The development and maintenance of multicellular organisms depend not only on

cell growth and cell division but also on cell death. The maintenance of tissue size
in adult animals often requires that cells die at the same rate as they are
produced—a process called cell turnover.
For normal cell death we intend a sequence of molecular events that destroy the
cell from within: the apoptosis. A cell dying by apoptosis undergoes morphological
and biochemical changes. A cell dying by apoptosis undergoes characteristic
morphological and biochemical changes, in part, to be eaten and digested quickly
by a nearby cell.
MORPHOLOGICAL CHANGES OF APOPTOTIC CELLS
- ADESE (non so se è giusto come ha scritto la prof) CELLS BECOME ROUNDED.
- THE PLASMATIC MEMBRANE UNDERGOES CHANGES INCLUDING EXPOSURE OF
PARTICULAR LIPIDS, LOSS OF EXTENSIONS AND CONTACTS
- CHROMATINE CONDENSES, DNA IS FRAGMENTED AND THE NUCLEAR
ENVELOPE IS LOST.
- CYTOSKELETON IS DESTROYED
MOLECULAR EVENTS THAT DESTROY THE CELL FROM WITHIN:
1) CELL SHRINK AND CONDENSES
2) THE CYTOSKELETON COLLAPSES
3) THE NUCLEAR ENVELOPE DISASSEMBLES
4) THE NUCLEAR CHROMATINE CONDENSES
5) THE APOPTOTIC CELL SURFACE FORMS LARGE PROTRUSIONS CALLED BLEBS
and, if the cell is large, it breaks up into membraneenclosed fragments called
apoptotic bodies
6) THE SURFACE IS CHEMICALLY MODIFIED SO THAT THE NEIGHBORING CELLS
often a macrophage in vertebrates RAPIDLY ENGULF IT BEFORE IT CAN SPILL ITS
CONTENT
​ THE RAPID ENGULFMENT IS IMPORTANT BECAUSE THE DEAD CELL DOES
NOT CAUSE DAMAGING INFLAMMATORY RESPONSE
Because apoptotic cells are eaten and digested so quickly, there are often few dead
cells to be seen, even in a tissue where large numbers of cells have died by
apoptosis
Apoptosis eliminates unwanted cells at least a million cells die this way every
second in a healthy adult human (and are replaced by cell division). in the process
called cell necrosis, Cells die as a result of acute injury typically swell and burst,
spewing
their contents across their neighbors, (Figure 18−38A). This eruption triggers a
potentially damaging inflammatory response. By contrast, a cell that undergoes
apoptosis dies neatly, without damaging its neighbors. macrophages (see Figure
15–32B). engulf the apoptotic cell before its contents can leak out
Role of apoptosis during development,
​ during embryonic development it helps sculpt our hands
and feet: these appendages begin as spade-shaped
structures, and the individual toes separate only when the
cells between them die,.
​ In other cases, cells die by apoptosis when the structure they form is no
longer needed. When a tadpole metamorphoses into a frog, for example, the
cells in the tail die by apoptosis, and the tail, which is not needed in the frog,
disappears.
​ Apoptosis also functions IN THE
CENTRAL NERVOUS SYSTEM
adjusting the number of nerve cells
to the size of the target

Role of apoptosis: cellular homeostasis

apoptosis also behaves as a quality control and eliminates cells under pathological
conditions:
​ Apoptosis eliminates cells that are abnormal, nonfunctional or potentially
dangerous
​ Apoptosis eliminates cells carrying damages in the various organelles if
damage is great, cells can kill themselves
​ Apoptosis eliminates t and b lymphocytes that either fail to produce
potentially
useful receptors (quindi potrebbero essere autoreattivi contro il proprio corpo)

Role of apoptosis: immune system regulation

​ apoptosis eliminates autoreactive immune cells during their development,
preventing autoimmune diseases
 ​ apoptosis also clears infected, damaged, or cancerous cells, maintaining the
integrity of tissues and organs.

apoptosis: its role in pathology

​ reduction of apoptosis induces: cancer, cronic infections
​ increase of apoptosis induces: neurodegeneration cerebral ischemia,
myocardial infarction autoimmune pathologies
APOPTOSIS IS AN ACTIVE PROCESS OF CELL DEATH BECAUSE:
​ IT REQUIRES ENERGY
​ IT REQUIRES PROTEIC SYNTHESIS
​ IT INVOLVES A LARGE NUMBER OF GENES
IT CAN BE INDUCE BY:
✔​ INTERNAL SIGNALS
✔​ EXTERNAL SIGNALS
✔​ ABSENCE OF EXTERNAL SIGNALS OF CELL SURVIVAL

THE APOPTOTIC PROCESS CAN BE DIVIDED INTO SEVERAL TIME STAGES

- INITIATION: CELLS DETERMINED TO DIE
- EXECUTION: MORPHOLOGICAL CHANGES
- RECOGNITION AND PHAGOCYTOSIS: ELIMINATION OF THE CELLULAR
DEBRIS BY SPECIALIZED CELLS

Apoptosis Depends on an Intracellular Proteolytic Cascade Mediated by

Caspases,
These intracellular proteases, when are activated triggers apoptosis and cleave
numerous intracellular proteins at specific aminoacid sequences: cytoskeleton,
nuclear lamina, dnaase inhibitors, dna repair enzymes and this morphological
changes lead to cell death
Because these proteases have a cysteine at their active site and cleave at specific
aspartic acids, they are called caspases (c for cysteine and asp for aspartic acid).
Caspases are present into the cytosol in inactive form for all cell life, which are
activated during apoptosis. There are two major classes of these apoptotic
caspases: initiator caspases and executioner caspases.
INITIATOR CASPASES: CASPASE-8 AND CASPASE-9 STRUCTURAL
CHARACTERISTICS
The initiator caspases, are caspase-8 and caspase-9. An initiator caspase
contains a protease domain in its large C-terminal region and a smaller,
adaptor-protein-binding prodomain in its N-terminal region. The caspase is made as
an inactive monomer, which is activated by dimerization..

THE PRO-DOMAIN OF THE INTRINSIC INITIATOR CASPASES CONTAIN A SINGLE

DEATH FOLD KNOWN AS CASPASE RECRUITMENT DOMAIN (CARD), WHILE THE
PRODOMAIN OF THE EXTRINSIC INITIATOR CASPASES CONTAINS TWO DEATH
FOLDS KNOWN AS DEATH EFFECTOR DOMAINS (DED)

APOPTOTIC SIGNAL ACTIVATES CASPASES THAT TRIGGER APOPTOSIS

Dimerization and activation occur only when apoptotic signals trigger the assembly
of specific adaptor-protein
complexes, which bring
together a pair of initiator
caspases, which are then
activated, leading to the
cleavage of the
prodomain-adaptor complex
from the protease domains.

THE TWO SUBUNITS, ONCE

CLEAVED, ATTACH EACH OTHER
CREATING THE MATURE ACTIVE
INITIATOR CASPASE
Executioner
Caspases: CASPASE-3, CASPASE-6 AND CASPASE-7 are initially formed as
inactive dimers. After the activation, the mature initiator caspases activate
executioner through a cleavage
(prodomain removal). After cleavage
by an initiator caspase, the
executioner caspase dimer undergoes
an activating conformational change
similar to that of initiators. Executioner
caspases then cleave a variety of key
proteins, leading to apoptosis

Activation of the apoptotic program cannot turn back. EXECUTIONER CASPASES CLEAVE HUNDREDS OF
DIFFERENT PROTEINS AMPLIFYING A SIGNAL THAT IS IRREVERSIBLE!
The caspase-initiated proteolytic cascade is irreversible, so that once a cell starts
out along the path to apoptotic death, it cannot turn back.
Executioner caspases cleave hundreds of different cell proteins during apoptosis,
Ad esempio
1)The cleavage of nuclear lamins by caspase-6, causes the irreversible breakdown
of the nuclear lamina
2) THE CLEAVAGE OF DNA by by caspase-3 PROVOKES THE IRREVERSIBLE
BREAKAGE OF DNA
3) The cleavage of certain proteins that regulate the actin cytoskeleton in the cell
cortex FORMS LARGE PROTRUSIONS CALLED BLEBS .
4) The cleavage of some cell–cell adhesion proteins that attach cells to their
neighbors PROVOKES THAT CELL ROUND UP AND DETACHES FROM THE
NEIGHBORING CELLS making it easier for a neighboring cell to engulf it
5) the cleavage of two phospholipid transfer proteins in the plasma membrane
results in the exposure of phosphatidylserine on the surface of apoptotic cells,
where it serves as an “eat me” signal to neighboring phagocytic cells.

Importantly, preventing any one of these individual protein-cleavage steps does not
stop apoptotic cell death,

How is an initiator caspase first activated in response to an apoptotic signal?

Our cells use two main activation pathways:

 1)​ extrinsic pathway is signaled from outside the cell and membrane
receptors play a key role and it is mediated by caspase-8
2)​ intrinsic pathway is signaled from the inside and mitochondria play a key
role and is mediated by caspase-9

●​ extrinsic pathway

un death ligand si lega al death receptor che recruits adaptors che determina la
dimerization of initiator pro-caspases che a sua volta activation of initiator
caspase che through clavage activation of executioner caspases

Extracellular signal proteins (death ligand) binding to cell-surface death receptors

trigger the extrinsic pathway of apoptosis.
●​ DEATH RECEPTORS
are transmembrane proteins containing
an extracellular ligand-binding domain,
a single transmembrane domain,
and an intracellular death domain (DD) that is required for the receptors to activate
the apoptotic program. The receptors are homotrimers and belong to the tumor
necrosis factor (TNF) receptor family and the Fas death receptor
●​ DEATH LIGAND STRUCTURE
The ligands that activate the death receptors are also homotrimers, belonging to
the TNF (tumor necrosis factor) family of signal proteins and Fas

A example the activation of the Fas receptor on the surface of a target cell by Fas
ligand on the surface of a killer (cytotoxic) lymphocyte

✔​ Binding Fas ligand to Fas receptors exposes the death domains (DD) on the
cytosolic side of the receptor.
✔​ An adaptor protein called FADD binds here and recruits inactive monomeric
initiator caspases (mainly caspase-8) by binding to a DED on the prodomain
of the caspase.
✔​ Since each monomer of caspase-8 has two DEDs, the other one also
becomes exposed and can recruit another monomer of caspase-8, resulting
in a chain reaction
✔​ The end result is the assembly of a large death-inducing signaling complex
(DISC) composed of multiple copies of Fas, FADD, and caspase-8.
✔​ Within the DISC, neighboring caspase-8 monomers interact to form activated
dimers, which cleaving off the prodomain and forming the mature activated
dimers, which are released into the cytosol,
 ✔​ where they can cleave and activate executioner caspases 3 and 7 to induce
apoptosis.

Il legame fas ligando a recettori Fas espone i domini di morte (DD) sul lato
citosolico del recettore. Qui si lega una proteina adattatrice chiamata FADD che
recluta caspasi iniziatrice monomerica inattiva (principalmente caspasi-8)
legandosi a un DED sul prodominio della caspasi. Siccome ogni monomero della
caspasi-8 ha due DED , anche l'altro diventa esposto e può reclutare un altro
monomero della caspasi-8 determinando una reazione a catena
Il risultato finale è l'assemblaggio di un grande complesso di segnalazione che
induce la morte (DISC) composto da più copie di Fas, FADD e caspasi-8. All'interno
del DISC, i monomeri di caspasi-8 adiacenti interagiscono per formare dimeri
attivati, che scindono il prodominio e formano i dimeri attivati ​maturi, che vengono
rilasciati nel citosol, dove possono scindere e attivare le caspasi esecutrici per
indurre l'apoptosi

●​ The Intrinsic Pathway of Apoptosis

The Intrinsic Pathway of Apoptosis is activated from inside the cell, often in
response to developmental signals or to injury such as DNA damage. the intrinsic
pathway of apoptosis is also called the mitochondrial pathway, as it depends on the
release into the cytosol of mitochondrial proteins that normally reside in the
intermembrane space of these organelles (see Figure 12–47). The most important
of these released proteins is cytochrome c, which is a component of the
mitochondrial electron-transport chain and therefore has a central role in ATP
production by oxidative phosphorylation in mitochondria (see Figure 14–18).
When released into the cytosol (Figure 18–7), however, it takes on an entirely new
function inducing apoptosis.
Once released, into the cytosol, cytochrome c binds to an adaptor protein
called Apaf1 activating it.
APAF1 oligomerizes
forming an eptameric
structure, that recruits
inactive initiator caspase-9
monomers into the complex,
forming an even larger
structure called an
apoptosome (Figure 18–8)
(corrospondente al DISC del
percorso estrinseco).
Caspase-9 is activated by dimerization within the apoptosome,
Once activated, caspase-9 cleaves and activates the downstream executioner
caspases that mediate apoptosis.
What are the upstream signals that induce the release of cytochrome c from
mitochondria? the intrinsic pathway is regulated by bcl2 family proteins (b
cell lymphoma 2)
The intrinsic pathway is responsible for the great majority of apoptotic cell deaths in
vertebrates. The Intrinsic Apoptotic Death Program Is Regulated by the Bcl2 Family
of Intracellular Proteins , these interact between each other to control the
permeabilization of the outer mitochondrial membrane and thereby govern the
release into the cytosol of cytochrome c andother soluble proteins a process called
mitochondrial outer membrane permeabilization (MOMP).
Some members of this protein family promote caspase activation and cell death,
whereas others inhibit these processes
There are three
structural and
functional classes of
mammalian Bcl2 family
Proteins (on the
mitocondrial outer
membrane???) :

(1) Anti-apoptotic Bcl2 family proteins inhibit apoptosis by preventing MOMP.

(2) Pro-apoptotic Bcl2 family called Bax and Bak effectors can directly induce
MOMP by creating openings in the outer mitochondrial membrane.
(3) BH3-only proteins promotes apoptosis by regulating the other two classes.
The balance between the activities of these three classes largely determines
whether MOMP occurs or not and, therefore, whether a mammalian cell lives or
dies by the intrinsic pathway of apoptosis
How pro-apoptotic Bcl2 family effectors induce MOMP and how anti-apoptotic
Bcl2 family proteins block it.
(A) Most of the pro-apoptotic effector Bak
is already attached to the outer
mitochondrial membrane before the protein
is activated. When activated by an
apoptotic stimulus, the protein undergoes a
conformational change that both
exposes a BH3 domain and creates a
BH3-binding groove, allowing Bak–Bak
oligomerization in the outer membrane.
(B) The Bak oligomers induce MOMP by creating
openings in the outer membrane that allow
cytochrome c and other soluble proteins in the
intermembrane space to diffuse into the cytosol;.
into the cytosol, cytochrome c stimulates the
assembly of apoptosomes (see Figure 18–8).

(C) The anti-apoptotic Bcl2 family protein

BclxL, is normally bound to the outer
mitochondrial membrane, where it can interact
via its BH3-binding groove to the exposed BH3
domain on activated Bak, thereby blocking
Bak–Bak oligomerization, MOMP, and
apoptosis.

bcl2 and bclxl, as anti-apoptotic proteins, are present in the cytosolic surface of the
outer membrane of mitochondria, in endoplasmic reticulum and in nuclear envelope
All BclxL function:
1.inhibition of pro-apoptotic proteins:
2.maintenance of mitochondrial integrity:
1. protects mitochondrial membranes from permeabilization, maintaining cellular
energy
production and preventing cell death.
3.role in non-apoptotic functions:
1. regulates mitochondrial dynamics, including fusion and fission.
2. plays a role in calcium homeostasis, metabolic regulation, and cell cycle
progression.
 (D) One way BH3-only proteins such as
Bad are thought to indirectly induce MOMP
and apoptosis is by inhibiting certain
anti-apoptotic Bcl2 family proteins such as
BclxL.

OTHER BH3-ONLY PROTEINS, SUCH AS BAD, NOXA, AND PUMA, BIND TO

AND NEUTRALIZE ANTI-APOPTOTIC PROTEINS (E.G., BCL-2, BCL-XL, MCL-
1), FREEING BAX AND BAK TO EXECUTE APOPTOSIS

Since apoptosis is an irreversible process, cell possesses some controls to

ensure
that pathway has been activated in appropriate cells
Cells in the cytosol have caspase inhibitory proteins called inhibitors of apoptosis
(IAP)** (XIAP in mammals) that act as a defense against inappropriate caspase
activation. . It binds to and inhibits caspase-9, an initiator caspase, and the
executioner caspases, caspase-3 and caspase-7. These caspases are inactive until
the stimulus causes the mitochondrion to release
not only cytochrome C but also two anti-IAP
proteins that help control caspase activation in the
cytosol: OMI and SMAC that bind xiap preventing
its inhibition of caspases

**These proteins were first identified in certain

insect viruses, which encode
IAP proteins to prevent a host cell that is infected
by the virus from activating
caspases and killing itself by apoptosis, which
would curtail the virus’s replication.
●​ Survival factors suppress apoptosis
by helping to ensure that
cells survive only when and
where they are needed.
In due modi 1 solo chi riceve
il segnale sopravvive
1) survival signals Many
types of nerve cells, for
example, are overproduced in the developing nervous
system, but survival factors are secreted by the target
cells they need to contact. So only the nerve cells that
receive the survival factor live, while the others die by
apoptosis. In this way, the number of surviving nerve
cells is automatically adjusted to match the number of
cells they connect with.
Survival factors usually bind to cell-surface receptors,
which activate intracellular signaling pathways that
suppress the apoptotic program, for example, stimulate
the synthesis of anti-apoptotic Bcl2 family proteins such
as Bcl2 itself or BclxL
1) survival signals bind membrane receptors that
through different pathways regulate gene expression the
expression of genes such as bcl-2 or bclxl is increased
 2) survival signals can also suppress apoptosis
activate serine/threonine protein kinase AKT, this
works in two ways
a) phosphorylates and inactivates the proapoptotic
bh3-only bad protein.
b) akt can also suppress apoptosis by phosphorylating
and inactivating transcription regulatory proteins which
stimulate the transcription of genes that encode
proteins that promote apoptosis, such as the bh3-only
bim protein.
when not phosphorylated, bad promotes apoptosis by
binding and inhibiting an anti-apoptotic protein from the
bcl2 family such as bcl2 itself. Once phosphorylated,
bad dissociates, releasing bcl2 to suppress apoptosis

 Stimolo apoptotico: Danno al DNA, stress ossidativo, deprivazione di fattori di crescita.

 Alterazioni mitocondriali: Rilascio di citocromo c e altre molecole pro-apoptotiche

Dopo la morte cellulare avviene la Phagocitosis the elimination of apoptotic bodies
is a fundamental part of the in vivo process
- prevents the inflammatory response
- cleanses the tissue
- elimination is an active process
- phagocytosis

HOW OCCURS PHAGOCITOSIS

In healthy cells an ATP-dependent

aminophospholipid flippase (ATP11C) actively flips
phosphatidylserine (PS) and
phosphatidylethanolamine (PE) from the outer to
the inner leaflet of the plasma membrane lipid
bilayer, keeping these lipids mainly confined to the
inner leaflet

during apoptosis, activated executioner

caspases
(caspase-3, caspase-7, or both) cleave and
inactivate the ATP11C flippase, preventing its
lipid translocation activity

during apoptosis, the

activated executioner caspases cleave and
thereby activate a phospholipid scramblase
(xkr8) in the plasma membrane that
nonspecifically flips phospholipids between the
two lipid leaflets of

the membrane, scrambling them between the

leaflets With the flippase permanently inactivated
and the scramblase permanently activated, PS
and PE become rapidly and irreversibly exposed
on the apoptotic cell surface, where the PS serves
as an “eat me” signal to neighboring phagocytic
cells. Both of these caspase-dependent
mechanisms are required to create this rapid and effective “eat me” signal

La Fagocitosi dei Corpi Apoptotici:

La fagocitosi dei corpi apoptotici è un processo fondamentale per l'eliminazione

efficiente delle cellule che hanno subito la morte cellulare programmata (apoptosi)
senza innescare una risposta infiammatoria dannosa per l'organismo.

Cosa sono i corpi apoptotici? Quando una cellula decide di "suicidarsi"

attraverso l'apoptosi, si frammenta in piccole vescicole circondate da membrana,
chiamate corpi apoptotici. Questi "pacchetti" contengono il materiale cellulare da
eliminare in modo ordinato.

Come avviene la fagocitosi dei corpi apoptotici?

1.​ Segnali "Mangia me":

Fosfatidilserina: Una molecola normalmente presente all'interno della membrana

cellulare viene esposta all'esterno dei corpi apoptotici. Questo agisce come un
segnale "mangia me" per i fagociti, come i macrofagi.

2.​ Riconoscimento e Inglobamento:

Recettori sui fagociti: I fagociti possiedono recettori specifici che riconoscono le

molecole segnale presenti sui corpi apoptotici.

Inglobamento: Una volta riconosciuti, i corpi apoptotici vengono inglobati

all'interno del fagocita in una vescicola chiamata fagosoma. Che si fonde con un
lisosoma, ricco di enzimi digestivi.

●​ WHAT IS NECROSIS? Necrosis is the premature death of cells in living

tissue INDUCED BY NON-PHYSIOLOGICAL AGENTS ( injury or disease).
It's a passive and often destructive process, unlike apoptosis which is a
controlled form of cell death.

THE CELL SWELLS AND BURSTS, WITH CONSEQUENT RELEASE OF

MATERIAL CYTOPLASMIC into the surrounding tissues WITH AN
INFLAMMATORY response by the body.
UNDER THE UMBRELLA TERM OF CELL NECROSIS, WE INTEND ALL
NON-APOPTOTIC WAYS OF DEATH
●​ cell necrosis occurs when cells lyse after an acute tissue insult cell swell and
burst inflammatory response
●​ NECROPTOSIS PYROPTOSIS (importanti contro le infezioni virali e
batteriche . hanno caratteristiche intermedie tra la necrosi e l’apoptosi)

●​ CAUSES OF CELL NECROSIS

ISCHEMIA: LACK OF BLOOD SUPPLY TO TISSUES, OFTEN DUE TO BLOCKAGE IN

BLOOD VESSELS
(E.G., MYOCARDIAL INFARCTION, STROKE).
INFECTION: BACTERIA, VIRUSES, OR TOXINS CAUSING CELL DAMAGE (E.G.,
GANGRENE CAUSED BY CLOSTRIDIUM BACTERIA).
TRAUMA: PHYSICAL INJURY THAT DISRUPTS TISSUE STRUCTURE.
RADIATION: DAMAGE FROM RADIATION THERAPY OR EXCESSIVE EXPOSURE.
CHEMICAL/TOXIC EXPOSURE: HARMFUL SUBSTANCES, INCLUDING POISONS AND
DRUGS.
IMMUNE REACTIONS: AUTOIMMUNE DISEASES LEADING TO CELL DESTRUCTION

●​ WHAT IS autophagy?

IT CAN BE INDUCE BY:

SEVERAL MECHANISMS BECAUSE IT SERVES AS A SURVIVAL MECHANISM

DURING STRESS OR NUTRIENT DEPRIVATION. AUTOPHAGY (GREEK FOR
"SELF-EATING") IS A CELLULAR DEGRADATION PROCESS WHERE CYTOPLASMIC
COMPONENTS ARE DELIVERED TO LYSOSOMES FOR RECYCLING.
●​ ROLES OF AUTOPHAGY
1)​ CELL SURVIVAL : PROVIDES NUTRIENTS AND ENERGY DURING
STARVATION (When cells are deprived of nutrients, autophagy allows them to
recycle their own cellular components, such as damaged proteins and organelles,
for energy)
2)​ QUALITY CONTROL: REMOVES DAMAGED ORGANELLES AND MISFOLDED
PROTEINS. (XENOPHAGY) CELL DEATH EXCESSIVE AUTOPHAGY CAN LEAD
TO CELL DEATH (AUTOPHAGIC OR TYPE II
PROGRAMMED CELL DEATH).
3)​ IMMUNITY ELIMINATES PATHOGENS

WHEN DO WE HAVE THE NEED TO SELF-DIGEST

THE CONSTITUENTS OF OUR CELLS?
1) CELL MAINTENANCE: REPLACEMENT OF PARTS THAT “AGE” OVER A LIFETIME
(BASAL
AUTOPHAGY).
2) RESPONDING TO STRESS (INDUCED AUTOPHAGY).
--> RAPID ELIMINATION OF WHAT STRESS HAS DAMAGED (PROTEIN
AGGREGATES OR
MALFUNCTIONING ORGANELLES).
--> ELIMINATION OF FOREIGN BODIES WITHIN THE CELL (INFECTION CONTROL).
--> RECYCLING MATERIAL OBTAINED FROM DEGRADATION FOR USE IN NEW
SYNTHESIS IN
EMERGENCY SITUATIONS (NUTRIENT OR ENERGY SHORTAGES).

TO RESUME: DIFFERENCES BETWEEN APOPTOSIS, NECROSIS AND AUTOPHAGY