Appunti di Biologia di base e di genomica/genetica, Appunti di Biologia Genetica

Scarica Appunti di Biologia di base e di genomica/genetica e più Appunti in PDF di Biologia Genetica solo su Docsity! imgs/logo_unisa.png UNIVERSITÀ DEGLI STUDI DI SALERNO DIPARTIMENTO DI INGEGNERIA DELL’INFORMAZIONE ED ELETTRICA E MATEMATICA APPLICATA Corso Di Laurea in INFORMATION ENGINEERING FOR DIGITAL MEDICINE BIOLOGY AND GENOMICS Professori: Elena CIAGLIA Giovanni NASSA Autori: Mario Apicella Salvatore Bruno ANNO ACCADEMICO 2023/2024 2 CONTENTS 5 6.10.1 Lymphatic organs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98 6.11 Platelets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99 6.11.1 The platelet aggregation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100 6.11.2 Method for counting WBC, RBC and PLT . . . . . . . . . . . . . . . . . . . . . . 100 6.11.3 Coulter impedance principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100 6.11.4 Leucocytes Formula analysis through VCS Technology . . . . . . . . . . . . . . . . 101 6.11.5 Kind of CD markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103 6.12 Immunofluorescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104 6.12.1 Multiparametric Flow Cytometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106 6.12.2 The journey in summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108 6.12.3 Fluidics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109 6.12.4 Optical . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111 6.12.5 Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113 6.12.6 Spill Over Problem . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113 6.12.7 Electronics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115 6.12.8 Multicolor Flow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115 7 The cell cycle 117 7.1 The key roles of cell division . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117 7.1.1 Stages of the cell cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117 7.1.2 Chromosomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118 7.2 INTERPHASE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119 7.2.1 G1 phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119 7.2.2 S-phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119 7.2.3 G2 phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120 7.3 MITOSIS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121 7.3.1 Phase 1: Mitosis or Karyokinesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121 7.3.2 Cytokinesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125 7.4 MEIOSIS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126 7.4.1 MEIOSIS I: reduction division . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126 7.4.2 Crossing Over . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127 7.4.3 MEIOSIS II . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128 7.5 Intracellular Control of the cell cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129 7.5.1 Positive regulators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129 7.5.2 Negative Regulators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130 7.6 Mechanism of cycle regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131 7.6.1 I part . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131 7.6.2 II part . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132 II Genomics 133 8 Genetics 135 8.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135 8.2 Genitic materialis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136 8.3 Genetic code . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137 8.4 Genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137 8.5 Genomes Organization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141 8.5.1 The prokaryote genome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142 8.5.2 Eukaryotic genome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145 8.5.3 Human Genomic DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147 8.6 Some definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152 6 CONTENTS 9 Gene Expression Regulation 153 9.1 Gene Expression and regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153 9.2 Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154 9.2.1 HouseKeeping genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154 9.2.2 Cell type specific genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154 9.3 Hormones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154 9.4 Regulation of Gene expression in Prokaryoties . . . . . . . . . . . . . . . . . . . . . . . . . 156 9.4.1 Steps in the regulation of the expression . . . . . . . . . . . . . . . . . . . . . . . . 157 9.4.2 The example of the E.Coli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158 9.4.3 Adding Lactose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160 9.5 Regulation of gene expression in Eukaryotes . . . . . . . . . . . . . . . . . . . . . . . . . . 161 9.5.1 RNA polymerase II transcription . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162 9.5.2 The cis-acting regulatory regions . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162 9.5.3 The trans-acting proteins control transcription initiation . . . . . . . . . . . . . . . 163 9.5.4 Chromatin structure and Epigenitc effects . . . . . . . . . . . . . . . . . . . . . . . 167 9.5.5 Genomic imprinting results from transcriptional silencing . . . . . . . . . . . . . . 169 9.5.6 Regulation after Transcription . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169 10 Splicing 171 10.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171 10.1.1 The 5′ end of eukaryotic mRNA is capped . . . . . . . . . . . . . . . . . . . . . . . 171 10.2 Nuclear splice sites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172 10.2.1 The GU-AG rule . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172 10.3 Splicing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173 10.3.1 snRNAs Are Required for Splicing . . . . . . . . . . . . . . . . . . . . . . . . . . . 173 10.3.2 Commitment of Pre-mRNA to the Splicing Pathway . . . . . . . . . . . . . . . . . 174 10.3.3 The Spliceosome Assembly Pathway . . . . . . . . . . . . . . . . . . . . . . . . . . 175 10.3.4 Alternative splicing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177 10.3.5 Defects in Pre-mRNA Splicing Cause Human Disease . . . . . . . . . . . . . . . . 178 11 Non-coding RNAs 181 11.1 Non-Coding RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181 11.1.1 CRISPRs: arecord of infections survived resistance gained . . . . . . . . . . . . . . 182 11.1.2 The three main classe of small non-coding RNA . . . . . . . . . . . . . . . . . . . 182 11.1.3 Mechanisms of Action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183 11.1.4 Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184 12 Mutations 185 12.1 Classificatio of Mutations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 188 12.2 Point mutations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189 12.3 Frameshift mutation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 190 12.4 Loss of function and gain of function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 190 12.5 Induced and spontaneous functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 190 12.6 How natural process can change the information stored in DNA . . . . . . . . . . . . . . . 190 12.6.1 Mistake during DNA replication . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192 12.6.2 Transposable elements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193 12.6.3 Unstable trinucleotide repeats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193 12.6.4 How mutagens alter DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194 12.7 DNA repair mechanism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197 12.8 Human diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203 12.8.1 Alkaptonuria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203 12.8.2 Xeroderma Pigmentosum (XP): . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203 12.8.3 Red/Green Color Blindness: Unequal Crossing-Over . . . . . . . . . . . . . . . . . 204 CONTENTS 7 13 Cancer 205 13.1 Origin of the word . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205 13.2 Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205 13.3 Cancer can originate in various body locations . . . . . . . . . . . . . . . . . . . . . . . . 206 13.3.1 Loss of normal growth control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 206 13.3.2 The beginning of Cancerous growth . . . . . . . . . . . . . . . . . . . . . . . . . . 207 13.3.3 Malignat VS Begign Tumors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 208 13.3.4 Cancer detection and Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209 13.3.5 Hyperplasia, Dysplasia and Carcinoma in situ . . . . . . . . . . . . . . . . . . . . . 209 13.3.6 Classification of cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210 13.3.7 Genes and cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211 13.3.8 Oncogenes and Tumor Suppressor Genes . . . . . . . . . . . . . . . . . . . . . . . . 211 14 Epigenomics 215 14.1 Epigenetic Changes: How much these can be important for the phenotype ? . . . . . . . . 215 14.1.1 Epigenetic and chromatin structure . . . . . . . . . . . . . . . . . . . . . . . . . . 216 14.1.2 How do cells know what DNA to express? . . . . . . . . . . . . . . . . . . . . . . . 217 14.2 DNA structure and epigenetic modifications . . . . . . . . . . . . . . . . . . . . . . . . . . 221 14.3 Epigenetic Modifcation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221 14.3.1 DNA methylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222 14.3.2 One way in which DNA Methylation suppresses gene expression . . . . . . . . . . 223 14.4 Histone Modification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 224 14.4.1 Kind of Modification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225 14.4.2 Implication of DNA modification . . . . . . . . . . . . . . . . . . . . . . . . . . . . 226 14.4.3 Epigenetic therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 226 15 Epigenetics: Chip and Met-Seq 227 15.0.1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 230 15.0.2 PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231 15.0.3 macro-Array . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231 15.0.4 ChiP-Seq . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 232 15.0.5 Library preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 232 15.0.6 Analisi Bionformatica . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 232 15.0.7 Considerazioni finali . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233 15.1 DNA methylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 234 CHAPTER 1 THE BIOLOGY 1.1 Introduction Biology is a branch of natural science which deals with the study of life and living organisms. Organism is any contiguous living system like animals, plants. All organisms are made up of basic fundamental unit called cell. The cell is the basic unit of life. It is the smallest structural and functional unit of organism, which is typically microscopic and consists of cytoplasm and a nucleus enclosed in a membrane Many independent organism are composed only of single cells. Cell biology is a scientific discipline that studies cells, their physiological properties and their structures. 1.2 Hierarchical organization of living systems. Life forms a hierarchy of organization from atoms to complex multicellular organisms such as showed in picture 1.1. Atoms are joined together to form molecules, which are assembled into more complex structures such as organelles. These in turn form subsystems that provide different functions. Cells can be organized into tissues, then into organs and organ systems. This organization then extends beyond individual organisms to populations, communities, ecosystems, and finally the biosphere. imgs/01.jpg Figure 1.1: Hierarchical organization of living systems. 11 12 CHAPTER 1. THE BIOLOGY 1.3 Biomolecules of cells imgs/02.jpg Figure 1.2: Propane structural formula The framework of biological molecules consists predomi- nantly of carbon atoms bonded to other carbon atoms or to atoms of hydrogen, oxygen, nitrogen, sulfur, or phospho- rus. Because carbon atoms can form up to four covalent bonds, molecules containing carbon can form straight chains, branches, or even rings, balls, tubes, and coils. Molecules consisting only of carbon and hydrogen are called hydrocar- bons. Because the oxidation of hydrocarbon compounds re- sults in a net release of energy, hydrocarbons make good fuels such as propane gas (C3H8) . It consists of a chain of three carbon atoms with eight hydrogen atom bound to it. 1.3.1 Functional groups imgs/03.jpg Figure 1.3: The primary functional chemical groups. A functional group is a specific set of atoms or chemical bonds within an organic molecule that imparts distinctive chemical properties and reac- tivity to that molecule. Functional groups are re- sponsible for the chemical characteristics and in- teractions of organic molecules. Each functional group has a characteristic chemical structure and determines how the molecule will react with other molecules during chemical reactions. They provide functional characteristics to the compound. It de- scribes how the cloud of electrons disperses around the bond that forms due to the compound’s elec- tronegativity. Specifically, the carboxylic is acidic, the amino is neutral, while the hydroxyl and car- bonyl groups are polar. Figure 1.3 illustrates these biologically important functional groups and lists the macromolecules in which they are found. One such common functional group is —OH, called a hydroxyl group. Functional groups have definite chemical properties that they retain no matter where they occur. Both the hydroxyl and carbonyl (C=O) groups, for example, are polar because of the electronegativity of the oxygen atoms. Other common functional groups are the acidic carboxyl (COOH), the phosphate (PO4−), and the basic amino (NH2) groups. Many of these functional groups can also participate in hydrogen bonding 2.1. CARBOHYDRATES 15 imgs/Glucosio.png Figure 2.4: Structure of the glucose molecule. Glucose is a linear, 6-carbon molecule that forms a six- membered ring in solution. Ring closure occurs such that two forms can result: α-glucose and β-glucose. These structures differ only in the position of the —OH bound to carbon 1. The structure of the ring can be represented in many ways; shown here are the most common 2.1.1 Polisaccharides Disaccharides In order to talk about Polisaccharides is important a little part for disaccharides. Transport forms of sug- ars are commonly made by linking two monosaccharides together to form a disaccharide. Disaccharides serve as effective reservoirs of glucose because the enzymes that normally use glucose in the organism cannot break the bond linking the two monosaccharide subunits. Enzymes that can do so are typically present only in the tissue that uses glucose imgs/Disaccaridi.png Figure 2.5: Disaccharides Polysaccharides are longer polymers made up of monosaccharides that have been joined through dehydration reactions. Starch, a storage polysaccharide, consists entirely of α-glucose molecules linked in long chains. Cellulose, a structural polysaccharide, also consists of glucose molecules linked in chains, but these molecules are β-glucose. Because starch is built from α-glucose we call the linkages α linkages; cellulose has β linkages. 16 CHAPTER 2. MACROMOLECULES 2.2 Nucleic acids imgs/06.jpg Figure 2.6: The structure of a nucleic acid Nucleic acids carry information inside cells. Two main vari- eties of nucleic acids are deoxyribonucleic acid (DNA) and ri- bonucleic acid (RNA). Genetic information is stored in DNA, and short-lived copies of this are made in the form of RNA. Nucleic acids consist of long polymers of repeating subunits called nucleotides. Each nucleotide consists of three com- ponents: 1. a pentose, or 5-carbon, sugar; 2. a phosphate (−PO− 4 ) group; 3. an organic nitrogen-containing, or nitrogenous, base. In DNA, the sugar is deoxyribose and in RNA it is ri- bose. Polynucleotides are formed by joining the phosphate of one nucleotide to a hydroxyl group on the sugar of another nucleotide in a dehydration reaction. We call this a phospho- diester bond because the two sugars are linked by ester bonds to a phosphate. Nucleotides have five types of nitrogenous bases. Two of these are large, double-ring molecules called purines that are each found in both DNA and RNA: the two purines are adenine (A) and guanine (G). The other three bases are single-ring molecules called pyrimidines that in- clude cytosine (C, in both DNA and RNA), thymine (T, in DNA only), and uracil (U, in RNA only). When a nucleic acid polymer is formed, the phosphate group of one nucleotide binds the hydroxyl group of the pentose sugar of another nucleotide releasing water and forming a phosphodiester bond, via a dehydration reaction. 2.2.1 DNA DNA molecules in organisms exist as two polynucleotide chains wrapped about each other in a long linear molecule in eukaryotes, and a circular molecule in most prokaryotes. Such a spiral shape is called a helix, and a helix composed of two chains is called a double helix. Each step of DNA’s helical staircase is composed of a base-pair. The pair consists of a base in one chain attracted by hydrogen bonds to a base opposite it on the other chain. The base-pairing rules arise from the most stable hydrogen bonding configurations between the bases: Adenine pairs with thymine (in DNA) or with uracil (in RNA) with the formation of two hydrogen bounds, and cytosine pairs with guanine with three hydrogen bounds. The bases that participate in base-pairing are said to be complementary to each other. Organisms use sequences of nucleotides in DNA to encode the information specifying the amino acid sequences of their proteins. This method of encoding information is very similar to the way in which sequences of letters encode information in a sentence. A sentence written in English consists of a combi- nation of the 26 different letters of the alphabet in a certain order; the code of a DNA molecule consists of different combinations of the four types of nucleotides in specific sequences, such as CGCTTACG. DNA molecules in organisms exist as two polynucleotide chains wrapped about each other in a long linear molecule in eukaryotes, and a circular molecule in most prokaryotes. The two strands of a DNA polymer wind around each other like the outside and inside rails of a spiral staircase. Such a spiral shape is called a helix, and a helix composed of two chains is called a double helix. Each step of DNA’s helical staircase is composed of a base-pair. The pair consists of a base in one chain attracted by hydrogen bonds to a base opposite it on the other chain 2.2. NUCLEIC ACIDS 17 imgs/07.jpg (a) The structure of DNA imgs/Nucleotide.png (b) The structure of a Nucletide 2.2.2 RNA imgs/Purine.png Figure 2.8: The structure of a nucleic acid As we know the DNA forms a double helix and uses desoxyribose as the sugar in its sugar- phosphate backbone, and use thymin among its nitrogenus base. RNA is usually single stranded, uses ribose as the sugar in its sugar- phosphate backbone, and uses uracil in place of thymine. RNA is similar to DNA but with two major chemical differences. First, RNA molecules con- tain ribose sugar, in which the C-2 is bounded to hydroxyl group. (In DNA, a hydrogen atom replaces this hydroxyl group). Uracil is used in place of thymin as we said and has a similar struc- ture, except that one of its carbons lacks a methyl (−CH3 group). RNA is present in human and animal cells and its role is multiple: • mRNA: it is a single-stranded RNA molecule that carries genetic information from the DNA in the cell’s nucleus to the ribosomes, where protein synthesis occurs. It serves as a template for protein synthesis, providing the instructions for the sequence of amino acids that make up a protein. mRNA is transcribed from a gene in the DNA during a process called transcription and is then translated into a protein during translation. • tRNA: smaller RNA molecule that plays a crucial role in protein synthesis. It has a unique three- dimensional cloverleaf-shaped structure. Each tRNA molecule carries a specific amino acid and contains an anticodon region, which can base-pair with the complementary codon on the mRNA during translation. tRNA ”transfers” the correct amino acid to the growing polypeptide chain at the ribosome, ensuring that the protein is assembled in the correct order. • rRNA: is a major structural component of ribosomes, which are cellular organelles responsible for protein synthesis. It makes up a significant part of the ribosome’s structure, along with proteins. 20 CHAPTER 2. MACROMOLECULES • Polar uncharged amino acids, such as threonine, have R groups that contain oxygen (or —OH). • Charged amino acids, such as glutamic acid, have R groups that contain acids or bases that can ionize. • Aromatic amino acids, such as phenylalanine, have R groups that contain an organic (carbon) ring with alternating single and double bonds. These are also nonpolar • Other Amino acids that have special functions have unique properties. Some examples are methio- nine, which is often the first amino acid in a chain of amino acids; proline, which causes kinks in chains, if ther’s a proline stretch is going to be a foding; and cysteine, which links chains together. Each amino acid affects the shape of a protein differently, depending on the chemical nature of its side group. For example, portions of a protein chain with numerous nonpolar amino acids tend to fold into the interior of the protein by hydrophobic exclusion Peptide Bond In addition to its R group, each amino acid is ionized at physiological pH, with a positive amino (NH+ 3 ) group at one end and a negative carboxyl (COO−) group at the other. The amino groups on a pair of amino acids can undergo a dehydration reaction to form a covalent bond. The covalent bond that links two amino acids is called a peptide bond. imgs/PeptideBond.png Figure 2.11: Legame Peptidico The two amino acids linked by such a bond are not free to rotate around the N—C linkage because the peptide bond has a partial double-bond character. This is different from the N—C and C—C bonds to the central carbon of the amino acid. This lack of rotation about the peptide bond is one factor that determines the structural character of the coils and other regular shapes formed by chains of amino acids. A protein is composed of one or more long unbranched chains. Each chain is called a polypeptide and is composed of amino acids linked by peptide bonds. The terms protein and poly-peptide tend to be used loosely and may be confusing. For proteins that include only a single poly-peptide chain, the two terms are synonymous. Although many different amino acids occur in nature, only 20 commonly 2.3. PROTEINS 21 occur in proteins. Of these 20, 8 are called essential amino acids because humans cannot synthesize them and thus must get them from their diets: imgs/AminoTable.png Figure 2.12: Tabella Amino Acidi Obviously is not important to know every single Amino Acid, but it’s important to know their nature thanks to the side group. Indeed Every Amino Acid have the same backbone but they are differentiated by the R group. 22 CHAPTER 2. MACROMOLECULES 2.3.2 Proteins structres The shape of a protein determines its function. In every protein studied, essentially all the internal amino acids are nonpolar ones—amino acids such as leucine, valine, and phenylalanine. Water’s tendency to hydrophobically exclude nonpolar molecules literally shoves the nonpolar portions of the amino acid chain into the protein’s interior. This tendency forces the nonpolar amino acids into close contact with one another, leaving little empty space inside. The structure of proteins is usually discussed in terms of a hierarchy of four levels: primary, secondary, tertiary, and quaternary. Primary structure The primary structure of a protein is its amino acid sequence. The N-terminal amino-acid is written on the left side whereas the C-terminal amino-acids is written on the right side. The R groups that distinguish the amino acids play no role in the peptide backbone of proteins, a protein can consist of any sequence of amino acids. Thus, because any of 20 different amino acids might appear at any position, a protein containing 100 amino acids could form any of 20100 different amino acid sequences. This important property of proteins permits great diversity. Theoretical number of different polypetides: 20n where: 20 is number of different amino acids and n is lenght of polypeptide. Secondary structure Secondary structure results from hydrogen bonds forming between nearby amino acids. This produces two different kinds of structures: • beta pleated sheets: a hydrogen bounds between aligned laterally. • alpha-helix : a hydrogen bonds between peptides folded in a spiral. It is common in fibrous structural proteins (ketamine) for almost the entire length of the polypeptide. The bonds are between H of a group C = O and the group N −H of the next turn of the helix, at 4 amino acids distance These two kinds of secondary structure create regions of the protein that are cylindrical (α helices) and planar (β sheets). A protein’s final structure can include regions of each type of secondary structure. Tertiary structure The final folded shape of a globular protein is called its tertiary structure. This tertiary structure contains regions into particular secondary structure and determines how these are ordered in space to produce the general structure. The tertiary structure is stabilized by a number of forces including hydrogen bonding between R groups of different amino acids, electrostatic attraction between R groups with opposite charge (also called salt bridges), hydrophobic exclusion of nonpolar R groups, and covalent bonds in the form of disulfides. imgs/14.png Figure 2.13: Interactions that contribute to a protein’s shape. 2.3. PROTEINS 25 Effect of a point mutation on protein structure: sickle cell anemia The substitution of an amino acids with charged amino acids (Glu) diferent polarity/electrical charge characteristics or with steric hindrance can affect the structure and function of a protein. A point mutation in the gene that codes for hemoglobin causes sickle cell anemia. This mutation changes one amino acid in the hemoglobin protein chain, causing the normally biconcave red blood cells to assume a crescent or sickle shape. imgs/16.png Figure 2.16: Sickle cell anemia 26 CHAPTER 2. MACROMOLECULES 2.4 Lipids Lipids are a somewhat loosely defined group of molecules with one main chemical characteristic: They are insoluble in water but soluble in organic solvents (alcohol, ether). They are the main storage reserve of the body and they are the chief concentrated storage form of energy forming about 3.5% of the cell content. In fact, lipids are constituents of membrane structure and regulate the membrane permeability. They serve as source of fat soluble vitamins. Lipids are important cellular metabolic regulators. They protect the internal organs and serve as insulating materials. From a chemistry point of view, lipids are hydrocarburs insoluble in water due to their numerous non polar covalent bonds C-H and they cannot constitute polymers because they are not composed of repeated units (monomers) linked together in a long chain. Lipids can be oils and fats: Oils such as those from olives, corn, and coconut are also lipids, as are waxes such as beeswax and earwax. Fats are solid oil fluid they differ in their state We can classify lipids as in the picture 2.17. imgs/Lipids.png Figure 2.17: Lipids classify 2.4.1 Simple lipids Simple lipids are esters of fatty acids within alcohol. They are two type: 1. Neutral or true fats: esters of fatty acids with glycerol. 2. Waxes: esters of fatty acids with alcohol other than glycerol. True fats are made up of C, H and O but O is less. A fat molecule is made up of two components: Glycerol and fatty acid. Fatty acids are long-chain hydrocarbons with a carboxyl group (COOH) at one end. Glycerol is a 3-carbon polyalcohol (three −OH groups). imgs/glycerol.jpg Figure 2.18: The structure of glycerol The fatty acid consists of a long unbranched hydrocarbon chain at the end of which there is carboxyl (−COOH) group. If all of the internal carbon atoms in a fatty acid chain are bonded to two hydrogen atoms, we call this saturated. A saturated fat is composed of triglycerides that contain three saturated fatty acids (the kind that have no double bonds). A saturated fat therefore has the maximum number of hydrogen atoms bonded to its carbon chain. Most animal fats are saturated.. The molecules are rigid and pack together. Fats rich in saturated fatty acids tend to be solid at room temperature. A fatty acid with double bonds between one or more pairs of successive carbon atoms will have fewer hydrogen atoms, and thus is said to be unsaturated. Unsaturated fat is composed of triglycerides 2.4. LIPIDS 27 that contain three unsaturated fatty acids (the kind that have one or more double bonds). These have fewer than the maximum number of hydrogen atoms bonded to the carbon chain. The double bonds introduce kinks (pieghe) in the molecule. The folds prevent unsaturated fat molecules from aligning with adjacent molecules. Lipids containing unsaturated fatty acids tend to be fluid at room temperature (oils). Remember that the carbon is thtravalent, so it always needs 4 bonds. imgs/AcidiGrassi.jpg Figure 2.19: The structure of Fatty Acid imgs/S&U lipids.png Figure 2.20: Saturated and unsaturated fats Triglycerides imgs/MDT.jpg Triglycerides are the most commonly used glycerides present in animal and vegetable fats. They made up of a molecule of glycerol (three carbon alcohol) combined with one, two or three molecules of fatty acids. The three fatty acids of a triglyceride need not be identical, and often they are very different from one another. The hydrocarbon chains of fatty acids vary in length. The most common are even-numbered chains of 14 to 20 carbons. The many C—H bonds of fats serve as a form of long-term energy storage. 30 CHAPTER 2. MACROMOLECULES Lipoprotein Lipoproteins are complex molecules that play a critical role in transporting lipids (fats) in the blood- stream. They consist of a combination of lipids (mainly triglycerides and cholesterol) and proteins (known as apolipoproteins). Lipoproteins serve as vehicles for transporting hydrophobic (water-repellent) lipids through the hydrophilic (water-based) environment of the bloodstream. There are five types of lipoproteins, each with distinct functions and compositions, classified according to their functional and physical properties: 1. Chylomicrons are the largest and least dense lipoproteins. They transport dietary fats (triglyc- erides) from the intestines to various tissues, including the liver and adipose tissue, for storage or energy use. 2. Very Low-Density Lipoproteins (VLDL) transport triglycerides synthesized in the liver to other tissues. 3. Low-Density Lipoproteins (LDL) are often referred to as ”bad cholesterol” because they pri- marily carry cholesterol from the liver to peripheral tissues. High levels of LDL cholesterol in the blood are associated with an increased risk of atherosclerosis and cardiovascular disease. 4. High-Density Lipoproteins (HDL): HDLs are often referred to as ”good cholesterol” because they have a protective role in cardiovascular health. HDLs transport cholesterol from peripheral tissues back to the liver for excretion or reprocessing. 5. Free fatty acid albumin complex Lipoproteins are globular, micelle-like particles consisting of a hydrophobic core of triacylglycerols and cholesterol esters surrounded by an amphipathic coat of protein, phospholipid, and cholesterol. The major function of lipoproteins is to transport triacylglycerols, cholesterol, and phospholipids around the body. The apolipoproteins (apoproteins) on the surface of the lipoproteins help to solubilize the lipids and target the lipoproteins to the correct tissues. imgs/Lipoprotein.png Figure 2.23: Lipoprotein 2.4. LIPIDS 31 2.4.3 Derived lipids Derivative lipids, also known as derived lipids, are a category of lipids that are obtained through the hydrolysis or chemical modification of simple and complex lipids. They are often derived from more complex lipid molecules and serve various biological functions. Some well-known derivative lipids include steroids, terpenes, and prostaglandins Steroid Steroids are a class of lipid derivatives characterized by a four-ring hydrocarbon structure.The steroids do not contain fatty acids but are included in lipids as they have fat-like properties. They play crucial roles in various physiological processes. Common steroids include cholesterol, which is a component of cell membranes and a precursor for the synthesis of other steroids, such as sex hormones (e.g., testosterone and estrogen) and corticosteroids (e.g., cortisol), and vitamin D. The most common steroids are sterols Cholesterol Cholesterol is a constituent of biological membranes and regulates their fluidity. Reduces membrane fluidity at moderately elevated temperature by reducing phospholipid movement. On the contrary, at lower temperatures, it prevents the solidification of the membrane by reducing the regular packing of phospholipids. imgs/cholesterol.png Figure 2.24: Cholesterol & membrane fluidity Terpen Terpenes are hydrocarbons derived from a combination of isoprene units. They are widely found in nature and have various functions. Some terpenes are involved in the formation of pigments, such as chlorophyll in plants, while others serve as essential components of the respiratory chain in cellular respiration (e.g., ubiquinone). Terpenes are also responsible for the aromas and flavors of many plants and fruits. Vitamins A, E and K contain a terpenoid called phytol. Carotenoids are pigments found in plants and are precursors for the synthesis of vitamin A (retinol) in the human body. Lycopene is a red pigment found in tomatoes and several other red and pink fruits. Gibberellins are indeed terpenes, and they play a crucial role in plant growth and development. 32 CHAPTER 2. MACROMOLECULES 3.1. VARIABILITY OF CELLULAR DIMESIONS 35 3.1 Variability of Cellular Dimesions The dimension of the cell is not fixed. They are usually microscopoe in size. Altough there are exception. For example a typical eukaryotic cell has a diameter between 10 and 100 micrometers (um), while most prokaryotic cells have a diameter between 1 and 10 um. • Animal cells are smaller that Plant cells (10-40 um vs. 100-120 um) • Bacteria are even smaller (0.1-0.2 um) and their dimensions are just at the limit fo resolution of an optic microscope imgs/scala.png The eye has a limit to see small objects, but how can we see cells if they are too small. The res- olution is the minimum distance that two objects have in order to be identified and can be seen as separate When two objects are closer than about 100 micrometer the light reflected from each them hits the same photoreceptor in the back of the eye. Only when objects are further than 100 um away the light will hit different cells, allowing the eye to perceive them as distinct. In order to solve this problem we introduced the microscope: • The optical microscope resolves objects separated by at least 200nm • Electronic transmission microscope re- solves objects separated by only 0.2nm • Scanning electronic microscope resolve objects separated by only 0.2nm but provide 3D images which improve our understanding of biological and physical phenomena. 36 CHAPTER 3. THE CELL 3.2 The surface area to volume ratio Most cells are relatively small for reasons related to the diffusion of substances into and out of them. The rate of diffusion is affected by a number of variables, including (1) surface area available for diffusion, (2) temperature, (3) concentration gradient of diffusing substance, and (4) the distance over which diffusion must occur. As the size of a cell increases, the length of time for diffusion from the outside membrane to the interior of the cell increases as well. Larger cells need to synthesize more macromolecules, have correspondingly higher energy requirements, and produce a greater quantity of waste. Molecules used for energy and biosynthesis must be transported through the membrane. Any metabolic waste produced must be removed, also passing through the membrane. The rate at which this transport occurs depends on both the distance to the membrane and the area of membrane available. For this reason, an organism made up of many relatively small cells has an advantage over one composed of fewer, larger cells. imgs/Ratio.png The advantage of small cell size is readily ap- parent in terms of the surface area-to-volume ra- tio. As a cell’s size increases, its volume increases much more rapidly than its surface area. The cell surface provides the only opportunity for interac- tion with the environment, because all substances enter and exit a cell via this surface. • More cells are small for reasons related to the diffusion of vital substances into and out of the cell • As the size of the cell increases, the diffusion time also increases • The degree of diffusion is influenced by num- ber of variables, including: 1. the temperature 2. The concentration gradient of the sub- stances that must diffuse 3. The surface area available for diffusion 4. The distance over which diffusion must occur Before it goes too big, a cell divide itself; if a cell size increases, the surface area to volume ratio decreases and it can lead to the death of the cell. • Large cell: It takes more time for the nutrients to reach the center of the cell. • Smaller cell: It takes less tiem for the same nutrients to reach the center of the cell. Generally Larger cellsmust synthesize more macro-molecules. have greater energy demands and produce greater quantities of waste. Molecules used for energy and biosynthesis must be transported across the membrane. The speed with which this can happen depends on the distance of these molecules from the membrane and the available area For this reason an organism made up of many relatively small cells has an advanatage over an organism made up of a few large cells. 3.3. ADAPTIVE MECHANISMS OF LARGE CELLS 37 3.3 Adaptive mechanisms of large cells The cell give rise to adaptive mechanism when its size increases as in the muscle tissue. There are more nucleus per cell. Plurinucleotide cells can produce biomolecules sufficient for energetic request. imgs/MuscularTIssue.png Figure 3.2: Muscular Tissue A second adaptive mechanism of large cells are neurons. They are made up of central body and axon and receptor. It is strict in order that all points of the membrane are at the same distance, so the space of the diffusion is restrict. imgs/Neurone.png Figure 3.3: Celebral cell 40 CHAPTER 3. THE CELL 3.5.1 The Genetic Material Every cell contains DNA, the hereditary molecule. In prokaryotes, the simplest organisms, most of the genetic material lies in a single circular molecule of DNA. It typically resides near the center of the cell in an area called the nucleoid. This area is not segregated, however, from the rest of the cell’s interior by membranes. By contrast, eukaryotic cells, found in more complex organisms, have DNA seg- regated into a nucleus, which is surrounded by a double-membrane structure called the nuclear envelope. In both types of organisms, the DNA contains the genes that code for the proteins synthesized by the cell. In addition to the main DNA, bacteria can contain small circular DNA molecules, called plasmids, which code for catabolic enzymes, for resistance to antibiotics or linked to mechanisms for the exchange of genetic material between organisms. 3.5.2 The cell Wall Most prokaryotic cells have a cell wall outside the membrane, with a support and protection function, preventing their explosion due to osmotic pressure. The cell wall is made up of peptidoglycan, a complex polymer of amino sugars linked to short polypep- tides, forming a single molecule with immunogenic potential. The peptidoglycan is a vital component of bacterial cell walls that provides structural support, protection, and mechanical strength to the cell. It also serves as a target for antibiotics and plays a role in bacterial shape determination, cell division, and immune recognition. Its importance lies in its multifaceted contributions to bacterial physiology and survival. imgs/peptidoglicano.png Figure 3.5: Molecola di Peptidoglicano There are two kind of cell wall: • Gram-positive cell wall: thick peptidoglycan layer • Gram-negative cell wall: thin peptidoglycan layer Some bacteria have a mucilaginous capsule polysaccharides with funxtions of: • Protection from phagocitosis 3.5. PROKARYOTES: ARCHAEBACTERIA AND EUBACTERIA 41 • adhesion • anti-dehydration (hydrophilic sugars) Internal membranes, invaginations of the plasma membrane (photosynthetic bacteria). Some prokaryotes move by means of rotating flagella. imgs/Flagella.png (a) Flagella imgs/Flagella2.png (b) Flagella Flagella (singular, flagellum) are long, threadlike structures protruding from the surface of a cell that are used in locomotion. Prokaryotic flagella are protein fibers that extend out from the cell. There may be one or more per cell, or none, depending on the species. Prokaryotes categories based on its shape • Cocci(spherical): Diplococcus, Staphilococcus • Bacilli • Vibrios • Spirilli • Spirochete Metabolic Characteristic Of Bacteria Bacteria are chmosyntetic and photosyntetic autotrophs, heterotrophic, aerobic and facultative and ob- ligate anaerobic. 3.5.3 Asexual reproduction Prokaryotes reproduction occurs via binary fission, a process that produces two identical daughter cells as in the picture 3.7: The main difference between Archaeabacteria and Eubacteria are the following: • Archaea – without peptidoglycan – Metanogeni – Lipidi con legame etere – Insensibili alla rifamicina – Vivono in condizioni simili a quelle della terra primitiva – Alofili, termofili • Bacteria – Cell wall enriched for peptidoglycan – Sensibili alla rifamicina che blocca la trascrizione – Lipidi con legame estere 42 CHAPTER 3. THE CELL imgs/ReproductionP.png Figure 3.7: Prokaryotes Reproduction 3.6 Eukaryotes and their cell structure Eukaryotic cells (figures 4.6 and 4.7) are far more complex than prokaryotic cells. The hallmark of the eukaryotic cell is compartmentalization. This is achieved through a combination of an extensive endomem- brane system that weaves through the cell interior and by numerous organelles. These organelles include membrane-bounded structures that form compartments within which multiple biochemical processes can proceed simultaneously and independently. imgs/TabellaDifferenzeProkEuk.png Figure 3.8: Principali differenze tra i due tipi di cellule Summarizing all cells share numerous basic structure: • A membrane • The cytosol • The chromosomes (Packaged DNA) • Ribosomes 3.6. EUKARYOTES AND THEIR CELL STRUCTURE 45 3.6.4 The nuclear envelop The surface of the nucleus is bounded by two phospholipid bilayer membranes, which together make up the nuclear envelope. The outer membrane of the nuclear envelope is continuous with the cytoplasm’s interior membrane system, called the endoplasmic reticulum. Scattered over the surface of the nuclear envelope are what appear as shallow depressions in the electron micrograph but are in fact structures called nuclear pores. The pore allows ions and small molecules to diffuse freely between nucleoplasm and cytoplasm, while controlling the passage of proteins and RNA–protein complexes. Transport across the pore is controlled and consists mainly of the import of proteins that function in the nucleus, and the export to the cytoplasm of RNA and RNA–protein complexes formed in the nucleus. The inner surface of the nuclear envelope is covered with a network of fibers that make up the nuclear lamina. This is composed of intermediate filament fibers called nuclear lamins. Lamina is important to protect, is a shell, in order to give rigidity to the nucleus , in order to protect the genetic material. Nuclear Pore They are organized in a circle with a large central hole. The complex allows small molecules to diffuse freely between the nucleus, plasma and cytoplasm and also controls the passage of proteins in RNA- protein complexes. The passage si primarily restricted to two types of molecules: (1) proteins, which move into the nucleus to be incorporated into nuclear structures or to catalyze particular activities in the nucleus, and RNA and RNA- protein complexes, formed in the nucleus and exported to the cytoplasm. 3.6.5 The Endomembrane System The interior of a eukaryotic cell is packed with membranes that form an elaborate internal, or endomem- brane, system. This endomembrane system fills the cell, dividing it into compartments, channeling the passage of molecules through the interior of the cell, and providing surfaces for the synthesis of lipids and some proteins. The endomembrane system in eukaryotic cells is one of the fundamental distinctions between eukaryotes and prokaryotes. 46 CHAPTER 3. THE CELL 3.6.6 The endoplasmic reticulum (ER) The endoplasmic reticulum (ER) is the largest internal membrane. The ER is composed of a phos- pholipid bilayer embedded with proteins. The ER has functional subdivisions, described here, and forms a variety of structures, from folded sheets to complex tubular networks. It may be: imgs/ER.png Figure 3.12: Endoplasmic Reticulum • smooth: builds lipids and carbahydrates • rough: stores proteins made by attached ribosomes The ER may also be connected to the cytoskeleton, which can affect ER structure and growth. The two largest compartments in eukaryotic cells are the space inside the ER, called the cisternal space, or lumen, and the region exterior to it, the cytosol, which is the fluid component of the cytoplasm containing dissolved organic molecules and ion The rough ER The rough ER is made up of cisternae on whose surface the ribosomes are attached. It generally represents 2/3 of the endoplasmic reticulum and is fundamentally used for the synthesis of protein material that the cell sheds outside and which therefore serves for the general economy of the organism. gets its name from the pebbly appearance of its surface. The RER is not easily visible with a light microscope, but it can be seen using the electron microscope. It appears to consist primarily of flattened sacs with surfaces made bumpy by riboomes. he proteins synthesized on the surface of the RER are destined to be exported from the cell, sent to lysosomes or vacuoles (described later in this section), or embedded in the plasma membrane. These proteins enter the cisternal space as a first step in the pathway that will sort proteins to their eventual destinations. Region associated with protein biosynthesis and represents the starting point of the secretory pathway It is the site of the synthesis of all transmembrane and luminal proteins of the secretory pathway 3.6. EUKARYOTES AND THEIR CELL STRUCTURE 47 In synthesis: • Synthesis of secretory proteins • Synthesis of whole membrane proteins • Post-translational modifications of proteins (e.g. Glycosylation) • Protein folding • Quality control • Biosynthesis of membranes The smooth ER The smooth ER are regions of the ER with relatively few bound ribosomes. The membranes of the SER contain many embedded enzymes, involved in the synthesis of a variety of carbohydrates and lipids. Steroid hormones are also synthesized in the SER. The majority of membrane lipids are assembled in the SER and then sent to whatever parts of the cell need membrane components. Membrane proteins in the plasma membrane and other cellular membranes are inserted by ribosomes on the RER. It appears to be a tubular system, rather than a set of flat sacs, which are instead typical of the Rough ER. It constitutes about 1/3 of the ER and performs many complex functions: communication with the outside, communication between endoplasmic rough reticulum and Golgi apparatus It is a reservoir of Ca2+ and calcium binding proteins as signal molecule (muscle contraction), and in the liver, enzymes found in smooth endoplasmic reticulum are reshonsible of detoxification. Smooth ER produces almost all major lipids, Synthesis occurs in the cytosolic half of the membrane through specific enzymes. The smooth ER membrane contains phospholipid movers (flippers); in the ER occurs the synthesis of steroid hormones from cholesterol. imgs/SmoothRough.png Figure 3.13: Smooth and Rough Reticulum It is reached by several proteins with catalizer function, it is a reservoir of calcium è un secondo messaggero per regolare le funzioni biologiche, anche per le protein che lo regolano perché è un cofattore di proteine, Per far si che i filamenti possono scivolare tra loro il calcio deve essere presente THE SMOOTH ER IS THE SITE FOR DETOXIFICATION REACTIONS Reactions are performed by the cytochrome P450 enzyme (oxygenase) family. Reactions allow detoxifi- cation from organic substances that are transformed from hydrophobic to hydrophilic 50 CHAPTER 3. THE CELL The individual stacks of membrane are called cisternae (Latin, “collecting vessels”), and they vary in number within the Golgi body from 1 or a few in protists to 20 or more in animal cells, and to several hundred in plant cells. In vertebrates, individual Golgi are linked to form a Golgi ribbon. Materials arrive at the cis face in transport vesicles that bud off the ER and exit the trans face, where they are discharged in secretory vesicles The proteins and lipids synthesized on the membranes of the smooth and rough ER are transported to the Golgi apparatus and modified upon passage within it. In many cases, the enzymes of the Golgi apparatus modify pre-existing glycoproteins and glycolipids, produced in the ER by cleavage of a sugar from a chain or by modifying one or more sugars. 3.6.9 Lysosomes Lysosoms contain digestive enzyme (protein with a catalytic activity). They are part of endomembranus system. They are crucial to catalyze protein, nucleic acids and carbohydrates. Throughout the lives of eukaryotic cells, lysosomal enzymes break down old organelles and recycle their component molecules, allowing room for newly formed organelles. For example, mitochondria are replaced in some tissues every 10 day. Lysosomes era activated upon their fusion with the vescicles to be digested, which result from phagocytosis (a specific type of endocytosis) or by fusing with an old ordamaged organelle. imgs/Lysosoms.png Figure 3.18: Lysosom 3.6. EUKARYOTES AND THEIR CELL STRUCTURE 51 The fusion event activates the proton pumps present in the lysosomal membranes, resulting in a reduction in the internal pH. When the internal pH decreases, the arsenal of digestive enzymes contained in the lysosomes become actived and this leads to the degradation of the embedded macomolecules or organelle. imgs/BacteriaEndo.png Figure 3.19: Fagocitosi In particular: • Autophytosis (Autophagy): is a cellular process that involves the degradation and recycling of cellular components, such as damaged organelles or proteins. It is a crucial mechanism for maintaining cellular homeostasis and ensuring the removal of dysfunctional cellular structures. • Endocytosis: is a cellular process that involves the uptake of extracellular material by the cell through the formation of vesicles derived from the cell membrane. There are several types of endocytosis, Phagocytosis and Pinocytosis (or fluid-phase endocytosis): Uptake of dissolved solutes and fluids • Phagocytosis is a specific type of endocytosis in which cells, primarily immune cells like macrophages and neutrophils, engulf and digest large particles, such as bacteria, dead cells, or other foreign sub- stances. 52 CHAPTER 3. THE CELL imgs/Mitochondrion.png (a) Mitochondrion imgs/MitoAlto.png (b) Mitochondrion 3.6.10 Mitochondrion The Mitochondrion is useful to develop all chemical reaction and all functional activities of a cell. We have as number of mitochondrion as the energy we need. They’re small diameter about 0.5 to 3 um x 1.5 um and bounded by two membranes: a smooth outer membrane, and an inner folded membrane with numerous contiguous layers called cristae. Mitochondria represent the energy center of the cell; without them eukaryotes would depend exclusively on glycolysis for energy production. Mitochondria divide like bacteria and can change position, shape and number within the cells accordin to the tissue energy and their internal structure is preserved to ensure the production of ATP. The Glycolysis occurs in the cytosol. The subsequent oxidation of pyruvate occurs within the mito- chondria: 3.6. EUKARYOTES AND THEIR CELL STRUCTURE 55 3.6.11 Peroxisomes Another important cell structure is the peroxisomes that are microbodies and they are important for the synthesis for glucose starting by constituents. Thy are micorbodies of 0.5-1 um of diameters and they’re delimited by a single membrane and they’re rich in enzymes such as peroxidase and catalose, D amino acid, oxidase, uricoxidase. They partecipate in the formation of a-keto acids and play an important role in gluconeogenesis startin form lipids and other non-carbohydrate precursors. 3.6.12 Centrioles Centrioles are organelles that constitute the kinetic center of the cell and regulate both its movements towards the environment (cilia, flagella) and the movements that occur within it (movement of chromo- somes). A eukaryotic cell does not produce brand-new mitochondria each time the cell divides. Instead, the mitochondria themselves divide in two, doubling in number, and these are partitioned between the new cells. Most of the components required for mitochondrial division are encoded by genes in the nucleus and are translated into proteins by cytoplasmic ribosomes. Mitochondrial replication is, therefore, impossible without nuclear participation, and mitochondria thus cannot be grown in a cell-free culture. Together with an electron-dense material surrounding them, called ”pericentriolar material” (PCM), they constitute the centrosome, the most important”microtubule organizing center” (MTOC) for the cell. During cell division the centrioles duplicate, but remain united into a single centrosome. At the end of cell division the two pairs separate, migrating to opposite poles of the cell and giving rise to two distinct centrosomes. imgs/Centrioli.png Figure 3.22: Centrioli 56 CHAPTER 3. THE CELL imgs/Citoscheletro.png (a) Citoscheletro imgs/Microtubuli.png (b) Microtubuli 3.6.13 Citoskeleton Remembering that the Cytosol It’s the part of the cytoplasm that remains if we remove the plasma membrane and the membranous organelles and It’s comparable to an aqueous gel in which many small and large molecules are immersed. Let’s itroduce the Cytoskeleton The cytoplasm of all eukaryotic cells is crisscrossed (Incrociato) by a network of protein fibers that sup- ports the shape of the cell and anchors organelles to fixed locations. This network, called the cytoskeleton, is a dynamic system, constantly assembling and disassembled. The Cytoskeleton is made up of microtubules and gives the shape (can be very complex) and movement to the cell. It is involved in the special organization of the cytoplasm; important for segregation of chromosomes during cytokinesis cell division. It gives a high level of internal organization to the cell. Eukaryotic cells may contain the following three types of cytoskeletal fibers, each formed from a different kind of subunit (that don’t work alone but they’re associated with accessory proteins, essential fot the assembly of cytoskeleton structures and their functioning): • actin filaments, sometimes called microfilaments: Actin filaments are long fibers about 7 nm in diameter. Each filament is composed of two protein chains loosely twined together like two strands of pearls . Each “pearl,” or subunit, on the chain is the globular protein actin. Actin filaments exhibit polarity—that is, they have plus (+) and minus (-) ends. These designate the direction of growth of the filaments. Actin molecules spontaneously form these filaments, even in a test tube • microtubules: Microtubules, the largest of the cytoskeletal elements, are hollow tubes about 25 nm in diameter, each composed of a ring of 13 protein protofilaments (figure 4.19). Globular proteins consisting of dimers of α and β -tubulin subunits polymerize to form the 13 protofila- ments. The protofilaments are arrayed side by side around a central core, giving the microtubule its characteristic tube shape. • intermediate filaments: They are characteristically 8 to 10 nm in diameter—between the size of actin filaments and that of microtubules. Once formed, intermediate filaments are stable and usually do not break down. In summary cytoskeleton gives: • Spatial organization of the cytoplasm • Intracellular movement of organelles • Segregation of chromosomes during cytokinesis cell division 3.7. THE PLANT CELL 57 • Cell movement on the substate • Muscle contraction TIPICAL OF EUKARYOTIC CELLS. 3.7 The Plant Cell The plant cell is a type of eukaryotic cell, with several peculiarities that differentiate it from animal cells, fungal cells and other living kingdom cells: • The presence of the cell wall made up of (a polymer whose elementary unit isglucose, proteins and, as a result of modifications, lignin, suberin...) and its related plasmodesmas, channels in the cell wall through which plant cells arein communication with eachother. • Plastids, and especially chloroplasts which, thanks to chlorophyll, enable plant cells to produce sugar (glucose) and oxygen monomers from CO2 using solar energy, this process is defined as chlorophyll photosynthesis. • The characterizing presence of numerous vacuoles (not organelles but endocellular cavities, wrapped by a membrane, called tonoplast) that occupy a large part of the cell and whose main functions is to maintain the cell turgor. They are involved in the control of the passage of molecules from the lymph to the cytosol, in the maintenance of the optimal pH of the cytosol and perform reserve functions of various substances. imgs/PlantCell.png Figure 3.24: Cellula di una pianta 60 CHAPTER 3. THE CELL CHAPTER 4 BIOLOGICAL MEMBRANES The plasma membrane, also known as the cell membrane, is a fundamental component of all biological cells. The plasma membrane is primarily composed of a phospholipid bilayer embedded with various proteins and other molecules. The plasma membrane has several critical functions in the cell: 1. Selective Permeability: One of the most crucial roles of the plasma membrane is to regulate the passage of substances in and out of the cell. 2. Cell Signaling: Membrane proteins receive signals from the external environment or neighboring cells and transmit them into the cell, initiating various cellular responses. 3. Transport: Integral membrane proteins, including channels and transporters, facilitate the move- ment of ions and molecules across the membrane. 4. Flexibility: The plasma membrane allows the cell to move, expand and change shape. 5. Protection: The membrane acts as a barrier that protects the cell’s contents from harmful substances and pathogens in the external environment. 6. Exocytosis and Endocytosis: The membrane is involved in processes like exocytosis (release of cellular products) and endocytosis (uptake of materials into the cell) through vesicle formation and fusion with the membrane. imgs/3/3.3.jpg Figure 4.1: The plasma membranes Membranes, whatever their cellular location, are all composed of lipids and proteins. The lipids arrange themselves to form a lipid double layer which constitutes: basic structure and barrier im- permeable to most water-soluble molecules. Each membrane has its own set of proteins that allow it to carry out its numerous function. 61 62 CHAPTER 4. BIOLOGICAL MEMBRANES All lipid molecules in the plasma membranes are amphipathic molecules because they have hydrophilic or polar head (comes into contact with the aqueous solution ) and a hydrophobic or non polar tail (which avoids aqueous solution). The presence of these two components is crucial for the association of these lipids in bi-layers in aqueous environments. The shape and amphipathic nature of phospholipids allows them to spontaneously form lipid bi-layer in aqueous environments. imgs/3/3.4.png Figure 4.2: The shape of phospholipid in aqueous environments The lipid micelle can also act as a casket, keeping other molecules inside, performing the function of transporting molecules. They are important because they are able to cross the phospholipid bi-layer. An example is mRNA vaccines which use micelles to transport the gene sequence. The lipids of a membrane consist of: • Phospholipids (phosphatidilserine, phosphoglycerides and sphingolipids) • Sterols (cholesterol) • Glycolipids are lipids modified by the addition of carbohydrate. The most common are: cerebrosides and gangliosides. Protective effect on the apical membrane of epithelial cells (from changes in pH and/or degradation enzymes). They have also eletrical effect altering the eletric field and ion concetration. imgs/3/3.5.png Figure 4.3: The main component of plasma membrane 4.4. THE FLUID MOSAIC MODEL 65 imgs/3/3.10.jpg Figure 4.6: Different composition between two layer phospholipids 4.4 The fluid mosaic model The lipid layer that forms the foundation of a cell’s membranes is a bilayer formed of phospholipids. These phospholipids include primarily the glycerol phospholipids, and the sphingolipids such as sphingomyelin. An early model, proposed in 1935, portrayed the plasma membrane as a sandwich: a phospholipid bilayer between two layers of globular protein. In 1972, S. Jonathan Singer and Garth J. Nicolson revised the model in a simple but profound way: They proposed that the globular proteins are inserted into the lipid bilayer, with their nonpolar segments in contact with the nonpolar interior of the bilayer and their polar portions protruding out from the membrane surface. In this model, called the fluid mosaic model, a mosaic of proteins floats in or on the fluid lipid bilayer like boats on a pond. imgs/3/3.11.png Figure 4.7: The fluid mosaic model of cell membranes 66 CHAPTER 4. BIOLOGICAL MEMBRANES 4.5 Membrane proteins Although the lipid bilayer provides the basic structure of all cell membranes and serves as a permeability barrier to the hydrophilic molecules on either side of it, most membrane functions are carried out by membrane proteins. Membrane proteins serve many functions. Some transport particular nutrients, metabolites, and ions across the lipid bilayer. Others anchor the membrane to macromolecules on either side. Still others function as receptors that detect chemical signals in the cell’s environment and relay them into the cell interior, or work as enzymes to catalyze specific reactions at the membrane (4.8). Each type of cell membrane contains a different set of proteins, reflecting the specialized functions of the particular membrane. imgs/3/3.12.png Figure 4.8: Plasma membrane proteins have a variety of functions 4.5.1 Membrane proteins associate with the lipid bylayer in different ways Although the lipid bilayer has a uniform structure, proteins can interact with a cell membrane in a number of different ways.: A Many membrane proteins extend through the bilayer, with part of their mass on either side (4.9A). Like their lipid neighbors, these transmembrane proteins are amphipathic, having both hydrophobic and hydrophilic regions. Their hydrophobic regions lie in the interior of the bilayer, nestled against the hydrophobic tails of the lipid molecules. Their hydrophilic regions are exposed to the aqueous environment on either side of the membrane. 4.5. MEMBRANE PROTEINS 67 B Other membrane proteins are located almost entirely in the cytosol and are associated with the cytosolic half of the lipid bilayer by an amphipathic α helix exposed on the surface of the protein (4.9B). C Some proteins lie entirely outside the bilayer, on one side or the other, attached to the membrane by one or more covalently attached lipid groups (4.9C). D Yet other proteins are bound indirectly to one face of the membrane or the other, held in place only by their interactions with other membrane proteins (4.9D). imgs/3/3.13.png Figure 4.9: Membrane proteins can associate with the lipid bilayer in different ways Proteins that are directly attached to the lipid bilayer—whether they are transmembrane, associated with the lipid monolayer, or lipid-linked—can be removed only by disrupting the bilayer with detergents. Such proteins are known as integral membrane proteins. The remaining membrane proteins are classified as peripheral membrane proteins; they can be released from the membrane by more gentle extraction procedures that interfere with protein–protein interactions but leave the lipid bilayer intact. 4.5.2 Transmembrane Transport of Ions and Small Molecules In all cells, the plasma membrane forms the barrier that separates the cytoplasm from the exterior envi- ronment, thus defining a cell’s physical and chemical boundaries. By preventing the unimpeded movement of molecules and ions into and out of the cell, the plasma membrane maintains essential differences be- tween the composition of the extracellular fluid and that of the cytosol. All cellular membranes, both plasma membranes and organelle membranes, consist of a bilayer of phos- pholipids in which other lipids and specific types of proteins are embedded. It is this combination of lipids and proteins that gives cellular membranes their distinctive permeability qualities. If cellular membranes were pure phospholipid bilayers, they would be excellent chemical barriers, impermeable to virtually all ions, amino acids, sugars, and other water-soluble molecules. In fact, only a few gases and small, uncharged, water-soluble molecules can readily diffuse across a pure phospholipid bilayer (Figure 4.5.2). But cellular membranes must serve not only as barriers, but also as conduits, selectively transporting molecules and ions from one side of the membrane to the other. Energy-rich glucose, for example, must be imported into the cell, and wastes must be shipped out. 70 CHAPTER 4. BIOLOGICAL MEMBRANES Passive or Active Trensport In many cases, the direction of transport depends only on the relative concentrations of the solute on either side of the membrane. Substances will spontaneously flow “downhill” from a region of high concentration to a region of low concentration, provided a pathway exists. Such movements are called passive, because they need no additional driving force. If, for example, a solute is present at a higher concentration outside the cell than inside, and an appropriate channel or transporter is present in the plasma membrane, the solute will move into the cell by passive transport, without expenditure of energy by the membrane transport protein. All channels—and many transporters—act as conduits for such passive transport. To move a solute against its concentration gradient, however, a membrane transport protein must do work: it has to drive the flow of the substance “uphill” from a region of low concentration to a region of higher concentration. To do so, it couples the transport to some other process that provides an input of energy. The movement of a solute against its concentration gradient in this way is termed active transport, and it is carried out by special types of transporters called pumps, which harness an energy source to power the transport process (Figure 4.10). As discussed later, this energy can come from ATP hydrolysis, a transmembrane ion gradient, or sunlight. imgs//3/3.19.png Figure 4.10: Solutes cross cell membranes by either passive or active transport. Some small, nonpolar molecules such as CO2 can move passively down their concentration gradient across the lipid bilayer by simple diffusion, without the help of a membrane transport protein. Most solutes, however, require the assistance of a channel or transporter. Passive transport, which allows solutes to move down their concentration gradients, occurs spontaneously; Active transport against a concentration gradient requires an input of energy. Only transporters can carry out active transport, and the transporters that perform this function are called pumps. Passive Transport Ion channels Because of their charge, ions interact well with polar molecules such as water, but are repelled by nonpolar molecules such as the interior of the plasma membrane. Therefore, ions cannot move between the cytoplasm of a cell and the extracellular fluid without the assistance of membrane transport proteins. Ion channels possess a hydrated interior that spans the membrane. Ions can diffuse through the channel in either direction, depending on their relative concentration across the membrane. Some channel proteins can be opened or closed in response to a stimulus. These channels are called gated channels, and depending on the nature of the channel, the stimulus can be either chemical or electrical. Three conditions determine the direction of net movement of the ions: 1. their relative concentrations on either side of the membrane, 2. the voltage difference across the membrane and for the gated channels, and 4.5. MEMBRANE PROTEINS 71 3. the state of the gate (open or closed). A voltage difference is an electrical potential difference across the membrane called a membrane potential. imgs//3/3.20.png Ion channels are not simple membrane pores and they have two important properties: 1. selectivity that depend on channel diameter and shape and distribution of the amino acids that cover its walls. 2. controlled flow - Ion channels are not always open, but they open following a specific stimu- lus. imgs/3/3.21.png Compared to transporters, channels do not have to undergo a change in conformation every an ion passes. Transporter Transporters are responsible for the movement of most small, watersoluble, organic molecules and a handful of inorganic ions across cell membranes. Each transporter is highly selective, often transferring just one type of solute. An important example of a transporter that mediates passive transport is the glucose transporter in the plasma membrane of many mammalian cell types. Because glucose is uncharged, the electrical compo- nent of its electrochemical gradient is zero. Thus the direction in which it is transported is determined by its concentration gradient alone. When glucose is plentiful outside cells, as it is after a meal, the sugar binds to the transporter’s externally displayed binding sites; if the protein then switches conforma- tion—spontaneously and at random—it will carry the bound sugar inward and release it into the cytosol, where the glucose concentration is low. Conversely, when blood glucose levels are low—as they are when you are hungry—the hormone glucagon stimulates liver cells to produce large amounts of glucose by the breakdown of glycogen. As a result, the glucose concentration is higher inside liver cells than outside. This glucose can bind to the internally displayed binding sites on the transporter. imgs/3/3.22.png Figure 4.11: Conformational changes in a transporter mediate the passive transport of a solute such as glucose. The transporter is shown in three conformational states: in the outward-open state (left), the binding sites for solute are exposed on the outside; in the inward-open state (right), the sites are exposed on the inside of the bilayer; and in the occluded state (center), the sites are not accessible from either side 72 CHAPTER 4. BIOLOGICAL MEMBRANES Active transport Cells cannot rely solely on passive transport to maintain the proper balance of solutes. The active transport of solutes against their electrochemical gradient is essential to achieving the appropriate in- tracellular ionic composition and for importing solutes that are at a lower concentration outside the cell than inside. For these purposes, cells depend on transmembrane pumps, which can carry out active transport in three main ways: 1. gradient-driven pumps link the uphill transport of one solute across a membrane to the downhill transport of another; 2. ATP-driven pumps use the energy released by the hydrolysis of ATP to drive uphill trans- port; and 3. light-driven pumps, which are found mainly in bacterial cells, use energy derived from sun- light to drive uphill transport. imgs/3/3.23.png imgs/3/3.24.png ATP powered pumps imgs/3/3.25.png ATP-powered pumps (or simply pumps) are ATPases that use the energy of ATP hydrolysis to move ions or small molecules across a membrane against a chemical concentration gradient, an electric potential, or both. This process, referred to as active transport, is an example of coupled chemical reactions. In this case, transport of ions or small molecules “uphill” against an electrochemical gra- dient, which requires energy, is coupled to the hydrolysis of ATP, which releases energy. The overall reaction—ATP hydrolysis and the “uphill” movement of ions or small molecules—is energetically favorable. The ATP-driven Na+ pump plays such a central part in the energy economy of animal cells. This pump uses the energy derived from ATP hydrolysis to transport Na+ out of the cell as it carries K+ in. The pump is therefore sometimes called the Na+-K+ ATPase or the Na+-K+ pump. During the pumping process, the energy derived from ATP hydrolysis promotes the exchange of Na+ and K+ ions. CHAPTER 5 CELL COMMUNICATION Communication between cells is common in nature. Cell signaling occurs in all multi cellular organisms, providing an indispensable mechanism for cells to influence one another. Cells can communicate directly with one another and change their own internal workings in response by way of a variety of chemical and mechanical signals. Cells send but also receive signal to coordinate the actions of distant organs, tissues, and cells. 5.1 Why? I multicellular organisms, cels send and receive chemical messages constantly ot coordinate the actions of distant organs, tissues, and cells and the ability to send messages quickly and efficiently enables cells to coordinate and fine-tune their functions so finely modulate the response activity of a cell. 5.2 How? Any cell of a multi cellular organism is exposed to a constant stream of signals. At any time, hundreds of different chemical signals may be present in the environment surrounding the cell. Each cell responds only to certain signals, however, and ignores the rest, like a person following the conversation of one or two individuals in a noisy, crowded room. Some of these signals arrive thanks to hormones and neurotransmitter, which act like words and phrases, telling a cell about the environment around it or communicating messages. 5.3 Ligand and Receptors Cell to cell communication is common among pluricellular organisms and requires: • Ligand: the signaling molecule • Receptor protein: the molecule to which the ligand binds (can be on the plasma membrane or inside the cell) Ligand and receptors are strictly complementary as a key and a lock; it’s important to say that is important to obtain a specific cell response. 75 76 CHAPTER 5. CELL COMMUNICATION The process which starts is called signal trasduction; Ligand binding induces a change in the receptor protein’s shape, initiating a signal transduction pathway that will produce a cellular response. A given cell responds only to those signaling molecules that can bind to the particular set of receptor proteins it possesses and “ignores” those for which it lacks receptors. imgs/Signal Trasduction Pathway.png It’s important to say that there are lot of intermediate events that occur and all of these are the so said signal transduction pathway. We can see that the message pass from a molecule to another an this amplify the message. Furthemore, different cell types can respond differently to the same signal. 5.4 Type of Signaling Mechanism Cells can communicate through any of four basic mechanisms, depending primarily on the distance between the signaling and responding cells. We can distinguish: • Direct contact: They produce the ligand themselves and they receive it, it is autocrine, to amplify the process. • Paracrine signaling: Signaling molecules affect nearby target cells. • Endocrine Signaling: Hormones travel through the bloodstream to distant target cells. • Synaptic Signaling: Nerve cells release neurotransmitters at synapses for rapid communication. Let’s analyze each one. imgs/SignalCommunication2.png 5.4. TYPE OF SIGNALING MECHANISM 77 5.4.1 Direct Contact When cells are very close to one another, some of the molecules on the plasma membrane of one cell can be recognized by receptors on the plasma membrane of an adjacent cell, they are in contact thanks to junctions. Many of the important interactions between cells in early development occur by means of direct contact between cell surfaces. Cells also signal through gap junctions (figure 9.2a). Gap junctions in animals and plasmodesma in plants are connections between the plasma membranes of neighbouring cells ; they allow rapid signaling, such as in cardiac muscle cells where electrical signals are transmitted directly from one cell to the next. Contact inhibition Contact inhibition is a process of arresting cell growth (overgrow) when cells come in contact with each other, as a result normal cells stop proliferating. Contact inhibition is a powerful anticancer mechanism that is lost in cancer cells. In cell biology, contact inhibition refers to two different but closely related phenomena: Contact inhibition of locomotion and contact inhibition of proliferation. imgs/ContactInhibition.png cadherins are primarily responsible for mediating cell-cell adhesion and play a direct role in contact inhibition, while integrins are involved in cell-Extracellular Matric interactions, which indirectly influence cellular behavior and are interconnected with the regulation of cell density and tissue architecture. 80 CHAPTER 5. CELL COMMUNICATION 5.6.2 Receptors Cells must have a specific receptor that responds to a particular signal molecule. The interaction between ligand and receptor is an example of molecular recognition → The two molecules are complementary and fit perfectly because of their conformation. This interaction changes the receptor structure and leads to the beginning of transduction pathway. Receptors can be defined by their location: imgs/Types-of-Receptors.png • cell surface receptors/membrane receptors (located on the plasma membrane to bind a ligand outside the cell. • intracellular receptors (located within the cell) Membrane receptors There are 3 subclasses of membrane receptors: • channel linked receptors: ion channel that opens in response to a ligand. The channel is said to be chemically gated because it opens only when a chemical (theneurotransmitter) binds to it. • Enzymatic receptors: When a signal molecule binds to the receptor, it activates the enzyme. In almost all cases, these enzymes are protein kinases, enzymes that add phosphate groups to proteins. • G protein coupled receptors: A G-(GUANOSIN) protein (bound to GTP) assiststhe trans- mission of the signal. In details, they bind to ligands outside the cell and t G proteins inside the cell. The G protein then activates an enzyme or an ion channel, transmitting signals from the cell’s surface to its interior. 5.6. LIGANDS (TYPES OF LIGANDS) AND RECEPTORS (TYPES OF RECEPTOR) 81 imgs/Receptors.png Intracellular receptors Many cell signals are lipid-soluble or very small molecules that can readily pass through the plasma membrane of the target cell and into the cell, where they interact with an intracellular receptor. Some of these ligands bind to protein receptors located in the cytoplasm; others pass across the nuclear membrane as well and bind to receptors within the nucleus. Steroid hormones receptors The nonpolar structure allows these hormones to cross the membrane and bind to intracellular receptors. The location of steroid hormone receptors prior to hormone binding is cytoplasmic, but their primary site of action is in the nucleus. Binding of the hormone to the receptor causes the complex to shift from the cytoplasm to the nucleus , usually affects gene expression regulation. Before binding to its ligand, then it goes inside the nucleus. Three functional domains → hormone-binding domain, DNA binding domain, domain that interact with co-activators to affect gene expression. imgs/IntracellularSteroid.png 82 CHAPTER 5. CELL COMMUNICATION 5.7 Signal Transduction Through Receptor Kinases The receptor tyrosine kinases (RTKs) influence the cell cycle, cell migration, cell metabolism, and cell proliferation— virtually all aspects of the cell are affected by signaling through these receptors. Alter- ations to the function of these receptors and their signaling pathways can lead to cancers in humans and other animals. The receptor is a transmembrane protein with an extracellular ligand-binding domain and an intracellular kinase domain. Signal transduction pathways begin with response proteins binding to phosphotyrosine on a receptor, and by receptor phosphorylation of response proteins. The autophosphorylation event transmits across the membrane the signal that began with the binding of the ligand to the receptor. The next step, propagation of the signal in the cytoplasm, can take a variety of different forms. These forms include activation of the tyrosine kinase domain to phosphorylate other intracellular targets or interaction of other proteins with the phosphorylated receptor. imgs/RTKs.png Insuline receptor The role of insulin is to lower blood glucose, acting by binding to an RTK. Another protein called the insulin response protein binds to the phosphorylated receptor and is itself phosphorylated. The insulin response protein passes the signal on by binding to additional proteins that lead to the activation of the enzyme glycogen synthase, which converts glucose to glycogen. So it is an hormone that helps maintaining constant blood glucose levels, so it lowers blood glucose. CHAPTER 6 IMMUNOBIOLOGY 6.1 Introduction Immunobiology is the branch of biology that focuses on the study of the immune system, which is a complex network of cells, tissues, and molecules that work together to defend the body against infections and diseases. Immunobiology explores how the immune system recognizes and responds to pathogens (such as bacteria, viruses, and parasites), as well as how it distinguishes between foreign invaders and the body’s own cells and tissues. Hematology analyzer or a complete blood count (CBC) machine are used to determine the number and types of blood cells in a patient’s blood sample, including white blood cells (leukocytes), red blood cells (erythrocytes), and platelets (thrombocytes). They are commonly used in clinical laboratories and medical facilities to provide information about a patient’s overall health and diagnose various medical conditions, including infections, anemias, and certain blood disorders. Figure 6.1 shows a full blood test whith their reference ranges. imgs/5/2.png Figure 6.1: Blood test 85 86 CHAPTER 6. IMMUNOBIOLOGY 6.2 Coulter Impedance Principle The Coulter Principle is a method used in hematology analyzers, including Coulter counters, to count and size blood cells, including white blood cells (leukocytes), red blood cells (erythrocytes), and platelets (thrombocytes). It is based on the fact that cells are bad conductors of eloctricity while the diluent is excellent conductor. Cells diluted in an electrolyte solution capable of conducting electric current are forced to pass through an aperture to which an electric current of constant intensity is applied. The cells’ double lipid membrane acts as an insulator of the electric field, opposing the passage of current, which flows continuously. The increase in resistance is recorded as an electrical pulse and is directly proportional to the size of the cell that produced it, allowing accurate measurement of cell volume. 6.3 Connective tissues Connective tissues are a diverse group of tissues in the body that serve to provide structural support, connect and anchor various body parts, and maintain the integrity of other tissues. They have a wide range of functions, including supporting, protecting, and nourishing different organs and structures. Connective tissues are characterized by the presence of an extracellular matrix, which includes fibers and ground substance. Connective tissues can be classified into the following categories: • Connective Tissue Proper: This category includes several subtypes, and it forms the majority of the connective tissue in the body. Some of the subtypes within this category are: 1. Loose Connective Tissue: This tissue is composed of loosely arranged collagen and elastic fibers within a gel-like ground substance. It provides support and allows for the diffusion of nutrients and wastes. Examples include areolar tissue and adipose tissue. 2. Dense Connective Tissue: This type of tissue contains densely packed collagen fibers, making it strong and resistant to stretching. Dense connective tissue can be further divided into regular and irregular types. Regular dense connective tissue is found in tendons and ligaments, while irregular dense connective tissue is found in the dermis of the skin. • Supportive Connective Tissue: This category includes tissues that provide structural support to the body. The main subtypes are: 1. Cartilage: Cartilage is a firm, flexible tissue that is resistant to compression. It is found in areas like the nose, ears, and between bones in joints. Three common types of cartilage are hyaline, elastic, and fibrocartilage. 2. Bone: Bone is a dense and rigid connective tissue that forms the skeleton. It provides support, protection, and serves as a reservoir for minerals like calcium and phosphorus. • Fluid Connective Tissue: Fluid connective tissues consist of a liquid extracellular matrix. The primary example is blood. 1. Blood : Blood is composed of a liquid matrix called plasma and cellular elements (red blood cells, white blood cells, and platelets). It functions in the transport of oxygen, nutrients, waste products, and immune responses. 2. Lymph: Lymphatic tissue contains immune cells and is involved in the body’s immune re- sponse. It is found in lymph nodes, tonsils, and other lymphatic organs. 6.3.1 Blood It is a viscous fluid specialized connective tissue forming part of the cardiovascular system, it is a slightly alkaline with pH 7.4. Circulates within the vascular district in amounts that vary slightly between Male and Female. The main functions of the blood are: • Tansport of respiratory gases (02 to tissues and C02 to lungs) 6.3. CONNECTIVE TISSUES 87 • Transport and distribution of nutrients • Transport of waste and toxic substances from peripheral to excretion sites • Transport of hormones and electrolyte regulation (e.g., buffering effect (that is, to balance pH to avoid hyperacidic and hypoacidic situations.) vs. lactic acid produced by muscle) • Defense against pathogens (immunity) • Prevention of fluid loss through damaged vessels (coagulation) • Thermoregulation through heat absorption and distribution. Taking the bloods, allowing it to rest it will stratify into two layers from which we distinguish: 46%- 63% plasma (liquid portion) and 37%-54% cellular elements (red blood cells, white blood cells and platelets). imgs/5/4.jpg Figure 6.2: Blood composition 6.3.2 Plasma Like shows in picture 6.3, in the plasma founds: 7% proteins, 92% water and 1% other solutes. The proteins are: 1. 60% albumins - To maintain the osmolarity of the blood. The concentration of albumin in the blood is such that both lysis and cellular wrinkling are avoided. 2. 35% Globulins - To transport ions, hormones and lipids. They have a immune function. 3. 4% fibrinogen - Essential components of clot- ting syste. They can be converted to insoluble fibrin In the other solutes found the Electolytes, organic nutrients and organic wastes imgs/5/6.png 90 CHAPTER 6. IMMUNOBIOLOGY 6.4 Hemopoiesis Hemopoiesis, also known as hematopoiesis, is the process by which the body produces blood cells and it starts during the third week of embryonic development and colonize organs. The sites of hematopoiesis are different in the fetus than in adult. In The fetus, hemopoiesis occurs in yolk sac, liver, spleen, thymus and bone marrow, while in the adult occurs in bone marrow of flat bones and bone ends. All peripheral blood elements are the result of a pattern of cellular differentiation that proceeds in stages starting from the stem cell. Stem cell is a bone marrow cell that has the ability to differentiate into any part of the human body, and is capable of having different fates. The reverse process is called Hemocataresis. It imgs/5/8.png is the process that leads to the destruction of blood cells and taht occurs in liver and spleen. 6.5. IMMUNOBIOLOGY 91 6.5 Immunobiology The cells, molecules, tissues and organs responsible for immunity constitute the immune system, and their collective and coordinated response to the introduction of foreign substances is called the immune response. The physiologic function of the immune system are 1. defense against infectious microbes. 2. remove of dead or damaged tissue and cell and 3. recognition and removal of abnormal cells such as tumor, infected cells, ... Defense against microbes is mediated by the early reactions of innate immunity and the later responses of adaptive immunity. imgs/5/11.png 6.5.1 Innate Innate immunity (also called natural or native immunity) provides the early line of defense against microbes. It consists of cellular and biochemical defense mechanisms that are in place even before infection and are poised to respond rapidly to infections. These mechanisms react to microbes and to the products of injured cells, and they respond in essentially the same way to repeated infections: resistance not increase after infection. The principal components of innate immunity are: 1. Macrophages 2. Granulocytes: Neutrophils, Basophils and eosinophils 3. Natural killer 4. Soluble mediator: Component proteins, acute phase proteins and cytokines and chemokines 6.5.2 Adaptive In contrast to innate immunity, there are other immune responses that are stimulated by exposure to infectious agents and increase in magnitude and defensive capabilities with each successive exposure to a particular microbe. Because this form of immunity develops as a response to infection and adapts to the infection, it is called adaptive immunity. The main components of adaptive immunity are 1. Lymphocytes: B lynphocytes (Humoral immunity), T lynphocytes (cell-mediator immunity) 2. soluble mediators: antibodies, cytokines and chemokines 92 CHAPTER 6. IMMUNOBIOLOGY 6.6 Innate immunity Natural immunity is mediated by pre-existing molecules and cells in tge body, does not increase in the presence of the pathogeon, and is nonspecific; in practice it acts as the body’s first line of defense but at the same time also serves as a trigger and auxiliary ”work force” for the subsequent specific immune response coordinated by T-helper lymphocytes. The immune system makes a double attack; it is based on the innate reactions and the lately of the acquired one. Non-specific immunity, also known as innate immunity, is the body’s first line of defense against pathogens and does not target specific pathogens. It includes various components and mechanisms that help protect the body from a wide range of potential threats. they include: • Physical Barriers: Skin : The skin acts as a physical barrier that prevents pathogens from entering the body. Mucous Membranes: Mucous membranes, such as those lining the respiratory and digestive tracts, help trap and expel pathogens. • Chemical Barriers: Stomach Acid: The acidic environment in the stomach can kill many ingested pathogens. Saliva and Tears: Contain enzymes and proteins that help break down and inhibit the growth of pathogens. Antimicrobial Peptides: Small proteins that can kill or inhibit the growth of bacteria and other microbes. • Phagocytic Cells: Macrophages: Large white blood cells that engulf and digest pathogens. Neutrophils: Another type of white blood cell that engulfs and destroys pathogens. Dendritic Cells: Antigen-presenting cells that help initiate the adaptive immune response. • Natural Killer (NK) Cells: NK cells can recognize and destroy infected or abnormal cells, such as cancer cells. • Host microbial flora: natural antibodies • Cellular producs: such as mucus that traps micorganism • Environmental conditions: such as pH. 6.8. LYMPHOCYTES 95 In conclusion the image shows how the immune system works to protect the body from pathogens. Innate immunity provides a rapid and general response, while adaptive immunity provides a more specific and powerful response. Adaptive immunity is necessary for a complete and lasting immune response. It is important to denote that T lymphocytes (or T cells) have two arms of proteic nature and their aim is to scan the micro environment and increase the binding sites to possible pathogens. The macro-phage shows a functional part of the phatogen to th T-lymphocyte in order to give him the confidence with that one (It is known as presentation of the anti-gene). All the process takes 7 days in order to be finished. 6.8 Lymphocytes Lymphovytes are present in percentage 20-40% of all calculating white blood. They are (In appearance): • Eccentric, dense nucleus occupying about 90% of the cell. • Cytoplasm sparse, pole blue in color with a few bluish granules of 8-10um, We can find them in two conditions: • Functional rest • Activation Upon the activation, the lymphocyte undergoes a series of morphological changes: • The cytoplasm, becomes larger • the nucleus moves to the side • Organelles and the amount of cytoplasmic RNA increase In addition to the T and B cell classes, there is a non-T, non-B class known as natural killer cells. They are true and proper natural killers, they kill everything they encounter that is different. • They do not perform activities in the circulation. but In the connective They tissue. They migrate into the lymphnode’ and spleen.wherethey form clones of identical cells • After stimulation by antigen they proliferate and differentiate into two populations: – Memory cells. which do not participate in the immune response. but remain in the clone and are ready to respond to that antigen – Effector cells. immunocompetent lymphocytes that classified as B and T lymphocytes 96 CHAPTER 6. IMMUNOBIOLOGY • B lymphocytes: – Are formed and become immunocompetent in the bone marrow – Responsible for humoral immune response – Can differentiate into plasma cells and produce antibodies • T lymphocytes: – They migrate from the bone marrow to the thymus (That’s why T name) where they mature – the constitute the cells of specific immunity, or the body’s ability to respond to a specific pathogenic stimulus – T cells are divided into: ∗ T helper lymphocytes help to activate B lymphocytes and T killer cells. ∗ T reg cells: suppress immuno response helping to prevent the immune system from overreacting and causing damage to healthy tissues. This is important for maintaining immune tolerance and preventing autoimmune diseases. ∗ T killer cells (cytotoxic) kill infected cells. • NK cells: – important for the defense against viral infections. They can kill virus-infected cells and produce antiviral cytokines. – important for the fight against cancer. They can kill cancer cells and produce cytokines that activate other immune cells against cancer. – play a role in regulating the immune response. They can produce cytokines that activate or suppress other immune cells. imgs/ClassiLinfociti.png 6.9. ANTIBODIES 97 imgs/NK.png 6.9 Antibodies Also called immunoglobulinst,hey are a groupof glycoproteins found in blood and tissue fluids. They are the antigen- specific productsof B lymphocytes in their terminal differentiation or PLASMA CELL stage. The antibodies are not cells, they’re glycoprotein produced by B cells, and our adaptive immune system depends on having specific antibodies that bind the invader: • Neutralization: by binding to a toxin, an antibody can block its function and make it non-toxic • Opsonization: by coating an invader, the antibodies mark it as something to be destroyed, and it will be ingested and destroyed by a macrophage. They make it more palatable. • Complement activation: antibodies can activate the complement system, which is a set of blood proteins that can destroy an invader directly, and/or make it more likely to be eaten by a macrophage. • ADCC (antibody-dependent-cell-mediated-cytotoxicity). At the end of the contraction survives only memory cells.