. These publications are essential for sharing findings in cellular and molecular biology and other sciences.

• Submitted works are evaluated by editors who determine their relevance to the journal's theme. If deemed appropriate, they are sent for peer review by specialists in the relevant field.

• Peer reviewers assess the methodology and clarity of the submitted work, providing feedback on its suitability for publication. Rejection may occur if it does not meet standards or requires significant modifications.

Importance of Methodology

• The scientific paper must adhere to reproducible methodologies that allow others to verify findings. This critical approach ensures that contributions are credible and can be analyzed further by other researchers.

Structure of a Scientific Paper

• A typical scientific paper includes a title summarizing the discovery, author affiliations, an abstract outlining objectives and methods, keywords for searchability, an introduction presenting background information, materials and methods detailing experimental procedures, results with supporting figures, discussion interpreting findings, conclusions or perspectives on implications, references cited throughout the work, acknowledgments for contributions made during research, and conflict of interest declarations.

Conflict of Interest Considerations

• Researchers must declare any potential conflicts of interest that could influence study outcomes. For instance, involvement with pharmaceutical companies testing new drugs necessitates transparency regarding financial interests.

Types of Scientific Papers

Types of Scientific Work and Access to Research

Overview of Clinical Studies

• Clinical studies, such as clinical trials, test the efficacy of diagnostic systems or therapeutic interventions. They provide valuable insights into specific medications.

• Case descriptions highlight rare pathologies, offering initial insights into diseases despite their limited scope compared to broader studies.

Accessing Scientific Literature

• Various bibliographic search engines like Pavmed and Cielo Lilax facilitate access to scientific works online.

• To find relevant research, users can input keywords related to their topic of interest (e.g., "p53 mutations in breast cancer treatment resistance").

Understanding Research Accessibility

• Scientific articles are categorized into two types based on accessibility: open access and subscription-based. Open access allows free public access after publication.

• Subscription-based articles require payment for full access; libraries often subscribe to journals, allowing institutional access without direct costs to researchers.

The Importance of Reviews in Research

• Review articles synthesize recent findings on a particular topic by analyzing multiple studies, providing an updated overview for readers.

• Bibliographies at the end of chapters in textbooks reference these reviews and classic studies, underscoring the foundational role of scientific literature in academic writing.

Scientific Methodology and Experimental Models

Experimental Approaches in Biology

• In cellular and molecular biology research, experiments may involve animal models. Findings from a small number of animals can be generalized across species due to conserved cellular mechanisms.

Utilizing Different Organisms for Research

Molecular Mechanisms in Eukaryotic Cells

Conserved Molecular Processes

• The synthesis of proteins and transcription are highly conserved molecular mechanisms across eukaryotic organisms, yielding similar results in unicellular and multicellular models.

• These processes trace back to a single type of cell from which prokaryotes and eukaryotes diverged, indicating fundamental similarities despite some differences.

Experimental Models for Research

• Prokaryotes serve as simpler models for studying protein synthesis before findings are applied to more complex eukaryotic systems, revealing commonalities with added complexity in eukaryotes.

• Knowledge gained from these studies can be transferred to human applications, such as drug testing in clinical trials after initial animal testing.

Examples of Experimental Models

• Bacteria are utilized as experimental models due to their simplicity, allowing researchers to study basic molecular mechanisms like transcription and translation.

• Yeast serves as another model organism where genetic manipulation has elucidated cell cycle regulation mechanisms relevant to both yeast and humans.

Increasing Complexity in Model Organisms

• Invertebrates like C. elegans provide insights into genetic determination of cell fate within multicellular organisms while maintaining a relatively simple structure.

• Drosophila melanogaster (fruit fly) is crucial for genetic studies; mutations in homeotic genes can lead to significant developmental anomalies that inform vertebrate biology.

Advancing Towards Vertebrate Models

• Zebrafish offer transparency that aids the study of cellular movements during development, with findings applicable to human biology.

• Mice are commonly used mammalian models due to their rapid reproduction rates; they help investigate conditions like vitiligo by mirroring human pigmentation disorders.

Implications for Cellular and Molecular Studies

• The similarities between mouse models and human conditions highlight the utility of experimental models in understanding cellular processes throughout various stages of development.

Comparative Analysis of Drug Effects in Experimental Models

Comparing Treated vs. Untreated Groups

• The speaker discusses the importance of comparing responses in controlled groups, specifically between untreated and treated groups with a specific drug to assess its effects.

Investigating Drug Effects in Animal Studies

• Animal studies, referred to as "in vivo" studies, allow for the investigation of drug effects under physiological conditions but introduce numerous variables that can affect outcomes.

Advantages of Cell Cultures

• To minimize variable influence on cellular behavior, isolating cells from organisms and conducting cell cultures is beneficial. This method allows for controlled experimentation with drugs while reducing external signals affecting results.

Limitations of In Vitro Studies

• While in vitro studies provide better control over variables compared to in vivo studies, they may not accurately reflect physiological states due to the absence of complex organismal interactions. For example, mitochondrial respiration might be influenced by neighboring cells' signals in a culture setting.

Subcellular Fractionation Techniques

• The speaker explains subcellular fractionation techniques that isolate mitochondria or other cellular components for detailed study, enhancing variable separation but distancing from real physiological conditions.

Examples of In Vivo Studies Using Animal Models

Behavioral Assessments in Rats

• An example is provided where rats are subjected to behavioral tests under various stimuli; this includes surgical procedures like extracting fetuses to study maternal alcohol effects on fetal development.

Transgenic Mouse Models

• Transgenic mice are discussed as models where genetic modifications (e.g., insertion of a green fluorescent protein gene) allow researchers to observe specific traits such as fluorescence in retinal cells. This technique aids in understanding genetic influences on physiology and development.

Microscopy Techniques for Observing Biological Samples

Preparing Tissue Samples for Microscopy

• The process involves fixing tissues to preserve structure before slicing them into thin sections using a microtome for microscopic examination; staining is then applied to differentiate tissue components visually.

Optical vs Electron Microscopy

Microscopy and Cell Culture Techniques

Overview of Microscopy in Cellular Studies

• The use of optical microscopy is highlighted for observing specific molecules with high resolution, essential for cellular and molecular biology studies.

• In vivo studies involve taking tissue samples, cutting them into fragments, and using a solution to facilitate further analysis.

Tissue Preparation and Culturing Process

• Tissue samples are treated with enzymes to break intercellular connections, leading to disaggregation. This process is followed by centrifugation to separate cells from the liquid.

• Cells are placed in Petri dishes within incubators that maintain body temperature (37°C) and a high concentration of carbon dioxide to mimic physiological conditions.

Maintaining Sterility in Cultures

• The importance of maintaining sterility during cell culture procedures is emphasized, utilizing laminar flow hoods to prevent contamination by prokaryotic organisms.

• Once cultured cells proliferate sufficiently, they can be sub-cultured or frozen in liquid nitrogen at -160°C for long-term storage.

Advanced Microscopy Techniques

• An inverted microscope design allows observation of cells at the bottom of Petri dishes, contrasting with traditional histology microscopes where objectives are positioned above.

• Differential interference contrast (DIC) microscopy enables visualization of live cells without staining, providing relief-like images for better recognition.

Fluorescence Microscopy Applications

• Epifluorescence microscopy reveals fluorescently tagged nuclei due to introduced DNA coding for green fluorescent proteins; this technique aids in visualizing cellular components.

• Vital dyes that do not harm cells allow researchers to observe mitochondrial activity alongside fluorescent markers indicating protein expression.

Experimental Comparisons and Subcellular Fractionation

• Experiments compare treated versus control groups within cell cultures to assess drug effects on cellular behavior.

• Subcellular fractionation techniques aim to isolate pure fractions from cellular components for detailed study; this involves mechanical disruption followed by homogenization.

Steps in Subcellular Fractionation

• The initial step includes mechanically breaking down tissues (e.g., liver), often aided by enzymes or detergents that help disaggregate the material effectively.

• Homogenates contain mixed cellular components such as nuclei, mitochondria, ribosomes, and extracellular matrix elements after treatment with detergents.

Centrifugation and Cellular Component Separation

Overview of Centrifugation Process

• The process begins with the rupture of tissue, resulting in all components dissolved in a homogeneous solution. The next step is to separate these components using centrifugation.

• Centrifugation applies gravitational forces greater than normal gravity, causing denser components to move to the bottom while less dense ones float on top.

Steps in Centrifugation

• Initially, the homogenate is subjected to 1000g for 10 minutes, yielding a pellet of whole cells and nuclei at the bottom and a supernatant above.

• The supernatant undergoes further centrifugation at 20,000g for 20 minutes, producing another pellet containing heavier cellular components like mitochondria, lysosomes, and peroxisomes.

Advanced Separation Techniques

• The remaining supernatant is then ultracentrifuged at 80,000g for one hour to isolate microsomes from vesicles derived from rough and smooth endoplasmic reticulum.

• A subsequent ultracentrifugation at 100,000g for three hours results in a pellet containing ribosomes and large macromolecules.

Verification of Isolated Fractions

• To confirm the identity of isolated fractions (e.g., mitochondrial), enzymatic activity can be assessed using specific substrates that yield identifiable products when processed by target enzymes.

• If the expected product appears (indicating presence of mitochondria), it confirms successful isolation; otherwise, lower product yields suggest fewer mitochondria are present.

Microscopy as a Validation Tool

• Electron microscopy can also be employed to visually confirm subcellular fractions based on morphology—nuclear fractions appear distinct from mitochondrial or microsomal fractions.

Density Gradient Centrifugation

Alternative Methods for Component Separation

• Another method involves density gradient centrifugation using varying concentrations (e.g., sucrose), allowing separation based on density differences among cellular components.

Applications in Research

• This methodology enables researchers to generalize findings across species through experimental models ranging from unicellular organisms to complex multicellular systems.

Experimental Models in Biological Research

Importance of Experimental Models

Understanding Diagnostic Procedures in Medicine

The Role of Complementary Studies

• Medical professionals often request additional studies to diagnose patient conditions and predict disease progression, which aids in determining appropriate treatments.

Sample Collection Techniques

• Blood samples are commonly collected using a syringe and needle; anticoagulants can be used to separate blood components into plasma and cells.

• Bone marrow samples may be taken for blood-related issues, with methods including aspiration or biopsy to preserve tissue organization.

Tissue Sampling Methods

• Different sampling techniques yield varying results: aspirated cells lose tissue structure, while biopsies maintain cellular organization for microscopic examination.

• Endoscopic procedures can obtain biopsies from lesions, allowing for detailed microscopic analysis.

Conceptual Framework of Sample Acquisition

• A conceptual framework illustrates various sample acquisition methods from both experimental models and patient consultations, highlighting the importance of preserving sample integrity.

Analysis Techniques for Samples

• Samples may need fixation to retain structural details; histological or cytological techniques allow observation under a microscope with or without staining.

• Biochemical studies can analyze proteins or enzymes by homogenizing tissues or plasma without needing structural preservation.

Advanced Microscopy Techniques

• Histological techniques help identify cellular components like nuclei and cytoplasm; immunohistochemistry locates specific proteins within tissues.

• Messenger RNA localization can be achieved through hybridization techniques, enhancing understanding of gene expression in tissues.

Techniques for Analyzing Proteins and Nucleic Acids

Overview of Sample Preparation

• The process begins with obtaining tissue samples, which can either preserve their structure or be homogenized to extract components like plasma.

• From the homogenate, specific molecules such as DNA, RNA, or proteins can be purified for further analysis.

Analytical Techniques

• Various techniques are employed to analyze these purified molecules:

• Electrophoresis: Separates proteins or nucleic acids based on size and charge.

• Southern Blot: Identifies DNA presence and quantity.

• Northern Blot: Used for RNA analysis.

• Western Blot: Detects proteins through antigen-antibody reactions.

Amplification and Manipulation of Nucleic Acids

• Polymerase Chain Reaction (PCR) is utilized to amplify DNA, while reverse transcriptase can convert RNA into complementary DNA (cDNA).

• Modified nucleic acids can be introduced into experimental models to study their effects.

Genetic Modification Techniques

• CRISPR technology allows for targeted mutations in DNA, enabling researchers to evaluate the consequences of these changes on cellular functions.

Flow of Genetic Information

• The flow of genetic information from DNA to RNA to proteins is illustrated, highlighting various techniques available at each stage:

• Techniques exist for analyzing individual genes as well as entire genomes.

Advanced Genomic Analysis

• Methods such as PCR and sequencing allow for detailed examination of genetic material.

• Whole-genome analysis techniques include microarray hybridization and massive parallel sequencing.

Transcriptomic Studies

• Individual RNA molecules can be analyzed using RT-qPCR and in situ hybridization.

• Mass sequencing followed by reverse transcription enables comprehensive profiling of all RNAs present in a sample.

Protein Analysis Techniques

• Immunohistochemistry and immunofluorescence detect specific proteins within tissues, revealing their distribution.

• Western Blotting quantifies protein levels while flow cytometry separates cells based on protein expression.

• Advanced methods like X-ray crystallography provide insights into protein structures; nuclear magnetic resonance spectroscopy also aids in structural determination.

Electrophoresis and Mass Spectrometry Techniques

Overview of Protein Analysis Techniques

• The process begins with electrophoresis or chromatography, which separates proteins from tissue samples. Following this, mass spectrometry is employed to analyze these proteins, providing insights into all proteins present in a cell or tissue.

DNA, RNA, and Protein Manipulation Techniques

• Various techniques exist for analyzing and manipulating DNA, RNA, and proteins. On the left side of the discussed diagram are methods that operate at the single-molecule level (e.g., detecting individual molecules of DNA, RNA, or protein), while the right side encompasses techniques that provide information about groups of molecules within a cell or tissue.

Summary of Class Content

• The class covered levels of organization in matter and how different scientific disciplines utilize various instruments to study these levels. This includes discussions on methodologies used in scientific research.

Scientific Methodologies Discussed

• The lecture addressed scientific methodology including inductive reasoning and experimental models. It also highlighted studies conducted both in vivo (within a living organism) and in vitro (outside a living organism).

Deductive Methodology in Medical Practice

• A focus was placed on deductive methodologies used by medical teams to apply general scientific knowledge to specific patient cases. This included an overview of study methodologies related to tissues, cells, and macromolecules based on sample processing methods and gene expression levels.